Oryzae, R. Solanacearum, and Pectobacterium Parmentieri That Carried Up To 13, 12, and 13

ABSTRACT
GÓMEZ ANDREA. Abundance Diversity and Function of Rhs Polymorphic Toxins in

Plant Pathogenic Bacteria (Under the direction of Dr. Alejandra Huerta).
Competition for limited resources and space is an activity between and among populations
of the natural world, including phytopathogenic bacteria. Polymorphic toxins (PT) are
proteinaceous multicomponent toxins produced by bacteria and used as weapons to kill or
inhibit the growth of neighboring competitor cells. Among these PT, Rearrangement hot
spot toxins (Rhs-toxins) are distinguished by their ubiquity in bacterial genomes, and they
are hypothesized to play an important role in bacterial communities dynamics across
diverse ecological niches. The research presented here aims to establish a comprehensive
database on the abundance, diversity, and potential functions of Rhs-toxins in four major
plant pathogenic bacterial genera: Xanthomonas, Ralstonia, Pectobacterium, and Dickeya.
Within the four genera tested, almost all species carry at least one Rhs-toxin in their
genome and have a variety of predicted functions. A small percentage of species did not
code for any Rhs-toxins in their genome. The majority of species carried anywhere between
three to five toxins in their genomes with the exception of some strains in Xanthomonas
oryzae, R. solanacearum, and Pectobacterium parmentieri that carried up to 13, 12, and 13
Rhs-toxins respectively. Furthermore, we found that some Rhs-toxins are shared among
strain in a species, between species, and even between genera. Understanding the ecological
implications of Rhs- toxin medicated competition in phytopathogenic bacteria offers
promising insights into bacterial survival strategies in their natural microbiomes and get us
one step closer toward innovative strategy for plant disease management.
© Copyright 2023 by Andrea Gómez
All Rights Reserved

Abundance Diversity and Function of Rhs Polymorphic Toxins in Plant Pathogenic
Bacteria
By
Andrea Gómez Cabrera
Thesis submitted to the Graduate

Faculty of North Carolina State
University in partial fulfillment of
the requirements for the degree of
Master of Science
Plant Pathology
Raleigh, North Carolina

2023
APPROVED BY:
Dr. Alejandra Huerta Dr. David Ritchie

Chair of Advisory Committee
Dr. Ignazio Carbone

DEDICATION
This work is dedicated with immense love to my grandmother, Yolanda, or as called her, "Mami
Yoli." She raised me, she waited for me every day to come home after school. She is the hands
that fed me and cared for me when I was sick. She is a part of the beauty in me and in so many
memories. She is the birds, she is the sea, she is a cup of cinnamon tea with a soft voice saying
"China quieres té de candela?”. She is in the picture on my desk, she is the necklace that I wear
when I need luck on an important day. My grandma passed away in April 2020 as a result of
COVID-19, and since that day, nothing has been the same. Every action, every goal, and my entire
future is driven by my desire to make her proud and happy. Life is unpredictable, and things change
all the time, but the love and memories we have for our loved ones are something that life cannot
take away from us. Before I left home to pursue my studies in the United States, I received her last
blessing and heartfelt hug. The last words she said to me, are: "Yo lo único que le pido es que siga
estudiando y que siempre agradezca a Dios por las buenas oportunidades que le brinda, con eso
yo soy feliz." ("All I ask of you is to keep studying and always thank God for the good opportunities
He provides; that will make me happy."). I remind myself of this every day.
I want to express my gratitude to God for giving me the gift of life and the privilege of growing
up in my beautiful country, Ecuador. I'm thankful for the chance to have known my grandmother
and be raised by her. My dear Mami Yoli, you live in my heart and my memories. I hope someday
we can meet again in a beautiful garden filled with jasmine flowers and birds.
To my mother Hercilia, who has always sacrificed everything she has for my well-being and
happiness, thank you for always making me feel that I can achieve everything I want and
supporting me in all my big dreams.
To my father Javier, who keeps me close to home through a phone call and constantly reminds me
how proud he is of me.
To my beloved sisters, Diana, and Estefania, the greatest sources of joy and pride in my life.
To my partner, Roshni, who has supported me with love and care through the challenges and
triumphs of this degree, meeting you has been my biggest blessing. Thank you to the Panwala
family for giving me a second home in this country.
To my guiding light, Dr. Alejandra Huerta, who believes in me and has been my constant support,
from the very first day of this scientific journey.
To my friends Kalina, Valeria, Katie, Ying-Yu, and Anna, for their true friendship that keeps me
grounded.
I want to express my heartfelt gratitude to all of you for your support, patience, guidance, and love.
ii
BIOGRAPHY
Andrea Mariel Gómez Cabrera was born in Guayaquil, Ecuador, raised by her mother, Hercilia,
her grandmother, Yolanda, and her older sisters, Diana, and Estefania. She developed a love for
agriculture at a young age, living on a banana tree plantation for the first eight years of her life.
Her father, who is an agricultural engineer, taught her the significance of this profession and its
wide-reaching impact. Andrea earned a scholarship from the Ecuadorian government to pursue her
Bachelor of Science degree in Agricultural Engineering at Zamorano University, Honduras in
2020. In her last year of undergraduate she completed an internship with Dr. Robert Beckstead's
lab at NC State University. There, she developed an interest in microbiology studying the vertical
transmission of the protozoan, Histomonas meleagridis, the cause of histomoniasis (blackhead) in
turkeys. Her experience as an intern in poultry health and a general plant pathology course led
Andrea to decide to pursue a master's degree in plant pathology at North Carolina State University
under the guidance of Dr. Alejandra I. Huerta. As a master's student, she has led important work
on characterizing Rhs-toxin abundance, diversity, and function within four of the most important
bacterial plant pathogens. Her work has generated multiple hypothesis and will be the base for
many research projects to come.
iii
TABLE OF CONTENTS
LIST OF TABLES ......................................................................................................................... v

LIST OF FIGURES....................................................................................................................... vi
LIST OF SUPPLEMENTAL FIGURES ..................................................................................... viii
Chapter I: Review of the Literature ........................................................................................... 1

Introduction .................................................................................................................................... 1
Ecology of foliar bacterial plant pathogens .................................................................................... 3
Ecology of soil borne bacterial plant pathogen .............................................................................. 5
Bacterial competition ..................................................................................................................... 7
Rhs-toxins and their delivery into target hosts............................................................................................... 8
Function of Rhs-toxins domains .................................................................................................. 10
Cited literature .............................................................................................................................. 12
Chapter II: Abundance and diversity of Rhs-toxins in four genera of plant pathogenic
bacteria ........................................................................................................................................ 21
Abstract ........................................................................................................................................ 21
Introduction .................................................................................................................................. 22
Materials and methods ................................................................................................................. 26
Results and discussion .................................................................................................................. 28
Cited literature .............................................................................................................................. 55
Chapter III: Concluding Remarks and Future Studies on Rhs-toxin Diversity and Function
in Four Genera of Plant Pathogenic Bacteria ........................................................................... 61
Cited literature ............................................................................................................................... 66
Appendices ................................................................................................................................. 205

Appendix A ................................................................................................................................. 206
iv
LIST OF TABLES
Table 1 Summary of Rhs-toxins dataset used for abundance and diversity study for four plant
pathogenic bacterial genera ...................................................................................... 69
Table 2 Bacterial genomes used in this study ........................................................................ 70
Table 3 Strains represented in this study with their non-curated Rhs-toxin repertoire.......... 81
Table 4 Strains represented in this study with their curated Rhs-toxins repertoire ............... 92
Table 5 Plant pathogenic genera and their species represented in this study with their
complementary N-terminal clade ........................................................................... 102
Table 6 Rhs-toxin protein length with their corresponding toxic domain and putative
predicted function. .................................................................................................. 122
v
LIST OF FIGURES
Figure 1.1 Organization and structure of a Rhs-toxin system in bacteria ................................ 173
Figure 1.2 Mechanism of Rhs-toxin delivery by the Type 6 secretion system ........................ 174
Figure 2.1 Abundance and diversity of Rhs-toxin sequences in plant pathogenic Xanthomonas
.................................................................................................................................................. .175
Figure 2.2 Extensive variation in protein sequence length among Rhs-toxin repertoires within
Xanthomonas genomes ........................................................................................... 176
Figure 2.3 Nine conserved Rhs-toxin system loci in genomes of Xanthomonas species ........ 177
Figure 2.4 A total of 181 unique Rhs C-Terminal tips in Xanthomonas species ..................... 178
Figure 2.5 Unique, general, and large unknown functional diversity among Rhs-toxins in
Figure 3.1 Abundance and diversity of Rhs-toxin sequences in plant pathogenic Ralstonia
......................................................................................................................................... .….….180
Ralstonia genomes.................................................................................................. 181
Figure 3.3 Three conserved Rhs-toxin system loci in genomes of Ralstonia species.............. 182
Figure 3.4 A total of 76 unique Rhs C-Terminal tips in Ralstonia species.............................. 183
Ralstonia species .................................................................................................... 184
Figure 4.1 Abundance and diversity of Rhs-toxin sequences in plant pathogenic Pectobacterium
................................................................................................................................................... 185
Pectobacterium genomes ........................................................................................ 186
Figure 4.3 Five conserved Rhs-toxin system loci in genomes of Pectobacterium species ...... 187
Figure 4.4 A total of 106 unique Rhs C-Terminal tips in Pectobacterium species .................. 188
Pectobacterium species .......................................................................................... 189
vi
Figure 5.1 Abundance and diversity of Rhs-toxin sequences in plant pathogenic Dickeya .... 190
Dickeya genomes .................................................................................................... 191
Figure 5.3 Three conserved Rhs-toxin system loci in genomes of Dickeya species................ 192
Figure 5.4 A total of 46 unique Rhs C-Terminal tips in Dickeya species ................................ 193
Figure 5.5 Unique, general, and large unknown functional diversity among Rhs-toxins in Dickeya
species..................................................................................................................... 194
Figure 6 Rhs-toxin abundance and diversity across species and genera of plant pathogenic
bacteria ................................................................................................................... 195
vii
LIST OF SUPPLEMENTAL FIGURES
Figure 2.1 Abundance and diversity of Rhs-toxin sequences in plant pathogenic Xanthomonas...
................................................................................................................................ 196
Figure 3.1 Abundance and diversity of Rhs-toxin sequences in plant pathogenic Ralstonia .. 198
Ralstonia genomes .................................................................................................. 199
Figure 4.1 Abundance and diversity of Rhs-toxin sequences in plant pathogenic Pectobacterium
................................................................................................................................ 200
Pectobacterium genomes ........................................................................................ 201
Figure 5.1 Abundance and diversity of Rhs-toxin sequences in plant pathogenic Dickeya .... 202
Dickeya genomes .................................................................................................... 203
Figure 6 Example of Rhs-toxin flanked by transposases ...................................................... 204
viii
Chapter I: Review of the Literature
Introduction
Bacteria are everywhere, and they can come together to establish a resilient microbial
community in the phytobiome. In these complex microbial communities, bacterial cells can
perform different roles and establish unique relationships with their surrounding biotic and
abiotic environment [1,2]. The individual needs of a species can dominate a microbial
community if the species is environmentally fit and there are environmental conditions that
support that species. Both beneficial and harmful interactions occur among and between bacterial
cells in these microbiomes [3]. For example, Yurtsev et al. (2016) demonstrated that two E. coli
strains can form a symbiotic mutualistic relationship and protect each other from ampicillin and
chloramphenicol inhibition, while Koskiniemi et al. (2013) revealed that Dickeya dadantii cells
can inhibit the growth of neighboring cell using proteinaceous toxins [4,5,6]. A diversity of
antagonistic relationships have been reported for multiple bacterial species in natural
environments such as soils, rhizospheres, and plant tissues [7,8,9,10]. These interactions drive
bacterial evolution and fitness [11].
Horizontal gene transfer (HGT) is one of the evolutionary mechanisms that bacteria employ to
acquire new genetic material through three principal modes: 1) conjugation, when bacteria
transfers genes directly to another cell, 2) transformation, when bacteria take up DNA from their
surroundings; and 3) transduction, when a bacteriophage injects DNA directly into a host
bacterial cell [12]. During conjugation, mobile genetic elements play an important role in the
integration of the new DNA fragments into the host genome, enhancing the transfer of DNA
inside the same cell or between cells [13]. Incorporation of new DNA into a bacterial genome
1
can expand a bacterial cell's metabolic capabilities and influence their survival through the
acquisition of fitness factors unique to a specific ecological niche [14,15].
As previously mentioned, bacteria can engage in competitive interactions with cells
from the same species, different species, and even different genera via the delivery of
inhibitory molecules such as antibiotics, bacteriocins, and PT. The secretion of these toxins is
facilitated through various bacterial mechanisms. However, their size and function ultimately
determine the mechanism of secretion [16,17]. For PT, -like Rhs-toxins, the type six
secretion system (T6SS) have been shown to play an important role in the secretion [18].
Rhs-toxins typically consist of highly conserved N-terminal regions responsible for toxin
translocation from the producing strain to the target strain. This region is often conserved
among genera and determines Rhs-toxin families [19]. A variable C-terminal tip forms the
second part of the polymorphic Rhs-toxin and contains the killing toxin domain, which is
hypervariable and can vary among and between strains, species, and genus [17]. Each C-
terminal tip toxin has a highly specific cognate immunity protein directly downstream of the
C-terming tip open reading frame, this protects the producing cell from self- killing and
guards against other bacterial cells that might carry the same toxin tip [20]. Meaning that the
positive selection of this toxin-immunity system is likely an outcome of cell-to-cell
competition (Figure 1.1) [19]. Genome mining studies from Zhang et al. in 2012
demonstrated that Rhs-toxins are abundant and diverse among bacteria and that the C-
terminal domains can be shared even between distantly related strains, suggesting that these
toxins may have evolved from an ancient reservoir of shared domains [20]. Multiple studies
have explored the abundance, diversity, and delivery of Rhs-toxins in animal bacterial
2
pathogens, but little is known about their importance and abundance in plant pathogenic
bacteria in agricultural ecosystems [21,22,23,24].
Ecology of foliar bacterial plant pathogens
A diversity of microorganisms typically inhabit the above-ground parts of plants, with bacteria
being the second most predominant microbe in the phyllosphere, preceded only by bacteriophage
[25,26]. Foliar bacteria can originate from soil, seeds, and air before adapting to life within the
leaf tissue [27]. Various environmental factors, including UV light, water, and nutrients, can
influence their community interactions at the genus and species levels [27, 28,29]. A previous
study by Massoni et al. (2020) suggested that the aerial parts of plants are dominated by bacteria
with evolutionarily conserved traits that can quickly and successfully adapt to abrupt
environmental changes, both biotic and abiotic [30]. Certain species of foliar bacteria can modify
their surrounding environment to enhance nutrient uptake or can develop endospores to protect
themselves against harsh environmental conditions [30, 31]. Many bacteria can adhere to
surfaces, leading to the formation of bacterial biofilms over time [32]. These biofilms are known
to protect bacterial cells from external factors, host defenses, and the effect of antimicrobial
compounds [33,34]. Leaf architecture and morphology can protect bacterial cells and
communities through rain water accumulation on the leaf surface. These tiny water bodies on the
leaf surface can provide cells with nutrients and water for their survival. Water not only provides
bacteria with life but also induces their dispersal, leading to bacterial cells in more
microenvironments ready to cause disease under favorable environmental conditions
[35,36,37,38].
3
One bacterial genus that displays a phyllosphere lifestyle is Xanthomonas, which include
many bacterial species that are plant pathogens and causal agents of bacterial spot and bacterial
blight disease on more than 400 different crops, including citrus, tomato, rice, pepper, beans,
peaches, and ornamental plants [39,40]. Xanthomonas spp. are widely distributed throughout
different geographical regions across the world [41]. The genus was first described and named
Bacterium vesicatorium by Doidge in 1914 when it was first isolated on tomatoes in South
Africa [42]. The primary dispersal mode for these pathogens are through seed in nursery
production facilities, where high temperatures, humidity, plant density, and excessive irrigation
create an ideal environment for their pathogen growth and transmission [40,41]. Xanthomonas
can survive epiphytically in the phyllosphere for extended periods of time, and under the right
circumstances form latent infections, promoting the spread of the pathogen [42,43,44].
Ecology of soilborne bacterial plant pathogens
Soil ecosystems house a large diversity of organisms including bacterial, fungi, nematodes, and
viruses. A single gram of soil can contain up to 10 billion bacterial cells with a species
diversity of 4,000 to 50,000 species [45]. It is estimated that there are approximately 1.5
million fungal species globally and populations in soil can vary from 1 to 10 million individuals
per square meter [45,46]. Moreover, it has been estimated that one gram of soil may harbor
anywhere from 10 million to 1 billion virus particles [47]. Many of these microorganisms can
be found in both the rhizosphere and bulk soil. The bulk soil encompasses the area surrounding
the rhizosphere, and the microbial communities found here tend to be more generalized and less
specialized to those found in the rhizosphere endophytically. The rhizosphere is a narrow soil
region between the plant roots and the bulk soil that is highly influenced by plant root exudates.
Within the rhizosphere, specialized plant-associated microbes like Bacillus, Azotobacter, and
4
other nitrogen-fixing bacteria are abundant [48]. These bacteria possess the ability to capture
inorganic nitrogen and convert it into a biologically available form that plants can absorb and
utilize for their growth. In exchange, the bacteria receive carbohydrates and other essential
nutrients from the plant, this mutually beneficial relationship enhances plant growth and
nutrient cycling in the soil [49, 50, 51].
Not all interactions are beneficial, some plant pathogenic bacteria can overwinter in bulk soil,
often associated with crop residue, and under favorable environmental conditions and a
susceptible plant host colonize the rhizosphere and host. These bacterial pathogens can often
overcome plant defenses and colonize the host, establishing a parasitic relationship that
ultimately leads to plant death [52]. Soil-borne bacterial plant pathogens are diverse and depend
on unique mechanisms to infect their host, employ an array of survival strategies, and can adapt
to changes in soil conditions or chemical applications [53,54]. Dissemination of soilborne
bacterial pathogens, includes seeds movement, contaminated tools, irrigation water, trade [55].
Ralstonia solanacearum, the causal agent of bacterial wilt, is one of the most destructive
soilborne bacterial plant pathogens worldwide. The disease was first reported in Brazil in 1922
[56]. A century later, this pathogen is still recognized as a major threat to agricultural
production systems largely due to its wide geographic distribution and large host range. among
its most significant hosts are solanaceous, leguminous, and tree species [57]. Under high
humidity, warm weather, acidic soils, and a susceptible host this pathogen infects and
multiplies in the vascular tissue of the plant, blocking the transportation of water and nutrients
through the xylem resulting the symptoms that gives it its name, wilt [58,59,60,61].
Bacterial wilt is a challenging disease to manage. This is largely due to the pathogen's
ability to persist in the soil for extended periods of time and genotypic diversity [62]. Cells of
5
the pathogen can be found in aquatic systems for several years after its introduction [63].
Furthermore, there is no chemical or biological treatment for the disease [54]. Instead,
traditional cultural practices can be applied to manage this disease including crop rotation with
non-hosts and amending the soil to disfavor bacterial viability [64, 65]. The strains in the R.
solanacearum species complex (RSSC) are categorized into different taxonomic levels such as
species, phylotypes, sequevars, races, and biovars. The most recent classification divided the
species complex into three different species: R. solanacearum, R. pseudosolanacearum, and R.
syzygii [63,64]. Prior to this designation, strains were grouped into four phylotypes (phy) (I, II,
III, and IV) which roughly correlated to a strains geographic origin, Phy II the America, Phy I
Asia, Phy III Africa and Phy IV Indonesia.
Pectobacterium spp. and Dickeya spp., are also soilborne plant pathogens that infect
multiple tuberous crops, leading to soft rot and wilting of plants [66]. Pectobacterium
carotovorum subsp. carotovorum (Pcc) is known for having the broadest host range among the
seventeen species in the genus, however, potatoes are their primary host, and when infected,
potato plants can show wilt, soft rot, and necrotic stem symptoms. [67] Soft-rot symptoms in
tubers are frequently observed after harvest, especially when tubers are bruised or damaged
during storage, these wounds act as infection courts for PCC to enter the host. Moreover,
favorable conditions like elevated moisture levels and moderate temperatures helps with the
dispersion and persistence of this pathogen. [68,69]. Like Pcc, Dickeya spp. also cause soft-rot,
stem wilt, and blight diseases in potatoes, carrots, and cabbage. Infections by D. dadantii can
produce localized symptoms consisting of blotches, blight, and rotting on multiple plant tissues,
including tubers, stems, leaves, and flowers [70]. Pectobacterium and Dickeya are frequently
6
encountered together in agricultural fields, and their co-infection can have a synergistic effect
on the host. In 2021, Hao et al. observed that when D. dianthicola and P. parmentieri were co-
infected, it resulted in heightened disease severity for black leg disease and an increased
occurrence of soft rot on tubers [71,72].
Bacterial competition
Bacteria have multiple mechanisms that enable them to detect friends and foes within their
ecological niche (e.g., microbiome), Bacterial competition can be categorized into two primary
types: exploitative and interference competition [73]. Exploitative competition is dependent on
cells/species ability to deplete available nutrients from the environment thus blocking access to
“invader” cells. Interference competition is when a cell employs antagonistic tactics by
producing a molecule that directly damages the competitor cell. Both competition strategies can
have detrimental effects on their target bacterial host making competition a strong factor
pushing bacteria to evolve a diversity of strategies to outcompete their rivals [21,74 ,75].
Numerous studies have shown that bacterial competition plays a vital role in the proliferation
and survival of bacterial pathogens [18, 76, 77], For instance, Pseudomonas aeruginosa and
Burkholderia cepacia, two gram-negative bacterial pathogens responsible for human respiratory
infections, in vitro studies revealed that B. cepacia isolates could outcompete P. aeruginosa
[78,79]. These two pathogens produce various secondary metabolites that enable them to
compete against each other, including the production of antibiotics, like streptomycin, and
proteinaceous toxins like bacteriocins [80]. Bacteriocins have antibacterial properties, similar to
antibiotics. However, bacteriocins can be produced by both gram-positive and negative
bacteria, and their only difference is that bacteriocins only inhibit closely related strains of
7
bacteria [81,82]. Bacteriocin families can encompass a wide array of proteins varying in size,
mode of action, and specificity [83]. It is predicted that 99% of all bacteria have the capability
to produce at least one bacteriocin, [84]. Similarly, Rearrangement hot spot toxins (Rhs-toxins)
another group of polymorphic toxins are ubiquitous in bacterial genomes but unlike
bacteriocins they are secreted via the T6SS, are not induced by UV irradiation of mitomycin C
[85]. Extensive research is available on the diversity and function of bacteriocins; however,
little is known about the abundance, diversity, and function of Rhs-toxins [83,84,85].
Rhs-toxins were first identified in the 1900s by CW. Hill and colleagues, initially it was
thought that they served as recombination elements in E. coli and named Rhs elements [21,86].
However, Koskiniemi and colleagues elucidated the true function of the Rhs elements to be
proteinaceous toxins used by bacteria for intraspecies competition [6,23]. These toxins have
been shown to correlate with the presence of a vgrG and hcp genes. These two genes linked to
the T6SS, known for its contractile needle capable of puncturing the cell wall of target bacterial
cells, delivering Rhs-toxins that either kill or inhibit bacterial replication [87,88].
Rhs-toxins and their delivery into target hosts
Rhs-toxins are one of many strategies employed by bacteria to compete against closely related
strains in a microbiome [89,90,91]. They have been shown to be translocated from the producing
strain into the target cell via the T6SS [92]. Unlike many bacterial secreted proteins, Rhs-toxins
are surprisingly large with an average sequence length of 1,500 amino acids (aa) long, and are
characterized by a three-component architecture. The initial part of the protein consists of an N-
terminal segment responsible for encoding the protein translocation, adhesion, and anchoring of
the toxin produced. For instance, molecular studies conducted with Aeromonas dhakensis have
8
revealed that when the N-terminal region is deleted there is a disruption in toxin secretion. [93,
94]. The second architectural motif that defines Rhs-toxins is a highly conserved ten-amino-acid
motif that separates the N-terminal from the C-terminal tip. The C-terminal is the third and final
component of the polymorphic protein that codes for a hypervariable region containing the toxic
domain which may degrade DNA, RNA, or proteins [19,95,96,97]. Genes that encode Rhs-toxins
are typically located upstream from an immunity protein that protects the bacteria from
autoinhibition. Occasionally, but not always, downstream of the immunity protein, one or more
orphan modules can be found encoding an alternative toxin domain, along with their cognate
immunity proteins. It is believed that each orphan module carries an alternative toxin domain and
thus may target a a unique strain (Figure 1.1 A) [98, 99, 100]. The structure of these Rhs-toxins
and their numerous homologues in bacterial genomes suggest that Rhs-toxins have a propensity
for genetic recombination, allowing for the replacement of the existing C-termina toxin with an
alternative orphan module. Jackson and colleagues (2009) suggest that this recombination plays a
major role in driving the diversity of Rhs-toxin in bacteria [101,102].
Research shows that polymorphic toxins, which house the unique Rhs-toxin, can be exported
through more than one secretion system [102, 103, 104]. Notably, the Type 6 secretion system
(T6SS) is the only one that has been observed to translocate Rhs-toxins [105]. This secretion
system was originally discovered in Vibrio cholerae and P. aeruginosa [89]. Recent research has
highlighted its essential role in bacterial competition. For example, a competition study involving
Shigella and Salmonella infections in the mouse intestine showed that their interactions rely on a
functional T6SS [106, 107]. This molecular nano weapon comprises a complex assembly of
various proteins, each of them serves a unique and crucial function in facilitating the effective
translocation of the Rhs-toxin through an injectable needle (see Figure 1.2). the protein groups
9
include: a hemolysin coregulated protein (Hcp), which makes up the main tube and is secreted
along with the toxin and protects it from degradations; a PAAR protein, forms the injectable tip
that carries the toxin outside the bacterial cell; and a valine-glycine repeat protein G (VgrG), acts
as a mediator enabling the toxin to move through this Type VI secretion apparatus [108]. Each of
the three protein groups Hcp, PAAR, and VgrG play a significant role in bacteria-bacteria
interactions, as they can be detected in the extracellular environment by bacteria in a microbiome
[109,110]. While multiple studies have been conducted on the diversity and secretion of Rhs-
toxins in bacterial pathogens affecting humans and animals, there is a knowledge gap on the
distribution of Rhs-toxins in plant pathogenic bacteria [22,23,26, 111, 112, 113,114,115].
Function of Rhs-toxins domains
Polymorphic toxins are distinguished by their unique structure, featuring a toxin domain in their
C-terminal tip, that is crucial for competition and works to disrupt biological processes within
target cells. They can carry diverse range of enzymatic activities, including Dnases for DNA
cleavage, Rnases causing RNA breakdown, glycosylases that are capable of disrupting and
modifying nucleic acid bases and peptidases that cleavage vital protein targets in other bacterial
cells [17, 92, 105, 116, 117, 118]. According to bioinformatic analysis from Zhang et al. (2012),
nucleases are the most widespread predicted function among toxin domain of polymorphic toxins
[20]. Previous studies on E. coli strain EC869 have demonstrated the functional activity of a C-
terminal domain encoding a potent Dnase capable of degrading genomic DNAA and killing
neighboring cells [119]. Similarly, Rnases can degrade RNA and inhibit cell growth while
peptidases cleave proteins and disrupt translocation mechanisms [21,92,120,121,122]. A study
performed on a Salmonella enterica Rhs-toxin found that this toxins inhibited protein synthesis in
E. coli through ADP- ribosylation, a chemical modification that can alter the activity and function
10
of proteins in bacterial cells [113,118]. Recently studies have found deaminases encoded in C-
terminal toxin domains, these enzymes can catalyze deamination in nucleic acids or proteins to
make them unfunctional [116,123,124,125]. Interestingly, research indicates that deaminases can
induce mutagenesis in some bacteria and inhibit or kill others [124]. Toxin-domains in
polymorphic toxins groups are diverse and this diversity can be observed even when comparing
closely related bacterial strains, hence the term “polymorphic” [125]. Zhang et al (2012) have
made significant progress in the prediction of numerous toxin domain families. However, the
large abundance, diversity, and predicted function of many C-terminal types remain a challenge.
Many polymorphic toxins, including Rhs-toxins remain unknown. [19,20,96 ,125]. The
abundance, diversity, and function of Rhs-toxins in gram-negative plant pathogenic bacteria,
remain unexplored. To elucidate the role of Rhs-toxins in the ecology and evolution of plant
pathogenic bacteria and the diseases they cause, we first need to understand the abundance and
diversity of Rhs-toxins in the genomes of economically significant bacterial plant pathogens. To
this end, my overarching hypothesis is that soilborne plant pathogenic bacteria exhibit a
higher abundance and greater functional diversity of Rhs-toxins in their genomes
compared to their foliar counterparts. To test this hypothesis, I proposed the following three
major aims:
Aim 1. Determine the abundance of Rhs-toxins in for the following bacterial genera:
Xanthomonas, Ralstonia, Pectobacterium, and Dickeya.
Aim 2. Explore the diversity and predicted function of Rhs-toxins in the genomes of
foliar and soilborne bacterial plant pathogens.
Aim 3. Analyze Rhs-toxins repertoires across the previous mentioned bacterial
genera according to the pathogen lifestyle.
11
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3. Maitra, A., and Dill, K. A. (2015). Bacterial growth laws reflect the evolutionary importance
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Chapter II: Abundance and diversity of Rhs-toxins in four genera of
plant pathogenic bacteria
Abstract
Rearrangement Hot Spot (Rhs) are proteinaceous toxins produced by bacteria that can kill or
inhibit the growth of neighboring competing cells. They are widespread throughout the bacterial
kingdom and play important roles in intra- and interspecies competition. However, little is known
about Rhs abundance, diversity, and function in plant pathogenic bacteria. To this end, we applied
a Hidden Markov Model (HMM) to extract and characterize the Rhs-toxin repertoires in genomes
of foliar and soilborne phytopathogens in: Xanthomonas, Pectobacterium, Dickeya, and Ralstonia.
Among the 343 genomes mined across the four genera, the abundance of Rhs-toxins per genome
varied from 0 to 13, depending on the genera and species. A protein functional analysis of all Rhs-
toxins analyzed in this study identified Rhs-toxins domains clustered into DNases, RNases,
proteases, and deaminases. Interestingly, the killing domain-containing region of some Rhs-toxins,
Rhs-toxin C-termina tips, were shared among and between bacterial genera and species. These
findings provide insights into the distribution and diversity of Rhs-toxins among plant pathogenic
bacteria, which may have implications for understanding niche specification and exclusion of
certain microbes from specific agricultural microbiomes.
Keywords: Soilborne, Foliar, Hidden Markov Model, Rhs-toxins, competition.
21
Introduction
The scientific study of bacterial toxins emerged a century ago from the pioneering research of
Klebs and Löffler in the 1880s. They not only identified the bacterial strain Corynebacterium
diphtheriae but also proposed that this bacterium produced a "poison" responsible for causing
diphtheria disease in humans [1]. Subsequently, Roux and Yersinia supported this hypothesis
by cultivating the bacterium, extracting the toxin, and introducing the toxin into guinea pigs,
which resulted in similar symptoms to those seen in diphtheria patients [2]. Currently, there is a
diversity of bacterial toxins known, each with distinct and some with overlapping mechanisms
of action, including cell membrane disruption, degradation of key molecular components
involved in cell replication, protein synthesis, and DNA replication or stability [3]. One of the
most extensively studied polymorphic toxins are those with an "A-B" architecture, in which two
distinct subunits: "A" and "B" comprise the complete functional unit. The "A" subunit carries
the toxic function of the protein, whereas the "B" subunit determines target cell specificity by
binding to the complementary cell surface (i.e., determines target host). The binding of subunit
“B” to the target cell facilitates the entry of the A-B toxin into the cell, once inside, the "A"
component is released into the target cell cytoplasmic melee to perform its function. Similar
mechanisms of action are observed in the two human bacterial pathogens that cause pulmonary
infections and cholera, Pseudomonas aeruginosa and Vibrio cholera, respectively [3,4].
In 2005, a novel polymorphic toxin, Rearrangement hot spot toxin (Rhs-toxins), was described
in Escherichia coli isolate EC93 [5]. However, back then the function of these genes were
predicted to play a role in genome rearrangement. It was a decade later that Rhs-toxin function
was functionally confirmed in the plant pathogenic bacterium Dickeya dadantii 3937 [6]. Rhs-
toxin mechanism of action was described to be contact-dependent, meaning that inhibition of
the target cell was only achieved when two cells were in direct contact, and termed contact-
22
dependent cell inhibition (CDI). The CDI system, described originally in gram positive
bacteria, works on the basis of a two-partner secretion mechanism in which a subfamily of
proteins named CdiB work with the type 5 secretion system (T5SS) to secrete and translocating
CdiA to the target cell surface. The CdiA protein encodes the toxin domain that inhibits the
growth of target bacterial cells [7]. The CDI is very much like the “A-B toxins” described
above. Unlike the above-mentioned “A-B toxin” systems the polymorphic toxin is followed by
a small protein that protects the toxin-producing cell from autoinhibition [7]. Aoki et al (2005)
showed both in vitro and in vivo that the C-terminal region of the Rhs-toxins identified in D.
dadantii strain 3937 and uropathogenic E. coli strain 536 encode a similar toxin function but
different nuclease toxin domains and that the cognate immunity protein that followed each C-
terminal tip was unique to each toxin domain [6]. Similarly, to the CDI mechanism, the Rhs
protein family is also known for its antibacterial activity, and it can be found across various
bacterial genera [8].
The polymorphic Rhs-toxin system protein family consists of a multi-domain rhs gene that can
encode variable toxin domains. The majority of functional Rhs-toxin system loci in bacterial
genomes have similar structures and architecture. The Rhs-toxin protein has an N-terminal
region characterized by tyrosine/aspartate (YD) peptide repeats that mediate Rhs-toxin-protein
interaction in the cell cytoplasm of the producing strain, enabling the translocation of the toxin
through the inner and outer membrane; and a C-terminal region demarcated by a conserved ten
amino acid peptide motif (PxxxxDPxGL) [8]. Directly downstream of the C-terminal tip there is
a cognate immunity protein that protects the producing bacterium from autoinhibition and
enables killing of competing cells (Figure 1B) [7,9]. Poole et al (2011) demonstrated that CdiA
and Rhs-toxin families share sequence similarity at the C-terminal, implying homology between
23
the toxin domain [10]. Similarly, to the CdiA systems, additional C-terminal and immunity
protein “genes” known as orphan modules can be found directly downstream of the full
polymorphic Rhs-toxin (Figure 1A). Orphan modules can encode for alternate toxin domains,
and thus, expand the target host range for a bacterial cell. C-terminal tips are always paired with
their cognate immunity protein. Despite lacking a complete N-terminal, orphan modules do
include the previously stated ten-amino-acid peptide motif that demarcates the end of the N-
terminal and beginning of the C-terminal tip; however, they do not have the N-terminal
conserved region [5,7]. In vivo studies performed in S. enterica serovar Typhimurium strain
LT2, confirmed that homologous recombination between an orphan module C-terminal tip and a
full polymorphic Rhs-toxin C-terminal tip can lead to an active protein and may provide the
producing strain with a competitive advantage [11]. Like the CDI system that uses the T5SS to
translocate toxins from the producing cell to the target cell, Rhs-toxins can be translocated by
the T6SS [12,13,14]. The T6SS is an expandable apparatus that injects Rhs-toxins directly from
the producing cell into cytoplasm of the target cell, and often referred to as a micro-weapon.
Composed of 13 to 14 core proteins (i.e., TssA, Tssl, Hcp), the T6SS builds a syringe-like
apparatus in a step-by-step manner to deliver and secrete proteins (Figure 1.2) [13,14]. To
facilitate the export of Rhs-toxins through the T6SS an association with a valine-glycine repeat
protein G (VgrG) is required [15]. The VgrG proteins are predicted to serve as molecular
chaperones for Rhs-toxins during the secretion process. Studies performed in D. dadantii, E.
cloacae, and P. aeruginosa have demonstrated that the deletion of the vgrG gene disrupts the
secretion of Rhs-toxins via the T6SS [16,17,18]. Bioinformatic analysis like the one conducted
by Zhang et al (2011) have begun to elucidate the predicted functional diversity of polymorphic
toxin families, from which Rhs-toxins are only one member [19]. Rhs-toxin are predicted to
24
have a diversity of toxic domains embedded in their C-terminal tips, and thus, perform various
functions including forming pores in the cell membrane of target cells, degrading phospholipids,
breaking down DNA, attacking peptide bonds with amino groups or metals ions (zinc, cobalt,
manganese, nickel, or copper) using water molecules to break chemical bonds (e.g. deaminases
and metallopeptidases, respectively), and disrupting key cellular processes like cell growth and
transcription regulations using ADP-ribosylases [15]. However, the function of many Rhs-toxins
remains unknown. More importantly, the role of Rhs-toxins in niche-specific colonization and
microbial structure in human, animal, and agricultural microbiomes remain a mystery.
Advancements in genomic and sequencing technologies have enhanced and broadened the
scope of bioinformatics, empowering researchers to study complex organisms and microbial
communities [20]. These advancements have enabled fast throughput genome sequencing and
their upload to publicly accessible databases, revealing the biochemical diversity, mechanisms,
structure, and predicted functions of toxin molecules. [21]. One of the most accurate methods to
identify toxin families is through sequence similarity analysis which helps determine sequence
homology between a known and an unknown protein sequence [22]. An initial Rhs-toxin
alignment of full sequences allows for the visualization of the conserved N-terminal and the
hypervariable nature of the C-terminal tips, which is one of the main characteristics of
polymorphic toxins [23] After creating an alignment, novel sequences can be detected using
BLAST and SSEARCH, and other probabilistic models like Hidden Markov Models (HMM)
[24,25, 26].
Numerous studies offer insights into the role of polymorphic toxins like those in the Rhs-toxin
family in human and animal bacterial pathogens [23,27,28]. However, there is little information
25
concerning the diversity and function of Rhs-toxins in bacterial plant pathogens, one of the
most destructive agents of plants that limit maximum attainable yields in many crops. Our
research aim is to build a Rhs-toxin user-friendly database through an online portal that can
serve the scientific community to 1) extract and identify Rhs-toxin in their favorite bacterial
genomes and 2) develop a comprehensive understanding of Rhs-toxins in bacteria. Through the
research and tools developed with the data collected and analyzed here, we will better
understand the diversity and function of Rhs-toxins, their ecological role, and evolution. A
long-term goal is to apply the knowledge gained from basic functional studies and ecological
niche theory to develop targeted on-demand disease management tools for biological control
of destructive bacterial pathogens using Rhs-toxins.
Materials and Methods
Data sources
Whole genome sequences for Xanthomonas, Ralstonia, Pectobacterium, and Dickeya genera
were downloaded on June 25, 2023, from the National Center for Biotechnology Information
(NCBI) public genome database (http://www.ncbi.nlm.nih.gov) in GenBank format
(Supplemental Table 1) Additionally, two genomes from Escherichia coli K-12, and P.
aeruginosa PAO1 were also retrieved and used as outgroups in phylogenetic analysis.
in-silico identification of Rhs-toxins
An Average Nucleotide Identity (ANI) analysis for all complete genomes from each of the four
bacterial genera and associated species were generated [29]. The results were used to remove
duplicate bacterial whole genome sequence (WGS) (i.e., 100% similar at the nucleotide level).
Only one representative WGS was chosen at random to mitigate selection bias in Rhs-toxin
26
abundance and diversity analysis [29]. The final WGS used are listed in Supplemental Table 2
with their respective characteristics. A perl script was used to convert nucleotide WGS
GenBank files into protein CDS (Appendix 1). A HMM-based method developed by Ameen et
al. (2021), named Rhs-HMM, was used to extract putative Rhs-toxin protein sequence using a
43 amino acids (aa) sequence that included the conserved 10 aa motif PxxxxDPxGL. All
putative Rhs-toxin sequences were downloaded in FASTA format for each bacterial genus. All
these sequences were passed through the NCBI Conserved Domains database to confirm the
presence of RHS domains [30,31].
Data Curation
All-putative Rhs-toxin sequence files for each bacterial genera were aligned, visually inspected,
and manually curated in Geneious v10.1(www.geneious.com) following published methods
[8,59]. Briefly, protein sequences that only had the 43 aa sequence were excluded from further
study. Custom bash scripts were applied on the final curated Rhs-toxin data files to identify
Rhs-toxin abundance per genome and their protein length (Appendix 1).
N-terminal phylogeny
To determine the number of Rhs-toxin families per genus all Rhs-toxin protein sequences from
each bacterial strain for each genus were aligned and analyzed. The sequence beyond the
demarcated 10 aa motif that demarcates the end of the conserved N-terminal and the start of the
hypervariable C-terminal tip was removed for all sequences for this analysis [8]. Final Rhs N-
terminal aa sequences were aligned using MAFFT [32] and the auto-alignment strategy
implemented in the DeCIFR toolkit (https://tools.cifr.ncsu.edu/mafft). Protein sequence
alignments were visually inspected in Geneious v10.1.3 for the Rhs core domain motifs
27
“RVxxxxxxxG '' which are typically 776-888 amino acids upstream of the PxxxxDPxGL motif
[8]. This 750-900 bp region is considered the “core region” and is housed within the N-terminal
of the Rhs-toxin architecture. Once identified, the core region was extracted and realigned to
infer a maximum likelihood tree using RAxML v8.2.12, based on a GTR matrix-based model of
amino acid substitution rates and 1,000 bootstrap replicates [33]. The phylogenies were
visualized using the upload tree option of the Tree-Based Alignment Selector (T-BAS, version
2.3) toolkit (https://tbas.hpc.ncsu.edu/) [34,35]. The clades for each genus defined a Rhs family.
C-terminal tip putative function determination and clustering
Similar to what was described for N-terminal analysis was done for C-terminal tips, except that
any sequence before the 10 aa motif, PxxxxDPxGL, was removed following previously
described methods [59]. The final curated C-terminal tip aa sequences were aligned and used to
run an all vs all blastP search to create a similarity network analysis (SNA). The SNA was
saved in xgmml format and visualized in Cytoscape v3.3.0. The Pfam database was used to
assign predicted functions to all C-terminal tip sequences in this study [36,37,38].
Results and Discussion
Rhs-toxins in plant pathogenic Xanthomonas
High abundance and diverse Rhs-toxin repertoire
Xanthomonas is a significant genus of plant-pathogenic bacteria that comprises 27 different
species Each species can have multiple pathovars and together cause vascular foliar diseases on
approximately 400 plant hosts, including agricultural crops and ornamentals [39, 40]. As of
June 2023, 509 complete genome sequences from 22 Xanthomonas species were downloaded
from the NCBI public database. These genomes were sequenced using Illumina, MinION, and
28
PacBio technologies [41]. Among these, the majority of closed genomes belong to X.
campestris, X. citri, and X. oryzae. Strains in these three species have been extensively studied
and sequenced since the 1980s due to their economic importance on agronomic hosts [42,43].
X. campestris causes Black rot disease in brassicas, including cabbage, broccoli, brussels
sprouts, and cauliflower. X. citri is the causal agent of citrus canker, affecting important fruit
crops such as orange, lime, and grapefruit. X. oryzae infects rice causing bacterial blight and
bacterial leaf streak on rice, as well as other perennial weeds [44,45,46,47].
To gain a comprehensive understanding of the true abundance of Rhs-toxins in all the
genera studied we had to ensure data accuracy, decrease bias, and eliminate redundancy in
WGS. This was partially accomplished using an Average Nucleotide Identity (ANI) analysis
where identical (100% nucleotide identity), mislabeled, and duplicated genomes were removed
from the original 509 downloaded genomes leaving a total of 207 genomes in the Xanthomonas
database. This was key for our analysis given the recent study that reports that 36% of genomes
from Aeromonas in GenBank were inaccurately categorized or duplicates [48]. Similarly, of the
509 Xanthomonas WGS downloaded from NCBI for this study 59% were duplicates. Prior to
data curation, the Rhs-HMM developed by Ameen et al 2021 retrieved a total of 707 Rhs-toxin
sequences among 20 Xanthomonas species (Supplemental Table 1). Of these, the number of
Rhs-toxin found in one genome ranged from as little as one to as many as 12 toxins. Notably,
34 genomes among X. phaseoli, X. arboricola, X. campestris, X. translucens, and X. vesicatoria
did not have any Rhs-toxin sequences according to our Rhs-HMM (Supplemental Table 3,
Supplemental Figure 2.1). The multiple sequence alignment with the 707 Rhs-toxin sequences
enabled visualization of the variation among Rhs-toxin sequence lengths (Supplemental Figure.
2.2). The majority of sequences consisted of the full Rhs-toxin which is defined as toxins with
29
the N-terminal and C-terminal tip. A second group consisted of the orphan C-terminal tips,
while other groups housed several sequences that had incomplete N-terminals or lacked C-
terminal tips entirely. A small group of sequences consisted of the 43 aa motif only. Among all
the Xanthomonas protein sequences only one, from strain AM6, was larger than 2000 aa.
Xanthomonas strain AM6 had no species designation and was isolated from water in the
Shandong province of China [49]. The NCBI conserved domain database identified a TcdB
pore-forming domain within the sequence, known to play a role as an insecticidal toxin [50]. A
total of 258 of the 604 Rhs-toxin sequences were in the range of 1400-1700 aa long. This is in
accordance with previous research that indicates that full-length Rhs-toxins are approximately
1400 aa an above (as depicted in Figure 1.1 B) [11]. Sequences that were approximately half
the size of a full length Rhs-toxin, in the range 400-700 aa were also found. These sequences
have a shorter N-terminal or C-terminal compared to the full-length toxins. Interestingly, some
of these half size Rhs-toxin sequences are flanked by transposable elements (Supplemental
Figure 6). Short Rhs-toxin sequences ranging from 120 to 350 aa were also identified in this
dataset indicating the presence of orphan Rhs C-terminal tips, however, any protein sequence of
<90 aa was considered inconsequential due to size and removed from further analysis. (Figure
1.1 B, Supplemental Figure 2.2). This analysis further excluded 34 genomes that did not have
any predicted Rhs-toxin in their genomes and eight sequences where the only toxins retrieved
matched the 43 aa Rhs HMM motif. The final curated Xanthomonas genomes dataset included
165 genomes encompassing 19 species.
A total of 604 Rhs-toxins remained in the Xanthomonas Rhs-toxin dataset (Supplemental Table
1 and Figure 2.1). Of these 122 were classified as orphan module C-terminal tips because they
30
lacked a corresponding Rhs N-terminal sequence. X. citri, X. prunicola, and X. oryzae were
found to house the highest number of predicted orphan module C-terminal tips, up to five, while
other species had none (Figure. 2.2). An alignment of the 604 Rhs-toxin sequences across 19
Xanthomonas species revealed extensive diversity in Rhs-toxin architecture and sequence
length supporting the hypothesis that Rhs-toxins are undergoing strong positive selection.
Furthermore, the high number of orphan module C-terminal tips, reservoirs for toxin diversity,
encoded in the genomes of Xanthomonas spp. indicate extensive competition among the species
and enhanced horizontal gene transfer.
Functional diversity deduced from Rhs-toxin N-terminal and C-terminal Clusters.
Rhs-toxins, categorized as polymorphic toxins, consist of two general components. (Figure 1.1
B). The N-terminal facilitates Rhs-toxin secretion from the producing strain to the target strain,
whereas the C-terminal region carries the active toxin domain [19,58]. Given that rhs genes are
predicted to undergo recombination through the exchange of their C-terminal tips, full length
Rhs-toxin sequences were separated into N-terminal and C-terminal sequences. The N-terminal
was used to group conserved N-terminals into families for each genus, and the C-terminal tip to
determine the predicted killing function. These analyses together provide significant insights
into Rhs-toxin functional diversity [18].
The two different 10 aa motifs RVxxxxxxxG and PxxxxDPxGL (Figure. 1.1 B) were
used to extract the Rhs-core sequence from the Rhs-toxin N-terminal. Any sequence that did not
contain these two motifs was excluded from the N-terminal alignment (<700 aa). Ultimately,
405 Rhs N- terminal sequences from different Xanthomonas species, five sequences from E.
coli, and one from P. aeruginosa were used to generate an Rhs-toxin phylogeny (as detailed in
Supplemental Table 5 and represented in Figure 2.3 A). A total of nine distinct clades, named I-
31
IX, were identified (Figure 2.3 B). Each clade contained a unique set of Rhs-toxin N-terminal
sequences that originated from a diversity of strains within the genera, indicating shared
homology in their secretion mechanism. Many studies have demonstrated that Rhs-toxins are
translocated from the producing strain to the target strain through the T6SS with the help of a
chaperone or adaptor protein that enables their translocation [51,52, 53]. Many of the T6SS
chaperone proteins are directly upstream of the Rhs-toxin N-terminal and are currently
categorized with domains of unknown function (DUF). However, these DUF proteins are linked
to VgrG and Hcp proteins, two important architectural components of the T6SS [54]. This
correlation indicates the involvement of the T6SS in the translocation of Rhs-toxins that have a
DUF protein upstream of full Rhs-toxin in Rhs-Toxin system loci.
To better understand the association between DUF proteins in the secretion of Rhs-
toxins, the upstream regions of the Rhs-toxin proteins were examined to enable the
identification of putative chaperone proteins. Intriguingly, each of the nine families defined by
our phylogenetic tree exhibited a unique Rhs-toxin system locus protein arrangement in the
upstream region of the Rhs- toxin (Figure 2.3 B). This leads us to hypothesize that the well
supported clades are also functionally distinct. Clade I has a hypothetical protein between the
Rhs-toxin N-terminal sequence and a tssl protein, known to be required for T6S activity [55].
Clades II and III have IPT/TIG domain containing proteins upstream of the toxin that are
known to play a role in as transcription factors, however, the chaperone toxin immediately
upstream of the Rhs protein and after the IPT/TIG are different [56]. Both clades IV and VI had
the full Rhs-toxin and their cognate immunity protein flanked by transposases, indicating the
potential for genetic mobility and rearrangements. Toxins in Clade VIII are associated with a
Type II secretion system (T2SS) structural protein that was 300 aa upstream of the Rhs-toxin
32
and followed by a predicted chaperone protein, suggesting that Rhs-toxins may be secreted by
more than one secretion system. Clade IX on the other hand is associated with a T5SS
autotransporter protein, known for playing a role in bacterial interactions, cytotoxicity, and
immunoregulation [57]. All of these Rhs-toxin system loci architectures hint at the importance
the environment may play in regulating the expression and release of Rhs-toxins, future work
on genomic syntony and functional studies will further elucidate the role of these protein in the
secretion and translocation of Rhs-toxins. Steele and colleagues (2021) discovered a group of
Rhs-toxins carried by Apibacter spp. were closely associated with proteins of the Type IX
secretion system (T9SS) and autotransporters. However, functional studies have not confirmed
this observation. This is further evidence that Rhs- Toxins may have alternative secretion and
delivery mechanisms to the T6SS [58]. Despite the diversity in predicted delivery strategies
observed in Xanthomonas spp., the exact mechanisms behind Rhs-toxin translocation remain
unknown, and only future functional characterization will help elucidate if and how Rhs-toxins
reach their target cell.
Rhs C-terminal tip killing domains in Xanthomonas species.
To comprehend the diversity of Rhs-toxins C-terminal tips and their killing mechanisms we
focused on the C-terminal tips, which harbor the toxin mode of action in this family of
polymorphic proteins. First, to identify homologous C-terminal tips, a total of 604 Rhs-toxin C-
terminal sequences with their 10-amino-acid peptide motif "PxxxxDPxGL” were used to perform
an all-vs- all BlastP search. C-terminal tips were considered homologous with members of the
same clade with strong bootstrap support. A total of 181 unique C-terminal tips, named rhs1,
rhs2, rhs3…rhs181, were identified within the genus. Of these 64 unique C-terminal tips formed
33
clusters that encompassed X to two tips sequences and the remaining 42 were singletons. Each
of the 64 clusters housed a unique toxin domain family and for the 42 singletons, no toxin
domain was found. Across bacterial species, numerous full-length Rhs-toxins C-terminal tips
shared homology with C-terminal tips from orphan modules (Figure. 2.4 B). These findings
suggest that Rhs-toxin diversification through C-terminal tip displacement is a phenomenon
occurring across different bacterial species in the genus and probably across genera in a
microbial community.
To explore the functional diversity of Rhs-toxin in Xanthomonas we used the C-terminal tip
sequences, known to carry the toxic domain, and subjected them to a blast search against the
Pfam database [59]. Out of the 604 C-terminal tip sequences, only 173 were successfully
recognized by Pfam and assigned a toxin domain/function. No predicted function was predicted
for the remaining 431 C-terminal tips (Figure 2.5). To determine if species, pathovar, or plant
host contributed to C- terminal sequence clustering a similarity network analysis (SNA) was
performed and revealed 96 cluster and 85 singletons (Fig. 2.4 B). The Pfam clusters correlated
to the C-terminal-tip unique Rhs-toxins, rhs1-rsh181. As expected, C-terminal tip sequences
clustered together based on shared toxin domains, and thus, function. However, our data
revealed instances where C-terminal tip sequences had the same predicted function and may
carry the same predicted toxin domain but were not homologous as seen with clusters l, 27, and
37. Each of those cluster are composed of sequences that encode predicted Tox-REase-7 toxin
domains. This domain is known to be able to identify and cleave DNA at specific target
sequences [23]. However, upon aligning one representative sequence from each cluster, they
were found to share <40% identity (Figure 2.5). We hypothesize that despite sharing the same
34
toxic domain, these sequences might employ distinct mechanisms of action or target specific
molecular pathways, just like the plant-microbe T3SS effectors proteins can be redundant, so
can the Rhs-toxins.
Various toxin domains were found to share a similar function, such as DNases, Rnases,
proteases, and deaminases, and different Rhs-toxin C-terminal tips have been found to be shared
among different species in the genus. For example, toxin domains MafB19-deam and Xoo_2897-
deam are only found in Rhs C-terminal tips rhs4 and rhs12, and their respective clusters: 4 and 12.
The sequences that make us these clusters originated from genomes in X. translucens and X.
oryzae, both of which are pathogens of cereals; wheat and rice, respectively. Clusters rhs1, rhs2,
rhs5, and rhs7 encompassed C-terminal tip sequences from genomes of four species. In contrast,
some clusters were more species-specific; like Cluster rhs4, containing 17 full-length Rhs-toxin
from X. oryzae and X. translucens, both of which infect cereals. Among these 96 clusters, 40
contain anywhere between six to twelve Rhs-toxin C-terminal sequences from different genomes,
while 27 clusters housed only three to four Rhs-toxins from different genomes. Interestingly, 26
clusters were unique to just two species within the Xanthomonas genus, X. euroxanthea, X. oryaze,,
X. translucens, X. sacchari, X. campestris, X. axonopodis, X. fragarie and Xanthomonas sp.
(Figure 2.4 B). Notably, some clusters were exclusively comprised full length Rhs-toxins, like
rhs2, rhs4, and rhs12. Other smaller and less common clusters exclusively contained orphan C-
terminal tips, like rhs18, rhs19, and rhs30. Additionally, we noticed that some orphan module C-
terminal tips shared homology with C-terminals tips of full length Rhs-toxin sequences, clusters
rhs1, rhs7, rhs8, rhs11, among others. This means that the same full length Rhs-toxins C-terminal
tip and orphan modules C-terminal tip can be found in various genomes of different strains across
different species as a full toxin or orphan module (Fig. 2.4 B, Supplemental Table 6). This data
35
supports the natural occurrence of C-terminal displacement given that the same Rhs C-terminal
can be found linked to full Rhs-toxins and as orphan C-terminal tips.
Rhs-toxins of plant pathogenic Ralstonia
Highly abundant and diverse Rhs-toxin repertoires
Ralstonia is a genus of gram-negative bacteria commonly found in soil and water sources in
warm temperate regions. Currently, six species have been identified within this genus: R.
insidiosa, R. mannitolilytica, R. pickettii, R. pseudosolanacearum, R. solanacearum, and R.
syzygii. While most Ralstonia species are commonly found in soil and considered environmental
microbes, R. solanacearum, R. pseudosolanacearum, and R. syzygii are known destructive plant
pathogens and are the causal agents of bacterial wilt on over 200 plant species. Economically
significant hosts of these pathogens are tomato, eggplant, pepper, banana, potato, and peanuts, as
well as various ornamental plants and trees [60, 61, 62]. Some strains of R. insidiosa, R.
mannitolilytica and R. picketii are considered human pathogens and mostly affect immuno
compromised individuals [63].
Because of their soilborne nature and xylem dwelling lifestyle our research objective was to
investigate how pathogen life cycle impacts the abundance and diversity of Rhs-toxins in
various species of Ralstonia. Toward this goal, we downloaded 131 closed WGS. An ANI
analysis excluded 40 duplicated genomes and a total of 338 Rhs-toxin sequences were extracted
from 91 genomes that were mined with the Rhs-HMM. Anywhere between 1-16 Rhs-toxin
sequences were found per Ralstonia genome, with the highest predicted number of Rhs-toxins
found in R. solanacearum strain CFBP2957, a phylotype IIA, sequevar 36 strain isolated from
tomato in Martinique, French Indies [64]. Interestingly, the Rhs model did not detect any full-
36
length Rhs-toxin sequences in the genomes of R. mannitolilytica, R. insidiosa, and R. picketii.
This indicates enrichment of Rhs-toxins in plant pathogenic Ralstonia species compared to the
environmental species. Of note, however, is that the Rhs HMM did capture two sequences in
strain SN82F48 of R. mannitolilytica. One sequence was 27 aa and the other 134 aa long, both
included the 10vaa peptide motif that is characteristic of Rhs-toxins (PxxxxDPxGL), suggesting
that at some point these species may have had Rhs-toxin encoding genes and lost them or have
recently acquired them through horizontal gene transfer.
A sequence alignment allowed visualization of the 338 Rhs-HMM extracted sequences and
revealed that several Rhs-toxin sequences in the dataset had truncated N-terminals, were
missing C-terminal tips, or both, and only consisted of the conserved 43 aa peptide motif.
Furthermore, any predicted C-terminal tip that was <90 aa long was also removed from further
analysis. A total of 294 Rhs-toxin sequences comprised the Ralstonia Rhs-toxin dataset.
Multiple studies have reported that a full Rhs-toxin is 1,400-1,700 aa long [8, 11, 65]. In our
analysis we discovered 1,200 aa long sequences that display a complete architecture and were
also linked to the T6SS. A total of 122 sequences were found to be in the range of 100-350 aa
long. Investigation into the location of these smaller Rhs-toxin sequences in their respective
bacterial genomes revealed that they were positioned downstream of full-length Rhs-toxins.
This observation suggests that these shorter sequences are orphan modules (Figure 1A). A
comparison between Rhs-toxin repertoires in Xanthomonas and Ralstonia, revealed that
Ralstonia genomes had fewer predicted orphan C- terminal tips (Figures 2.2 & 3.2).
Additionally, there were numerous predicted Rhs-toxin sequences that ranged in size between
350-700 aa in Xanthomonas. These sequences appeared to have a truncated N-terminal but
retained the “core region” of the N-terminal and the C-terminal tip. Interestingly these truncated
37
Rhs-toxins were flanked by transposable elements, like clades IV and VI in Xanthomonas,
implying that they could potentially be pseudogenes or undergoing recombination [65]. This
result suggests that Rhs-toxin protein lengths can be extremely variable, and they can differ
from what has been stated in the literature [8, 69].
N-terminal and C-terminal Clusters of Rhs-Toxins
To better understand the functional diversity of Rhs-toxin we divided our data set into a file
with Rhs-toxin N-terminal sequences only and one with C-terminal tips. The N-terminal
sequences, which house the “core region” were used to cluster Rhs-toxins into clades (e.g.,
families) (Figure 3.3 A). The "core" region is defined by Jackson et al (2009) as the hyper-
conserved region within the N-terminal of the Rhs-toxins that is found between two conserved
peptide motif RVxxxxxxxG and PxxxxDPxGL. The latter motif also demarcates the start of the
C-terminal tips. The former can be found 750-900 aa upstream of 10 aa motif [8]. Any sequences
that were shorter than 700 aa from the N-terminal to the 10 aa peptide motifs were removed
based on the observations described above. A total of 114 sequences remained after our final
data curation and were used to build the Rhs-toxins phylogeny of Rhs-toxin N-terminal
sequences for Ralstonia. This analysis revealed three primary Rhs-toxins clades (i.e., families),
outgroup E. coli and P. aeruginosa (Figure 3.3 A). Significant diversity was observed among
and between Rhs-N-terminal clades. Clade I housed Rhs-toxins from genomes that
corresponded to strains of R. solanacearum only, whereas Clades 2 and 3 housed Rhs-toxins
from genomes of R. pseudosolanacearum, R. syzygy, and R. wenshanensis. Furthermore, Clades
1 and 3 were linked to a Vgr and Tssl protein, respectively, indicating an associated with the
T6SS. A correlation was found between Rhs-toxins N-terminal sequences that were in cluster I
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and a DUF1795 protein that was directly upstream of the N- terminal and downstream of the
vgr linked gene (Figure 3.3 B). The correlation, between a DUF and a T6SS was not only found
in this genus but in other genera as well. Like in Xanthomonas we hypothesize that this DUF is
directing the Rhs-toxin into one of the several secretion mechanisms. Unlike clade I, clade III
has a DcrB-related protein upstream of N-terminal sequence and downstream of a Tssl protein
(Figure. 3.3 B). Interestingly, DcrB proteins have been found to participate in phage DNA
transport pathways suggesting that they could also be participating in the transport of Rhs-
toxins in bacteria via an alternative mechanism [66]. Cluster II differs structurally from clades 1
and II in that in the upstream of the N-terminal a gspG protein, known for having an active role
in the Type II secretion system (TIISS), is found along with a protein named Type II secretion
system F family protein. These results support our findings in Xanthomonas above where it is
predicted that the Rhs-toxins in clade VIII are secreted via the T2SS.
To explore the diversity of Rhs-toxin killing modes of action, we conducted an all-vs-all
blastP analysis to identify homologous and unique Rhs-toxin C-terminal tips. A total 294 C-
terminal tip sequences that included the 10 aa peptide motif were used for this analysis.
Sequences exhibiting >85% similarity were designated arbitrarily as rhs-rhs76.) (Figure 3.4 A). A
presence and absence plot of Rhs-C-terminal tips revealed that several C-terminal tip sequences
were shared among and between strain of same species, while 32 C-terminal tip sequences
displayed no similarity to any other sequence in this dataset, and thus, considered Rhs singleton
tips. The C-terminal tips rhs1 and rhs2 were found in 34 of the 86 complete genomes mined.
Interestingly, both toxins were found in genomes from strains from the three plant pathogenic
species (Figure 3.4 A). The C- terminal tip sequence represented in rhs2 was unique to R.
solanacearum strains from R. solanacearum and pseudosolanacearum. Next, we constructed a
39
Sequence Similarity Network (SNA) with the 294 C-terminal sequences to better understand
their clustering patterns. The SNA results confirmed that C-terminal tips sequences were not
only clustering by sequence homology but also their predicted toxin domain. A total of 44 Rhs-
toxin C-terminal tips clusters and 22 C-terminal tip singletons were found. Clusters rhs1 and
rhs2 housed the majority of the C-terminal tips in our dataset and corresponded to genomes from
strain in the solanacearum species. In contrast, clusters like rhs3, rhs6 and rhs9 housed C-
terminal tips from genomes of different Ralstonia species, such as R. solanacearum, R.
pseudosolanacerum, Ralstonia syzygii and Ralstonia sp. Notably, certain clusters exclusively
comprised C-terminal tips from full length Rhs-toxins, as observed in clusters rhs3, rhs10-12,
and rhs14. Smaller and less common clusters exclusively composed from orphan module C-
terminal tip were also observed, like rhs13 and rhs43. Additionally, we noticed like in
Xanthomonas, that C-terminal tip sequences from orphan modules shared homology with C-
terminal tips from full length Rhs-toxin sequences, clusters rhs6, rhs9 and rhs26 (Figure 2.4 B).
This means that the same hypervariable tip of the full Rhs-toxins and orphan modules can be
found among various strains and species (Figure 3.4 B, Supplemental Table 6). These results
provide insight on the diversity of Rhs-toxins in Ralstonia species; however, it also shows that a
soilborne plant pathogen has a lower abundance and diversity of Rhs-toxin compared to a foliar
plant pathogen.
Diversity of Rhs toxin domains and their function in Ralstonia species
A total of 294 C-terminal tip sequences were submitted to Pfam to predict functional domains
that provide insight into function of the Rhs-toxin C-terminal tips in our dataset. A total of 18
distinct toxin domains were predicted for 96 C-terminal tips while the remaining 200 sequences
had no unique protein domain or motif that enable prediction of function. Dnases, Rnases, and
40
Peptidases were among the leading predicted function for the Rhs-toxin C-terminal tips domains
predicted in Ralstonia. Interestingly, the majority of C-terminal tip sequences clustered into rhs2
which encode a GH-E toxin domain that is believed to degrade nucleic acids (Figure 3.4 B). In
addition, clusters rhs3 and rhs5 were found to encode an AHH toxin domain, predicted to also
function as a Dnase. Like clusters rhs11, rhs27, and rhs37 from Xanthomonas that encode a
Tox-Rease-7 domain, without sharing C-terminal tip homology at the aa level, rhs2 and rhs3
from Xanthomonas also share the same toxin domain but not homology. This is further
evidence that different Rhs-toxin can have the same killing domain but are not identical and
may have different target mechanistic targets.
Unique toxins and toxin domains, rhs66 from R. wenshanensis, were also identified
through our analysis. This rhs66 did not share homology with any other C-terminal sequences
in our datasets. According to Pfam results, it encodes a CdiA-CT domain associated with the
contact- dependent growth inhibition (CDI) system. This domain is believed to encode
ribonucleases and when expressed can effectively inhibit the growth of neighboring cells
(Figure 3.5) [67]. Another interesting find was the presence of the peptidase_C80 toxin domain
among various C-terminal tips of different strains of R. solanacearum, cluster rhs;11 (Figure.
3.4 B). These toxins are recognized for containing a cysteine protease domain, which degrades
proteins. Similar domains have been found in toxins from bacterial species such as Clostridium
difficile and Vibrio cholera [68]. These findings shed light on diversity and predicted function
of Rhs-toxins in Ralstonia, however, like in Xanthomonas we were only able to predict function
for <33% of Rhs-toxin being the question: What are the remaining 66% of Rhs-toxins doing?
41
Rhs-toxins of Pectobacterium
Pectobacterium is a destructive plant pathogen that causes significant losses on various
vegetables. There are 18 recognized species in the Pectobacterium genus, of which 14 are plant
pathogens and the remaining four; P. fontis, P. aquaticum, P. versatile, and P. polonicum are
environmental microbes isolated from water sources [69,70,71]. Together the plant pathogenic
species are known as the soft rot Pectobacteriaceae (SRP) and are the most common and widely
studied causal agent of soft rot of potato [72]. The primary virulence factors of SRP are
pectinolytic enzymes that are secreted by the T2SS and degrade pectin in the middle lamella and
primary plant cell walls, which results in soft, wet, rotted, diseased tissue [73, 74]. The T2SS is a
multi-protein two-step molecular machine composed of 12-15 different proteins that translocate
folded proteins from the periplasm, through the outer membrane, into the extracellular milieu of
bacterial cells. In addition to the delivery of proteases, lipases, and carbohydrates-active enzymes
to the extracellular space of Gram-negative bacteria, they also release toxins [75]. The SRP can
cause multiple diseases of potato, including tuber soft rot, blackleg, aerial stem rot, and lenticel
rot and can be a destructive disease at every stage of potato production, including planting,
harvest, transport, and storage [76]. Potatoes are produced by planting tubers from the previous
year’s crop. Vascular pathogens like SRP cannot be excluded from tubers, thus seed potatoes
harvested from infected plants will carry inoculum into the next growing season [77].
Unlike Xanthomonas and Ralstonia, which largely depend on the T3SS to inject effector
protein directly into the plant host cells, Pectobacterium pathogens largely depend on the T2SS
[78]. Given that the T2SS has been implicated in toxin release and they are linked to a few Rhs-
toxin clusters in our study, our research objective was to investigate the presence of Rhs-toxins in
42
various species of the genus Pectobacterium to understand their function and the role of the T2SS
in the secretion of these toxins. Towards this goal, 95 complete WGS were downloaded from
NCBI on June 25, 2023. An average Nucleotide Identity (ANI) analysis identified 33 duplicated
genomes which were removed from further analysis. The Rhs-HMM was able to extract 255 Rhs-
toxin sequences from the 62 mined complete WGS (Supplemental Table 1) [79]. Anywhere
between 1-14 Rhs-toxin sequences were found per genome for this genus, with the highest
number observed in P. parmentieri Strain QK-5, which was isolated from a solanaceous weed in
Poland and deposited in NCBI in 2019. The Rhs-HMM failed to detect any Rhs-toxins in the
genomes of P. punjabense and Pectobacterium spp. (Supplemental Figure 4.1 A and
Supplemental Table 3).
A multiple sequence alignment of 255 Rhs-toxin sequences was used to pinpoint the 10
amino acid motifs that demarcate the N-terminal and C-terminal regions. The alignment enabled
visualization of the variations among Rhs-toxin sequence lengths (Supplemental Figure. 4.2).
Several sequences had truncated N-terminals, lacked a C-terminal tip or consisted of the 43 aa
motif used by Rhs HMM. However, the majority consisted of the full Rhs-toxin or were a
putative orphan module C-terminal tip. To our surprise a few genomes from strains of P.
brasiliense, P. carotovorum, P. odoriferum, P. parvum, P. versatile, and Pectobacterium spp.
encoded Rhs-toxin proteins that exceeded 2000 aa in length. These were similar in size to the one
found in Xanthomonas spp. strain AM6, rhs181 (Figure 2.2, Supplemental Figure 4.2). A NCBI
conserved domain database search on these long Rhs-toxins showed that the additional sequence
also contained the RHS repeats that characterizes Rhs-toxin. In addition to the rare long Rhs-
toxin sequences we also found sequences with the typical length range of 1400-1700 aa
(Supplemental Figure 4.2). Sequences spanning 350-700 aa also appeared in the Pectobacterium
43
dataset displaying truncated N-terminals. Of the total number of Rhs-toxin extracted from the
genomes, 63 are predicted to be Rhs-toxin C-terminal tip sequences, 120-350 aa long
(Supplemental Figure 4.2). Any protein sequence <90 aa long was considered a truncated C-
terminal tip or a Rhs-HMM artifact and removed from further analysis (Figure 1, Supplemental
Figure 4.2).
An alignment of the 255 Rhs-toxin sequences across Pectobacterium species revealed
considerable diversity in sequence length. The average median for Rhs-toxin per Pectobacterium
genome was between two to four, except for P. parmentieri, which had a median of 12 Rhs-
toxins per genome in our study (Figure 4.1). Interestingly, this same species housed the largest
number of predicted orphan module C-terminal tips (Figure. 4.2) while other species carried 0
orphan modules: P. Polaris, P. actinidiae, P. cacticida, P. parvum and P. aquaticum. The
majority of the toxin repertoire in Pectobacterium encoded full Rhs-toxin sequences from 1000-
1700aa (Supplemental Figure 4.2 and Figure 4.2). This result suggests that Rhs-toxin protein
lengths can be extremely variable, and that some species can encode more Rhs-toxins than others,
suggesting suitable fitness for competition.
To better understand the diversity of Rhs-toxins among various Pectobacterium species, the
curated Rhs-toxin dataset for this genus was divided into two files, as described above. The “core
region” of 165 N-terminal sequences was used to generate a phylogenetic tree that identified four
Rhs-toxins N-terminal families for this genus, named family I, II, III, and V (Figure 4.3).
Interestingly, all six clades had similar Rhs-toxin system loci architecture in that upstream of the
full length Rhs-toxin there was a DUF protein associated with the T6SS. For example, Family II
44
and VI had a DUF1795 flanked by the full-length Rhs-toxin protein and a type VI secretion
protein, however a protein alignment showed that these two DUF were not homologues. Family 1
and IV have a PAAR domain upstream of the Rhs- toxin in addition to more protein linked to the
T6SS. Of all the families in Pectobacterium, the Rhs- toxins that were found in clade III had no
correlation to any type of secretion system and were exceedingly long 2000 aa toxins. In the
upstream region of Rhs-toxin in this clade, a ssDNA-binding protein (SSB1) was found. The
SSB1 recognizes and repair DNA damage [78].
To explore the diversity of Rhs-toxin C-terminal tip killing mechanisms we conducted an all-
vs- all blastP analysis as described above with a total of 255 C-terminal tip sequences from 60
distinct genomes of 14 Pectobacterium species. Sequences exhibiting >85% similarity were
designated arbitrarily as rhs1-rhs106 (Fig. 4.4 A) Each identifier represents a different C-terminal
tip sequence. A C-terminal tip sequence presence-absence plot revealed that several C-terminal
tips are shared between Pectobacterium species, while 42 C-terminal tips sequence had no
similarity to any other toxin, and thus, unique and a singleton (Fig 4.4 A). A SNA was built with
the 255 C-terminal tip sequences to better understand the sharing of C-terminal tips among strain
and species in the genus. The SNA results confirmed that C-terminal sequences were clustering
according to their toxin domain. A total of 64 distinct Rhs-toxin C-terminal tip clusters were
elucidated. The most abundant Rhs C-terminal tip was rhs6, with representative C-terminal tip
sequences in genomes from strains in P. quasiaquaticum, P. polaris, P. parvum, P. odoriferum,
and P. carotovorum. Interestingly, this C- terminal tip was only found among one species of the
plant pathogens and was more abundant in the environmental species. Cluster rhs3, rhs7-rhs9,
and rhs11 were formed by C-terminal tips that originated from genomes of four different species
among P. aquaticum; P. atrosepticum; P. brasiliense; P. carotovorum; P, odoriferum; P.
45
parmentieri; P. Polaris; and P. versatile. Twenty- one rhs clusters were composed of 2-3 Rhs
sequences and were dominated by sequences in genomes from a single species (Figure 4.4 B).
Similarly, to Rhs C-terminal tip clusters in Xanthomonas and Ralstonia some clusters in
Pectobacterium also had C-terminal tips associated with both full-length Rhs- toxin and orphan
module C-terminal tips, cluster rhs2-rhs7. (Fig. 4.4 B, Supplemental Table 5).
Predicted function diversity of Rhs toxin C-terminal tips in Pectobacterium species
A blast analysis of 255 C-terminal (CT) sequences against the Pfam database assigned a toxin
domain to 28 of the C-terminal tip rhs clusters from a total of 102 tip sequences while 153 CT
sequences remained undetermined. Putative functions predicted from sequence motifs embedded
in the C-terminal tips include: Dnases, Rnases, peptidases and deaminases (Fig. 4.5). The
majority of species within the Pectobacterium genera encode a GH-E, AHH, and ParB toxin
domains, all described as DNases in the literature [23]. Cluster 11 C-terminal tip sequences were
predicted to encode an unusual Ntox30 toxin domain, which is a known RNase (Fig. 4.4B). Not
surprisingly, a cdiA-CT toxin domain was predicted to be encoded in Rhs-toxin tips that formed
clusters rhs41, rhs49, rhs55, and rhs63 from genomes of P. parmentieri, P. aquaticum, and P.
brasiliense. This toxin domain has the ability to inhibit the growth of neighboring bacterial cells,
a function previously observed in Rhs-toxin of Dickeya dadantii strain 3937, a cousin genus of
Pectobacterium [11]. Another interesting finding is the toxin domain TNT encoded by C-
terminal sequences in P. parmentieri strain HC and P. versatile strain SR1. This toxin domain is
known as the tuberculosis necrotizing toxin and while it is presumed to act as a protease, the
specific mechanisms of action remain unknown [80]. The predicted function for most of the Rhs-
toxin C- terminal tips in Pectobacterium, Ralstonia, and Xanthomonas represent but the tip of the
iceberg with respect to determining their diversity and killing action as only <30% of all
46
identified Rhs- toxin C-terminal tips can be assigned a predicted function and an even smaller
group have been functionally characterized.
Rhs-toxins of Dickeya
The only Rhs-toxins that have been somewhat functionally characterized in plant pathogenic
bacteria are rhsA and rhsB from D. dadantii and rhs12 and rhs14 from R. solanacearum [18,81].
Dickeya spp., like some Pectobacterium, are plant pathogens that cause blackleg and soft rot of
potato. Together, these two soft rotting genera are known as the SRP and their main virulence
strategy is the deployment of cell-wall-degrading enzymes that breaks down the plant cell wall
leading to maceration and rot of plant tissue. This genus of plant pathogens comprises 12 species
that affect various economically important vegetables [82, 83, 84, 85]. As a closely related
pathogen of Pectobacterium, Dickeya species are worthy of study for the same reason described
above for Pectobacterium. Furthermore, D. dadantii strain 3937 was the model strain in which
the true function of Rhs-toxins was functionally characterized. This strain and family are
furthermore in the order Enterobacteria, used as the first model for the analysis of Rhs-toxin
phylogeny and the determination of Rhs N-terminal families (e.g., clades) [8]. Much effort has
been invested in understanding the functional role of Rhs-toxin in human pathogens like
Salmonella, Escherichia coli, Klebsiella, and Shigella. However, little is known about Rhs-toxin
in plant pathogenic bacteria.
To narrow this knowledge gap, we also downloaded 34 complete WGS of Dickeya
species from NCBI (Supplemental Table 1). The ANI revealed four duplicated genomes in this
dataset that were removed leaving a total of 31 unique genomes to run the Rhs-HMM. A total of
144 putative Rhs-toxins were extracted from these genomes, from which we noted that the lowest
47
number of Rhs-toxin sequences found per genome among all strains was two and the highest was
nine. The nine Rhs-toxins sequences were traced back to strain ZXC1 of D. fangzhongdai,
however after further sequence analyses three WGS were removed from further analysis. The
Rhs HMM did not detect any Rhs-toxins in bacterial genomes of D. aquatica, D. lacustris, and
D. poaceiphila (Supplemental Fig 5.1). Interestingly, D. aquatica, D. lacustris were both recently
described as pectolytic bacteria isolated from waterways in France. D. poaceiphila was also
recently isolated from sugarcane and described [86]. Visualization of all the Rhs-toxin for the
genus was used to remove any remaining sequence that could further bias our analysis as
described above. This led to a total of 113 Rhs-proteins being retained. After data curation strains
of D. fangzhongdai, D. dadantii, D. chrysanthemi were found to have the highest number of Rhs-
toxin in the genus, whereas D. zeae, D. dianthicola, and D. parazeae had the lowest number with
2 Rhs-toxin per genome (Figure 5.1). According to the literature, Rhs-toxins typically range in
length from 1,400 to 1,700 aa [8,17]. A total of 77 sequences were in this range for this genus.
Sequences spanning 400- 700 aa were also found, just like they were also found in Xanthomonas,
Ralstonia, and a few Pectobacterium species (Figures 2.2, 3.2, 4.2, 5.2). Unlike the other genera,
Dickeya spp. did not have as many Rhs-toxin sequences in this range. This may be an artifact of
the smaller number of genomes in this dataset, and thus, Rhs-sequences retrieved from them.
Twenty-eight sequences were found to be in the range of 100 to 350 amino acids and further
investigation revealed that they were positioned downstream of full-length Rhs proteins
confirming our hypothesis that these are orphan module-C-terminal tips. (Figure 5.2).
48
To better understand the functional diversity of Rhs-toxins, N-terminal and C-terminal regions
were divided into two files as described above (Fig. 1). Three major families were identified for
this genus and all of them house Rhs-toxins N-terminal tips from all Dickeya spp. (Fig. 5.3A).
Family I was the smallest clade, comprising 6 Rhs-sequences from D. zeae, D. solani, D.
dadantii, and D. dianthicola. Family II had N-terminal sequences from almost all species, with
the exception of D. dianthicola, while Family III was the largest family and exhibits the largest
species diversity. Closer examination of the genomic loci that encompassed the Rhs-toxin N-
terminal sequences for each clade, revealed that all three clades were associated with the T6SS
and had very similar Rhs-toxin system loci (Figure 5.3 B). Despite their association with the
TS6S protein, the predicted DUF chaperon protein that immediately preceded the Rhs
polymorphic toxin differs for the three females. Family I and III encode proteins annotated as
DUF1795, but a sequence alignment indicates no shared homology. This is the same DUF1795
that was found upstream of the Rhs-toxin in Pectobacterium clades II and VI, and clade I of
Ralstonia (Figures 3.3, 4.3, & 5.3). Suggesting that these Rhs-toxin employ the same
mechanisms to exit the producing cell or are subjected to the same environmental stimuli that
triggers expression. In contrast, family II of Dickeya N-terminal sequences encodes two proteins
annotated as DUF4123 back-to-back upstream of the Rhs-toxin. This same locus architecture was
also observed in family IV of Pectobacterium. Despite having the same annotation, they also
differ in their aa identity. These results correlate with similar observations from Rhs-toxins of
Ralstonia and Pectobacterium that are also linked to the T6SS.
49
An all-vs- all BlastP analysis was performed with 113 C-terminal tips to identify homologous and
unique C- terminal tips (Supplemental Table 6). C-terminal tips exhibiting >85% similarity were
categorized into arbitrary groups and designated as rhs1-rhs46 (Fig. 5.4 A). A C-terminal tip
presence-absence plot revealed that several C-terminal tips were shared among Dickeya strains
and species, while 16 sequences were unique, and thus, considered Rhs singleton tips (Fig. 5.4A).
A SNA of 113 C- terminal sequences confirmed the results from the Rhs-toxin C-terminal tip
sequences rhs1-rhs46 to form 30 clusters and 16 singleton tips (Figure 5.4 A&B). Like all the
other bacterial genera investigated here, the same C-terminal tips sequence was found as both
full-length-C-terminal tips and orphan modules C-terminal sequence cluster 1, 2, 4, and 5 (Figure
5.4 B).
Diversity of Rhs toxin domains and their functions in Dickeya species
A BlastP analysis of the 113 C-terminal tip sequences against the Pfam database revealed
13 distinct toxin domains among 63 Rhs-toxin sequences. The predominant toxin domain
among rhs clusters for this genus encoded for DNases and RNases. Most species in the genus
were found to possess toxin domains such as AHH, TOX-HNH-EHH, and NUC, all having the
same predicted function, degrading DNA, (DNases). Notably, Rhs-toxins from D. fangzhondai
and D. Zeae were identified to encode the toxic domain Ntox8, a known RNase. Similar to
Pectobacterium and Ralstonia, we also noticed a CdiA-CT toxic domain in D. Zeae that is
known to be capable of inhibiting the growth of competing bacterial cells.
Koskiniemi et al. (2013) revealed that two out of the three Rhs proteins found in D. dadantii
strain 3937 had the ability to inhibit the growth of neighboring cells. In our in-silico research,
The Rhs HMM identified 6 rhs-toxins in the genome of D. dadantii strain 3937 (Supplemental
Table 3). Out of these, four were predicted to be DNases just like Koskiniemi and colleagues
50
functionally confirmed. (Supplemental Table 6). A putative function was not assigned to the two
remaining C-terminal tips in strain 3937, however, they are assigned a locus tag. One of the Rhs-
toxin sequences, DDA3937_RS06980, was 1,658 aa long. The second was a C-terminal tip
sequence, DDA3937_RS06995, that was 346 aa long. Visual analysis of the locus where these
two proteins are found in the genome confirmed that the former is a predicted full-length Rhs-
protein and the latter the associated orphan module C-terminal tip.
Toxin domains shared among different bacterial genera.
Rhs-toxins are diverse and can recombine within bacterial genomes to form new active full-
length Rhs-toxin. It is predicted that this high functional diversity among Rhs-toxin in bacterial
genera are acquired through HGT among closely related species [8, 87]. Here we show how both
full-length Rhs-toxins and orphan module C- terminal tips sequences encoding specific toxin
domains are shared among strains of the same species and between species. The findings shared
thus far emphasize the complexity and diversity within Rhs-toxin systems and their secretion
patterns across plant pathogenic bacteria. To add to the complexity, we compared Rhs-toxin
repertoires among bacterial strains, from different species, among the four bacterial genera
investigated in this study. A total of 165, 87, 60, and 31 complete genomes from Xanthomonas,
Ralstonia, Pectobacterium, and Dickeya, respectively, were used in our study to look at the
abundance and diversity of Rhs-toxin in plant pathogenic bacteria. From these a total of 1266
Rhs-toxin sequences were analyzed (Supplemental Table 1). Briefly, 604, 294, 255, and 113
Rhs-toxin were found among Xanthomonas, Ralstonia, Pectobacterium, and Dickeya species,
respectively. Most genomes within a species and genera had at least one Rhs-toxin encoded in
their genomes, with some exceptions. There were 34 genomes among five species of
Xanthomonas that our Rhs-model was not able to detect any Rhs-genes of significance;
51
Ralstonia had 4 genomes among three species; Pectobacterium 2, and Dickeya 3 each from a
different species. The majority of genomes that were predicted to have no Rhs-toxin were traced
back to species that are either environmental soil or water bacteria. Furthermore, our data
indicate that Rhs-toxin are enriched in plant pathogenic bacteria, although additional complete
genomes from environmental samples will help support these findings.
The genus Xanthomonas has the highest abundance of Rhs-toxins, but this can be an
artifact of the higher number of complete WGS that are available for some of the species in this
genus. The highest number of Rhs-toxins per genome was found in two strains of P.
parmentieri, WC19161 and QK-5, each encoding 13 putative Rhs-toxins. Through this data set,
we also discovered that nearly 50% of the genomes in each species contained anywhere
between one and three orphan modules, with some exception of some genomes. For example,
strain CFBP 1156 of X. hyacinthi is predicted to encode a total of 10 Rhs-toxins in its genome;
Of these, two are full- length Rhs-toxins (~1400-1700aa), while the remaining eight appear to
be orphan module C- terminal tips (Supplemental Table 3 and Fig. 2.2). This Xanthomonas
pathogen is the causal agent of yellow disease of Hyacinthus, Scilla, and other related
ornamental plants and reported across multiple countries in Europe, Australia, and the USA
[88].
The diversity among Rhs-toxin system architecture and the N-termini that make up the
different clades that are associated with the mode of toxin secretion for these four genera is
extensive. Once again, the N-termini from Rhs-toxins of Xanthomonas exhibit the highest
diversity with nine distinct clades that correlated to unique Rhs-toxin system architecture.
Among the nine different clades identified in Xanthomonas, only Clade I was linked to the
T6SS. This clade was largely made up of N-terminal sequences that originated from X.
52
fragarie, X. translucens, and X. oryzae genomes, the causal agents of an angular spot of
strawberries, bacterial streak of wheat, (and other cereals), and bacterial blight and streak of
rice and other weeds, respectably. Clade II of Xanthomonas was the most diverse, with
representative N-terminal sequences from the majority of species in the genus. This same clade
was linked to genes of the T2SS (Fig. 2.3). The appearance of genes associated with structural
components of the T2SS in Rhs-toxin system architecture was unexpected given the extensive
body of literature that reports the T6SS as the mechanism of Rhs-toxin secretion via contact-
dependent manner [89]. Genes associated with the T2SS were also found in Rhs-toxin system
architectures of Ralstonia. However, the T2SS was not associated with Rhs-toxin systems in
Pectobacterium and Dickeya, plant pathogens that cause soft rot in potatoes (Fig. 3.3, 4.3, 5.3).
As such, these two pathogenic genera rely on the T2SS for delivery of cell-wall-degrading
enzyme, their primary virulence mechanism against their plant host.
A substantial number of C-terminal sequences were identified among the total 1267
C- terminal tips analyzed in this study. These sequences were clustered into 233 groups based
on sequence similarity (85%). Once again, Xanthomonas exhibited the majority of clusters with
a total of 96, followed by Pectobacterium, Ralstonia, and Dickeya with 44, 64, and 30,
respectively. Many of these clusters were composed of sequences that originated from a single
species, a few species, or multiple species such as Ralstonia clusters 5 and 12-15; Dickeya
cluster 1-15; and Xanthomonas cluster 2, 4, and 5, respectively (Fig. 2.4). Pfam was able to
assign functional toxin domain to <30% of all clusters from each genus leaving many C-
terminal tip sequence functions a mystery. Xanthomonas encoded the highest number of
predicted toxin domains, and a significant portion of these domains are unique to the genus. For
instance, the two deaminases named XOO_2897-deam and MafB19-deam, which are
53
exclusively found in the genomes of X. translucens and X. oryzae, two bacterial pathogens of
cereals. Notably, we only detected CdiA- CT domains in Ralstonia, Pectobacterium, and
Dickeya, but not in Xanthomonas. CdiA-CT are dependent on cell-to-cell contact, such as that
enabled by the T6SS, to inhibit the target bacterial cells [11].We observed a consistent trend
among these four genera, where C-terminal tip sequences were shared among different species.
This observation is interesting, if a target cell has a very similar C-terminal tip it is assumed
they also carry the cognate immunity protein to protect them from autoinhibition. This implies
that two cells that carry the same Rhs-toxins will be immune to the killing function for that
toxin. An all-vs-all BLASTP analysis of all 1267 C-terminal tips from all four genera elucidated
four clusters of Rhs-toxin C-terminal tips that are shared between Xanthomonas and Ralstonia
at >50% identity. Ralstonia and Dickeya shared one C-terminal cluster where the sequence
shared over 90% identity. Another cluster made up of C-terminal tip sequences from Ralstonia
and Pectobacterium showed 60% identity. The majority of shared clusters were found between
Pectobacterium and Dickeya, with 19 clusters exhibiting more than 80% identity (see Figure.
6). These results confirm our hypothesis that Rhs-toxins are found in different genera and
species of plant pathogenic bacteria. However, this data rejects my original hypothesis that
soilborne plant pathogens have greater abundance and diversity than foliar pathogens.
54
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60
Chapter III: Concluding Remarks and Future Studies on Rhs-toxin Diversity and
Function in Four Genera of Plant Pathogenic Bacteria
The significant amount of genomic, functional, in-vitro, and in-vivo studies accentuate the
importance of competition mechanisms as a phenomenon that drives evolution at macro and
micro scales across different environments. In response to competitive pressures exerted on
bacteria by both abiotic and biotic factors (e.g., microbes), bacteria have evolved a diversity of
molecular mechanisms to ensure their survival. The diversity of mechanisms a bacterial cell
employs to suppress competitor cell is vast and has been extensively reviewed in the literature
(1,2,3,4). The diversity and redundancy of microbial competition mechanisms provide insight
into the vast number of bacteria and other microbes in a microbial community. This large
microbial diversity in the environment might explain the diversification observed in Rhs-toxins
from pathogenic bacteria in their quest to survive and reach a suitable host. Like the plant-
microbe evolutionary arms-race, bacteria are also in an arms race with other microbes in their
community [5, 6, 7,].
Rhs-toxins have been studied for over 30 years, since CW Hill and colleagues identified
them as accessory elements in E. coli, however, it was only in the last 12 years that in-silico
analysis demonstrated their widespread presence across the bacterial kingdom and their true
function as interspecies bacterial toxins were elucidated [5,8,9,10]. Recent studies have indicated
that deletion of rhs genes are affects a bacterium's competitive abilities, suggesting that these
genes play a crucial role in bacterial fitness [11,12,13].
Rhs-toxin research has centered on bacterial pathogens that infect humans, insects, and
mammals but research on their diversity and role in plant pathogenic bacteria has lagged
61
behind [14,15,16,17]. Many studies that have focused on plant pathogenic organisms have
highlighted the little we know about competition mechanisms in the phytobiome [18,19].
1. What are the relationships between microbes in the soil, rhizosphere, phyllosphere,
flower, and fruit microbiomes?
2. What species/genera act synergistically to outcompete a pathogen or vice versa?
3. How do competition mechanisms influence microbial structure in microbiomes?
4. How can microbial competition mechanism be exploited for pathogen management.
This last one has been a goal for more plant pathologist in the last century with very little
success. To achieve biological control of bacterial plant pathogens, we first need to understand
bacterial ecology at a deeper level. A recent study showed that R. solanacearum strain K60
outcompetes two different strains in from the same genus, one in the same species, strain
UW551, and on from a R. pseudosolanacearum a closely related species in tomato stems and
rhizospheres [20,21]. All three strains are capable of infecting the same host but when together
strain K60 has a colonization advantage compared to the other two in tropical conditions.
Research from Ameen et al. in 2021 investigated Rhs-toxin diversity within the Ralstonia
solanacearum species complex, where they found that strain K60 from Ralstonia solanacearum
encodes a higher number of Rhs-toxins than GMI1000 and UW551. These results correlate with
our findings where K60 encodes nine Rhs- toxins, GMI1000 has 6, and UW551 3, all after
curation. Together these findings suggest that due to a higher abundance and a diverse Rhs-
toxins repertoire, strain K60 has great capability to compete and succeed against other closely
related strains.
62
Rhs-toxin abundance and diversity research was limited and incomplete due to scarcity of
high-quality WGS sequencing. Our data is in agreement with published literature that shown
rhs genes tend to appear at the end of contigs, which might contribute to Rhs-toxin abundance
bias [22]. Thus, a complete WGS is essential to understand the true abundance, diversity, and
function of Rhs-toxins in plant pathogenic bacteria.
In this study we hypothesized that soilborne plant pathogens have a higher abundance and
diversity of Rhs-toxins repertoires in comparison to pathogens that affect foliar tissue. To test
this hypothesis, an in- silico analysis using the Rhs-HMM developed by Yaken et al 2021 was
used to mine WGS of four different bacterial genera: Xanthomonas, a foliar pathogen; Ralstonia,
a soilborne vascular pathogen; Pectobacterium, a soilborne sot rot pathogen; and Dickeya, also a
soilborne soft rot pathogen. The data set included a total of 343 genome from these four genera.
Rhs-toxin abundance comparisons between bacterial pathogens based on soil or foliar lifestyles
show that the foliar pathogen, Xanthomonas, had a higher number of Rhs-toxins in the genus
compared to soilborne pathogens. This evidence rejects our primary hypothesis at the genus
level. However further analysis of this dataset comparing species that infect the same host and
have different lifestyles may indicate differently. Among the genus, there are 21 Xanthomonas
species, each can infect one to multiple hosts. A study from Zhang et al in 2019 revealed that
Xanthomonas is one of the most abundant bacterial genera within the seed microbiome [23]. The
high genome plasticity of Xanthomonas spp. suggesting a higher diversity of competitive
mechanisms suggesting that Xanthomonas as a whole may be interaction with a higher diversity
of microbes under different environmental conditions. The evolution of virulence for
Xanthomonas, like for many bacteria, has been influences by horizontal gene transfer, it is
estimated that 5-25% of Xanthomonas genomes was gained through recombination [24,25,26].
63
This may explain why our data elucidates more sharing of Rhs-toxins gens by multiple
Xanthomonas species [Figure. 2.4, 2.5].
Although Xanthomonas presented a greater diversity and abundance in its Rhs-toxin
repertoire in comparison to soilborne bacterial genera, we show that each genus is functionally
diverse within species and even between strains in a species. Most genomes mined have between
1-13 Rhs-toxins encoded in their genomes with various protein lengths. We observed that the
majority of genomes encode at least one full-Rhs toxin structure that includes the N-terminal and
C-terminal tip. At least 80% percent of genomes housed at least one orphan module C-terminal
tip, with the highest genome displaying eight putative orphan modules. We were surprised to
find that in each bacterial genera, there were genomes that were predicted to not encode any
orphan modules with the methods we used (Table 2). In Ralstonia there were two genomes with
zero Rhs- toxins, R. picketii and R. insidiosa. The two strains in the species are classified as
environmental bacteria are found in soil and water sources. Both have been reported as pathogens
in humans that are immunocompromised [27,28]. Since these two species are not true plant
pathogens and have a different lifestyle than plant pathogenic species, we believe that their lack
of Rhs-toxins is due the lack of competition with other microbes in the niche. Similar results
were found in D. lacustris and D. aquatica. Strain from both species were isolated from water
systems [29,30,31]
Koskiniemi et al in 2013, suspected that bacteria were share Rhs-toxin domains, our findings
confirm that indeed, Rhs-toxin C-terminal tips sequences, and this, domains are being shared
within, between, and among bacterial genera. A SNA shows that Ralstonia and Xanthomonas
share one Rhs-toxin, both bacteria can colonize the vascular tissue and continue their life cycle
inside the plant [32,33,34]. Rhs-toxin sharing was also observed among the soilborne pathogens.
64
Ralstonia shared one Rhs-toxin with both Pectobacterium and Dickeya. While Pectobacterium
and Dickeya shared 38 Rhs-toxins. Predicted function of for some of the Rhs-toxin C-terminal
tips that are shared among bacterial genera include endonucleases: AHH, LHH, NUC, PARB
and GIY-YIG. These toxin domains break the nucleotide chain into two or more pieces
provoking cell damage [33].
The secretion mechanism of Rhs-toxins into target cells or the extracellular millue remains
unexplored. Much of the Rhs-toxin literature links Rhs-toxin secretion to the type six secretion
system (T6SS), but recent studies propose, including us, that they may be secreted through other
mechanisms [8,15]. A of Rhs-toxins in Apibacter spp. found that the N-terminal housed SpvB
and TcdB domains instead of a typical T6SS PAAR domain. Interestingly, SpvB and TcdB
are predicted to be linked with a novel pathway of secretion, the type nine secretion (T9SS). The
T9SS was first reported in 2020 by Jana and colleagues in Bacteroidetes. They found that this
secretion system was exporting polymorphic toxins across the outer membrane of bacterial cells
[15,35]. Similarly, most of the N-terminal sequences that formed the nine clades, families, of
Xanthomonas showed nine different Rhs-system loci architectures, Clade I was linked to
the T2SS. This association between Rhs-toxin and the T2SS was also observed in Ralstonia
with Clade II. Here we propose that Rhs-toxin may be secreted via alternative secretion systems
and not only the T6SS.
The Rhs-toxin polymorphic system is still not completely understood and with every study
more of their diversity and function is known. However, many questions remain unanswered,
specifically those related to the role of Rhs-toxin in microbial community ecology and pathogen
evolution. This thesis is the first step for many more studies to come that precisely study these
knowledge gaps.
65
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Table 1. Summary of Rhs-toxins dataset used for abundance and diversity study for four plant pathogenic bacterial genera.
# of
# of Total # of Total # of
Rhs- Total # Total #
# of # of Rhs- N- C-
Genera toxin of of
Genomes species toxins/ terminal terminal
domains clusters singletons
genome sequences sequences
found
Before data curation
Xanthomonas 207 21 707
Ralstonia 91 8 338
Pectobacterium 62 15 491
Dickeya 34 11 144
After data curation
Xanthomonas 165 20 604 412 604 33 96 85
Ralstonia 87 6 294 144 294 18 44 32
Pectobacterium 60 14 255 165 255 28 64 42
Dickeya 31 8 113 85 113 13 30 16
69
Table 2. Bacterial genomes used in this study.
Xanthomonas
Assembly Number Species Strain Source
GCF_009931595.1 X. albilineans Xa-FJ1 NCBI
GCF_007724205.1 X. arboricola SL2098 NCBI
GCF_000972745.1 X. arboricola 17 NCBI
GCF_008761555.1 X. arboricola 15-088 NCBI
GCF_020002335.2 X. arboricola R1 NCBI
GCF_022930885.1 X. arboricola YchA NCBI
GCF_023375075.1 X. arboricola CITA 33 NCBI
GCF_905220745.1 X. arboricola 1314c NCBI
GCF_905220805.1 X. arboricola CFBP 6600 NCBI
GCF_905367745.1 X. arboricola CPBF 765 NCBI
GCF_023375035.1 X. arboricola IVIA 1317 NCBI
GCF_023374975.1 X. arboricola IVIA 3978 NCBI
GCF_905220695.1 X. arboricola 1311a NCBI
GCF_018141705.1 X. arboricola A7 NCBI
GCF_000225915.1 X. axonopodis F1 NCBI
GCF_003698225.1 X. axonopodis LMG26789 NCBI
GCF_013177355.1 X. axonopodis NCPPB 796 NCBI
GCF_000007145.1 X. campestris ATCC 33913 NCBI
GCF_000070605.1 X. campestris B100 NCBI
GCF_000277895.2 X. campestris NCPPB 4379 NCBI
GCF_000403575.2 X. campestris CN15 NCBI
GCF_001854165.1 X. campestris 85-10 NCBI
GCF_012848175.1 X. campestris NEB122 NCBI
GCF_013388375.1 X. campestris MAFF106181 NCBI
GCF_014841015.1 X. campestris M28 NCBI
GCF_025666215.1 X. campestris CFBP 8444 NCBI
GCF_028749645.1 X. campestris CFBP1371 NCBI
70
Table 2 (continued).
GCF_028749685.1 X. campestris 719 NCBI

GCF_030053615.1 X. campestris GBBC 3077 NCBI
GCF_028750335.1 X. campestris 16-Oct NCBI
GCF_021777455.1 X. campestris BJSJQ20200612 NCBI
GCF_000221965.1 X. campestris 756C NCBI
GCF_001186415.1 X. campestris ICMP 21080 NCBI
GCF_009177345.1 X. campestris MAFF302021 NCBI
GCF_021777195.1 X. campestris GSXT20191014 NCBI
GCF_028749805.1 X. campestris 13108G NCBI
GCF_000961195.1 X. citri NT17 NCBI
GCF_000961475.1 X. citri AW15 NCBI
GCF_001610815.1 X. citri FDC 1609 NCBI
GCF_001610915.1 X. citri 1566 NCBI
GCF_001719145.1 X. citri MSCT NCBI
71
GCF_002139955.1 X. citri TX160149 NCBI

GCF_002218245.1 X. citri CFBP7111 NCBI
GCF_002224525.1 X. citri XcmH1005 NCBI
GCF_002224545.1 X. citri XcmN1003 NCBI
GCF_003999505.1 X. citri ISO12C3 NCBI
GCF_006228425.1 X. citri LMG7439 NCBI
GCF_007567665.1 X. citri 8ra NCBI
GCF_007724385.1 X. citri K2 NCBI
GCF_016495605.1 X. citri UnB-XtecTG02-2 NCBI
GCF_016801635.1 X. citri DAR73886 NCBI
GCF_016801675.1 X. citri DAR72029 NCBI
GCF_017742815.1 X. citri 6165R(delta)tal22B NCBI
GCF_018831285.1 X. citri M12 NCBI
GCF_018831345.1 X. citri T21 NCBI
GCF_020525465.1 X. citri GZ09 NCBI
GCF_027186325.1 X. citri CFBP 2036 NCBI
GCF_028752035.1 X. cucurbitae OH_261 NCBI
GCF_028752055.1 X. cucurbitae MI_359 NCBI
GCF_028752095.1 X. cucurbitae IL_234 NCBI
GCF_903989455.1 X. euroxanthea 1 NCBI
GCF_905187425.1 X. euroxanthea CPBF 424 NCBI
GCF_001908795.1 X. euvesicatoria LMG930 NCBI
GCF_017724035.1 X. euvesicatoria CFBP3836 NCBI
GCF_001705545.1 X. fragariae Fap29 NCBI
GCF_022117535.1 X. fragariae SHQP01 NCBI
GCF_026967555.1 X. fragariae YLX21 NCBI
GCF_030286005.1 X. fragariae LMG 703 NCBI
GCF_900183985.1 X. fragariae NBC2815 NCBI
GCF_900183995.1 X. fragariae PD5205 NCBI
GCF_001908755.1 X. hortorum JS749-3 NCBI
GCF_001908775.1 X. hortorum ICMP 7383 NCBI
GCF_002285515.1 X. hortorum B07-007 NCBI
72
GCF_008728175.1 X. hortorum VT106 NCBI

GCF_014338485.1 X. hortorum LM16734 NCBI
GCF_024339125.1 X. hortorum jj2001 NCBI
GCF_024499015.1 X. hortorum OSU493 NCBI
GCF_026651895.1 X. hortorum Oregano 108 NCBI
GCF_028370135.1 X. hortorum 305 NCBI
GCF_903978195.1 X. hortorum CFBP 498 NCBI
GCF_009769165.1 X. hyacinthi CFBP 1156 NCBI
GCF_002850175.1 X. oryzae MAI134 NCBI
GCF_004355485.1 X. oryzae CFBP7337 NCBI
GCF_000019585.2 X. oryzae PXO99A NCBI
GCF_000212755.2 X. oryzae X11-5A NCBI
GCF_001021915.1 X. oryzae YM15 NCBI
GCF_001276975.2 X. oryzae NCPPB4346 NCBI
GCF_001466505.1 X. oryzae AXO1947 NCBI
GCF_001746635.1 X. oryzae PXO211 NCBI
GCF_004319445.1 X. oryzae NJ611 NCBI
GCF_004319465.1 X. oryzae BB156-2 NCBI
GCF_004319485.1 X. oryzae BB151-3 NCBI
GCF_004319505.1 X. oryzae BAI23 NCBI
GCF_004355745.1 X. oryzae PXO404 NCBI
GCF_004355785.1 X. oryzae AUST2013 NCBI
GCF_004355845.1 X. oryzae KXO85 NCBI
GCF_009660205.1 X. oryzae 0-9 NCBI
GCF_009707765.1 X. oryzae ITCCBB0002 NCBI
GCF_021729945.1 X. oryzae HGA4 NCBI
GCF_022406815.1 X. oryzae YN01 NCBI
GCF_022559705.1 X. oryzae NJ01 NCBI
GCF_028752415.1 X. oryzae JS49-6 NCBI
GCF_030284465.1 X. oryzae YNJC NCBI
GCF_001908855.1 X. perforans LH3 NCBI
GCF_018831425.1 X. phaseoli Px100 NCBI
GCF_002759095.2 X. phaseoli CFBP412 NCBI
73
GCF_018831405.1 X. phaseoli PR1 NCBI

GCF_018831445.1 X. phaseoli Xcp25 NCBI
GCF_020866885.1 X. phaseoli CHN01 NCBI
GCF_025987685.1 X. phaseoli BB013a NCBI
GCF_025987785.1 X. phaseoli CFBP 7423 NCBI
GCF_025263745.1 X. prunicola MAI5069 NCBI
GCF_025263785.1 X. prunicola MAI5037 NCBI
GCF_025266285.1 X. prunicola CIX383 NCBI
GCF_024266585.1 X. sacchari DD13 NCBI
GCF_025765795.1 X. sacchari YT9-19-2 NCBI
GCF_025765815.1 X. sacchari LT6-2 NCBI
GCF_025765835.1 X. sacchari LT6-16-1 NCBI
GCF_025765895.1 X. sacchari JR3-14 NCBI
GCF_025765935.1 X. sacchari HR3-46 NCBI
GCF_025765955.1 X. sacchari HR1-32 NCBI
GCF_029761895.1 X. sacchari DJ16 NCBI
GCF_014236795.1 X. theicola CFBP 4691 NCBI
GCF_008330965.1 X. translucens P3 NCBI
GCF_001021935.1 X. translucens Xtu 4699 NCBI
GCF_003050685.1 X. translucens ICMP11055 NCBI
GCF_008365355.1 X. translucens LW16 NCBI
GCF_017301655.1 X. translucens XtKm33 NCBI
GCF_017301775.1 X. translucens XtLr8 NCBI
GCF_021497045.1 X. translucens MAI5034 NCBI
GCF_021903275.1 X. translucens CFBP 2055 NCBI
GCF_021903515.1 X. translucens LMG 728 NCBI
GCF_021906995.1 X. translucens ICMP 16317 NCBI
GCF_023221635.1 X. translucens Xtu-UPB513 NCBI
GCF_030168915.1 X. translucens NCPPB 3711 NCBI
GCF_021903395.1 X. translucens UPB458 NCBI
GCF_030168955.1 X. translucens ART-Xtg2 NCBI
74

GCF_030168975.1 X. translucens ART-Xtg29 NCBI
GCF_000770355.2 X. vasicola NCPPB 2649 NCBI
GCF_003949975.1 X. vasicola Xv1601 NCBI
GCF_001908815.1 X. vesicatoria LM159 NCBI
GCF_001908725.1 X. vesicatoria LMG911 NCBI
GCF_014236815.1 Xanthomonas sp. GW NCBI
GCF_014236835.1 Xanthomonas sp. SS NCBI
GCF_014236855.1 Xanthomonas sp. SI NCBI
GCF_015224665.2 Xanthomonas sp. WG16 NCBI
GCF_025665335.1 Xanthomonas sp. AM6 NCBI
GCF_025666195.1 Xanthomonas sp. CFBP 8443 NCBI
GCF_025666235.1 Xanthomonas sp. CFBP 8445 NCBI
GCF_903989465.1 Xanthomonas sp. 2 NCBI
Ralstonia
GCF_001653935.1 R. insidiosa FC1138 NCBI
GCF_001663855.1 R. insidiosa ATCC 49129 NCBI
GCF_001628775.1 R. mannitolilytica SN83A39 NCBI
GCF_000954135.1 R. mannitolilytica SN82F48 NCBI
GCF_016466415.2 R. pickettii K-288 NCBI
R. pseudosolanacearum
GCF_000009125.1 GMI1000 NCBI
GCF_002220465.1 CQPS-1 NCBI
GCF_003595305.1 RS 476 NCBI
GCF_022625045.1 SL1931 NCBI
GCF_023518395.1 PeaFJ1 NCBI
GCF_024925465.1 LMG 9673 NCBI
GCF_025859515.1 Cw717 NCBI
GCF_025859635.1 Sw698 NCBI
GCF_029220045.1 RUN2340 NCBI
GCF_000215325.1 R. solanacearum Po82 NCBI
75
GCF_000283475.1 R. solanacearum PSI07 NCBI

GCF_001299555.1 R. solanacearum UY031 NCBI
GCF_001586135.1 R. solanacearum KACC 10722 NCBI
GCF_001587135.1 R. solanacearum UW163 NCBI
GCF_001587155.1 R. solanacearum IBSBF1503 NCBI
GCF_001663415.1 R. solanacearum YC40-M NCBI
GCF_001708525.1 R. solanacearum KACC 10709 NCBI
GCF_002162015.1 R. solanacearum SEPPX05 NCBI
GCF_002501565.1 R. solanacearum RS 488 NCBI
GCF_002549815.1 R. solanacearum RS 489 NCBI
GCF_002894285.1 R. solanacearum RSCM NCBI
GCF_003515145.1 R. solanacearum T51 NCBI
GCF_003515185.1 R. solanacearum SL3175 NCBI
GCF_003590605.1 R. solanacearum IBSBF 2571 NCBI
GCF_003999715.1 R. solanacearum HA4-1 NCBI
GCF_011290405.1 R. solanacearum B2 NCBI
GCF_011290425.1 R. solanacearum 204 NCBI
GCF_012562465.1 R. solanacearum CIAT_078 NCBI
GCF_013306015.1 R. solanacearum FJAT91.F50 NCBI
76
Table 2 (continued)

GCF_013375735.1 R. solanacearum FJAT91-F8 NCBI
GCF_013704765.1 R. solanacearum FJAT15244-F8 NCBI
GCF_014490805.1 R. solanacearum FJAT454.F50-1 NCBI
GCF_014884685.1 R. solanacearum RUN2474 NCBI
GCF_014884705.1 R. solanacearum RUN2279 NCBI
GCF_014884745.1 R. solanacearum Rs5 NCBI
GCF_015098135.1 R. solanacearum MAFF 211479 NCBI
GCF_018733955.1 R. solanacearum RS24 NCBI
GCF_024802605.1 R. solanacearum Bs715 NCBI
GCF_025884395.1 R. solanacearum Wj644 NCBI
GCF_029219985.1 R. solanacearum MolK2 NCBI
GCF_029220025.1 R. solanacearum CFBP2957 NCBI
GCA_002251655.1 R. solanacearum UW551 NCBI
GCA_002251695.1 R. solanacearum K60 NCBI
GCF_016743075.1 R. syzygii UGMSS_Db01 NCBI
GCF_018243215.1 R. syzygii LLRS-1 NCBI
GCF_029219945.1 R. syzygii BDBR229 NCBI
GCF_029219965.1 R. syzygii R24 NCBI
GCF_021173085.1 R. wenshanensis 56D2 NCBI
GCF_026016705.1 Ralstonia sp. RS642 NCBI
77
Pectobacterium
GCF_029027905.1 P. actinidiae GX-Pa1 NCBI
GCF_003382565.3 P. aquaticum A212-S19-A16 NCBI
GCF_015689195.1 P. aroidearum L6 NCBI
GCF_020181375.1 P. aroidearum LJ2 NCBI
GCF_024498715.1 P. aroidearum AK042 NCBI
GCF_024498755.1 P. aroidearum QJ313 NCBI
GCF_000011605.1 P. atrosepticum SCRI1043 NCBI
GCF_000740965.1 P. atrosepticum 21A NCBI
GCF_018634035.1 P. atrosepticum Green1 NCBI
GCF_001932635.1 P. brasiliense BC1 NCBI
GCF_002068115.1 P. brasiliense SX309 NCBI
GCF_002764035.1 P. brasiliense BZA12 NCBI
GCF_009873295.1 P. brasiliense 1692 NCBI
GCF_016864975.1 P. brasiliense ZLMLSHJ5 NCBI
GCF_016944255.1 P. brasiliense IPO:4132 NAK:239 NCBI
GCF_016944315.1 P. brasiliense IPO:4062 NAK:237 NCBI
GCF_020683105.1 P. brasiliense TS20HJ1 NCBI
GCF_022220705.1 P. brasiliense 130 NCBI
GCF_026723725.1 P. brasiliense 21PCA_AGRO2 NCBI
GCF_025946765.1 P. cacticida CFCC10813 NCBI
GCF_009931215.1 P. carotovorum JR1.1 NCBI
GCF_000023605.1 P. carotovorum PC1 NCBI
GCF_013488025.1 P. carotovorum WPP14 NCBI
GCF_015277635.1 P. carotovorum PCCS1 NCBI
GCF_016415585.1 P. carotovorum 2A NCBI
GCF_016864995.1 P. carotovorum XP-13 NCBI
GCF_020520265.1 P. carotovorum A077-S18-O15 NCBI
GCF_021049225.1 P. carotovorum 25.1 NCBI
GCF_022392325.1 P. carotovorum RC5297 NCBI
GCF_022392805.1 P. carotovorum ZM1 NCBI
GCF_028736315.1 P. carotovorum ZJ-4-2 NCBI
GCF_000769535.1 P. odoriferum BC S7 NCBI
GCF_009931295.1 P. odoriferum JK2.1 NCBI
GCF_000024645.1 P. parmentieri WPP163 NCBI
GCF_000260925.1 P. parmentieri SCC3193 NCBI
78
GCF_003628575.1 P. parmentieri IFB5408 NCBI

GCF_009931535.1 P. parmentieri HC NCBI
GCF_016126895.1 P. parmentieri WC19161 NCBI
GCF_016864935.1 P. parmentieri QK-5 NCBI
GCF_024722495.1 P. parvum YT22221 NCBI
GCF_020971565.1 P. parvum FN20211 NCBI
GCF_002288545.1 P. polaris NIBIO1392 NCBI
GCF_002307355.1 P. polaris NIBIO1006 NCBI
GCF_020047135.1 P. polaris QK413-1 NCBI
GCF_014946775.2 P. quasiaquaticum A477-S1-J17 NCBI
GCF_014946825.2 P. quasiaquaticum A398-S21-F17 NCBI
GCF_002921235.2 P. versatile F131 NCBI
GCF_003031305.1 P. versatile 3=2 NCBI
GCF_003932035.1 P. versatile 14A NCBI
GCF_018141505.1 P. versatile ECC15 NCBI
GCF_020423065.1 P. versatile SR12 NCBI
GCF_020423085.1 P. versatile SR1 NCBI
GCF_020865565.1 P. versatile A73-S18-O15 NCBI
GCF_012427845.1 P. punjabense SS95 NCBI
GCF_022477975.1 Pectobacterium sp. PL64 NCBI
GCF_023272715.1 Pectobacterium sp. 21LCBS03 NCBI
GCF_025643735.1 Pectobacterium sp. F1-1 NCBI
GCF_028335745.1 Pectobacterium sp. A5351 NCBI
Dickeya
GCF_000023565.1 D. chrysanthemi Ech1591 NCBI
GCF_000147055.1 D. dadantii 3937 NCBI
GCF_003049785.1 D. dadantii DSM 18020 NCBI
GCF_018904205.1 D. dadantii S3-1 NCBI
GCF_020047155.1 D. dadantii M2-3 NCBI
GCF_025643375.1 D. dadantii FZ06 NCBI
GCF_030168535.1 D. dadantii XJ12 NCBI
GCF_014893095.1 D. dianthicola 67-19 NCBI
GCF_018361085.1 D. dianthicola 16ME22T NCBI
GCF_022871065.1 D. dianthicola LAR.16.03.LID NCBI
GCF_000758345.1 D. fangzhongdai ND14b NCBI
GCF_002812485.1 D. fangzhongdai DSM 101947 NCBI
79
GCF_003628775.1 D. fangzhongdai PA1 NCBI

GCF_009873225.2 D. fangzhongdai AP6 NCBI
GCF_022385255.1 D. fangzhongdai 643b NCBI
GCF_024971955.1 D. fangzhongdai Onc5 NCBI
GCF_029320885.1 D. fangzhongdai ZXC1 NCBI
GCF_000025065.1 D. parazeae Ech586 NCBI
GCF_000511285.2 D. solani RNS 08.23.3.1.A NCBI
GCF_001401705.2 D. solani RNS 05.1.2A NCBI
GCF_000382585.2 D. zeae MS1 NCBI
GCF_000816045.1 D. zeae EC1 NCBI
GCF_002887555.1 D. zeae MS2 NCBI
GCF_012278405.1 D. zeae EC2 NCBI
GCF_012278555.1 D. zeae CE1 NCBI
GCF_019444095.1 D. zeae JZL7 NCBI
GCF_019464635.1 D. zeae PL65 NCBI
GCF_020520245.1 D. zeae A586-S18-A17 NCBI
GCF_021614795.1 D. zeae MS_2018 NCBI
GCF_023212925.1 D. zeae A5272 NCBI
GCF_900095885.1 D. aquatica 174/2 NCBI
GCF_029635795.1 D. lacustris LMG30899 NCBI
GCF_007858975.2 D. poaceiphila NCPPB 569 NCBI
GCF_003049805.1 Dickeya. sp. Secpp 1600 NCBI
Outgroup
GCF_025643435.1 Escherichia coli K-12 NCBI
Pseudomonas
GCF_000006765.1 aeruginosa PAO1 NCBI
80
Table 3. Strains represented in this study with their non-curated Rhs-toxins repertoire.
Xanthomonas
Species Strain # Rhs per genome
X. albilineans Xa-FJ1 5
X. arboricola SL2098 1
X. arboricola 17 4
X. arboricola 15-088 3
X. arboricola R1 3
X. arboricola YchA 4
X. arboricola CITA 33 3
X. arboricola 301 3
X. arboricola 1314c 4
X. arboricola CFBP 6600 2
X. arboricola CPBF 765 3
X. arboricola 3 1
X. arboricola IVIA 1317 1
X. arboricola 1311a 1
X. arboricola A7 0
X. axonopodis F1 2
X. axonopodis LMG26789 2
X. axonopodis NCPPB 796 3
X. campestris ATCC 33913 2
X. campestris B100 2
X. campestris NCPPB 4379 3
X. campestris CN15 4
X. campestris 85-10 6
X. campestris NEB122 5
X. campestris MAFF106181 2
X. campestris M28 3
X. campestris CFBP 8444 5
X. campestris CFBP1371 2
81
X. campestris 719 2
X. campestris 8284 2
X. campestris GBBC 3077 2
X.campestris 576 1
X. campestris 16-Oct 1
X. campestris BJSJQ20200612 1
X. campestris 756C 0
X. campestris ICMP 21080 0
X. campestris GSXT20191014 0
X. campestris 609 0
X. campestris 13108G 0
X. campestris 792 0
X. campestris 659 0
X. campestris 793 0
X. citri NT17 4
X. citri AW15 5
X. citri FDC 1609 4
X. citri 1566 2
82
X. citri MSCT 9
X. citri TX160149 4
X. citri CFBP7111 5
X. citri CFBP7112 5
X. citri XcmH1005 7
X. citri XcmN1003 8
X. citri CFBP6166 4
X. citri CFBP6975 5
X. citri CFBP6990 8
X. citri CFBP6991 7
X. citri ISO12C3 5
X. citri LMG7439 6
X. citri 8ra 9
X. citri K2 9
X. citri UnB-XtecTG02-2 11
X. citri DAR73886 4
X. citri DAR72029 4
X. citri CFBP7119 11
X. citri 6165R(delta)tal22B 2
X. citri M12 4
X. citri T21 10
X. citri GZ09 2
X. citri CFBP 2036 7
X. citri CFBP7113 1
X. cucurbitae OH_261 3
X. cucurbitae MI_359 2
X. cucurbitae IL_234 2
X. euroxanthea 1 3
X. euroxanthea CPBF 424 4
X. euvesicatoria LMG930 5
X. euvesicatoria CFBP3836 4
X. fragariae Fap29 3
X. fragariae SHQP01 8
X. fragariae YLX21 10
X. fragariae LMG 703 4
X. fragariae NBC2815 3
X. fragariae PD5205 3
X. hortorum JS749-3 4
X. hortorum ICMP 7383 3
83
X. hortorum B07-007 2
X. hortorum VT106 2
X. hortorum LM16734 3
X. hortorum jj2001 4
X. hortorum OSU493 4
X. hortorum Oregano 108 7
X. hortorum 305 4
X. hortorum CFBP 498 3
X. hyacinthi CFBP 1156 11
X. oryzae MAI134 1
X. oryzae CFBP7337 1
X. oryzae PXO99A 10
X. oryzae X11-5A 7
X. oryzae YM15 3
X. oryzae NCPPB4346 7
X. oryzae AXO1947 2
X. oryzae PXO211 6
X. oryzae NJ611 11
X. oryzae BB156-2 11
X. oryzae BAI23 3
X. oryzae PXO404 6
X. oryzae AUST2013 5
X. oryzae KXO85 7
X. oryzae 0-9 5
X. oryzae ITCCBB0002 4
X. oryzae HGA4 6
X. oryzae YN01 3
X. oryzae NJ01 2
X. oryzae JS49-6 6
X. oryzae YNJC 5
X. perforans LH3 1
X. phaseoli Px100 1
X. phaseoli CFBP412 0
84
X. phaseoli PR1 0
X. phaseoli Xcp25 0
X. phaseoli CHN01 0
X. phaseoli BB013a 0
X. phaseoli CFBP 7423 0
X. prunicola MAI5069 4
X. prunicola CIX383 10
X. sacchari DD13 4
X. sacchari YT9-19-2 11
X. sacchari LT6-2 5
X. sacchari LT6-16-1 8
X. sacchari JR3-14 5
X. sacchari HR3-46 9
X. sacchari DJ16 3
X. theicola CFBP 4691 7
X. translucens P3 1
X. translucens Xtu 4699 1
X. translucens ICMP11055 3
X. translucens LW16 2
X. translucens XtKm33 2
X. translucens XtLr8 2
X. translucens MAI5034 2
X. translucens CFBP 2055 3
X. translucens LMG 728 8
X. translucens ICMP 16317 4
X. translucens Xtu-UPB513 3
X. translucens NCPPB 3711 4
X. translucens UPB458 1
X. translucens ART-Xtg2 1
85

X. vasicola NCPPB 2649 4
X. vasicola Xv1601 3
X. vesicatoria LM159 1
X. vesicatoria LMG911 0
Xanthomonas sp. GW 4
Xanthomonas sp. SS 4
Xanthomonas sp. SI 6
Xanthomonas sp. WG16 4
Xanthomonas sp. AM6 9
Xanthomonas sp. CFBP 8443 4
Xanthomonas sp. 2 8
Ralstonia
R. insidiosa FC1138 0
R. insidiosa ATCC 49129 0
R. mannitolilytica SN83A39 2
R. mannitolilytica SN82F48 0
R. pickettii K-288 0
R. pseudosolanacearum CQPS-1 2
R. pseudosolanacearum RS 476 7
R. pseudosolanacearum SL1931 2
R. pseudosolanacearum PeaFJ1 2
R. pseudosolanacearum LMG 9673 7
R. pseudosolanacearum Cw717 3
R. pseudosolanacearum Sw698 3
R. pseudosolanacearum RUN2340 6
R. pseudosolanacearum GMI1000 7
R. solanacearum T101 1
R. solanacearum RSCM 1
R. solanacearum Po82 3
86
R. solanacearum UY031 7
R. solanacearum KACC 10722 5
R. solanacearum UW163 3
R. solanacearum IBSBF1503 2
R. solanacearum YC40-M 2
R. solanacearum SEPPX05 2
R. solanacearum RS 488 7
R. solanacearum SL3175 11
R. solanacearum IBSBF 2571 3
R. solanacearum HA4-1 2
R. solanacearum B2 3
R. solanacearum 204 2
R. solanacearum CIAT_078 3
R. solanacearum FJAT91.F50 2
87
R. solanacearum FJAT454.F50-1 2
R. solanacearum RUN2474 6
R. solanacearum Rs5 7
R. solanacearum MAFF 301560 2
R. solanacearum RS24 2
R. solanacearum Bs715 2
R. solanacearum Wj644 2
R. solanacearum K60 15
R. solanacearum FJAT15244-F8 1
R. solanacearum PSI07 4
R. solanacearum MolK2 10
R. solanacearum CFBP2957 16
R. syzygii LLRS-1 14
R. syzygii BDBR229 5
R. syzygii UGMSS_Db01 5
R. syzygii R24 1
R. wenshanensis 56D2 5
Ralstonia sp. RS642 5
Pectobacterium
P. actinidiae GX-Pa1 3
P. aquaticum A212-S19-A16 3
P. aroidearum L6 6
88
P. aroidearum LJ2 2
P. aroidearum AK042 3
P. aroidearum QJ313 2
P. atrosepticum SCRI1043 5
P. atrosepticum 21A 1
P. atrosepticum Green1 4
P. brasiliense BC1 3
P. brasiliense SX309 3
P. brasiliense BZA12 2
P. brasiliense 1692 3
P. brasiliense ZLMLSHJ5 2
P. brasiliense IPO:4132 NAK:239 2
P. brasiliense IPO:4062 NAK:237 3
P. brasiliense TS20HJ1 4
P. brasiliense 21PCA_AGRO2 4
P. cacticida CFCC10813 3
P. carotovorum JR1.1 1
P. carotovorum PC1 2
P. carotovorum WPP14 3
P. carotovorum PCCS1 4
P. carotovorum 2A 4
P. carotovorum XP-13 2
P. carotovorum A077-S18-O15 3
P. carotovorum 25.1 3
P. carotovorum RC5297 7
P. carotovorum ZM1 4
P. carotovorum ZJ-4-2 5
P. odoriferum BC S7 3
P. odoriferum JK2.1 5
P. parmentieri WPP163 8
P. parmentieri SCC3193 12
P. parmentieri IFB5408 12
P. parmentieri HC 9
P. parmentieri WC19161 13
89
P. parmentieri QK-5 14
P. parvum YT22221 2
P. parvum FN20211 1
P. polaris NIBIO1392 5
P. polaris QK413-1 2
P. punjabense SS95 0
P. quasiaquaticum A477-S1-J17 3
P. quasiaquaticum A398-S21-F17 4
P. versatile F131 4
P. versatile 2-Mar 4
P. versatile 14A 6
P. versatile ECC15 2
P. versatile SR12 2
P. versatile SR1 4
P. versatile A73-S18-O15 4
Pectobacterium sp. PL64 2
Pectobacterium sp. 21LCBS03 5
Pectobacterium sp. F1-1 1
Pectobacterium sp. A5351 0
Dickeya
D. aquatica 174/2 0
D. chrysanthemi Ech1591 7
D. dadantii DSM 18020 3
D. dadantii S3-1 6
D. dadantii M2-3 5
D. dadantii FZ06 5
D. dadantii XJ12 6
D. dadantii 3937 6
D. dianthicola 67-19 3
D. dianthicola 16ME22T 2
D. dianthicola LAR.16.03.LID 3
D. fangzhongdai ND14b 6
D. fangzhongdai DSM 101947 4
D. fangzhongdai PA1 6
D. fangzhongdai AP6 3
D. fangzhongdai 643b 6
D. fangzhongdai Onc5 5
90
D. fangzhongdai ZXC1 9
D. lacustris LMG30899 0
D. parazeae Ech586 2
D. poaceiphila NCPPB 569 0
D. solani RNS 05.1.2A 4
D. solani RNS 08.23.3.1.A 4
D. zeae MS1 5
D. zeae EC1 4
D. zeae MS2 5
D. zeae EC2 6
D. zeae CE1 4
D. zeae JZL7 4
D. zeae PL65 4
D. zeae A586-S18-A17 5
D. zeae MS_2018 6
D. zeae A5272 2
Dickeya. sp. Secpp 1600 4
Outgroup
Species Strain # Rhs per
genome
Escherichia coli K-12 7
Pseudomonas aeruginosa PAO1 1
91
Table 4: Strains represented in this study with their curated Rhs-toxins repertoire.
Xanthomonas
X. albilineans Xa-FJ1 5
X. arboricola SL2098 1
X. arboricola 17 4
X. arboricola YchA 3
X. arboricola 1314c 4
X. arboricola 1311a 1
X. arboricola 15-088 3
X. arboricola R1 3
X. arboricola CITA 33 3
X. arboricola 301 2
X. arboricola 3 1
X. axonopodis F1 2
X. axonopodis LMG26789 1
X. axonopodis NCPPB 796 3
X. campestris M28 3
X. campestris GBBC 3077 2
X. campestris 16-Oct 1
X. campestris ATCC 33913 1
X. campestris B100 2
X. campestris NCPPB 4379 3
X. campestris NEB122 4
X. campestris CFBP 8444 5
X. campestris 719 2
92
X. campestris 576 1
X. campestris BJSJQ20200612 1
X. citri NT17 4
X. citri AW15 5
X. citri FDC 1609 2
X. citri MSCT 7
X. citri TX160149 3
X. citri CFBP7111 5
X. citri CFBP7112 5
X. citri XcmH1005 5
X. citri XcmN1003 6
X. citri CFBP6166 2
X. citri CFBP6975 3
X. citri CFBP6990 7
X. citri CFBP6991 6
X. citri ISO12C3 4
X. citri LMG7439 5
X. citri 8ra 7
X. citri K2 7
X. citri UnB-XtecTG02-2 11
X. citri DAR73886 4
X. citri DAR72029 4
X. citri CFBP7119 9
X. citri M12 2
X. citri T21 9
X. citri GZ09 2
X. citri CFBP 2036 5
X. cucurbitae OH_261 3
X. cucurbitae MI_359 2
X. cucurbitae IL_234 2
X. euroxanthea 1 3
93

X. euvesicatoria LMG930 5
X. euvesicatoria CFBP3836 4
X. fragariae Fap29 2
X. fragariae SHQP01 6
X. fragariae YLX21 9
X. fragariae LMG 703 3
X. fragariae NBC2815 3
X. fragariae PD5205 2
X. hortorum B07-007 2
X. hortorum VT106 2
X. hortorum jj2001 3
X. hortorum Oregano 108 7
X. hortorum JS749-3 4
X. hortorum ICMP 7383 3
X. hortorum LM16734 3
X. hortorum OSU493 3
X. hortorum 305 4
X. hortorum CFBP 498 3
X. hyacinthi CFBP 1156 10
X. oryzae X11-5A 5
X. oryzae NCPPB4346 5
X. oryzae NJ611 9
X. oryzae BAI23 3
X. oryzae NJ01 2
X. oryzae JS49-6 5
X. oryzae MAI134 1
X. oryzae PXO99A 8
X. oryzae YM15 2
X. oryzae AXO1947 2
X. oryzae PXO211 4
94
X. oryzae PXO404 5
X. oryzae AUST2013 3
X. oryzae KXO85 3
X. oryzae 0-9 4
X. oryzae ITCCBB0002 4
X. oryzae HGA4 5
X. oryzae YN01 3
X. oryzae YNJC 2
X. perforans LH3 1
X. sacchari DD13 4
X. sacchari YT9-19-2 7
X. sacchari LT6-2 4
X. sacchari LT6-16-1 5
X. sacchari JR3-14 5
X. sacchari DJ16 3
X. sp. GW 4
X. sp. CFBP 8445 3
X. theicola CFBP 4691 7
X. translucens P3 1
X. translucens Xtu 4699 1
X. translucens ICMP11055 3
X. translucens LW16 2
X. translucens XtLr8 2
X. translucens MAI5034 2
X. translucens ICMP 16317 4
95
X. translucens Xtu-UPB513 2
X. translucens NCPPB 3711 4
X. translucens UPB458 1
X. vasicola Xv1601 3
X. vesicatoria LM159 1
Xanthomonas sp. SS 4
Xanthomonas sp. SI 6
Xanthomonas sp. WG16 4
Xanthomonas sp. AM6 7
Xanthomonas sp. 2 5
Ralstonia
R. mannitolilytica SN83A39 1
R. pseudosolanacearum CQPS-1 2
R. pseudosolanacearum RS 476 6
R. pseudosolanacearum SL1931 1
R. pseudosolanacearum PeaFJ1 2
R. pseudosolanacearum LMG 9673 7
R. pseudosolanacearum Cw717 1
R. pseudosolanacearum Sw698 2
R. pseudosolanacearum RUN2340 6
R. pseudosolanacearum GMI1000 6
R. solanacearum Po82 3
R. solanacearum UY031 5
R. solanacearum IBSBF1503 2
R. solanacearum YC40-M 2
R. solanacearum SEPPX05 2
96
R. solanacearum RSCM 1
R. solanacearum IBSBF 2571 3
R. solanacearum HA4-1 2
R. solanacearum B2 3
R. solanacearum CIAT_078 3
97
R. solanacearum FJAT454.F50-1 2
R. solanacearum Rs5 6
R. solanacearum RS24 2
R. solanacearum Bs715 2
R. solanacearum Wj644 2
R. solanacearum PSI07 4
R. solanacearum MolK2 9
R. solanacearum CFBP2957 12
R. solanacearum K60 9
R. syzygii LLRS-1 9
R. syzygii BDBR229 4
R. syzygii UGMSS_Db01 4
R. syzygii R24 1
R. wenshanensis 56D2 4
Pectobacterium
P. actinidiae GX-Pa1 2
P. aquaticum A212-S19-A16 3
P. aroidearum AK042 3
P. aroidearum LJ2 2
98
P. aroidearum L6 6
P. atrosepticum SCRI1043 5
P. atrosepticum 21A 1
P. atrosepticum Green1 4
P. brasiliense BC1 3
P. brasiliense ZLMLSHJ5 2
P. brasiliense IPO:4132 2
P. brasiliense 21PCA_AGRO2 3
P. brasiliense BZA12 2
P. brasiliense SX309 3
P. brasiliense IPO:4062 3
P. brasiliense TS20HJ1 4
P. cacticida CFCC10813 3
P. carotovorum JR1.1 1
P. carotovorum XP-13 2
P. carotovorum WPP14 3
P. carotovorum A077-S18-O15 3
P. carotovorum 25.1 3
P. carotovorum PCCS1 4
P. carotovorum 2A 4
P. carotovorum ZM1 4
P. carotovorum RC5297 7
P. carotovorum PC1 2
P. carotovorum ZJ-4-2 5
P. odoriferum BC S7 3
P. odoriferum JK2.1 5
P. parmentieri WPP163 8
P. parmentieri SCC3193 12
P. parmentieri WC19161 13
P. parmentieri QK-5 13
P. parmentieri HC 9
P. parvum YT22221 2
P. parvum FN20211 1
P. polaris QK413-1 2
99
P. quasiaquaticum A477-S1-J17 3
P. quasiaquaticum A398-S21-F17 4
Pectobacterium sp. PL64 2
Pectobacterium sp. F1-1 1
Pectobacterium sp. 21LCBS03 5
P. versatile A73-S18-O15 4
P. versatile ECC15 2
P. versatile SR12 2
P. versatile F131 4
P. versatile 2-Mar 3
P. versatile SR1 4
P. versatile 14A 6
Dickeya
D. chrysanthemi Ech1591 6
D. dadantii DSM 18020 3
D. dadantii S3-1 5
D. dadantii M2-3 3
D. dadantii FZ06 5
D. dadantii XJ12 5
D. dadantii 3937 6
D. dianthicola 67-19 2
D. dianthicola 16ME22T 2
D. dianthicola LAR.16.03.LID 2
D. fangzhongdai ND14b 5
D. fangzhongdai DSM 101947 3
D. fangzhongdai PA1 4
D. fangzhongdai AP6 3
D. fangzhongdai 643b 4
D. fangzhongdai Onc5 4
D. fangzhongdai ZXC1 6
D. parazeae Ech586 2
D. solani RNS 05.1.2A 3
D. solani RNS 08.23.3.1.A 4
Dickeya sp. Secpp 1600 4
D. zeae MS1 3
D. zeae EC1 3
100
D. zeae MS2 4
D. zeae EC2 4
D. zeae CE1 2
D. zeae JZL7 3
D. zeae PL65 2
D. zeae A586-S18-A17 4
D. zeae MS_2018 5
D. zeae A5272 2
Outgroup
Escherichia coli K-12 5
Pseudomonas aeruginosa PAO1 1
101
Table 5. Plant pathogenic genera and their species represented in this study with their
complementary N-terminal clade.
Xanthomonas
Species Strain Rhs_locus Clade
X. oryzae MAI134 BVV20_RS16895 I
X. oryzae CFBP7331 ACU16_RS16515 I
X. oryzae BB156-2 EYC55_RS06280 I
X. oryzae NCPPB4346 EYR26_RS16965 I
X. oryzae NJ01 MML47_RS05895 I
X. oryzae YNJC QN060_RS16955 I
X. oryzae PXO404 EBA23_RS08555 I
X. oryzae AUST2013 EBA17_RS16090 I
X. oryzae JS49-6 LL928_RS15920 I
X. oryzae PXO211 ATY44_RS15850 I
X. translucens XtKm33 ISN33_RS07930 I
X. translucens CFBP 2055 KCU58_RS05760 I
X. translucens LW16 F0H32_RS14580 I
X. translucens MAI5034 LTC53_RS14350 I
X. oryzae CFBP7337 EBA11_RS16380 I
X. oryzae AXO1947 AXO1947_RS05255 I
X. oryzae CFBP7342 BE73_RS04950 I
X. oryzae PXO99A PXO_RS07175 I
X. translucens XtLr8 ISN38_RS15700 I
X. translucens XtKm15 ISN36_RS12090 I
X. oryzae X11-5A EYR27_RS02355 I
X. oryzae AXO1947 AXO1947_RS21135 I
X. oryzae BAI23 EYC57_RS05720 I
X. oryzae NJ611 EYC54_RS06110 I
X. translucens UPB458 KFS86_RS08990 I
102
X. oryzae YN01 IYN96_RS05820 I

X. oryzae NJ01 MML47_RS05905 I
X. oryzae YNJC QN060_RS16945 I
X. translucens P3 F0H33_RS14415 I
X. oryzae 0-9 GHV42_RS05860 I
X. fragariae LMG 703 OW158_RS12015 I
X. fragariae Fap29 BER93_RS11435 I
X. fragariae PD5205 PD5205_RS11720 I
X. translucens ICMP 16317 K8O61_RS11885 I
X. translucens LMG 843 KFS85_RS06145 I
X. fragariae NBC2815 NBC2815_RS20935 I
X. fragariae Fap29 BER93_RS20935 I
X. fragariae PD5205 PD5205_RS11680 I
X. oryzae 0-9 GHV42_RS05875 I
X. oryzae X11-5A EYR27_RS02345 I
X. oryzae PXO404 EBA23_RS08545 I
X. oryzae CFBP7342 BE73_RS27715 I
X. translucens CFBP 2541 KHA79_RS11980 I
X. fragariae NBC2815 NBC2815_RS11835 I
X. translucens LMG 843 KFS85_RS06170 I
X. translucens Xtu 4699 FD63_RS13525 I
X. translucens CFBP 2055 KCU58_RS05740 I
X. translucens Xtu-UPB513 MZO50_RS08935 I
103
X. translucens ART-Xtg2 KM563_RS15915 I

X. translucens LW16 F0H32_RS14615 I
X. oryzae HGA4 IZG20_RS13450 I
X. translucens CFBP 2541 KHA79_RS11990 I
X. translucens ICMP11055 NZ30_RS06515 I
X. translucens ICMP11055 NZ30_RS06505 I
X. hyacinthi CFBP 1156 FZ025_RS20375 II
X. arboricola CPBF 766 KHN80_RS03670 II
X. oryzae PXO99A PXO_RS12195 III
X. oryzae JS49-6 LL928_RS20780 III
X. fragariae YLX21 OZ429_RS17695 III
X. arboricola R1 K9U01_RS02055 III
X. sacchari JR3-14 NG824_RS09410 III
X. oryzae PXO211 ATY44_RS24970 III
X. oryzae ITCCBB0002 GKO49_RS02190 III
X. sacchari LT6-16-1 NG829_RS11660 III
X. sacchari YT9-19-2 NG828_RS12090 III
X. sacchari HR1-32 NG831_RS12155 III
X. albilineans Xa-FJ1 XaFJ1_RS11700 III
X. oryzae BB156-2 EYC55_RS20950 III
X. axonopodis NCPPB 796 XAV_RS01910 III
X. campestris MAFF106181 AD14011_RS21420 III
X. oryzae CFBP7342 BE73_RS24035 III
Xanthomonas sp. SS HEP74_RS06905 III
Xanthomonas sp. SI HEP75_RS07115 III
X. hortorum JS749-3 BJD10_RS00030 III
X. oryzae NJ611 EYC54_RS02420 III
X. fragariae SHQP01 K4A87_RS01730 III
104
X. axonopodis NCPPB 796 XAV_RS01895 III

X. citri T21 DGN02_RS02115 III
X. fragariae SHQP01 K4A87_RS01745 III
Xanthomonas sp. AM6 OCJ37_RS10125 III
X. vasicola Xv1601 CXP37_RS23425 IV
X. prunicola MAI5037 M0D47_RS15265 IV
X. prunicola CIX383 M0D48_RS03750 IV
X. vasicola NCPPB 2649 NX80_RS23480 IV
X. campestris NCPPB 4379 KWO_RS23195 IV
X. arboricola 17 XB05_RS11070 IV
Xanthomonas sp. 2 H7A87_RS06480 IV
X. arboricola SL2098 FPL04_RS16495 IV
X. arboricola 15-088 F6Y24_RS05500 IV
X. arboricola R1 K9U01_RS06725 IV
X. cucurbitae MI_359 K6982_RS05345 IV
X. citri DAR73886 H8Z71_RS14935 IV
X. citri DAR72029 H8Z73_RS06720 IV
X. citri NT17 AMD13_RS07130 IV
Xanthomonas sp. WG16 IG630_RS06660 IV
X. citri AW15 AMD07_RS14915 IV
X. citri TX160149 B7L66_RS03335 IV
X. citri AW15 AMD07_RS26300 IV
X. citri CFBP7111 XcvCFBP7111P_RS17725 IV
X. citri 8ra FPK90_RS25350 IV
X. citri K2 FPL05_RS24910 IV
X. citri CFBP7119 XcgCFBP7119R_RS24575 IV
X. cucurbitae IL_234 K6979_RS13135 IV
X. hortorum LM16734 XHV734_RS14570 IV
X. arboricola 3 H5027_RS08090 IV
X. arboricola 17 XB05_RS24585 IV
X. citri UnB-XtecTG02-2 G3566_RS06640 IV
X. oryzae NCPPB4346 EYR26_RS09400 IV
105

X. citri XcmN1003 APY30_RS17325 IV
X. cucurbitae OH_261 K6978_RS05695 IV
X. citri CFBP6990 XcfCFBP6990P_RS06790 IV
X. citri CFBP6991 XcfCFBP6991P_RS06810 IV
X. citri T21 DGN02_RS06730 IV
X. citri LMG7439 XapA_RS24135 IV
X. prunicola MAI5069 M0D45_RS12480 IV
X. hortorum ICMP 7383 BI317_RS16880 IV
X. campestris M28 IFJ81_RS22255 IV
X. euvesicatoria CFBP3836 XeaCFBP3836p_RS16095 IV
X. campestris CFBP6690 JH261_RS05475 IV
X. fragariae YLX21 OZ429_RS05545 IV
X. euvesicatoria CFBP3836 XeaCFBP3836p_RS02340 IV
X. citri MSCT BGK55_RS14880 IV
X. citri XcmH1005 APY29_RS16285 IV
X. citri XcmN1003 APY30_RS06770 IV
X. citri CFBP 2036 LPY96_RS16390 IV
X. campestris MAFF106181 AD14011_RS05040 IV
X. campestris 719 JH264_RS16040 IV
X. euroxanthea 1 H7A86_RS06100 IV
X. translucens CFBP 2541 KHA79_RS18900 IV
X. theicola CFBP 4691 G4Q83_RS21605 IV
X. oryzae 0-9 GHV42_RS13390 IV
X. oryzae CFBP7342 BE73_RS10025 IV
X. sacchari HR1-32 NG831_RS05640 V
X. translucens LMG 728 KM539_RS12985 V
X. hortorum Oregano 108 OEG85_RS14840 VI
X. arboricola YchA LZZ50_RS07680 VI
X. euroxanthea 1 H7A86_RS08655 VI
X. arboricola 1314c LOK39_RS10655 VI
X. hortorum 305 PML25_RS09245 VI
X. fragariae YLX21 OZ429_RS07145 VI
X. vesicatoria LM159 BI313_RS24690 VI
X. euroxanthea CPBF 424 XTG_RS08390 VI
106
X. euvesicatoria CFBP3836 XeaCFBP3836p_RS05615 VI

Xanthomonas sp. 2 H7A87_RS08515 VI
X. prunicola MAI5037 M0D47_RS05415 VI
X. prunicola CIX249 M0D43_RS05650 VI
X. prunicola CIX383 M0D48_RS15735 VI
X. campestris CFBP5824 JH314_RS16820 VI
X. hortorum OSU493 NDY25_RS04850 VI
X. hortorum 305 PML25_RS16215 VI
X. euroxanthea CPBF 424 XTG_RS11135 VI
Xanthomonas sp. 2 H7A87_RS05400 VI
X. citri CFBP7112 XcvCFBP7112P_RS00780 VII
X. citri CFBP6990 XcfCFBP6990P_RS00645 VII
X. citri CFBP6991 XcfCFBP6991P_RS00645 VII
X. prunicola MAI5069 M0D45_RS09610 VII
X. prunicola CIX97 M0D46_RS09630 VII
X. euvesicatoria LMG930 BJD11_RS24465 VII
X. axonopodis F1 XACM_RS19450 VII
X. campestris 85-10 BHE83_RS13365 VII
X. citri UnB-XtecTG02-2 G3566_RS00655 VII
X. prunicola MAI5037 M0D47_RS12515 VII
X. arboricola 17 XB05_RS25205 VII
X. arboricola YchA LZZ50_RS02835 VII
X. euroxanthea 1 H7A86_RS00675 VII
X. arboricola CPBF 1494 KHN93_RS00775 VII
X. cucurbitae OH_261 K6978_RS00120 VII
X. campestris GBBC 3077 QMY63_RS01220 VII
Xanthomonas sp. 2 H7A87_RS00685 VII
X. campestris CFBP1371 JH299_RS20680 VII
X. campestris NEB122 HG421_RS19535 VII
X. euroxanthea CPBF 766 KHO05_RS00695 VII
X. hortorum Oregano 108 OEG85_RS00950 VII
X. euroxanthea CPBF 424 XTG_RS00650 VII
X. campestris ATCC 33913 XCC_RS00685 VII
X. campestris M28 IFJ81_RS00725 VII
X. campestris 8284 JH306_RS00675 VII
X. campestris 10103 JH282_RS00725 VII
X. campestris 576 JH277_RS08765 VIII
107
X. campestris M28 IFJ81_RS09090 VIII

X. campestris GBBC 3077 QMY63_RS11040 VIII
X. campestris 12049 JH293_RS08480 VIII
X. campestris 16-Oct JH307_RS12435 VIII
X. euvesicatoria LMG930 BJD11_RS05975 VIII
X. euroxanthea CPBF 424 XTG_RS15535 VIII
X. arboricola 1314c LOK39_RS12170 VIII
X. campestris 85-10 BHE83_RS09435 VIII
X. citri DAR73886 H8Z71_RS08810 VIII
X. hortorum Oregano 108 OEG85_RS05880 VIII
X. arboricola 1311a LOK40_RS12405 VIII
X. citri NT17 AMD13_RS13340 VIII
Xanthomonas sp. WG16 IG630_RS13345 VIII
X. citri AW15 AMD07_RS10975 VIII
X. citri CFBP7111 XcvCFBP7111P_RS10715 VIII
X. hortorum jj2001 NMB96_RS19530 VIII
X. hortorum JS749-3 BJD10_RS12280 VIII
X. hortorum LM16734 XHV734_RS08375 VIII
X. hortorum ICMP 7383 BI317_RS10180 VIII
X. hortorum B07-007 XJ27_RS06820 VIII
X. hortorum CFBP 498 CFBP8129_RS13300 VIII
X. hortorum VT106 DYQ48_RS13035 VIII
X. oryzae PXO99A PXO_RS06855 VIII
X. oryzae CFBP2286 ACU11_RS25435 VIII
X. arboricola 301 P3C56_RS06145 VIII
X. prunicola MAI5037 M0D47_RS16575 VIII
X. vasicola NCPPB 1060 NX81_RS13995 VIII
X. prunicola CIX97 M0D46_RS13530 VIII
X. citri ISO12C3 AC612_RS07065 VIII
X. citri M12 DGN11_RS06735 VIII
X. citri FDC 1609 TP50_RS18950 VIII
X. citri CFBP6166 XcfCFBP6166P_RS06935 VIII
X. campestris NEB122 HG421_RS21100 VIII
X. oryzae NJ611 EYC54_RS21870 VIII
X. oryzae BB156-2 EYC55_RS09335 VIII
X. oryzae KXO85 EBA20_RS17030 VIII
108
X. oryzae PXO99A PXO_RS06285 VIII

X. oryzae PXO404 EBA23_RS08175 VIII
X. oryzae JS49-6 LL928_RS15550 VIII
X. oryzae PXO211 ATY44_RS16160 VIII
X. oryzae AUST2013 EBA17_RS16470 VIII
X. cucurbitae OH_261 K6978_RS14005 VIII
X. cucurbitae IL_234 K6979_RS05550 VIII
X. citri UnB-XtecTG02-2 G3566_RS22055 VIII
X. oryzae X11-5A EYR27_RS05005 VIII
X. citri MSCT BGK55_RS25635 VIII
X. fragariae SHQP01 K4A87_RS04825 VIII
X. arboricola 301 P3C56_RS09790 VIII
X. cucurbitae MI_359 K6982_RS13340 VIII
X. campestris 29-5 JH291_RS13450 VIII
X. citri NT17 AMD13_RS16920 VIII
Xanthomonas sp. WG16 IG630_RS16920 VIII
X. citri AW15 AMD07_RS05675 VIII
X. citri TX160149 B7L66_RS04365 VIII
X. fragariae YLX21 OZ429_RS14290 VIII
X. citri ISO12C3 AC612_RS14010 VIII
X. citri T21 DGN02_RS22685 VIII
X. campestris NCPPB 4379 KWO_RS16610 VIII
X. citri 8ra FPK90_RS25030 VIII
X. citri K2 FPL05_RS24570 VIII
X. citri CFBP7119 XcgCFBP7119R_RS18080 VIII
X. oryzae ITCCBB0002 GKO49_RS15840 VIII
X. oryzae 0-9 GHV42_RS05550 VIII
X. oryzae HGA4 IZG20_RS13760 VIII
X. campestris 85-10 BHE83_RS26150 VIII
X. citri XcmH1005 APY29_RS09475 VIII
X. citri XcmN1003 APY30_RS13510 VIII
109
X. citri CFBP 2036 LPY96_RS09700 VIII

X. euvesicatoria LMG930 BJD11_RS09260 VIII
X. arboricola 1314c LOK39_RS09320 VIII
X. hortorum ICMP 7383 BI317_RS13745 VIII
X. hortorum VT106 DYQ48_RS09410 VIII
X. hortorum LM16734 XHV734_RS12125 VIII
X. arboricola YchA LZZ50_RS14280 VIII
X. campestris CN15 XCCCN15_RS09225 VIII
X. campestris CN03 XCCCN03_RS08915 VIII
X. arboricola CPBF 765 KHN80_RS22080 VIII
X. oryzae CFBP7342 BE73_RS24340 VIII
X. oryzae CFBP7341 ACU17_RS24240 VIII
X. arboricola 17 XB05_RS25140 VIII
X. campestris B100 XCCB100_RS24355 VIII
X. axonopodis F1 XACM_RS08075 VIII
X. hortorum OSU493 NDY25_RS20705 VIII
X. hortorum 305 PML25_RS10040 VIII
X. hortorum JS749-3 BJD10_RS15725 VIII
X. citri T21 DGN02_RS13315 VIII
X. arboricola IVIA 3978 KPG65_RS18010 VIII
X. citri GZ09 LCZ91_RS03705 VIII
X. arboricola IVIA 1317 KP727_RS13905 VIII
X. citri XcmH1005 APY29_RS05890 VIII
X. citri XcmN1003 APY30_RS17055 VIII
X. citri CFBP 2036 LPY96_RS06290 VIII
110
X. translucens LMG 728 KM539_RS10370 VIII

X. campestris CFBP 8444 NRY95_RS12615 VIII
Xanthomonas sp. GW HEP73_RS20695 VIII
X. campestris NEB122 HG421_RS10280 VIII
X. oryzae X11-5A EYR27_RS01770 VIII
X. prunicola MAI5069 M0D45_RS05685 VIII
X. oryzae NJ611 EYC54_RS21155 VIII
X. citri CFBP7111 XcvCFBP7111P_RS26910 VIII
Xanthomonas sp. SS HEP74_RS19970 VIII
Xanthomonas sp. AM6 OCJ37_RS19440 VIII
X. sacchari DD13 NKJ47_RS02030 VIII
X. citri LMG7439 XapA_RS09500 VIII
Xanthomonas sp. SI HEP75_RS10700 VIII
Xanthomonas sp. SI HEP75_RS20380 VIII
X. citri UnB-XtecTG02-2 G3566_RS22005 VIII
X. translucens MAI5034 LTC53_RS05350 IX
X. hyacinthi CFBP 1156 FZ025_RS15345 IX
X. campestris CFBP 8444 NRY95_RS06510 IX
X. translucens XtKm33 ISN33_RS16900 IX
X. translucens Xtu-UPB513 MZO50_RS18285 IX
X. translucens ICMP 16317 K8O61_RS05725 IX
Xanthomonas sp. CFBP 8443 NUG20_RS21735 IX
X. sacchari DD13 NKJ47_RS07105 IX
X. translucens ICMP11055 NZ30_RS21745 IX
X. translucens CFBP 2541 KHA79_RS05675 IX
X. sacchari HR3-46 NG825_RS12405 IX
X. translucens XtLr8 ISN38_RS20150 IX
X. translucens NCPPB 3711 KHF85_RS05665 IX
X. sacchari DJ16 QBE03_RS00945 IX
X. sacchari LT6-16-1 NG829_RS20780 IX
X. sacchari JR3-14 NG824_RS20860 IX
Xanthomonas sp. SS HEP74_RS05875 IX
Xanthomonas sp. SI HEP75_RS06045 IX
111
X. sacchari JR3-14 NG824_RS14860 IX

X. translucens CFBP 2055 KCU58_RS15120 IX
X. sacchari LT6-16-1 NG829_RS06270 IX
X. theicola CFBP 4691 G4Q83_RS22720 IX
X. sacchari YT9-19-2 NG828_RS21750 IX
X. sacchari LT6-2 NG827_RS20835 IX
X. theicola CFBP 4691 G4Q83_RS22980 IX
Xanthomonas sp. AM6 OCJ37_RS05745 IX
X. sacchari DJ16 QBE03_RS07645 IX
X. campestris CFBP 8444 NRY95_RS05470 IX
Xanthomonas sp. GW HEP73_RS22405 IX
X. sacchari LT6-2 NG827_RS06235 IX
X. sacchari YT9-19-2 NG828_RS10195 IX
X. translucens LMG 843 KFS85_RS14540 IX
X. translucens LMG 728 KM539_RS06285 IX
X. albilineans Xa-FJ1 XaFJ1_RS11545 IX
Ralstonia
R. solanacearum IBSBF 2571 C2124_RS18965 I
R. solanacearum RS 488 CCY86_RS18665 I
R. solanacearum RS 489 CDC59_RS18560 I
R. solanacearum CIAT_078 G8D25_RS02275 I
R. solanacearum CFBP2957 PG903_RS23825 I
R. solanacearum IBSBF1503 RALBFv3_RS16990 I
R. solanacearum Po82 RSPO_RS18555 I
R. solanacearum UY031 RSUY_RS18170 I
R. solanacearum UW163 UW163_RS18365 I
R. solanacearum UW551 B7R79_19675 I
R. solanacearum Rs5 HF906_RS22570 I
R. solanacearum K60 B7R77_24635 I
R. solanacearum MolK2 PG909_RS22915 I
112
R. solanacearum YC40-M A3768_RS18470 II

R. pseudosolanacearum CQPS-1 BC350_RS21085 II
R. solanacearum HA4-1 CFM90_RS22255 II
R. solanacearum T117 CJO74_RS17965 II
R. solanacearum SL3730 CJO90_RS17890 II
R. solanacearum 204 G7939_RS19800 II
R. solanacearum 202 G7969_RS18430 II
R. solanacearum FJAT454.F50-1 H9X76_RS24320 II
R. solanacearum FJAT1452.F50 HI796_RS17655 II
R. solanacearum FJAT91-F8 HWE47_RS22540 II
R. solanacearum 362200 IMF25_RS24870 II
R. pseudosolanacearum PeaFJ1 JNO62_RS22440 II
Ralstonia sp. RS647 LGV81_RS21355 II
R. solanacearum Bs715 NQ321_RS04685 II
R. solanacearum Wj644 NQ322_RS19390 II
Ralstonia sp. RS650 LGV82_RS19850 II
R. solanacearum UW386 E7Z57_RS20465 II
R. solanacearum K60 B7R77_18740 II
R. pseudosolanacearum LMG 9673 NY025_RS04795 II
R. pseudosolanacearum RUN2340 PG907_RS18555 II
R. solanacearum IBSBF1503 RALBFv3_RS22655 II
113
R. solanacearum RUN2279 HF908_RS21940 II

R. solanacearum RUN2474 HF909_RS21545 II
R. solanacearum CFBP2957 PG903_RS16825 II
R. solanacearum UW763 HF907_RS22995 II
R. solanacearum MAFF 301560 JK146_RS18420 II
R. solanacearum RS24 KM864_RS24520 II
R. wenshanensis 56D2 KOL96_RS09195 II
R. pseudosolanacearum RS 476 CDC45_RS18395 II
R. solanacearum B2 G7968_RS24875 II
R. pseudosolanacearum GMI1000 RS_RS17875 II
R. solanacearum IBSBF 2571 C2124_RS17000 II
R. solanacearum CIAT_078 G8D25_RS07370 II
R. solanacearum Po82 RSPO_RS16740 II
R. solanacearum UW163 UW163_RS23250 II
R. pseudosolanacearum LMG 9673 NY025_RS04740 II
R. solanacearum MolK2 PG909_RS09385 II
R. solanacearum RS 489 CDC59_RS16560 II
R. syzygii R24 PG906_RS24625 II
R. pseudosolanacearum CQPS-1 BC350_RS23285 III
R. solanacearum T117 CJO74_RS23195 III
R. solanacearum SL3730 CJO90_RS22770 III
R. solanacearum RSCM CYD94_RS25805 III
R. solanacearum 204 G7939_RS25520 III
R. solanacearum B2 G7968_RS19500 III
R. solanacearum 202 G7969_RS20605 III
R. solanacearum FJAT454.F50-1 H9X76_RS21700 III
R. solanacearum FJAT1303.F50 HI793_RS23775 III
114

R. solanacearum MAFF 301560 JK146_RS23060 III
R. solanacearum MAFF 311693 JL096_RS19885 III
R. solanacearum RS24 KM864_RS21360 III
R. solanacearum KACC 10709 LBM341_RS17810 III
Ralstonia sp. RS642 LGV80_RS22445 III
R. solanacearum Bs715 NQ321_RS01715 III
R. solanacearum Wj644 NQ322_RS21745 III
R. pseudosolanacearum Cw717 NQS35_RS21795 III
R. pseudosolanacearum Sw698 NQS37_RS22645 III
R. solanacearum YC40-M A3768_RS23425 III
R. pseudosolanacearum RS 476 CDC45_RS23365 III
R. solanacearum FJAT15244-F8 H1A20_RS25825 III
R. solanacearum UW763 HF907_RS18100 III
R. syzygii UGMSS_Db01 JK151_RS13280 III
R. pseudosolanacearum SL1931 MNY32_RS19885 III
R. syzygii BDBR229 PG904_RS11850 III
R. pseudosolanacearum GMI1000 RS_RS22605 III
R. wenshanensis 56D2 KOL96_RS01525 III
R. solanacearum KACC 10722 LBM2029_RS21285 III
R. solanacearum SEPPX05 RSSE_RS24345 III
R. syzygii LLRS-1 GO998_RS24545 III
115
R. solanacearum CFBP2957 PG903_RS22375 III

R. solanacearum PSI07 RPSI07_RS26455 III
R. solanacearum HA4-1 CFM90_RS25210 III
R. solanacearum 362200 IMF25_RS21770 III
R. pseudosolanacearum PeaFJ1 JNO62_RS19340 III
Pectobacterium
P. parmentieri IFB5441 C5E20_RS12580 I
P. parmentieri SCC3193 W5S_RS11680 I
P. parmentieri QK-5 IG623_RS11480 I
P. parmentieri WPP163 PECWA_RS11940 I
P. parmentieri WC19161 JBL47_RS10785 I
P. carotovorum RC5297 F9W95_RS04445 II
P. versatile 14A EIP93_RS21960 II
P. brasiliense ZLMLSHJ5 IHJ54_RS00290 II
P. aroidearum QJ313 L0Y30_RS00315 II
P. aroidearum AK042 L0Y25_RS00295 II
P. carotovorum PCCS1 IQ281_RS05730 II
P. parmentieri WPP163 PECWA_RS01740 II
P. parmentieri WC19161 JBL47_RS20215 II
P. parmentieri IFB5408 C5E17_RS01945 II
P. aroidearum QJ021 N5056_RS00290 II
P. brasiliense SX309 B5S52_RS00340 II
P. parmentieri HC GMW39_RS17190 II
Pectobacterium sp. 21LCBS03 MYB54_RS00315 II
P. versatile A73-S18-O15 LLE50_RS11935 II
P. parmentieri QK-5 IG623_RS01900 II
P. aroidearum QJ011 L0Y21_RS00280 II
P. brasiliense 130 MBA20_RS00310 II
P. polaris NIBIO1006 BJJ97_RS05660 II
P. parmentieri HC GMW39_RS16120 II
P. brasiliense TS20HJ1 LJQ72_RS00325 II
P. carotovorum ZJ-4-2 PSR30_RS00285 II
116
P. aroidearum LJ2 LCF43_RS00295 II

P. carotovorum RC5297 F9W95_RS21780 III
P. versatile SR1 LGL96_RS06335 III
P. versatile 14A EIP93_RS18515 III
P. versatile A73-S18-O15 LLE50_RS07535 III
P. versatile ECC15 IMY97_RS05045 III
P. versatile F131 F131LOC_RS17460 III
P. versatile 2-Mar OA04_RS18695 III
P. brasiliense TS20HJ1 LJQ72_RS17400 III
P. odoriferum BC S7 BCS7_RS18250 III
P. parvum YT22221 NV347_RS17715 III
P. parvum FN20211 LOZ86_RS17700 III
Pectobacterium sp. F1-1 NAL19_RS17595 III
P. odoriferum JK2.1 EO763_RS18400 III
P. carotovorum 2A JFY74_RS18195 III
P. carotovorum RC5297 F9W95_RS01055 III
P. brasiliense TS20HJ1 LJQ72_RS16070 IV
P. polaris NIBIO1006 BJJ97_RS21700 IV
P. aroidearum QJ021 N5056_RS16410 IV
P. aquaticum A212-S19-A16 DMB82_RS05180 IV
P. polaris NIBIO1392 BJK05_RS11620 IV
P. aroidearum LJ2 LCF43_RS05215 IV
P. atrosepticum Green1 HLB43_RS04945 IV
P. brasiliense 1692 GT391_RS12385 IV
P. parmentieri WC19161 JBL47_RS21180 IV
P. quasiaquaticum A477-S1-J17 IG609_RS04575 IV
P. aroidearum QJ313 L0Y30_RS05190 IV
P. aroidearum L6 I2D83_RS05600 IV
P. brasiliense 130 MBA20_RS04915 IV
P. versatile ECC15 IMY97_RS06365 IV
P. aroidearum QJ011 L0Y21_RS17240 IV
P. carotovorum 2A JFY74_RS05245 IV
P. carotovorum XP-13 IHJ55_RS05130 IV
P. carotovorum JR1.1 EH204_RS16510 IV
P. carotovorum ZM1 GBN63_RS00750 IV
Pectobacterium sp. PL64 HP572_RS14765 IV
P. brasiliense BZA12 CTV95_RS20455 IV
P. brasiliense SX309 B5S52_RS22765 IV
117
P. carotovorum PCCS1 IQ281_RS00565 IV

P. carotovorum PC1 PC1_RS16100 IV
P. versatile F131 F131LOC_RS16115 IV
Pectobacterium sp. 21LCBS03 MYB54_RS04870 IV
P. carotovorum RC5297 F9W95_RS20515 IV
P. parmentieri HC GMW39_RS20680 IV
P. aroidearum AK042 L0Y25_RS05390 IV
P. versatile A73-S18-O15 LLE50_RS06165 IV
P. versatile 14A EIP93_RS17085 IV
P. brasiliense IPO:4062 NAK:237 H5A40_RS00715 IV
P. parmentieri IFB5408 C5E17_RS21685 IV
P. cacticida CFCC10813 OI450_RS15625 IV
P. odoriferum JK2.1 EO763_RS17045 IV
P. parmentieri QK-5 IG623_RS23445 IV
P. carotovorum WPP14 HER17_RS04950 IV
P. carotovorum ZJ-4-2 PSR30_RS16850 IV
P. versatile SR12 LGL95_RS05075 IV
P. carotovorum 25.1 LQF52_RS16850 IV
P. carotovorum A077-S18-O15 LHL03_RS16705 IV
P. atrosepticum SCRI1043 ECA_RS16870 IV
P. polaris QK413-1 KSL88_RS05045 IV
P. brasiliense IPO:4132 NAK:239 H5A38_RS05120 IV
P. actinidiae GX-Pa1 M9782_RS18655 IV
P. brasiliense ZLMLSHJ5 IHJ54_RS15735 IV
P. cacticida CFCC10813 OI450_RS00335 IV
P. parmentieri SCC3193 W5S_RS21520 IV
P. odoriferum BC S7 BCS7_RS16795 IV
P. quasiaquaticum A398-S21-F17 IG605_RS04455 IV
P. versatile 2-Mar OA04_RS17325 IV
P. quasiaquaticum A398-S21-F17 IG605_RS04545 IV
P. brasiliense 21PCA_AGRO2 OWC53_RS17105 IV
P. versatile SR1 LGL96_RS05030 IV
P. atrosepticum SCRI1043 ECA_RS21200 V
P. atrosepticum Green1 HLB43_RS00830 V
P. parmentieri SCC3193 W5S_RS00700 V
P. parmentieri IFB5408 C5E17_RS00710 V
P. carotovorum A077-S18-O15 LHL03_RS00330 V
118
P. carotovorum 2A JFY74_RS20110 V
P. aroidearum QJ011 L0Y21_RS13090 V
P. brasiliense BC1 NC16_RS12970 V
P. parvum YT22221 NV347_RS12470 V
P. brasiliense SX309 B5S52_RS09345 V
P. quasiaquaticum A477-S1-J17 IG609_RS11695 V
P. brasiliense IPO:4132 NAK:239 H5A38_RS00515 V
P. carotovorum 25.1 LQF52_RS00325 V
P. atrosepticum 21A GZ59_RS20890 V
P. aroidearum QJ011 L0Y21_RS14315 V
P. aroidearum QJ021 N5056_RS13645 V
Pectobacterium sp. 21LCBS03 MYB54_RS08925 V
P. brasiliense 21PCA_AGRO2 OWC53_RS08265 V
P. versatile 14A EIP93_RS14330 V
P. atrosepticum SCRI1043 ECA_RS14080 V
P. aroidearum L6 I2D83_RS14360 V
P. parmentieri QK-5 IG623_RS07860 V
P. carotovorum ZJ-4-2 PSR30_RS13630 V
P. quasiaquaticum A477-S1-J17 IG609_RS07235 V
P. quasiaquaticum A398-S21-F17 IG605_RS07275 V
P. versatile F131 F131LOC_RS00100 V
P. versatile SR1 LGL96_RS10940 V
P. carotovorum PC1 PC1_RS00250 V
P. versatile 2-Mar OA04_RS22165 V
P. parmentieri WPP163 PECWA_RS13170 V
P. parmentieri QK-5 IG623_RS12785 V
P. odoriferum JK2.1 EO763_RS00285 V
P. carotovorum WPP14 HER17_RS00295 V
P. brasiliense IPO:4062 H5A40_RS06580 V
P. parmentieri WC19161 JBL47_RS00905 V
P. versatile SR12 LGL95_RS09080 V
119
P. polaris NIBIO1392 BJK05_RS07590 V

P. parmentieri WC19161 JBL47_RS09455 V
P. polaris QK413-1 KSL88_RS00315 V
P. polaris NIBIO1392 BJK05_RS16395 V
P. aquaticum A212-S19-A16 DMB82_RS00260 V
P. carotovorum ZM1 GBN63_RS05270 V
P. actinidiae GX-Pa1 M9782_RS01740 V
P. brasiliense BZA12 CTV95_RS15660 V
P. carotovorum XP-13 IHJ55_RS00260 V
P. brasiliense 1692 GT391_RS07590 V
P. cacticida CFCC10813 OI450_RS03605 V
P. carotovorum PCCS1 IQ281_RS03370 V
Dickeya
D. solani RNS 08.23.3.1.A D083_RS20790 I
D. zeae MS2 C1O30_RS03620 I
D. solani RNS 05.1.2A RS71_RS17570 I
D. zeae MS_2018 HJ586_RS19390 I
D. dadantii M2-3 KTF62_RS03905 I
D. dianthicola LAR.16.03.LID E4659_RS04125 I
D. fangzhongdai ZXC1 PQ617_RS14045 II
D. zeae EC2 DWV07_RS06335 II
D. dadantii S3-1 KNV89_RS16960 II
Dickeya sp. Secpp 1600 CPA59_RS06965 II
D. fangzhongdai AP6 FXN80_RS07265 II
D. solani RNS 08.23.3.1.A D083_RS19120 II
D. parazeae Ech586 DD586_RS06410 II
D. fangzhongdai 643b MJO48_RS07475 II
D. dadantii FZ06 MUB29_RS07370 II
D. zeae A5272 FGI00_RS14475 II
D. zeae JZL7 H7F34_RS06275 II
120
D. zeae PL65 FGI21_RS07385 II

D. fangzhongdai DSM 101947 CVE23_RS07450 II
D. fangzhongdai Onc5 K0H75_RS06810 II
D. chrysanthemi Ech1591 DD1591_RS13970 II
D. chrysanthemi Ech1591 DD1591_RS13945 II
D. fangzhongdai ND14b LH89_RS05155 II
D. zeae CE1 DWG24_RS14315 II
D. zeae MS2 C1O30_RS06820 II
D. zeae A586-S18-A17 LHK94_RS03085 II
D. dadantii M2-3 KTF62_RS07090 II
D. fangzhongdai PA1 B6N31_RS06925 II
D. dadantii 3937 DDA3937_RS06980 II
D. dadantii XJ12 OL445_RS16860 II
D. dadantii DSM 18020 CO076_RS07105 II
D. zeae MS_2018 HJ586_RS10110 II
D. solani RNS 05.1.2A RS71_RS15945 II
D. zeae EC1 W909_RS06285 II
D. zeae MS1 J417_RS06755 II
D. fangzhongdai ZXC1 PQ617_RS07225 III
D. solani RNS 08.23.3.1.A D083_RS16290 III
D. dianthicola 67-19 HGI48_RS08570 III
D. dadantii 3937 DDA3937_RS04095 III
D. dadantii FZ06 MUB29_RS04230 III
D. dadantii DSM 18020 CO076_RS08595 III
D. fangzhongdai PA1 B6N31_RS08490 III
D. dadantii XJ12 OL445_RS18575 III
D. zeae A586-S18-A17 LHK94_RS01390 III
D. chrysanthemi Ech1591 DD1591_RS05850 III
D. dadantii S3-1 KNV89_RS18970 III
D. fangzhongdai ND14b LH89_RS11675 III
D. dadantii 3937 DDA3937_RS13755 III
D. parazeae Ech586 DD586_RS07320 III
Dickeya sp. Secpp 1600 CPA59_RS07690 III
D. fangzhongdai AP6 FXN80_RS08800 III
D. zeae MS1 J417_RS03680 III
D. fangzhongdai Onc5 K0H75_RS00290 III
121

D. dianthicola LAR.16.03.LID E4659_RS07965 III
D. zeae MS_2018 HJ586_RS09155 III
D. dianthicola 67-19 HGI48_RS07200 III
D. zeae MS2 C1O30_RS07635 III
D. dadantii DSM 18020 CO076_RS08825 III
D. zeae CE1 DWG24_RS06035 III
D. fangzhongdai DSM 101947 CVE23_RS00290 III
D. fangzhongdai AP6 FXN80_RS00275 III
Dickeya sp. Secpp 1600 CPA59_RS00315 III
D. dianthicola 16ME22T JRZ88_RS07490 III
D. zeae PL65 FGI21_RS00575 III
D. zeae JZL7 H7F34_RS08170 III
D. fangzhongdai 643b MJO48_RS00270 III
D. zeae EC2 DWV07_RS07660 III
D. fangzhongdai 643b MJO48_RS08915 III
D. dadantii M2-3 KTF62_RS08735 III
D. fangzhongdai PA1 B6N31_RS00265 III
Outgroup
Pseudomonas aeruginosa PAO1 PA2684 Outgroup
Escherichia coli K-12 NAG70_RS16640 Outgroup
122
Table 6. Rhs-toxin protein length with their corresponding toxic domain and putative predicted function.
Xanthomonas
Putative
Rhs toxin Protein
Toxin domain predicted
Species strain Rhs locus Identifier Length function
X. citri LMG7439 XapA_RS12625 rhs1 Tox-REase-7 303 Dnase
X. citri NT17 AMD13_RS09980 rhs1 Tox-REase-7 335 Dnase
Xanthomonas sp. WG16 IG630_RS09960 rhs1 Tox-REase-7 335 Dnase
X. citri DAR72029 H8Z73_RS09560 rhs1 Tox-REase-7 335 Dnase
X. citri AW15 AMD07_RS12525 rhs1 Tox-REase-7 350 Dnase
X. citri TX160149 B7L66_RS12545 rhs1 Tox-REase-7 350 Dnase
X. citri DAR73886 H8Z71_RS12115 rhs1 Tox-REase-7 350 Dnase
X. citri XcmN1003 APY30_RS09720 rhs1 Tox-REase-7 335 Dnase
X. citri MSCT BGK55_RS11855 rhs1 Tox-REase-7 329 Dnase
X. citri CFBP 2036 LPY96_RS13485 rhs1 Tox-REase-7 329 Dnase
X. citri XcmH1005 APY29_RS13305 rhs1 Tox-REase-7 335 Dnase
X. vasicola Xv1601 CXP37_RS23425 rhs1 Tox-REase-7 1042 Dnase
X. campestris NCPPB 4379 KWO_RS19495 rhs1 Tox-REase-7 367 Dnase
X. vasicola NCPPB 2649 NX80_RS03165 rhs1 Tox-REase-7 333 Dnase
X. prunicola MAI5037 M0D47_RS15265 rhs1 Tox-REase-7 1033 Dnase
X. prunicola CIX383 M0D48_RS03750 rhs1 Tox-REase-7 1033 Dnase
X. prunicola CIX249 M0D43_RS15440 rhs1 Tox-REase-7 1033 Dnase
X. campestris NCPPB 4379 KWO_RS23195 rhs1 Tox-REase-7 1033 Dnase
X. vasicola Xv1601 CXP37_RS01435 rhs1 Tox-REase-7 333 Dnase
XcvCFBP7112P_R
X. citri CFBP7112 rhs1 Tox-REase-7 325 Dnase
S10110
X. citri UnB-XtecTG02- 2 G3566_RS09615 rhs1 Tox-REase-7 334 Dnase

Unknown
X. hortorum Oregano 108 OEG85_RS14840 rhs2 Undetermined 1459 function
Unknown
X. arboricola YchA LZZ50_RS07680 rhs2 Undetermined 1589 function
Unknown
X. euroxanthea 1 H7A86_RS08655 rhs2 Undetermined 1589 function
Unknown
X. translucens MAI5034 LTC53_RS05350 rhs2 Undetermined 1349 function
123
CFBP 1156 FZ025_RS15345 rhs2 Undetermined 1551 Unknown
X. hyacinthi function
Unknown
X. campestris CFBP 8444 NRY95_RS06510 rhs2 Undetermined 1565
function
XtKm33 ISN33_RS16900 rhs2 Undetermined 1352 Unknown
X. translucens function
Unknown
X. translucens Xtu-UPB513 MZO50_RS18285 rhs2 Undetermined 1323
function
Unknown
X. translucens ICMP 16317 K8O61_RS05725 rhs2 Undetermined 979
function
Unknown
X. campestris 576 JH277_RS08765 rhs2 Undetermined 1594 function
Unknown
X. campestris 16-Oct JH307_RS12435 rhs2 Undetermined 1594
function
Unknown
X. euvesicatoria LMG930 BJD11_RS05975 rhs2 Undetermined 1357
function
Unknown
X. euroxanthea CPBF 424 XTG_RS15535 rhs2 Undetermined 1520
function
Unknown
X. campestris M28 IFJ81_RS09090 rhs2 Undetermined 1329
function
Unknown
X. campestris GBBC 3077 QMY63_RS11040 rhs2 Undetermined 1329
function
Unknown
X. campestris 12049 JH293_RS08480 rhs2 Undetermined 1329
function
Unknown
X. arboricola 1314c LOK39_RS12170 rhs2 Undetermined 1337
function
Unknown
X. campestris 85-10 BHE83_RS09435 rhs2 Undetermined 893
function
Unknown
X. citri DAR73886 H8Z71_RS08810 rhs3 DUF4329 1612
function
Unknown
X. arboricola 1314c LOK39_RS10655 rhs3 DUF4329 1588
function
Unknown
X. hortorum 305 PML25_RS09245 rhs3 DUF4329 1210 function
Unknown
X. hortorum Oregano 108 OEG85_RS05880 rhs3 DUF4329 1550
function
Unknown
X. hortorum jj2001 NMB96_RS19550 rhs3 DUF4329 386
function
Unknown
X. arboricola 1311a LOK40_RS12405 rhs3 DUF4329 1592
function
Unknown
X. citri DAR72029 H8Z73_RS13160 rhs3 DUF4329 1592
function
Unknown
X. citri NT17 AMD13_RS13340 rhs3 DUF4329 1592
function
Unknown
Xanthomonas sp. WG16 IG630_RS13345 rhs3 DUF4329 1592
function
124
Unknown
X. citri AW15 AMD07_RS10975 rhs3 DUF4329 1592
function
Unknown
CFBP7112 XcvCFBP7112P_ rhs3 DUF4329 538
X. citri function
RS13110
Unknown
X. campestris 85-10 BHE83_RS06255 rhs3 DUF4329 492 function
Unknown
X. citri 8ra FPK90_RS14470 rhs3 DUF4329 459
function
Unknown
X. citri K2 FPL05_RS13800 rhs3 DUF4329 459
function
Unknown
CFBP7119 XcgCFBP7119R_ rhs3 DUF4329 459
X. citri function
RS14225
Unknown
CFBP7111 XcvCFBP7111P_ rhs3 DUF4329 904
X. citri function
RS10715
Unknown
CFBP6990 XcfCFBP6990P_R rhs3 DUF4329 413
X. citri function
S13035
CFBP6991 XcfCFBP6991P_R rhs3 DUF4329 413 Unknown
X. citri function
S13070
X. oryzae MAI134 BVV20_RS16895 rhs4 XOO_2897-deam 1528 Protease
X. oryzae CFBP7331 ACU16_RS16515 rhs4 XOO_2897-deam 1528 Protease
X. oryzae BB156-2 EYC55_RS06280 rhs4 XOO_2897-deam 1528 Protease
X. oryzae NCPPB4346 EYR26_RS16965 rhs4 XOO_2897-deam 1527 Protease
X. oryzae NJ01 MML47_RS05895 rhs4 XOO_2897-deam 1527 Protease
X. oryzae YNJC QN060_RS16955 rhs4 XOO_2897-deam 1527 Protease
X. oryzae HGA4 IZG20_RS13390 rhs4 XOO_2897-deam 811 Protease
X. oryzae PXO404 EBA23_RS08555 rhs4 XOO_2897-deam 1129 Protease
X. oryzae AUST2013 EBA17_RS16090 rhs4 XOO_2897-deam 1527 Protease
X. oryzae JS49-6 LL928_RS15920 rhs4 XOO_2897-deam 1527 Protease
X. oryzae CFBP7331 ACU16_RS16535 rhs4 XOO_2897-deam 1564 Protease
X. oryzae NCPPB4346 EYR26_RS16995 rhs4 XOO_2897-deam 1180 Protease
X. oryzae PXO211 ATY44_RS15850 rhs4 XOO_2897-deam 1564 Protease
X. translucens XtKm33 ISN33_RS07930 rhs4 XOO_2897-deam 1611 Protease
X. translucens CFBP 2055 KCU58_RS05760 rhs4 XOO_2897-deam 1611 Protease
X. translucens LW16 F0H32_RS14580 rhs4 XOO_2897-deam 1156 Protease
X. translucens MAI5034 LTC53_RS14350 rhs4 XOO_2897-deam 1607 Protease
Unknown
X. hortorum jj2001 NMB96_RS19530 rhs5 Undetermined 1598
function
Unknown
X. hortorum JS749-3 BJD10_RS12280 rhs5 Undetermined 1340
function
Unknown
LM16734 XHV734_RS0837 rhs5 Undetermined 1598
X. hortorum function
5
125
Unknown
X. hortorum ICMP 7383 BI317_RS10180 rhs5 Undetermined 1598
function
Unknown
X. hortorum B07-007 XJ27_RS06820 rhs5 Undetermined 1598
function
Unknown
CFBP 498 CFBP8129_RS1330 rhs5 Undetermined 1598
X. hortorum function
0
Unknown
X. hortorum VT106 DYQ48_RS13035 rhs5 Undetermined 1237
function
Unknown
X. oryzae PXO99A PXO_RS06855 rhs5 Undetermined 1493
function
Unknown
X. oryzae CFBP2286 ACU11_RS25435 rhs5 Undetermined 1493
function
Unknown
X. arboricola 301 P3C56_RS06145 rhs5 Undetermined 1524
function
Unknown
X. prunicola MAI5037 M0D47_RS16575 rhs5 Undetermined 1524
function
Unknown
X. oryzae PXO404 EBA23_RS08200 rhs5 Undetermined 477
function
Unknown
X. oryzae KXO85 EBA20_RS16975 rhs5 Undetermined 477
function
Unknown
X. oryzae JS49-6 LL928_RS15575 rhs5 Undetermined 477
function
Unknown
X. oryzae AUST2013 EBA17_RS16445 rhs5 Undetermined 502
function
Unknown
X. vasicola NCPPB 1060 NX81_RS13995 rhs6 Undetermined 1347 function
Unknown
X. prunicola CIX97 M0D46_RS13530 rhs6 Undetermined 1510
function
Unknown
X. citri ISO12C3 AC612_RS07065 rhs6 Undetermined 1506
function
Unknown
X. citri M12 DGN11_RS06735 rhs6 Undetermined 1318
function
Unknown
X. vasicola NCPPB 2649 NX80_RS11085 rhs6 Undetermined 1502
function
Unknown
X. citri FDC 1609 TP50_RS18950 rhs6 Undetermined 1395
function
Unknown
X. citri CFBP6166 XcfCFBP6166P_RS rhs6 Undetermined 840
function
06935
Unknown
function
06830
Unknown
function
Unknown
function
Unknown
function
126
Unknown
function
Unknown
X. citri CFBP7112 XcvCFBP711 rhs7 Undetermined 1552
function
2P_RS00780
Unknown
X. citri CFBP6990 XcfCFBP699 rhs7 Undetermined 1576
function
0P_RS00645
Unknown
X. citri CFBP6991 XcfCFBP699 rhs7 Undetermined 1576
function
1P_RS00645
Unknown
function
Unknown
function
Unknown
X. axonopodis LMG26789 Xcom_RS02415 rhs7 Undetermined 333
function
X. euvesicatoria LMG930 BJD11_RS24465 rhs7 Undetermined 1096 Unknown
function
Unknown
X. axonopodis F1 XACM_RS19450 rhs7 Undetermined 1557
function
Unknown
function
Unknown
X. euvesicatoria CFBP3836 XeaCFBP3836p rhs7 Undetermined 853
function
Unknown
X. theicola CFBP 4691 G4Q83_RS22755 rhs7 Undetermined 324
function
UnB-XtecG02- Unknown
X. citri G3566_RS00655 rhs7 Undetermined 913
2 function
X. campestris NEB122 HG421_RS21100 rhs8 Ntox16 1529 Rnase
X. oryzae NJ611 EYC54_RS21870 rhs8 Ntox16 1487 Rnase
X. oryzae BB156-2 EYC55_RS09335 rhs8 Ntox16 1496 Rnase
X. oryzae HGA4 IZG20_RS13790 rhs8 Ntox16 378 Rnase
X. oryzae KXO85 EBA20_RS17030 rhs8 Ntox16 1496 Rnase
X. oryzae PXO99A PXO_RS06285 rhs8 Ntox16 1496 Rnase
X. oryzae PXO404 EBA23_RS08175 rhs8 Ntox16 1496 Rnase
X. oryzae JS49-6 LL928_RS15550 rhs8 Ntox16 1496 Rnase
X. oryzae PXO211 ATY44_RS16160 rhs8 Ntox16 920 Rnase
X. oryzae AUST2013 EBA17_RS16470 rhs8 Ntox16 1003 Rnase
X. oryzae X11-5A EYR27_RS04990 rhs8 Ntox16 214 Rnase
Unknown
function
Unknown
function
Unknown
function
127
Unknown
X. cucurbitae OH_261 K6978_RS14005 rhs9 Undetermined 1523
function
Unknown
X. cucurbitae IL_234 K6979_RS05550 rhs9 Undetermined 1523
function
Unknown
X. arboricola 17 XB05_RS25205 rhs9 Undetermined 1541
function
Unknown
X. arboricola YchA LZZ50_RS02835 rhs9 Undetermined 1567
function
Unknown
Xanthomonas sp. 2 H7A87_RS20740 rhs9 Undetermined 410
function
Unknown
X. euroxanthea 1 H7A86_RS00675 rhs9 Undetermined 1548
function
Unknown
X. arboricola CPBF 1494 KHN93_RS00775 rhs9 Undetermined 1548
function
Unknown
Xanthomonas sp. CFBP 8443 NUG20_RS21735 rhs9 Undetermined 1572
function
Unknown
X. oryzae BB156-2 EYC55_RS01340 rhs10 Undetermined 1527
function
Unknown
X. citri UnB- rhs10 Undetermined 1367
G3566_RS22055 function
XtecTG02- 2
Unknown
X. oryzae X11-5A EYR27_RS05005 rhs10 Undetermined 1465
function
Unknown
X. citri MSCT BGK55_RS25635 rhs10 Undetermined 1494
function
Unknown
X. oryzae NJ611 EYC54_RS21145 rhs10 Undetermined 584
function
Unknown
function
Unknown
function
12670
Unknown
function
12645
X. citri ISO12C3 AC612_RS14020 rhs10 Undetermined 426 Unknown
function
Unknown
X. citri M12 DGN11_RS10075 rhs10 Undetermined 426
function
Unknown f
X. translucens XtKm7 ISN30_RS08370 rhs11 Undetermined 1358
unction
Unknown
X. sacchari DD13 NKJ47_RS07105 rhs11 Undetermined 1603 function
Unknown
X. fragariae SHQP01 K4A87_RS04825 rhs11 Undetermined 1489
function
Unknown
function
Unknown
function
128
Unknown
X. arboricola 301 P3C56_RS09790 rhs11 Undetermined 1487
function
Unknown
function
Unknown
function
Unknown
function
Unknown
X. sacchari YT9-19-2 NG828_RS06410 rhs11 Undetermined 297
function
Protease
X. oryzae CFBP7337 EBA11_RS16380 rhs12 MafB19-deam 1579
Protease
X. oryzae AXO1947 AXO1947_RS05255 rhs12 MafB19-deam 1579
Protease
X. oryzae CFBP7342 BE73_RS04950 rhs12 MafB19-deam 1579
Protease
X. oryzae BB156-2 EYC55_RS06235 rhs12 MafB19-deam 1579
Protease
X. oryzae BB151-3 EYC56_RS17670 rhs12 MafB19-deam 1579
Protease
X. oryzae PXO99A PXO_RS07175 rhs12 MafB19-deam 1579
Protease
X. oryzae NCPPB4346 EYR26_RS16955 rhs12 MafB19-deam 1178
Protease
X. translucens XtLr8 ISN38_RS15700 rhs12 MafB19-deam 1612
Protease
X. translucens XtKm15 ISN36_RS12090 rhs12 MafB19-deam 1612
Unknown
X. citri UnB-XtecTG02- 2 G3566_RS21935 rhs13 Undetermined 394
function
Unknown
X. citri T21 DGN02_RS22560 rhs13 Undetermined 242
function
Unknown
function
Unknown
X. citri CFBP7111 XcvCFBP7111P_RS rhs13 Undetermined 299
function
27080
Unknown
function
Unknown
X. arboricola CITA 33 KQR53_RS03210 rhs13 Undetermined 241
function
Unknown
X. arboricola SL2098 FPL04_RS16495 rhs13 Undetermined 1324
function
Unknown
X. arboricola 15-088 F6Y24_RS05500 rhs13 Undetermined 1155
function
Unknown
X. arboricola R1 K9U01_RS06725 rhs13 Undetermined 1155
function
Unknown
X. cucurbitae MI_359 K6982_RS13340 rhs14 Undetermined 1509
function
129
Unknown
X. campestris 29-5 JH291_RS13450 rhs14 Undetermined 1586
function
Unknown
X. citri DAR72029 H8Z73_RS16735 rhs14 Undetermined 1507
function
Unknown
X. citri DAR73886 H8Z71_RS05205 rhs14 Undetermined 1507
function
Unknown
X. citri NT17 AMD13_RS16920 rhs14 Undetermined 1507
function
Unknown
Xanthomonas sp. WG16 IG630_RS16920 rhs14 Undetermined 1507
function
Unknown
X. citri AW15 AMD07_RS05675 rhs14 Undetermined 1507
function
Unknown
X. citri TX160149 B7L66_RS04365 rhs14 Undetermined 1507
function
Unknown
X. fragariae YLX21 OZ429_RS14290 rhs14 Undetermined 859
function
Unknown
X. citri ISO12C3 AC612_RS14010 rhs15 Undetermined 1334
function
Unknown
function
Unknown
function
Unknown
function
Unknown
function
Unknown
function
Unknown
function
Unknown
X. citri CFBP6990 XcfCFBP6990P_R rhs15 Undetermined 327
function
S22380
Dnase
X. cucurbitae MI_359 K6982_RS05345 rhs16 HNH 1184
Dnase
X. citri DAR73886 H8Z71_RS14935 rhs16 HNH 1061
Dnase
X. citri DAR72029 H8Z73_RS06720 rhs16 HNH 1061
Dnase
X. citri NT17 AMD13_RS07130 rhs16 HNH 1061
Dnase
Xanthomonas sp. WG16 IG630_RS06660 rhs16 HNH 1061
Dnase
X. citri AW15 AMD07_RS14915 rhs16 HNH 1061
Dnase
X. citri TX160149 B7L66_RS03335 rhs16 HNH 1061
Dnase
X. citri GZ09 LCZ91_RS13550 rhs16 HNH 506
Unknown
function
130
Unknown
function
Unknown
X. oryzae AXO1947 AXO1947_RS2113 rhs17 Undetermined 1563
function
5
Unknown
function
Unknown
X. oryzae BAI23 EYC57_RS05720 rhs17 Undetermined 1567
function
Unknown
function
Unknown
function
Unknown
X. translucens UPB458 KFS86_RS08990 rhs17 Undetermined 1601
function
Unknown
X. hyacinthi CFBP 1156 FZ025_RS00660 rhs18 Undetermined 241
function
Unknown
function
Unknown
X. citri LMG7439 XapA_RS21055 rhs18 Undetermined 238
function
Unknown
X. citri 8ra FPK90_RS22325 rhs18 Undetermined 238
function
Unknown
X. citri K2 FPL05_RS22220 rhs18 Undetermined 238
function
Unknown
X. citri CFBP7119 XcgCFBP7119R_R rhs18 Undetermined 238
function
S21955
Unknown
X. citri CFBP7112 XcvCFBP7112P_R rhs18 Undetermined 238
function
S06845
UnB-XtecTG02 Unknown
function
-2
Unknown
X. hortorum jj2001 NMB96_RS07235 rhs19 Undetermined 303
function
Unknown
X. campestris B100 XCCB100_RS2325 rhs19 Undetermined 303
function
5
Unknown
X. campestris CN15 XCCCN15_RS2137 rhs19 Undetermined 303
function
5
Unknown
function
5
Unknown
function
Unknown
X. campestris BJSJQ20200 JM952_RS21260 rhs19 Undetermined 303
function
612
Unknown
function
131
Unknown
function
Unknown
X. vesicatoria LM159 BI313_RS24690 rhs20 Undetermined 1590
function
Unknown
function
Unknown
function
Unknown
X. hortorum OSU493 NDY25_RS19930 rhs20 Undetermined 525
function
Unknown
X. hortorum 305 PML25_RS09255 rhs20 Undetermined 525
function
Unknown
X. euvesicatoria CFBP3836 XeaCFBP3836p_R rhs20 Undetermined 1589
function
S05615
Unknown
X. campestris NCPPB 4379 KWO_RS16610 rhs21 Undetermined 1629
function
Unknown
function
Unknown
function
Unknown
function
S18080
X. oryzae ITCCBB0002 GKO49_RS15840 rhs21 Undetermined 1496 Unknown
function
Unknown
X. oryzae 0-9 GHV42_RS05550 rhs21 Undetermined 1496
function
Unknown
X. oryzae HGA4 IZG20_RS13760 rhs21 Undetermined 1496
function
Unknown
function
Unknown
function
Unknown
X. citri XcmH1005 APY29_RS09475 rhs22 Undetermined 1262
function
Unknown
X. citri XcmN1003 APY30_RS13510 rhs22 Undetermined 1262
function
Unknown
X. citri CFBP 2036 LPY96_RS09700 rhs22 Undetermined 1262
function
Unknown
function
Unknown
function
Unknown
function
Unknown
X. campestris GBBC 3077 QMY63_RS01220 rhs23 Undetermined 1513
function
132
Xanthomonas sp. 2 H7A87_RS00685 rhs23 Undetermined 1532 Unknown

function
X. campestris NEB122 HG421_RS19545 rhs23 Undetermined 319 Unknown
function
X. campestris CFBP1371 JH299_RS20680 rhs23 Undetermined 1533 Unknown
function
X. oryzae YN01 IYN96_RS05820 rhs24 GIY-YIG_SF 1539 Dnase
X. oryzae NJ01 MML47_RS05905 rhs24 GIY-YIG_SF 1138 Dnase
X. oryzae YNJC QN060_RS16945 rhs24 GIY-YIG_SF 1138 Dnase
X. oryzae YM15 FE36_RS22260 rhs24 GIY-YIG_SF 444 Dnase
X. translucens P3 F0H33_RS14415 rhs24 GIY-YIG_SF 1603 Dnase
X. translucens CFBP 2541 KHA79_RS11965 rhs24 GIY-YIG_SF 538 Dnase
X. arboricola 1314c LOK39_RS09320 rhs25 Undetermined 1512 Unknown
function
X. hortorum CFBP 498 CFBP8129_RS16560 rhs25 Undetermined 1512 Unknown
function
X. hortorum ICMP 7383 BI317_RS13745 rhs25 Undetermined 1512 Unknown
function
X. hortorum VT106 DYQ48_RS09410 rhs25 Undetermined 1512 Unknown
function
X. hortorum LM16734 XHV734_RS12125 rhs25 Undetermined 1512 Unknown
function
X. hortorum B07-007 XJ27_RS02650 rhs25 Undetermined 249 Unknown
function
X. translucens ICMP11055 NZ30_RS21745 rhs26 Undetermined 1325 Unknown
function
X. translucens CFBP 2541 KHA79_RS05675 rhs26 Undetermined 1342 Unknown
function
X. sacchari HR3-46 NG825_RS12405 rhs26 Undetermined 1287 Unknown
function
X. translucens XtLr8 ISN38_RS20150 rhs26 Undetermined 1316 Unknown
function
X. translucens XtKm15 ISN36_RS20150 rhs26 Undetermined 1418 Unknown
function
X. translucens NCPPB 3711 KHF85_RS05665 rhs26 Undetermined 926 Unknown
function
X. citri AW15 AMD07_RS26300 rhs27 Tox-REase-7 1122 Dnase
X. citri CFBP7111 XcvCFBP7111P_R rhs27 Tox-REase-7 1207 Dnase
S17725
X. citri 8ra FPK90_RS25350 rhs27 Tox-REase-7 1036 Dnase
X. citri K2 FPL05_RS24910 rhs27 Tox-REase-7 1036 Dnase
X. citri CFBP7119 XcgCFBP7119R_RS rhs27 Tox-REase-7 1036 Dnase
24575
133
X. cucurbitae IL_234 K6979_RS13135 rhs27 Tox-REase-7 1154 Dnase

X. oryzae BB156-2 EYC55_RS12190 rhs28 Undetermined 261 Unknown
function
X. oryzae NJ611 EYC54_RS12385 rhs28 Undetermined 234 Unknown
function
function
X. oryzae PXO99A PXO_RS12195 rhs28 Undetermined 1138 Unknown
function
function
X. oryzae JS49-6 LL928_RS20780 rhs28 Undetermined 1746 Unknown
function
function
X. arboricola YchA LZZ50_RS14280 rhs29 Undetermined 1341 Unknown
function
X. campestris CN15 XCCCN15_RS0922 rhs29 Undetermined 1283 Unknown
5 function
X. campestris CN03 XCCCN03_RS0891 rhs29 Undetermined 1280 Unknown
5 function
X. campestris 29-5 JH291_RS16890 rhs29 Undetermined 1288 Unknown
function
X. campestris 30-1 JH303_RS13035 rhs29 Undetermined 848 Unknown
function
X. oryzae PXO99A PXO_RS30110 rhs30 Ntox37 152 Rnase
X. oryzae PXO211 ATY44_RS23900 rhs30 Ntox37 152 Rnase
X. oryzae PXO404 EBA23_RS24925 rhs30 Ntox37 152 Rnase
X. oryzae KXO85 EBA20_RS25335 rhs30 Ntox37 152 Rnase
X. oryzae ITCCBB0002 GKO49_RS00155 rhs30 Ntox37 152 Rnase
X. oryzae JS49-6 LL928_RS08110 rhs30 Ntox37 152 Rnase
X. oryzae CFBP7331 ACU16_RS16550 rhs31 Undetermined 1546 Unknown
function
Unknown
function
Unknown
X. oryzae YN01 IYN96_RS05805 rhs31 Undetermined 1389
function
Unknown
X. oryzae YM15 FE36_RS23255 rhs31 Undetermined 467
function
Unknown
function
Unknown
X. fragariae LMG 703 OW158_RS12015 rhs31 Undetermined 1526
function
134
Unknown
function
Unknown
X. oryzae CFBP7342 BE73_RS24340 rhs32 Undetermined 1462
function
Unknown
function
Unknown
function
Unknown
function
Unknown
function
Unknown
function
Unknown
function
5
Unknown
function
0
Unknown
X. campestris B100 XCCB100_RS2435 rhs33 Undetermined 1250
function
5
Unknown
X. campestris NEB122 HG421_RS19535 rhs34 Undetermined 1600
function
Unknown
function
Unknown
function
Unknown
function
Unknown
X. hortorum Oregano 108 OEG85_RS00980 rhs34 Undetermined 545
function
Unknown
function
X. hortorum Oregano 108 OEG85_RS00950 rhs35 Undetermined 1537 Unknown
function
Unknown
function
Unknown
function
Unknown
function
Unknown
function
Unknown
X. hortorum LM16734 XHV734_RS14570 rhs36 Undetermined 1175
function
Unknown
X. arboricola 3 H5027_RS08090 rhs36 Undetermined 1175
function
135
Unknown
function
Unknown
function
Dnase
X. citri UnB-XtecTG02- 2 G3566_RS06640 rhs37 Tox-REase-7 1176
Dnase
X. oryzae NCPPB4346 EYR26_RS09400 rhs37 Tox-REase-7 1191
Dnase
X. citri CFBP7111 XcvCFBP7111P_R rhs37 Tox-REase-7 1015
S03540
Dnase
X. vasicola NCPPB 902 NX08_RS17220 rhs37 Tox-REase-7 1052
Dnase
X. citri XcmN1003 APY30_RS17325 rhs37 Tox-REase-7 1186
Unknown
X. citri FDC 1609 TP50_RS15830 rhs38 Undetermined 347
function
Unknown
function
S09985
Unknown
X. axonopodis F1 XACM_RS08075 rhs38 Undetermined 1412
function
Unknown
function
Unknown
function
Dnase
X. fragariae Fap29 BER93_RS11435 rhs39 Tox-SHH 1530
Dnase
X. fragariae PD5205 PD5205_RS11720 rhs39 Tox-SHH 1530
Dnase
X. fragariae LMG 703 OW158_RS12005 rhs39 Tox-SHH 1018
Dnase
X. translucens ICMP 16317 K8O61_RS11885 rhs39 Tox-SHH 1530
Dnase
X. translucens LMG 843 KFS85_RS06145 rhs39 Tox-SHH 1530
Unknown
X. translucens ICMP 16317 K8O61_RS11900 rhs40 Undetermined 1553
function
Unknown
X. fragariae NBC2815 NBC2815_RS2093 rhs40 Undetermined 1112
function
5
Unknown
X. fragariae Fap29 BER93_RS20935 rhs40 Undetermined 1408
function
Unknown
X. fragariae PD5205 PD5205_RS11680 rhs40 Undetermined 1408
function
Unknown
X. fragariae LMG 703 OW158_RS11970 rhs40 Undetermined 1534
function
Unknown
X. sacchari JR3-14 NG824_RS04165 rhs41 Undetermined 195
function
Unknown
function
136
Unknown
X. sacchari LT6-2 NG827_RS16975 rhs41 Undetermined 195
function
Unknown
X. sacchari LT6-16-1 NG829_RS16855 rhs41 Undetermined 195
function
Unknown
X. sacchari HR1-32 NG831_RS17630 rhs41 Undetermined 535
function
Unknown
function
Unknown
function
Unknown
function
Unknown
function
Unknown
function
Unknown
function
Unknown
function
Unknown
function
Unknown
function
Unknown
X. oryzae YN01 IYN96_RS05795 rhs43 Undetermined 1612
function
Unknown
function
Unknown
function
Unknown
function
Unknown
function
06790
X. citri CFBP6991 XcfCFBP6991P_RS rhs44 Undetermined 1044 Unknown
function
06810
Unknown
X. campestris CFBP5824 JH314_RS16820 rhs45 Undetermined 1576
function
Unknown
X. hortorum OSU493 NDY25_RS04850 rhs45 Undetermined 857
function
Unknown
X. hortorum 305 PML25_RS16215 rhs45 Undetermined 857
function
Unknown
function
Unknown
function
137
Unknown
function
Unknown
X. citri CFBP7112 XcvCFBP7112P_R rhs46 Undetermined 1203
function
S16595
Unknown
function
Unknown
function
Unknown
X. vasicola NCPPB 1060 NX81_RS24560 rhs47 DUF4258 292
function
Unknown
X. vasicola Xv1601 CXP37_RS23430 rhs47 DUF4258 344
function
Unknown
X. arboricola 15-088 F6Y24_RS23010 rhs48 COG5529 264
function
Unknown
X. arboricola R1 K9U01_RS02070 rhs48 COG5529 264
function
Unknown
X. arboricola CITA 33 KQR53_RS07875 rhs48 COG5529 264
function
Unknown
function
22370
Unknown
function
23120
Unknown
function
Unknown
X. sacchari DJ16 QBE03_RS00945 rhs50 DUF4329 1560
function
Unknown
X. sacchari LT6-16-1 NG829_RS20780 rhs50 DUF4329 1560
function
Unknown
X. sacchari JR3-14 NG824_RS20860 rhs50 DUF4329 1560
function
Dnase
X. oryzae NJ611 EYC54_RS06145 rhs51 LHH 1590
Dnase
X. oryzae BB151-3 EYC56_RS17620 rhs51 LHH 323
Dnase
X. oryzae BB151-3 EYC56_RS17600 rhs51 LHH 1103
Rnase
X. hortorum OSU493 NDY25_RS20705 rhs52 Ntox34 1488
Rnase
X. hortorum 305 PML25_RS10040 rhs52 Ntox34 1488
Rnase
X. hortorum JS749-3 BJD10_RS15725 rhs52 Ntox34 1489
Unknown
Xanthomonas sp. SS HEP74_RS05875 rhs53 Undetermined 1565
function
Unknown
Xanthomonas sp. SI HEP75_RS06045 rhs53 Undetermined 1454
function
Unknown
function
138
Unknown
function
Unknown
function
Unknown
X. sacchari DJ16 QBE03_RS01275 rhs54 Undetermined 330
function
Dnase
X. hortorum ICMP 7383 BI317_RS16880 rhs55 PDDEXK_nuclease 1197
Dnase
X. citri CFBP6975 XcfCFBP6975P_R rhs55 PDDEXK_nuclease 665
S22530
Dnase
X. campestris M28 IFJ81_RS22255 rhs55 PDDEXK_nuclease 1183
Dnase
X. oryzae X11-5A EYR27_RS02345 rhs56 AHH 1512
Dnase
X. oryzae NCPPB4346 EYR26_RS17005 rhs56 AHH 1526
Dnase
X. oryzae PXO404 EBA23_RS08545 rhs56 AHH 1526
Dnase
X. oryzae BB156-2 EYC55_RS24855 rhs56 AHH 320
Unknown
X. campestris ATCC 33913 XCC_RS00685 rhs57 Undetermined 1501
function
Unknown
X. campestris M28 IFJ81_RS00725 rhs57 Undetermined 1501
function
Unknown
function
Unknown
function
Unknown
X. euvesicatoria CFBP3836 XeaCFBP3836p_R rhs58 Undetermined 1169
function
S16095
Unknown
function
Unknown
X. campestris CFBP6690 JH261_RS05475 rhs58 Undetermined 1170
function
Unknown
function
Unknown
function
Unknown
X. citri XcmH1005 APY29_RS00650 rhs59 Undetermined 241
function
Unknown
X. citri XcmN1003 APY30_RS00665 rhs59 Undetermined 241
function
Unknown
X. citri CFBP 2036 LPY96_RS01375 rhs59 Undetermined 241
function
Unknown
X. arboricola 15-088 F6Y24_RS23015 rhs60 Undetermined 265
function
Unknown
X. arboricola CITA 33 KQR53_RS07890 rhs60 Undetermined 265
function
Unknown
X. arboricola R1 K9U01_RS02055 rhs60 Undetermined 1808
function
139
Unknown
function
Unknown
X. CFBP3836 XeaCFBP3836p_ rhs61 Undetermined 1017
function
euvesicatoria RS02340
Unknown
function
Unknown
function
Unknown
function
Unknown
function
Unknown
X. oryzae BAI23 EYC57_RS05735 rhs62 Undetermined 1547
function
Unknown
X. translucens CFBP 2541 KHA79_RS1198 rhs62 Undetermined 1587
function
0
Unknown
X. fragariae NBC2815 NBC2815_RS118 rhs62 Undetermined 1587
function
35
Unknown
X. arboricola IVIA 3978 KPG65_RS18010 rhs63 Undetermined 1326
function
Unknown
function
Unknown
X. citri CFBP7119 XcgCFBP7119R_ rhs63 Undetermined 1473
function
RS14210
X. citri K2 FPL05_RS13785 rhs63 Undetermined 1439 Unknown
function
Unknown
X. translucens LMG 843 KFS85_RS06170 rhs64 DUF2380 1027
function
Unknown
X. translucens Xtu 4699 FD63_RS13525 rhs64 DUF2380 1543
function
Unknown
X. translucens CFBP 2055 KCU58_RS05740 rhs64 DUF2380 1539
function
Unknown
X. translucens ICMP 16317 K8O61_RS11920 rhs64 DUF2380 1539
function
Unknown
function
Unknown
X. translucens CFBP 2055 KCU58_RS15120 rhs65 Undetermined 1476
function
Unknown
function
Unknown
X. translucens LMG 728 KM539_RS06295 rhs65 Undetermined 512 function
Unknown
function
Unknown
X. sacchari YT9-19-2 NG828_RS21750 rhs66 Undetermined 1572 function
140
X. sacchari LT6-2 NG827_RS20835 rhs66 Undetermined 1572 Unknown

function
X. theicola CFBP 4691 G4Q83_RS22980 rhs66 Undetermined 930 Unknown
function
X. oryzae PXO211 ATY44_RS24970 rhs67 Undetermined 1264 Unknown
function
function
function
X. oryzae ITCCBB0002 GKO49_RS02190 rhs67 Undetermined 1760 Unknown
function
X. citri 8ra FPK90_RS25305 rhs68 Undetermined 307 Unknown
function
X. citri K2 FPL05_RS24850 rhs68 Undetermined 307 Unknown
function
X. citri CFBP7119 XcgCFBP7119R_R rhs68 Undetermined 307 Unknown
S24505 function
X. citri GZ09 LCZ91_RS03705 rhs68 Undetermined 1498 Unknown
function
X. citri CFBP6990 XcfCFBP6990P_R rhs69 Undetermined 1350 Unknown
S16400 function
X. citri CFBP6991 XcfCFBP6991P_R rhs69 Undetermined 1350 Unknown
S16435 function
X. vasicola NCPPB 1060 NX81_RS17510 rhs69 Undetermined 1011 Unknown
function
X. citri T21 DGN02_RS16635 rhs69 Undetermined 424 Unknown
function
X. hortorum CFBP 498 CFBP8129_RS251 rhs70 Undetermined 1562 Unknown
30 function
X. arboricola IVIA 1317 KP727_RS13905 rhs70 Undetermined 1562 Unknown
function
X. citri CFBP7119 XcgCFBP7119R_R rhs70 Undetermined 1561 Unknown
S24395 function
Xanthomonas sp. AM6 OCJ37_RS05745 rhs70 Undetermined 1486 Unknown
function
X. citri MSCT BGK55_RS14880 rhs71 Undetermined 1223 Unknown
function
141
X. citri XcmH1005 APY29_RS16285 rhs71 Undetermined 1223 Unknown

function
X. citri XcmN1003 APY30_RS06770 rhs71 Undetermined 1223 Unknown
function
X. citri CFBP 2036 LPY96_RS16390 rhs71 Undetermined 1223 Unknown
function
X. citri XcmH1005 APY29_RS05890 rhs72 Undetermined 1384 Unknown
function
X. citri XcmN1003 APY30_RS17055 rhs72 Undetermined 1358 Unknown
function
X. citri T21 DGN02_RS16640 rhs72 Undetermined 847 Unknown
function
X. citri CFBP 2036 LPY96_RS06290 rhs72 Undetermined 1331 Unknown
function
X. campestris MAFF106181 AD14011_RS0504 rhs73 ParB 1164 Dnase
X. campestris 719 JH264_RS16040 rhs73 ParB 1164 Dnase
X. sacchari DJ16 QBE03_RS07645 rhs74 Undetermined 1499 Unknown
function
X. sacchari HR3-46 NG825_RS06490 rhs74 Undetermined 1288 Unknown
function
X. fragariae SHQP01 K4A87_RS01750 rhs75 Undetermined 546 Unknown
function
X. fragariae NBC2815 NBC2815_RS0239 rhs75 Undetermined 450 Unknown
0 function
X. translucens Xtu-UPB513 MZO50_RS08935 rhs76 Tox-URI2 1563 Dnase
X. translucens ART-Xtg2 KM563_RS15915 rhs76 Tox-URI2 1560 Dnase
X. sacchari LT6-2 NG827_RS20540 rhs77 Undetermined 329 Unknown
function
X. translucens NCPPB 3711 KHF85_RS19690 rhs77 Undetermined 322 Unknown
function
X. campestris CFBP 8444 NRY95_RS05470 rhs78 Undetermined 1547 Unknown
function
Xanthomonas sp. CFBP 8445 NUG21_RS11915 rhs78 Undetermined 1362 Unknown
function
function
X. oryzae HGA4 IZG20_RS13435 rhs79 Undetermined 243 Unknown
function
142
Unknown
X. translucens LMG 728 KM539_RS10370 rhs80 Undetermined 1524
function
Unknown
X. campestris CFBP 8444 NRY95_RS12615 rhs80 Undetermined 1530
function
Unknown
function
Unknown
function
Unknown
function
Unknown
function
Xanthomonas sp. GW HEP73_RS20695 rhs82 Tox-MPTase3 1521 Protease
X. euroxanthea CPBF 424 XTG_RS11135 rhs82 Tox-MPTase3 1549 Protease

Unknown
X. albilineans Xa-FJ1 XaFJ1_RS11700 rhs83 Undetermined 1812
function
Unknown
X. oryzae BB156-2 EYC55_RS20950 rhs83 TraT 1798
function
Unknown
Xanthomonas sp. GW HEP73_RS22405 rhs84 Undetermined 1417
function
Unknown
X. campestris NEB122 HG421_RS10280 rhs84 Undetermined 1593
function
Unknown
X. axonopodis NCPPB 796 XAV_RS01910 rhs85 YkuD 1774
function
Unknown
X. fragariae YLX21 OZ429_RS17740 rhs85 YkuD 502 function
X. sacchari HR1-32 NG831_RS05640 rhs86 Tox-REase-9 1593 Dnase
Unknown
function
X. campestris MAFF106181 AD14011_RS21420 rhs87 DNase_NucA_NucB 1817 Dnase
X. campestris 719 JH264_RS19020 rhs87 DNase_NucA_NucB 274 Dnase
Unknown
function
Unknown
X. sacchari HR1-32 NG831_RS12130 rhs88 Undetermined 251 function
X. oryzae NJ611 EYC54_RS02450 rhs89 Ntox37 445 Rnase
X. oryzae BB156-2 EYC55_RS20925 rhs89 Ntox37 1777 Rnase
Unknown
X. sacchari LT6-2 NG827_RS06235 rhs90 Undetermined 1540
function
Unknown
Xanthomonas sp. GW HEP73_RS22415 rhs90 Undetermined 1528
function
Unknown
function
Unknown
function
143
Xanthomonas sp. SS HEP74_RS06905 rhs92 Undetermined 1816 Unknown

function
Xanthomonas sp. SI HEP75_RS07115 rhs92 Undetermined 1816 Unknown
function
X. perforans LH3 BJD13_RS17860 rhs93 Undetermined 290 Unknown
function
X. arboricola 1314c LOK39_RS19830 rhs93 Undetermined 290 Unknown
function
X. oryzae ITCCBB0002 GKO49_RS23560 rhs94 GIY-YIG_SF 615 Dnase
X. translucens LW16 F0H32_RS14615 rhs94 GIY-YIG_SF 1598 Dnase
X. hortorum JS749-3 BJD10_RS00030 rhs94 Undetermined 1751 Unknown
function
function
X. sacchari YT9-19-2 NG828_RS12055 rhs95 FinO_conjug_rep 316 Protease
X. sacchari HR1-32 NG831_RS12085 rhs95 FinO_conjug_rep 200 Protease
function
X. oryzae BAI23 EYC57_RS05750 rhs96 Undetermined 1124 Unknown
function
X. fragariae SHQP01 K4A87_RS01730 rhs97 Undetermined 1756 Unknown
function
X. euroxanthea 1 H7A86_RS06100 rhs98 LpoA 1033 Protease
Unknown
function
S13005
Unknown
X. hortorum Oregano 108 OEG85_RS21165 rhs100 Undetermined 567 function
Unknown
function
X. sacchari HR3-46 NG825_RS08785 rhs102 Tae4 1773 Protease
Unknown
X. translucens NCPPB 3711 KHF85_RS19670 rhs103 Undetermined 232
function
Unknown
function
X. oryzae HGA4 IZG20_RS13450 rhs105 Tox-SHH 1523 Dnase
Unknown
function
Unknown
function
Unknown
X. fragariae YLX21 OZ429_RS17750 rhs108 Undetermined 1774 function
Unknown
function
144
Unknown
function
Unknown
function
S13040
Unknown
function
Unknown
Xanthomonas sp. CFBP 8443 NUG20_RS15640 rhs113 DUF4329 1556
function
Unknown
function
Unknown
X. citri CFBP7119 XcgCFBP7119R_ rhs115 Undetermined 738
function
RS08365
Dnase
X. sacchari HR1-32 NG831_RS21295 rhs116 Tox-REase-9 317
Unknown
function
Unknown
function
Unknown
function
Rnase
X. campestris CFBP 8444 NRY95_RS01070 rhs120 Ntox21 329
Unknown
function
Xanthomonas sp. SI HEP75_RS15370 rhs122 Undetermined 320 Unknown
function
Unknown
function
Unknown
function
Unknown
X. translucens NCPPB 3711 KHF85_RS19705 rhs125 Undetermined 325
function
Unknown
function
Xanthomonas sp. SI HEP75_RS09565 rhs127 FHA 342 Protease
Unknown
function
X. axonopodis NCPPB 796 XAV_RS01895 rhs129 PMT1 1759 Protease
Unknown
function
Unknown
Xanthomonas sp. AM6 OCJ37_RS20860 rhs131 Undetermined 1558
function
Unknown
function
Unknown
function
145
Unknown
X. sacchari DD13 NKJ47_RS07755 rhs134 Undetermined 263
function
Unknown
function
Unknown
function
Unknown
function
Unknown
function
Unknown
X. translucens ICMP11055 NZ30_RS06515 rhs139 Undetermined 1365
function
Protease
X. citri T21 DGN02_RS02115 rhs140 CHAP 1787
Unknown
function
Unknown
X. citri CFBP7111 XcvCFBP7111P_ rhs142 Undetermined 1332
function
RS26910
Unknown
function
Unknown
function
Unknown
function
Dnase
X. citri T21 DGN02_RS06740 rhs146 HNH 648
Unknown
function
Dnase
X. campestris CFBP 8444 NRY95_RS15825 rhs148 HNH 304
Unknown
function
Unknown
function
Unknown
X. translucens ICMP11055 NZ30_RS06505 rhs151 Undetermined 1584
function
Unknown
function
Unknown
function
Unknown
function
Unknown
function
Unknown
X. translucens LMG 843 KFS85_RS14540 rhs156 Undetermined 1581
function
Unknown
X. citri ISO12C3 AC612_RS11240 rhs157 Undetermined 233 function
146
Unknown
Xanthomonas sp. CFBP 8443 NUG20_RS00825 rhs158 Undetermined 332 function
Unknown
X. hortorum JS749-3 BJD10_RS00020 rhs159 Undetermined 562 function
Unknown
X. oryzae BB151-3 EYC56_RS02655 rhs160 Undetermined 1773 function
Unknown
function
Unknown
Xanthomonas sp. GW HEP73_RS22295 rhs162 DUF4157 1539
function
X. euvesicatoria LMG930 BJD11_RS09230 rhs163 Undetermined 493 Unknown
function
Unknown
X. axonopodis NCPPB 796 XAV_RS0 rhs164 Undetermined 604
function
1920
Unknown
function
Unknown
function
Unknown
function
Unknown
function
Unknown
function
X. arboricola CPBF 766 KHN80_RS03670 rhs170 PT-HINT 1623 Protease
Unknown
function
Unknown
function
Unknown
function
X. theicola CFBP 4691 G4Q83_RS00275 rhs174 Peptidase_C97 427 Protease
Unknown
function
Unknown
function
UnB-XtecTG02- Unknown
2 function
Unknown
X. oryzae CFBP7342 BE73_RS10025 rhs178 Undetermined 1334 function
Unknown
function
Unknown
function
Unknown
function
147
Ralstonia
Rhs Putative
Toxin domain Protein
Species strain Rhs locus Toxin predicted
Length
identifier function
Unknown
R. solanacearum YC40-M A3768_RS18470 rhs1 Undetermined 1718
function
R. Unknown
pseudosolanacearum CQPS-1 BC350_RS21085 rhs1 Undetermined 1706
function
Unknown
R. solanacearum HA4-1 CFM90_RS22255 rhs1 Undetermined 1698
function
Unknown
R. solanacearum T117 CJO74_RS17965 rhs1 Undetermined 1716 function
Unknown
R. solanacearum T78 CJO80_RS19245 rhs1 Undetermined 1716
function
Unknown
function
R. solanacearum T42 CJO83_RS17890 rhs1 Undetermined 1740 Unknown
function
Unknown
R. solanacearum SL3730 CJO90_RS17890 rhs1 Undetermined 1740
function
Unknown
function
Unknown
R. solanacearum UW386 E7Z57_RS20405 rhs1 Undetermined 506
function
Unknown
R. solanacearum 204 G7939_RS19800 rhs1 Undetermined 1706
function
Unknown
R. solanacearum B2 G7968_RS24855 rhs1 Undetermined 370
function
Unknown
R. solanacearum 202 G7969_RS18430 rhs1 Undetermined 1706
function
Unknown
R. solanacearum FJAT454.F50-1 H9X76_RS24320 rhs1 Undetermined 1706
function
Unknown
R. solanacearum FJAT1303.F50 HI793_RS18750 rhs1 Undetermined 418 function
Unknown
R. solanacearum FJAT1452.F50 HI796_RS17655 rhs1 Undetermined 1718
function
Unknown
function
Unknown
function
Unknown
function
Unknown
function
Unknown
function
Unknown
function
148
Unknown
function
Unknown
function
Unknown
function
Unknown
function
Unknown
function
Unknown
R. solanacearum FJAT91-F8 HWE47_RS2254 rhs1 Undetermined 1704
function
0
Unknown
R. solanacearum 362200 IMF25_RS24870 rhs1 Undetermined 1698
function
R. Unknown
pseudosolanacearum PeaFJ1 JNO62_RS22440 rhs1 Undetermined 1698
function
Unknown
Ralstonia sp. RS647 LGV81_RS21355 rhs1 Undetermined 1718
function
Unknown
R. solanacearum Bs715 NQ321_RS04685 rhs1 Undetermined 1716 function
Unknown
R. solanacearum Wj644 NQ322_RS19390 rhs1 Undetermined 1704
function
Unknown
R. solanacearum MolK2 PG909_RS09415 rhs1 Undetermined 414
function
R.
pseudosolanacearum CQPS-1 BC350_RS23285 rhs2 GH-E 1522 Dnase
R. solanacearum T117 CJO74_RS23195 rhs2 GH-E 1522 Dnase
R. solanacearum SL3730 CJO90_RS22770 rhs2 GH-E 1522 Dnase
Unknown
function
Unknown
R. solanacearum RSCM CYD94_RS25805 rhs2 Undetermined 1471
function
R. solanacearum 204 G7939_RS25520 rhs2 GH-E 1522 Dnase
R. solanacearum B2 G7968_RS19500 rhs2 GH-E 1522 Dnase
R. solanacearum 202 G7969_RS20605 rhs2 GH-E 1522 Dnase
R. solanacearum FJAT454.F50-1 H9X76_RS21700 rhs2 GH-E 1522 Dnase
R. solanacearum FJAT1303.F50 HI793_RS23775 rhs2 GH-E 1522 Dnase
149

R. solanacearum MAFF 301560 JK146_RS23060 rhs2 GH-E 1522 Dnase
R. solanacearum MAFF 311693 JL096_RS19885 rhs2 GH-E 1522 Dnase
R. solanacearum RS24 KM864_RS21360 rhs2 GH-E 1522 Dnase
R. solanacearum KACC 10709 LBM341_RS17810 rhs2 GH-E 1522 Dnase
Ralstonia sp. RS642 LGV80_RS22445 rhs2 GH-E 1522 Dnase
Unknown
function
R. solanacearum Bs715 NQ321_RS01715 rhs2 GH-E 1522 Dnase
R. solanacearum Wj644 NQ322_RS21745 rhs2 GH-E 1522 Dnase
R.
pseudosolanacearum Cw717 NQS35_RS21795 rhs2 GH-E 1522 Dnase
R.
pseudosolanacearum Sw698 NQS37_RS22645 rhs2 GH-E 1522 Dnase
R. solanacearum YC40-M A3768_RS23425 rhs3 AHH 1518 Dnase
R.
pseudosolanacearum RS 476 CDC45_RS23365 rhs3 AHH 1518 Dnase
R. solanacearum FJAT15244-F8 H1A20_RS25825 rhs3 AHH 1518 Dnase
R. solanacearum UW763 HF907_RS18100 rhs3 AHH 1518 Dnase
R. solanacearum FJAT1452.F50 HI796_RS22565 rhs3 AHH 1518 Dnase
R. syzygii UGMSS_Db01 JK151_RS13280 rhs3 AHH 1524 Dnase
Ralstonia sp. RS647 LGV81_RS26510 rhs3 AHH 1518 Dnase
Ralstonia sp. RS650 LGV82_RS22835 rhs3 AHH 1518 Dnase
R.
pseudosolanacearum SL1931 MNY32_RS19885 rhs3 AHH 1518 Dnase
150
R. syzygii Dnase
BDBR229 PG904_RS11850 rhs3 AHH 1524
R. Dnase
pseudosolanacearum GMI1000 RS_RS22605 rhs3 AHH 1518
Unknown
function
Unknown
function
Unknown
R. solanacearum RUN2279 HF908_RS21900 rhs4 Undetermined 641
function
Unknown
R. solanacearum RUN2474 HF909_RS21515 rhs4 Undetermined 641
function
Unknown
function
Unknown
function
Unknown
function
Unknown
function
Unknown
R. syzygii UGMSS_Db01 JK151_RS22250 rhs4 Undetermined 763
function
R. Unknown
pseudosolanacearum LMG 9673 NY025_RS04810 rhs4 Undetermined 642
function
Unknown
R. syzygii BDBR229 PG904_RS23545 rhs4 Undetermined 763
function
R. Unknown
pseudosolanacearum RUN2340 PG907_RS18570 rhs4 Undetermined 641
function
Unknown
R. solanacearum PSI07 RPSI07_RS23215 rhs4 Undetermined 763 function
Unknown
R. solanacearum K60 B7R77_18740 rhs4 Undetermined 1,732
function
R. solanacearum IBSBF 2571 C2124_RS18965 rhs5 AHH 1433 Dnase
R. solanacearum RS 488 CCY86_RS18665 rhs5 AHH 1433 Dnase
R. solanacearum RS 489 CDC59_RS18560 rhs5 AHH 1433 Dnase
R. solanacearum CIAT_078 G8D25_RS02275 rhs5 AHH 1433 Dnase
R. solanacearum CFBP2957 PG903_RS23825 rhs5 AHH 1433 Dnase
R. solanacearum IBSBF1503 RALBFv3_RS16990 rhs5 AHH 1433 Dnase
R. solanacearum Po82 RSPO_RS18555 rhs5 AHH 1433 Dnase
R. solanacearum UY031 RSUY_RS18170 rhs5 AHH 1433 Dnase
R. solanacearum UW163 UW163_RS18365 rhs5 AHH 1433 Dnase
R. solanacearum UW551 B7R79_19675 rhs5 AHH 1433 Dnase
Unknown
R. syzygii LLRS-1 GO998_RS16440 rhs6 Undetermined 778
function
151
R. solanacearum Unknown
UW763 HF907_RS21545 rhs6 Undetermined 779
function
Ralstonia sp. Unknown
RS642 LGV80_RS25870 rhs6 Undetermined 779
function
Ralstonia sp. Unknown
RS650 LGV82_RS18275 rhs6 Undetermined 779
function
CFBP2957 PG903_RS00780 rhs6 Undetermined 780
function
R. syzygii Unknown
BDBR229 PG904_RS22280 rhs6 Undetermined 779
function
SEPPX05 RSSE_RS25425 rhs6 Undetermined 779
function
K60 B7R77_25580 rhs6 Undetermined 193
function
IBSBF 2571 C2124_RS17055 rhs7 Undetermined 476
function
CIAT_078 G8D25_RS07315 rhs7 Undetermined 461
function
RUN2279 HF908_RS21925 rhs7 Undetermined 674
function
RUN2474 HF909_RS21530 rhs7 Undetermined 669
function
R. Unknown
function
R. Unknown
function
Unknown
R. solanacearum IBSBF1503 RALBFv3_RS2265 rhs7 Undetermined 1727
function
5
Unknown
R. solanacearum Po82 RSPO_RS16785 rhs7 Undetermined 476
function
Unknown
R. solanacearum UW163 UW163_RS23205 rhs7 Undetermined 476
function
R. Unknown
pseudosolanacearum RS 476 CDC45_RS20075 rhs8 Undetermined 742 function
R. solanacearum Rs5 HF906_RS14655 rhs8 ParB 745 Dnase
R. solanacearum UW763 HF907_RS21440 rhs8 ParB 742 Dnase
R. solanacearum RUN2279 HF908_RS21940 rhs8 ParB 1702 Dnase
R. solanacearum RUN2474 HF909_RS21545 rhs8 ParB 1726 Dnase
R.
pseudosolanacearum LMG 9673 NY025_RS04770 rhs8 ParB 524 Dnase
R. solanacearum CFBP2957 PG903_RS16825 rhs8 ParB 1699 Dnase
R.
pseudosolanacearum GMI1000 RS_RS19450 rhs8 ParB 742 Dnase
R. solanacearum K60 B7R77_06485 rhs8 ParB 648 Dnase
R. Unknown
pseudosolanacearum RS 476 CDC45_RS18105 rhs9 Undetermined 765
function
152
R. syzygii LLRS-1 GO998_RS25265 rhs9 Undetermined 171 Unknown

function
R. solanacearum UW763 HF907_RS23280 rhs9 Undetermined 765 Unknown
function
R. syzygii UGMSS_Db0 JK151_RS09130 rhs9 Undetermined 765 Unknown
1 function
R. syzygii BDBR229 PG904_RS07320 rhs9 Undetermined 765 Unknown
function
R. GMI1000 RS_RS17595 rhs9 Undetermined 765 Unknown
pseudosolanacearum function
R. solanacearum K60 B7R77_09795 rhs9 Undetermined 767 Unknown
function
function
function
function
R. solanacearum SL3175 CJO92_RS05000 rhs10 Undetermined 780 Unknown
function
function
R. solanacearum KACC 10722 LBM2029_RS06085 rhs10 Undetermined 778 Unknown
function
function
R. solanacearum T95 CJO78_RS21520 rhs11 Peptidase_C80 778 Protease
R. solanacearum T51 CJO82_RS21185 rhs11 Peptidase_C80 778 Protease
Unknown
function
R. solanacearum SL2064 CJO98_RS21545 rhs11 Peptidase_C80 778 Protease
R. solanacearum KACC 10722 LBM2029_RS20775 rhs11 Peptidase_C80 778 Protease
R. wenshanensis 56D2 KOL96_RS01525 rhs12 GH-E 1542 Dnase
R. solanacearum KACC 10722 LBM2029_RS21285 rhs12 GH-E 1541 Dnase
R. solanacearum SEPPX05 RSSE_RS24345 rhs12 GH-E 1540 Dnase
Unknown
function
Unknown
R. solanacearum T95 CJO78_RS25235 rhs13 Undetermined 249 function
153
Unknown
function
Unknown
function
Unknown
function
Unknown
R. solanacearum KACC 10722 LBM2029_RS25550 rhs13 Undetermined 249
function
Unknown
R. syzygii LLRS-1 GO998_RS00950 rhs14 DUF2380 776
function
Unknown
R. solanacearum KACC 10709 LBM341_RS13945 rhs14 DUF2380 776
function
R. Unknown
Sw698 NQS37_RS02985 rhs14 DUF2380 776
Unknown
R. solanacearum CFBP2957 PG903_RS16705 rhs14 DUF2380 777
function
Unknown
R. solanacearum MolK2 PG909_RS13555 rhs14 DUF2380 777
function
Unknown
R. solanacearum RS 488 CCY86_RS01325 rhs15 Undetermined 782
function
Unknown
R. solanacearum RS 489 CDC59_RS01315 rhs15 Undetermined 783
function
Unknown
function
Unknown
function
Unknown
R. solanacearum UY031 RSUY_RS01280 rhs15 Undetermined 782
function
Unknown
function
Unknown
function
Unknown
function
Unknown
function
Unknown
R. solanacearum CFBP2957 PG903_RS22375 rhs16 Undetermined 1474
function
R. Unknown
function
R. Unknown
function
R. solanacearum UW763 HF907_RS25250 rhs17 Undetermined 789 Unknown
function
R. Unknown
pseudosolanacearum GMI1000 RS_RS23445 rhs17 Undetermined 741
function
R. Unknown
GMI1000 RS_RS23575 rhs17 Undetermined 741
154
Unknown
R. solanacearum UW763 HF907_RS22995 rhs18 Undetermined 1695
function
Unknown
R. solanacearum MAFF 301560 JK146_RS18420 rhs18 Undetermined 1702
function
Unknown
R. solanacearum RS24 KM864_RS24520 rhs18 Undetermined 1702
function
Unknown
function
Unknown
function
Unknown
function
Dnase
R. solanacearum UW763 HF907_RS03305 rhs20 GIY-YIG_SF 775
Dnase
R. solanacearum MolK2 PG909_RS04760 rhs20 GIY-YIG_SF 777
Dnase
R. solanacearum K60 B7R77_09775 rhs20 GIY-YIG_SF 784
Unknown
function
Unknown
function
Unknown
R. solanacearum PSI07 RPSI07_RS26455 rhs21 Undetermined 1539
function
Unknown
R. solanacearum HA4-1 CFM90_RS25210 rhs22 Undetermined 1482
function
Unknown
R. solanacearum 362200 IMF25_RS21770 rhs22 Undetermined 1482
function
R. Unknown
PeaFJ1 JNO62_RS19340 rhs22 Undetermined 1482
Unknown
R. solanacearum RS 488 CCY86_RS16535 rhs23 Undetermined 758
function
Unknown
function
Unknown
R. solanacearum UY031 RSUY_RS16195 rhs23 Undetermined 758
function
Unknown
R. wenshanensis 56D2 KOL96_RS09195 rhs24 Undetermined 1887
function
Unknown
function
Unknown
function
R. Unknown
RS 476 CDC45_RS18395 rhs25 Undetermined 1711
Unknown
R. solanacearum B2 G7968_RS24875 rhs25 Undetermined 1713
function
R. Unknown
pseudosolanacearum GMI1000 RS_RS17875 rhs25 Undetermined 1711
function
155
Unknown
function
Unknown
function
Unknown
R. solanacearum K60 B7R77_06540 rhs26 Undetermined 752
function
Unknown
R. solanacearum T98 CJO77_RS05780 rhs27 HI1514 774 function
Unknown
R. solanacearum SL3175 CJO92_RS05780 rhs27 HI1514 774
function
Unknown
R. solanacearum PSI07 RPSI07_RS13970 rhs27 HI1514 774
function
Unknown
function
Unknown
function
Unknown
function
Unknown
R. solanacearum KACC 10722 LBM2029_RS07455 rhs28 Undetermined 799
function
R. solanacearum RS 488 CCY86_RS00760 rhs29 Ntox44 775 Rnase
R. solanacearum MolK2 PG909_RS12995 rhs29 Ntox44 776 Rnase
R. solanacearum UY031 RSUY_RS00730 rhs29 Ntox44 775 Rnase
R. solanacearum UW551 B7R79_15660 rhs29 Ntox44 775 Rnase
function
function
Ralstonia sp. RS642 LGV80_RS25775 rhs30 Undetermined 752 Unknown
function
Ralstonia sp. RS650 LGV82_RS18195 rhs30 Undetermined 752 Unknown
function
R. solanacearum RS 488 CCY86_RS17490 rhs31 DUF2778 764 Unknown
function
R. solanacearum RS 489 CDC59_RS17400 rhs31 DUF2778 764 Unknown
function
R. solanacearum UY031 RSUY_RS17095 rhs31 DUF2778 764 Unknown
function
R. solanacearum UW551 B7R79_18395 rhs31 DUF2778 764 Unknown
function
R. solanacearum IBSBF 2571 C2124_RS17000 rhs32 Undetermined 1716 Unknown
function
R. solanacearum CIAT_078 G8D25_RS07370 rhs32 Undetermined 1716 Unknown
function
156
Unknown
R. solanacearum Po82 RSPO_RS16740 rhs32 Undetermined 1716
function
Unknown
R. solanacearum UW163 UW163_RS23250 rhs32 Undetermined 1716
function
Unknown
function
Unknown
function
Unknown
function
Unknown
function
Unknown
R. solanacearum CFBP2957 PG903_RS18400 rhs35 NuoF 796
function
R. Unknown
pseudosolanacearum RUN2340 PG907_RS00945 rhs35 Undetermined 801 function
R. solanacearum Rs5 HF906_RS14570 rhs36 Undetermined 743 Unknown
function
R. Unknown
function
R. solanacearum Rs5 HF906_RS22570 rhs37 AHH 1420 Dnase
R. solanacearum K60 B7R77_24635 rhs37 AHH 1418 Dnase
Unknown
function
Unknown
function
Unknown
R. solanacearum UW763 HF907_RS23235 rhs39 Undetermined 630 function
R. Unknown
RUN2340 PG907_RS00925 rhs39 Undetermined 787 function
pseudosolanacearum
Unknown
R. syzygii LLRS-1 GO998_RS01050 rhs40 Undetermined 135 function
R. Unknown
RUN2340 PG907_RS19990 rhs40 Undetermined 661 function
pseudosolanacearum
Rnase
R. solanacearum T98 CJO77_RS15880 rhs41 Ntox37 768
Rnase
R. solanacearum SL3175 CJO92_RS15890 rhs41 Ntox37 768
Unknown
R. syzygii LLRS-1 GO998_RS01035 rhs42 DUF4258 760 function
Unknown
R. solanacearum UW763 HF907_RS11975 rhs42 DUF4258 760 function
Unknown
R. solanacearum RUN2279 HF908_RS21860 rhs43 Undetermined 195 function
Unknown
Unknown
R. syzygii LLRS-1 GO998_RS25125 rhs44 Undetermined 767 function
Unknown
157
R. solanacearum CFBP2957 PG903_RS08630 rhs45 Undetermined 798 Unknown

function
R. solanacearum PSI07 RPSI07_RS04725 rhs46 Undetermined 774 Unknown
function
R. solanacearum MolK2 PG909_RS22915 rhs47 Undetermined 1419 Unknown
function
R. solanacearum MolK2 PG909_RS09265 rhs48 Undetermined 703 Unknown
function
R. solanacearum K60 B7R77_09705 rhs49 Tox-GHH 803 Dnase
R. solanacearum CFBP2957 PG903_RS20055 rhs50 Ntox24 776 Rnase
R. Unknown
function
Unknown
R. solanacearum Rs5 HF906_RS15770 rhs52 Undetermined 753
function
Unknown
R. solanacearum FJAT15353.F8 HI815_RS18720 rhs53 Undetermined 485 function
Ralstonia sp. RS650 LGV82_RS01465 rhs54 HNH 801 Dnase
Unknown
function
Unknown
function
Unknown
function
0
R. Unknown
function
Unknown
R. solanacearum MolK2 PG909_RS09385 rhs59 Undetermined 1715
function
Unknown
R. mannitolilytica SN83A39 VZ52_RS23650 rhs60 Undetermined 134
function
Unknown
function
Unknown
function
Unknown
function
Unknown
function
Dnase
R. solanacearum CFBP2957 PG903_RS15080 rhs65 Tox-REase-7 768
Rnase
R. wenshanensis 56D2 KOL96_RS03740 rhs66 CdiA-CT 347
Unknown
R. solanacearum Rs5 HF906_RS18130 rhs67 Undetermined 698
function
R. Unknown
LMG 9673 NY025_RS04785 rhs68 Undetermined 456
Unknown
R. syzygii R24 PG906_RS24625 rhs69 DUF2778 1643
function
158
R. solanacearum Rs5 HF906_RS24280 rhs70 Unknown

Undetermined 248
function
R. wenshanensis 56D2 KOL96_RS24630 rhs71 Dnase
Tox-HNH-EHHH 345
R. solanacearum RS 489 CDC59_RS00750 rhs72 Unknown
Undetermined 775
function
R. solanacearum MolK2 PG909_RS12430 rhs73 Unknown
Undetermined 323
function
R. solanacearum MolK2 PG909_RS09395 rhs74 Unknown
Undetermined 626
function
R. LMG 9673 NY025_RS04755 rhs75 Unknown
Undetermined 483
R. solanacearum K60 B7R77_06465 rhs76 Unknown
Undetermined 759
function
Pectobacterium
Rhs- Putative
Toxin Protein
Species strain Rhs locus toxin predicted
domain Length
identifier function
Unknown
P. carotovorum RC5297 F9W95_RS21780 rhs1 Undetermined 2743
function
Unknown
P. versatile SR1 LGL96_RS06335 rhs1 Undetermined 2743
function
Unknown
P. versatile 14A EIP93_RS18515 rhs1 Undetermined 2743
function
Unknown
P. versatile A73-S18-O15 LLE50_RS07535 rhs1 Undetermined 2743 function
Unknown
P. versatile ECC15 IMY97_RS05045 rhs1 Undetermined 2743
function
Unknown
P. versatile F131 F131LOC_RS17460 rhs1 Undetermined 2743
function
Unknown
P. versatile 2-Mar OA04_RS18695 rhs1 Undetermined 2609
function
Unknown
P. brasiliense TS20HJ1 LJQ72_RS17400 rhs1 Undetermined 2510
function
Dnase
P. WPP14 HER17_RS00280 rhs2 ParB 538
carotovorum
Dnase
P. brasiliense IPO:4062 H5A40_RS06565 rhs2 ParB 537
NAK:237
Dnase
P. parmentieri WPP163 PECWA_RS22890 rhs2 ParB 115
Dnase
P. parmentieri HC GMW39_RS18205 rhs2 ParB 115
Dnase
P. parmentieri IFB5408 C5E17_RS00820 rhs2 ParB 115
Dnase
P. parmentieri IFB5441 C5E20_RS00960 rhs2 ParB 115
Dnase
P. parmentieri WC19161 JBL47_RS00930 rhs2 ParB 115
159
P. parmentieri QK-5 IG623_RS00755 rhs2 ParB 115 Dnase

Unknown
function
Unknown
function
Unknown
P. versatile A73-S18-O15 LLE50_RS11925 rhs3 Undetermined 483
function
Unknown
P. aroidearum QJ021 N5056_RS00280 rhs3 Undetermined 288
function
Unknown
P. parmentieri SCC3193 W5S_RS25520 rhs3 Undetermined 234
function
Unknown
P. parmentieri WC19161 JBL47_RS23105 rhs3 Undetermined 234
function
Unknown
P. parmentieri IFB5485 C5E21_RS23040 rhs3 Undetermined 234
function
Unknown
function
Unknown
P. atrosepticum SCRI1043 ECA_RS21200 rhs4 Undetermined 1427 function
Unknown
P. atrosepticum Green1 HLB43_RS00830 rhs4 Undetermined 1427
function
Unknown
function
P. parmentieri IFB5408 C5E17_RS00710 rhs4 Undetermined 920 Unknown
function
Unknown
P. atrosepticum SCRI1043 ECA_RS23505 rhs4 Undetermined 153
function
P. atrosepticum Green1 HLB43_RS00790 rhs4 Undetermined 153 Unknown
function
Unknown
function
Unknown
P. carotovorum A077-S18-O15 LHL03_RS00330 rhs5 Undetermined 1464
function
Unknown
P. aroidearum L6 I2D83_RS22465 rhs5 Undetermined 349
function
Unknown
function
Unknown
P. parmentieri QK-5 IG623_RS23235 rhs5 Undetermined 351
function
Unknown
function
Unknown
function
Unknown
P. carotovorum 2A JFY74_RS20120 rhs5 Undetermined 302
function
Dnase
P. polaris NIBIO1392 BJK05_RS23140 rhs6 AHH 394
160
P. carotovorum 2A JFY74_RS20110 rhs6 AHH 1461 Dnase

P. aroidearum QJ011 L0Y21_RS13090 rhs6 AHH 1448 Dnase
P. brasiliense BC1 NC16_RS12970 rhs6 AHH 1448 Dnase
P. parvum YT22221 NV347_RS12470 rhs6 AHH 1448 Dnase
P. brasiliense SX309 B5S52_RS09345 rhs6 AHH 1448 Dnase
P. quasiaquaticum A477-S1-J17 IG609_RS11695 rhs6 AHH 1448 Dnase
IPO:4132
P. brasiliense H5A38_RS00515 rhs7 NUC 1442 Dnase
NAK:239
P. carotovorum 25.1 LQF52_RS00325 rhs7 NUC 1442 Dnase
P. atrosepticum 21A GZ59_RS20890 rhs7 NUC 1429 Dnase
P. parmentieri WC19161 JBL47_RS03295 rhs7 NUC 668 Dnase
P. parmentieri SCC3193 W5S_RS00735 rhs7 NUC 308 Dnase
P. carotovorum 25.1 LQF52_RS00290 rhs7 NUC 145 Dnase
P. aroidearum QJ011 L0Y21_RS14315 rhs8 GH-E 1424 Dnase
P. aroidearum QJ021 N5056_RS13645 rhs8 GH-E 1424 Dnase
Pectobacterium sp. 21LCBS03 MYB54_RS08925 rhs8 GH-E 1424 Dnase
P. brasiliense BC1 NC16_RS14180 rhs8 GH-E 1424 Dnase
P. brasiliense 21PCA_AGRO2 OWC53_RS08265 rhs8 GH-E 1424 Dnase
P. versatile 14A EIP93_RS14330 rhs8 GH-E 1424 Dnase
Unknown
function
Unknown
function
Unknown
function
Unknown
function
Unknown
P. atrosepticum Green1 HLB43_RS05045 rhs9 Undetermined 138
function
Unknown
P. brasiliense ZLMLSHJ5 IHJ54_RS00290 rhs9 Undetermined 1399
function
Unknown
P. aroidearum QJ313 L0Y30_RS00315 rhs10 Undetermined 1380
function
Unknown
P. aroidearum AK042 L0Y25_RS00295 rhs10 Undetermined 1380
function
Unknown
P. carotovorum PCCS1 IQ281_RS05730 rhs10 Undetermined 1380
function
Unknown
P. parmentieri WPP163 PECWA_RS01740 rhs10 Undetermined 1379
function
Unknown
function
Unknown
function
161
Unknown
function
Unknown
function
Unknown
function
P. polaris NIBIO1006 BJJ97_RS21700 rhs11 Ntox30 1774 Rnase
P. aroidearum QJ021 N5056_RS16410 rhs11 Ntox30 1704 Rnase
P. aquaticum A212-S19-A16 DMB82_RS05180 rhs11 Ntox30 1729 Rnase
P. polaris NIBIO1392 BJK05_RS11620 rhs11 Ntox30 1729 Rnase
P. aroidearum LJ2 LCF43_RS05215 rhs11 Ntox30 1772 Rnase
P. atrosepticum Green1 HLB43_RS04945 rhs11 Ntox30 1647 Rnase
Unknown
function
Unknown
function
Unknown
function
Unknown
function
Unknown
function
Unknown
P. odoriferum BC S7 BCS7_RS18250 rhs13 Undetermined 1279
function
Unknown
P. parvum YT22221 NV347_RS17715 rhs13 Undetermined 2733
function
Unknown
P. parvum FN20211 LOZ86_RS17700 rhs13 Undetermined 2733
function
Unknown
Pectobacterium F1-1 NAL19_RS17595 rhs13 Undetermined 2704
function
sp.
Unknown
P. odoriferum JK2.1 EO763_RS18400 rhs13 Undetermined 2459
function
Unknown
function
Unknown
function
Unknown
function
Unknown
function
Unknown
function
Unknown
P. brasiliense 1692 GT391_RS12385 rhs15 Undetermined 1703
function
Unknown
function
Unknown
function
162
P. quasiaquaticum A477-S1-J17 IG609_RS04575 rhs16 Pertussis_S1 1767 Dnase

P. aroidearum QJ313 L0Y30_RS05190 rhs16 Pertussis_S1 1758 Dnase
P. aroidearum L6 I2D83_RS05600 rhs16 Pertussis_S1 1758 Dnase
Unknown
function
Unknown
function
Unknown
function
Unknown
P. brasiliense 130 MBA20_RS04915 rhs18 Undetermined 1754
function
Unknown
P. versatile ECC15 IMY97_RS06365 rhs18 Undetermined 1707 function
Unknown
P. aroidearum QJ011 L0Y21_RS17240 rhs18 Undetermined 1750
function
Unknown
P. parmentieri HC GMW39_RS25065 rhs19 Undetermined 311
function
Unknown
function
Unknown
function
P. carotovorum ZJ-4-2 PSR30_RS13630 rhs20 AHH 1421 Dnase
P. quasiaquaticum A477-S1-J17 IG609_RS07235 rhs20 AHH 1421 Dnase
P. quasiaquaticum A398-S21-F17 IG605_RS07275 rhs20 AHH 1421 Dnase
Unknown
function
Unknown
P. brasiliense 130 MBA20_RS04940 rhs21 Undetermined 697
function
Unknown
Pectobacterium sp. 21LCBS03 MYB54_RS04885 rhs21 Undetermined 697
function
P. versatile F131 F131LOC_RS00100 rhs22 ParB 1425 Dnase
P. versatile SR1 LGL96_RS10940 rhs22 ParB 1425 Dnase
P. carotovorum ZM1 GBN63_RS05245 rhs22 ParB 236 Dnase
Unknown
P. carotovorum XP-13 IHJ55_RS05130 rhs23 Undetermined 1710
function
Unknown
P. carotovorum JR1.1 EH204_RS16510 rhs23 Undetermined 1710
function
Unknown
P. carotovorum ZM1 GBN63_RS00750 rhs23 Undetermined 1710
function
P. carotovorum PC1 PC1_RS00250 rhs24 YwqJ-deaminase 1437 Dnase
P. versatile 2-Mar OA04_RS22165 rhs24 YwqJ-deaminase 1437 Dnase
P. versatile SR1 LGL96_RS10925 rhs24 YwqJ-deaminase 645 Dnase
P. versatile F131 F131LOC_RS00085 rhs24 YwqJ-deaminase 645 Dnase
P. parmentieri SCC3193 W5S_RS12980 rhs25 GH-E 1413 Dnase
163
Dnase
P. parmentieri IFB5485 C5E21_RS12620 rhs25 GH-E 1413
Dnase
P. parmentieri WPP163 PECWA_RS13170 rhs25 GH-E 1428
Dnase
P. parmentieri QK-5 IG623_RS12785 rhs25 GH-E 1428
Unknown
function
Unknown
function
Unknown
P. carotovorum WPP14 HER17_RS00295 rhs26 Undetermined 1451 function
IPO:4062 Unknown
P. brasiliense H5A40_RS06580 rhs26 Undetermined 1451
NAK:237 function
Unknown
function
P. parmentieri WPP163 PECWA_RS11940 rhs27 GIY-YIG_SF 1348 Dnase
P. parmentieri WC19161 JBL47_RS00905 rhs27 GIY-YIG_SF 1438 Dnase
P. carotovorum ZJ-4-2 PSR30_RS12465 rhs27 GIY-YIG_SF 1437 Dnase
Unknown
P. parmentieri IFB5485 C5E21_RS02065 rhs28 Undetermined 703 function
Unknown
function
Unknown
function
Unknown
function
Pectobacterium sp. PL64 HP572_RS14765 rhs29 dnaA 1748 Dnase
Unknown
P. brasiliense BZA12 CTV95_RS20455 rhs29 Undetermined 1750
function
P. brasiliense SX309 B5S52_RS22765 rhs29 Undetermined 1750 Unknown
function
Unknown
function
P. carotovorum PC1 PC1_RS16100 rhs30 Ribonuclease 1639 Rnase
P. versatile F131 F131LOC_RS16115 rhs30 Ribonuclease 1721 Rnase
P. polaris NIBIO1392 BJK05_RS11575 rhs30 Ribonuclease 737 Rnase
P. odoriferum JK2.1 EO763_RS17005 rhs30 Ribonuclease 566 Rnase
P. versatile SR12 LGL95_RS09080 rhs31 Ntox15 1429 Rnase
NES2-
P. polaris NIBIO1392 BJK05_RS07590 rhs31 NLS_ChREBP-like 1429 Dnase
Unknown
function
Unknown
function
Unknown
function
164
Unknown
function
Protease
P. parmentieri HC GMW39_RS20680 rhs34 TNT 1789
Protease
P. parmentieri HC GMW39_RS24965 rhs34 TNT 130
Unknown
P. aroidearum QJ021 N5056_RS00290 rhs35 Undetermined 1390
function
Unknown
P. brasiliense SX309 B5S52_RS00340 rhs35 Undetermined 1391
function
Unknown
P. parmentieri HC GMW39_RS17190 rhs36 Undetermined 1385 function
Unknown
function
Unknown
function
Unknown
function
P. parmentieri SCC3193 W5S_RS17045 rhs38 Undetermined 1423 Unknown
function
Unknown
function
P. aroidearum AK042 L0Y25_RS05390 rhs39 LHH 1690 Dnase
P. aroidearum AK042 L0Y25_RS05420 rhs39 LHH 150 Dnase
P. versatile A73-S18-O15 LLE50_RS06165 rhs40 LHH 1712 Dnase
P. versatile 14A EIP93_RS17085 rhs40 LHH 1712 Dnase
P. parmentieri IFB5408 C5E17_RS21815 rhs41 CdiA-CT 530 Rnase
P. parmentieri WC19161 JBL47_RS21230 rhs41 CdiA-CT 224 Rnase
P. parmentieri IFB5408 C5E17_RS13630 rhs42 Tox-URI2 1411 Dnase
P. parmentieri WC19161 JBL47_RS09455 rhs42 Tox-URI2 1411 Dnase
Unknown
function
Unknown
function
P. brasiliense BC1 NC16_RS00275 rhs44 HNHc 1454 Dnase
P. carotovorum A077-S18-O15 LHL03_RS00305 rhs44 HNHc 679 Dnase
Unknown
P. versatile A73-S18-O15 LLE50_RS11935 rhs45 Undetermined 1404
function
Unknown
function
Unknown
function
Unknown
function
P. parmentieri IFB5485 C5E21_RS02050 rhs47 Tox-ART-HYD1 675 Protease
P. parmentieri QK-5 IG623_RS02090 rhs47 Tox-ART-HYD1 675 Protease
165
Dnase
P. polaris QK413-1 KSL88_RS00315 rhs48 Tox-SHH 1453
Dnase
P. polaris NIBIO1392 BJK05_RS16395 rhs48 Tox-SHH 1453
Rnase
P. carotovorum PCCS1 IQ281_RS00550 rhs49 CdiA-CT 634
Rnase
P. brasiliense 21PCA_AGRO2 OWC53_RS17050 rhs49 CdiA-CT 136
Dnase
P. brasiliense IPO:4062 H5A40_RS00715 rhs50 Tox-URI2 1748
NAK:237
Dnase
P. parmentieri IFB5408 C5E17_RS21685 rhs50 Tox-URI2 1760
Unknown
function
Unknown
function
P. aroidearum QJ011 L0Y21_RS00280 rhs52 Tox-ART-HYD1 1400 Protease
P. brasiliense 130 MBA20_RS00310 rhs52 Tox-ART-HYD1 1401 Protease
Unknown
P. cacticida CFCC10813 OI450_RS15625 rhs53 Undetermined 1622
function
Unknown
function
Unknown
function
Unknown
function
P. brasiliense 1692 GT391_RS12395 rhs55 CdiA-CT 242 Rnase
P. aquaticum A212-S19-A16 DMB82_RS05195 rhs55 CdiA-CT 539 Rnase
P. parmentieri IFB5408 C5E17_RS12225 rhs56 Ntox30 1334 Rnase
P. parmentieri WC19161 JBL47_RS10785 rhs56 Ntox30 1334 Rnase
P. polaris NIBIO1006 BJJ97_RS05660 rhs57 ADPRTs_Tse2 1395 Dnase
P. carotovorum RC5297 F9W95_RS04415 rhs57 ADPRTs_Tse2 466 Dnase
P. aquaticum A212-S19-A16 DMB82_RS00260 rhs58 Aromatic_hydrox 1425 Dnase
Unknown
function
Unknown
function
Unknown
function
Unknown
function
Unknown
P. parmentieri QK-5 IG623_RS02165 rhs60 Undetermined 127 function
Unknown
function
Unknown
function
166
Unknown
function
Unknown
function
Rnase
P. parmentieri WPP163 PECWA_RS24630 rhs63 CdiA-CT 186
Rnase
P. parmentieri SCC3193 W5S_RS25910 rhs63 CdiA-CT 186
Dnase
P. actinidiae GX-Pa1 M9782_RS01740 rhs64 NUC 1451
Dnase
P. brasiliense BZA12 CTV95_RS15660 rhs64 Endonuclea_NS_2 1451
Unknown
P. carotovorum WPP14 HER17_RS04950 rhs65 Undetermined 1702
function
Unknown
P. quasiaquaticum A398-S21-F17 IG605_RS04515 rhs66 Undetermined 142
function
Unknown
function
Unknown
P. carotovorum ZJ-4-2 PSR30_RS16850 rhs68 COG4875 1780
function
Unknown
P. polaris NIBIO1006 BJJ97_RS21685 rhs69 Undetermined 784
function
Unknown
P. versatile SR12 LGL95_RS05075 rhs70 Undetermined 1706
function
P. parmentieri WPP163 PECWA_RS03155 rhs71 AHH 1422 Dnase
P. carotovorum 25.1 LQF52_RS16850 rhs72 LHH 1698 Dnase
Unknown
function
Unknown
P. odoriferum BC S7 BCS7_RS21710 rhs74 Undetermined 197
function
Unknown
function
Unknown
function
Unknown
function
Pectobacterium sp. PL64 HP572_RS20700 rhs78 GcvP2 138 Dnase
Unknown
function
P. carotovorum A077-S18-O15 LHL03_RS16705 rhs80 HNHc 1718 Dnase
Unknown
function
Unknown
P. carotovorum XP-13 IHJ55_RS00260 rhs82 Undetermined 1441
function
Unknown
function
Unknown
P. polaris QK413-1 KSL88_RS05045 rhs84 Undetermined 1697
function
167
Unknown
P. brasiliense IPO:4132 H5A38_RS05120 rhs85 Undetermined 1741
function
NAK:239
Unknown
P. actinidiae GX-Pa1 M9782_RS18655 rhs86 Undetermined 1718
function
Unknown
P. brasiliense ZLMLSHJ5 IHJ54_RS15735 rhs87 Undetermined 1622
function
Unknown
P. carotovorum ZJ-4-2 PSR30_RS00285 rhs88 Undetermined 1385
function
Unknown
P. cacticida CFCC10813 OI450_RS00335 rhs89 Undetermined 1759
function
Dnase
P. parmentieri SCC3193 W5S_RS21520 rhs90 NTP_transf_2 1776
Unknown
P. brasiliense 1692 GT391_RS07590 rhs91 Undetermined 1449
function
Unknown
P. carotovorum ZJ-4-2 PSR30_RS14000 rhs92 Undetermined 1448
function
Dnase
P. aroidearum LJ2 LCF43_RS00295 rhs93 ParB 1403
Dnase
P. cacticida CFCC10813 OI450_RS03605 rhs94 DNase_NucA_ 1416
NucB
P. parmentieri IFB5441 C5E20_RS19805 rhs95 Undetermined 1430 Unknown
function
Unknown
function
Pectobacterium sp. 21LCBS03 MYB54_RS04895 rhs97 Undetermined 311 Unknown
function
Unknown
function
Unknown
P. odoriferum BC S7 BCS7_RS16795 rhs99 DUF3990 1433
function
Unknown
function
Unknown
P. parmentieri IFB5408 C5E17_RS24140 rhs101 Undetermined 135 function
Unknown
P. quasiaquaticum A398-S21-F17 IG605_RS04455 rhs102 Undetermined 1735
function
Unknown
P. versatile 2-Mar OA04_RS17325 rhs103 Undetermined 1770
function
P. quasiaquaticum A398-S21-F17 IG605_RS04545 rhs104 Tox-HNH-HHH 1179 Dnase
Unknown
P. brasiliense 21PCA_AGRO2 OWC53_RS17105 rhs105 Undetermined 1769
function
P. versatile SR1 LGL96_RS05030 rhs106 TNT 1715 Protease
Dickeya
Rhs- Putative
Toxin Protein
Species strain Rhs locus toxin predicted
domain Length
identifier function
D. dadantii 3937 DDA3937_RS23790 rhs1 NUC 195 Dnase
168
Dnase
D. dadantii XJ12 OL445_RS18200 rhs1 NUC 179
Dnase
D. fangzhongdai ZXC1 PQ617_RS07225 rhs1 NUC 1449
RNS Dnase
D. solani D083_RS16290 rhs1 NUC 1448
08.23.3.1.A
Dnase
D. dianthicola 67-19 HGI48_RS08570 rhs1 NUC 1449
Dnase
D. dadantii 3937 DDA3937_RS04095 rhs1 NUC 1434
Dnase
D. dadantii FZ06 MUB29_RS04230 rhs1 NUC 1449
Dnase
D. dadantii DSM 18020 CO076_RS08595 rhs1 NUC 834
Dnase
D. zeae MS_2018 HJ586_RS10085 rhs2 Tox-HNH-EHHH 541
Dnase
D. dadantii 3937 DDA3937_RS06990 rhs2 Tox-HNH-EHHH 461
Dnase
D. dadantii XJ12 OL445_RS16875 rhs2 Tox-HNH-EHHH 461
Dnase
D. fangzhongdai Onc5 K0H75_RS06830 rhs2 Tox-HNH-EHHH 447
Dnase
D. fangzhongdai ZXC1 PQ617_RS14045 rhs2 Tox-HNH-EHHH 1686
Dnase
D. zeae EC2 DWV07_RS06335 rhs2 Tox-HNH-EHHH 1665
Unknown
D. fangzhongdai PA1 B6N31_RS08490 rhs3 Undetermined 1420
function
Unknown
D. fangzhongdai ZXC1 PQ617_RS14855 rhs3 Undetermined 1435
function
Unknown
D. dadantii XJ12 OL445_RS18575 rhs3 Undetermined 1435
function
Unknown
D. zeae A586-S18-A17 LHK94_RS01390 rhs3 Undetermined 1420
function
Unknown
D. dadantii FZ06 MUB29_RS14910 rhs3 Undetermined 1420
function
Unknown
D. zeae A586-S18-A17 LHK94_RS03045 rhs4 Undetermined 190
function
D. dadantii S3-1 KNV89_RS16960 rhs4 Undetermined 1657 Unknown
function
Unknown
Dickeya sp. Secpp 1600 CPA59_RS06965 rhs4 Undetermined 1657
function
D. fangzhongdai AP6 FXN80_RS07265 rhs4 Undetermined 1657 Unknown
function
Unknown
D. fangzhongdai ND14b LH89_RS05140 rhs4 Undetermined 459 function
D. fangzhongdai ND14b LH89_RS22050 rhs5 Ntox8 117 Rnase
D. zeae A5272 FGI00_RS14460 rhs5 Ntox8 649 Rnase
D. dadantii S3-1 KNV89_RS16990 rhs5 Ntox8 637 Rnase
D. zeae A586-S18-A17 LHK94_RS03030 rhs5 Ntox8 563 Rnase
D. fangzhongdai ZXC1 PQ617_RS14085 rhs5 Ntox8 343 Rnase
RNS
D. solani 08.23.3.1.A D083_RS19120 rhs6 Tox-HNH-EHHH 1660 Dnase
169
D. solani RNS D083_RS19135 rhs6 Tox-HNH-EHHH 302 Dnase

08.23.3.1.A
D. parazeae Ech586 DD586_RS06410 rhs6 Tox-HNH-EHHH 1657 Dnase
D. dadantii FZ06 MUB29_RS07405 rhs7 Undetermined 137 Unknown
function
D. fangzhongdai 643b MJO48_RS07475 rhs7 Undetermined 1686 Unknown
function
D. dadantii FZ06 MUB29_RS07370 rhs7 Undetermined 1685 Unknown
function
D. zeae A5272 FGI00_RS14475 rhs8 WHH 1661 Dnase
D. chrysanthemi Ech1591 DD1591_RS05850 rhs8 WHH 1423 Dnase
D. dadantii S3-1 KNV89_RS18970 rhs8 WHH 1422 Dnase
D. fangzhongdai ND14b LH89_RS11675 rhs9 HNHc 1451 Dnase
D. dadantii 3937 DDA3937_RS13755 rhs9 HNHc 1436 Dnase
D. dadantii FZ06 MUB29_RS08100 rhs9 HNHc 1436 Dnase
D. parazeae Ech586 DD586_RS07320 rhs10 Undetermined 1426 Unknown
function
Dickeya sp. Secpp 1600 CPA59_RS07690 rhs10 Undetermined 1441 Unknown
function
function
D. zeae MS1 J417_RS03680 rhs11 Undetermined 1459 Unknown
function
D. fangzhongdai Onc5 K0H75_RS00290 rhs11 Undetermined 1457 Unknown
function
D. chrysanthemi Ech1591 DD1591_RS07515 rhs11 GIY-YIG_SF 1457 Dnase
D. chrysanthemi Ech1591 DD1591_RS22030 rhs12 LHH 201 Dnase
D. zeae JZL7 H7F34_RS08230 rhs12 LHH 139 Dnase
D. dianthicola LAR.16.03.LID E4659_RS07965 rhs12 LHH 1443 Dnase
D. dianthicola 16ME22T JRZ88_RS07500 rhs12 LHH 317 Dnase
D. fangzhongdai 643b MJO48_RS08925 rhs13 AHH 205 Dnase
D. zeae MS_2018 HJ586_RS09155 rhs13 AHH 1439 Dnase
D. dadantii S3-1 KNV89_RS17735 rhs13 AHH 1440 Dnase
D. dianthicola 67-19 HGI48_RS07200 rhs13 AHH 1414 Dnase
D. zeae MS2 C1O30_RS07635 rhs14 HNHc 1460 Dnase
D. dadantii DSM 18020 CO076_RS08825 rhs14 HNHc 1446 Dnase
D. zeae CE1 DWG24_RS06035 rhs14 HNHc 1446 Dnase
D. zeae EC2 DWV07_RS20485 rhs14 HNHc 350 Dnase
Unknown
D. fangzhongdai ND14b LH89_RS05130 rhs15 Undetermined 230
function
170
D. zeae JZL7 H7F34_RS06275 rhs15 Undetermined 1643 Unknown

function
D. zeae PL65 FGI21_RS07385 rhs15 Undetermined 1654 Unknown
function
D. fangzhongdai DSM 101947 CVE23_RS07450 rhs15 Undetermined 1679 Unknown
function
D. fangzhongdai Onc5 K0H75_RS06810 rhs16 Undetermined 1691 Unknown
function
D. fangzhongdai PA1 B6N31_RS06955 rhs16 Undetermined 363 Unknown
function
Dickeya sp. Secpp 1600 CPA59_RS06985 rhs16 Undetermined 368 Unknown
function
function
D. chrysanthemi Ech1591 DD1591_RS13970 rhs17 Undetermined 1679 Unknown
function
D. dadantii 3937 DDA3937_RS0699 rhs17 Undetermined 346 Unknown
5 function
D. dadantii XJ12 OL445_RS16885 rhs17 Undetermined 346 Unknown
function
D. zeae EC1 W909_RS06320 rhs17 Undetermined 346 Unknown
function
D. zeae MS1 J417_RS06820 rhs18 Tox-HNH-EHHH 573 Dnase
D. chrysanthemi Ech1591 DD1591_RS13945 rhs18 Tox-HNH-EHHH 1673 Dnase
D. fangzhongdai ND14b LH89_RS05155 rhs19 WHH 1689 Dnase
D. zeae CE1 DWG24_RS14315 rhs19 WHH 1653 Dnase
D. solani RNS D083_RS20790 rhs20 Undetermined 1395 Unknown
08.23.3.1.A function
D. zeae MS2 C1O30_RS03620 rhs20 Undetermined 1392 Unknown
function
function
function
D. solani RNS 05.1.2A RS71_RS17570 rhs22 GIY-YIG_SF 1402 Dnase
D. zeae MS_2018 HJ586_RS19400 rhs22 Undetermined 511 Unknown
function
D. fangzhongdai Onc5 K0H75_RS00300 rhs23 NUC 196 Dnase
Dickeya sp. Secpp 1600 CPA59_RS00315 rhs23 NUC 1458 Dnase
D. solani RNS 05.1.2A RS71_RS17580 rhs24 AHH 171 Dnase
D. zeae MS_2018 HJ586_RS19390 rhs24 AHH 1395 Dnase
D. dadantii M2-3 KTF62_RS03905 rhs25 AHH 1396 Dnase
171
D. dianthicola LAR.16.03.LID E4659_RS04125 rhs25 AHH 1396 Dnase

Unknown
D. dianthicola 16ME22T JRZ88_RS07490 rhs26 Undetermined 1449
function
Unknown
function
D. zeae MS2 C1O30_RS06820 rhs27 AHH 1673 Dnase
D. zeae A586-S18-A17 LHK94_RS03085 rhs27 AHH 1667 Dnase
Unknown
D. dadantii M2-3 KTF62_RS07090 rhs28 Undetermined 1645
function
Unknown
D. fangzhongdai PA1 B6N31_RS06925 rhs28 Undetermined 1671
function
Unknown
D. zeae PL65 FGI21_RS00575 rhs29 Undetermined 1443
function
Unknown
function
Unknown
D. dadantii 3937 DDA3937_RS06980 rhs30 Undetermined 1658
function
172
Figure 1.1. Organization and structure of a Rhs-toxin system in bacteria. (A) Representation
of a Rhs-toxin system showing common flanking upstream and downstream regions to the toxin.
An associated vgr gene (green) that facilitates secretion of the toxin. Directly downstream of the
toxin is an immunity (Imm) (dark blue) protein that protects from self-killing. Occasionally, an
orphan module (grey) is found downstream of the Imm protein, encoding an alternative C-terminal
tip with its Imm protein. (B) The full Rhs-toxin is approximately 1,400 - 1,750 amino acids (aa)
long and consists of two main regions: 1) N-terminal region involved in both signaling and
translocation of the toxin. Within the N-terminal region is a core region flanked by two conserved
motifs (depicted in red) of 10 aa: RvxxxxxxxG and PxxxxDPxGL; and 2) C-terminal region
contains the toxin domain.
173
Figure 1.2. Rhs-toxin delivery by the Type 6 secretion system. The Type Six Secretion System
(T6SS) involves in the secretion of the Rhs-toxin. In the extended assembly phase (left), a
baseplate is formed, comprising components like vgrG, PAAR spike where the Rhs-toxin is
attached. During the contracted and delivery step (middle), a quick discharge process occurs, with
the Hcp-VgrG-PAAR exiting the cell and penetrating the outer membrane of a target cell. This
delivery process involves the transfer of an Rhs-toxin, which may act to disrupt essential functions
and inhibit its proliferation. On the right, the disassembled T6SS often relocates to start a new
assembly process for subsequent secretion events.
174
Figure 2.1. Abundance and diversity of Rhs-toxin sequences in plant pathogenic Xanthomonas. The x-axis show
Xanthomonas species, the y-axis number of Rhs-toxin per genome, and each dot represent a genome color coded by
species. The size of the circle corresponds to the number of Rhs-toxins per genome. The dotted line intercepting the y-
axis shows the genomes with the largest number of Rhs-toxins. Short black lines, interspersed among dots, represent
the median value of total Rhs-toxins found for each species. Data represents 165 genomes across 20 species.
175
Protein Length
1400
1700
2000
350
700
90
Xa
−F
13 J1
1
13 1a
15 14c
−0
88
17
3
CP CIT 301
B A
Figure
CP F 14 33
B 9
CP F 7 4
BF 65
IV 7
I 6
IV A 13 6
IA 17
39
78
SL R1
20
9
Yc 8
LM hA
NC G2 F1
PP 678
B 9
7
10 96
1
12 03
0
2.2.
12 49
16 112
−O
c
29 t
−
30 5
−
50 1
53
57
6
71
9
AT 8 8284
CC 5−
BJ 33 10
SJ 91
Q2
0 B 3
CF 2006100
BP 12
CF 8
4
CFBP1 44
3
CFBP5 71
BP 824
66
9
C 0
GB CN03
BC N1
pseudogenes, respectively.
M 30 5
A 77
NC FF1 M2
PP 061 8
B 81
NE4379
B1
22
CF A 8ra
B W
CF P 2 15
B 0
CF P6 36
1
CFBP6 66
9
CFBP6 75
9
CFBP6 90
9
CFBP7 91
1
CFBP7 11
1
DABP7 12
1
DAR72 19
R7 029
FD 38
C 86
16
09
IS GZ
O1 09
2C
LM 3
G7 K2
43
M9
M 12
SC
Un NT T
B− T 17
Xte X1 T2
cT 601 1
Xc G0 49
m 2
Xc H1 −2
mN 00
1 5
IL 003
M _234
OHI_35
_2 9
CP 61
CF BF 1
BP 424
LM383
G9 6
3
LMFap2 0
NB G 7 9
C 03
protein sequence length among Rhs-toxin

PD2815
SH 520
QP 5
YL 01
X2
B0 3 1
CF 7−0 05
IC BP 07
M 49
P7 8
3
jj2 83
JS 00
7 1
Or LM 49−3
eg 16
an 73
o 4
OS 108
U4
Strain
CF VT 93
BP 10
11 6
AU 56
S 0−
AXT20 9
O1 13
94
BB BAI 7
2
B 151 3
CFB15 −3
6
CFBP2 −2
2
CFBP7 86
B
CF P7 1
33
3
CFBP7 37
BP 341
IT 73
CC HG42
BB A4
JS 0002
4
KX9−6
NC M O8
PP AI1 5
B4 34
34
N 6
NJ J01
PX 61
1
PXO21
1
PX O40
O 4
X1 99A
1−
5
YM A
YN 15
YN 1 0
JC
CI LH3
X
CI 249
X3
8
M CIX 3
AI 97
M 50
AI 37
50
DD 69
13
HRDJ1
6
HR1−3
2
J 3−4
LT R3− 6
6− 14
16
YT T 1L −
9− 6−2
19
−2
CF
A 2
CFBP 8 M6
BP 44
84 3
45
GW
Protein length of Curated Rhs−toxins repertoires in Xanthomonas
SI
CF W SS
B G
AR P 46 16
CF T− 91
X
CFBP 2 tg2
IC BP 055
M 25
IC P 1 41
M 63
P 1
LM110 7
5
LMG 7 5
G 28
8
L 43
NC MA W1
PP I5 6
B 034
37
11
UP P
3
XtKB458
XtKm1
5
XtKm33
m7
X
Xtu Xtu tLr
NC −UP 469 8
9
NCPPB B51
P 1 3
NC PB 060
PP 264
B 9
Xv 902
1
LM601
15
9
citri
fragariae
hortorum
cucurbitae
arboricola
campestris
albilineans
axonopodis
euroxanthea
represents individual strains, y axis represents the protein sequence length in amino acids (aa), and each dot represents
and 1400 aa demarcate the orphan module C-terminal tip from full Rhs-toxins and full Rhs-toxins from putative
toxin; and 4) Any toxin >1900 aa also containing full length toxins. The dashed lines that intercept the y-axis at 350
this represents Rhs-toxins tips and parts of the core region; 3) Any toxin between 700-1900 aa, indicative of a full Rhs-
divided into four categories: 1) Any toxin <350 aa, this represents Rhs-toxin tips; 2) Any toxin between 350-699 aa,
an Rhs-toxin sequence color coded by species. The size of the dot represents the amino acid sequence length that are
repertoires within Xanthomonas genomes. The x axis
Extensive variation in
euvesicatoria
Species
<350aa
>2000aa
sp.
350−699aa
700−1886aa
oryzae
theicola
vasicola
sacchari
176
hyacinthi
perforans
prunicola
vesicatoria
translucens
Protein Length Range

177
Figure 2.3. Nine conserved Rhs-toxin system loci in genomes of Xanthomonas species. (A) A phylogeny was
shades of green were identified, with maximum likelihood. The white cluster correspond to outgroups from E. coli and P.
strip represents their respective species. Branches are supported by >90% with 1,000 bootstraps. (B) Representative
immediately upstream of the Rhs-toxin (purple); Immunity protein/hipothetical immunity proteins (bright green); T6SS-
aeruginosa. Branch labels indicate the Rhs-toxin locus tag from which the core regions where extracted, and the outer
structures of Rhs-toxin system loci from each of the nine different clades: Rhs-toxin (light green); Coding sequence
linked proteins (light blue); T2SS-linked proteins (pink); Transposases (yellow); and proteins not associated with
constructed from 412 Rhs-toxin core region sequences representing 19 species. Nine distinct clades depicted in various
100.0 PA2684
NAG70 RS16640
NAG70 RS11315
NAG70 RS15605
NAG70 RS15595
NAG70 RS00575
NAG70 RS01195
KFS86 RS08990
NBC2815 RS11835
KHA79 RS11980
K8O61 RS11900
OW158 RS12015
NZ30 RS06515
K8O61 RS11885
KFS85 RS06145
OW158 RS12005
BER93 RS11435
PD5205 RS11720
F0H32 RS14615
NZ30 RS06505
KHA79 RS11990
MZO50 RS08935
KM563 RS15915
F0H33 RS14415
ISN38 RS15700
100.0 ISN36 RS12090
NBC2815 RS20935
OW158 RS11970
PD5205 RS11680
BER93 RS20935
K8O61 RS11920
KCU58 RS05740
KFS85 RS06170
FD63 RS13525
F0H32 RS14580
KCU58 RS05760
ISN33 RS07930
LTC53 RS14350
EYR27 RS02355
EYC55 RS06265
EYC56 RS17650
AXO1947 RS21135
EYC57 RS05720
ACU17 RS24220
EYC54 RS06110
EYC57 RS05750
EYC56 RS17635
EYR27 RS02345
EYR26 RS17005
EBA23 RS08545
ACU17 RS16415
EYC55 RS06255
ACU11 RS16075
IYN96 RS05795
GHV42 RS05875
EYC54 RS23225
ACU16 RS16535
ATY44 RS15850
100.0
EYR26 RS16995
EYC55 RS06280
ACU16 RS16515
BVV20 RS16895
EYR26 RS16965
MML47 RS05895
QN060 RS16955
EBA23 RS08555
rhs loci structure of nine clades in Xanthomonas
EBA17 RS16090
LL928 RS15920
IZG20 RS13450
EYC54 RS06145
EYC56 RS17600
ACU16 RS16550
GHV42 RS05860
secretion (grey). Numbers to the left represent approximate full Rhs-toxin sequence length (aa).
IYN96 RS05805
MML47 RS05905
IYN96 RS05820
QN060 RS16945
EYC57 RS05735
BE73 RS27715
PXO RS07175
EYR26 RS16955
EYC56 RS17670
EYC55 RS06235
BE73 RS04950
100.0
AXO1947 RS05255
EBA11 RS16380
FZ025 RS20375
KHN80 RS03670
OCJ37 RS10125
HEP74 RS06905
HEP75 RS07115
100.0
K9U01 RS02055
AD14011 RS21420
NG831 RS12155
NG828 RS12090
NG831 RS12145
NG825 RS08785
NG824 RS09410
NG829 RS11660
XaFJ1 RS11700
XaFJ1 RS10230
XaFJ1 RS10215
GKO49 RS02190
90.0
ATY44 RS24970
PXO RS12220
PXO RS13150
LL928 RS20780
PXO RS13125
PXO RS12195
XAV RS01910
XAV RS01895
K4A87 RS01730
OZ429 RS17725
OZ429 RS17750
OZ429 RS17695
K4A87 RS01745
DGN02 RS02115
BJD10 RS00030
EYC55 RS20925
EYC54 RS02420
EYC56 RS02680
BE73 RS24035
EYC55 RS20950
EYC56 RS02655
100.0
KHA79 RS18900
BE73 RS10025
XeaCFBP3836p RS02340
H7A86 RS06100
NX81 RS24565
KWO RS23195
NX80 RS23480
M0D47 RS15265
M0D48 RS03750
M0D43 RS15440
G4Q83 RS21605
CXP37 RS23425
M0D45 RS12480
BI317 RS16880
H7A87 RS06480
FPL04 RS16495
XB05 RS11070
F6Y24 RS05500
K9U01 RS06725
M0D46 RS01270
OZ429 RS05545
XB05 RS24585
H5027 RS08090
XHV734 RS14570
IFJ81 RS22255
JH261 RS05475
JH264 RS16040
AD14011 RS05040
K6979 RS13135
K6978 RS05695
K6982 RS05345
AMD07 RS26300
XcvCFBP7111P RS03540
G3566 RS06640
NX08 RS22840
EYR26 RS09400
GHV42 RS13390
NX08 RS17220
APY30 RS17325
XcfCFBP6990P RS06790
XapA RS24135
DGN02 RS06730
LPY96 RS16390
APY29 RS16285
APY30 RS06770
BGK55 RS14880
FPK90 RS25350
FPL05 RS24910
XcgCFBP7119R RS24575
H8Z73 RS06720
H8Z71 RS14935
IG630 RS06660
AMD13 RS07130
B7L66 RS03335
100.0
~1,389 aa
~1,306 aa
~1,126 aa
~1,407 aa
~1,415 aa
~1,435 aa
~1,443 aa
~1,400 aa
AMD07 RS14915
~999 aa
KM539 RS12985
NG831 RS05640
100.0
XTG RS08390
BI313 RS24690
XTG RS11135
PML25 RS09245
LZZ50 RS07680
H7A86 RS08655
OEG85 RS14840
LOK39 RS10655
H7A87 RS08515
100.0 JH314 RS16820
H7A87 RS05400
M0D48 RS15735
M0D47 RS05415
M0D43 RS05650
OZ429 RS07145
NDY25 RS04850
PML25 RS16215
100.0
OEG85 RS00950
JH282 RS00725
XCC RS00685
IFJ81 RS00725
96.0
JH306 RS00675
QMY63 RS01220
JH299 RS20680
H7A87 RS00685
K6978 RS00120
HG421 RS19535
XTG RS00650
KHO05 RS00695
XB05 RS25205
B
H7A86 RS00675
LZZ50 RS02835
KHN93 RS00775
M0D45 RS09610
M0D46 RS09630
M0D43 RS12695
M0D47 RS12515
M0D48 RS00875
G3566 RS00655
XACM RS19450
BHE83 RS13365
BJD11 RS24465
HG421 RS10280
LOK40 RS12405
AMD07 RS10975
H8Z71 RS08810
H8Z73 RS13160
IG630 RS13345
AMD13 RS13340
JH277 RS08765
JH307 RS12435
NMB96 RS19530
BJD10 RS12280
BI317 RS10180
CFBP8129 RS13300
XJ27 RS06820
translucens
DYQ48 RS13035
vesicatoria
XHV734 RS08375
EYC54 RS21870
EYC55 RS09335
EYC54 RS21155
EYC56 RS22600
EYC55 RS01340
prunicola
hortorum
100.0
EYC55 RS01365
hyacinthi
EBA20 RS17030
LL928 RS15550
sacchari
EBA23 RS08175
vasicola
PXO RS06285
theicola
EBA17 RS16470
ATY44 RS16160
BE73 RS24340
ACU17 RS24240
GHV42 RS05550
oryzae
GKO49 RS15840
IZG20 RS13760
ACU11 RS25435
PXO RS06855
KWO RS16610
NX81 RS17510
NX80 RS04185
NX81 RS13995
NX80 RS11085
DGN02 RS13315
sp.
DGN02 RS22685
G3566 RS22005
G3566 RS22055
TP50 RS18950
AC612 RS14010
AC612 RS07065
DGN11 RS06735
HG421 RS21100
OEG85 RS05880
JH291 RS13450
M0D47 RS16575
P3C56 RS06145
KPG65 RS18010
LZZ50 RS14280
KHN80 RS22080
XB05 RS25140
EYR27 RS05005
P3C56 RS09790
M0D46 RS13530
M0D43 RS16650
M0D48 RS04960
M0D45 RS05685
Pseudomonas aeruginosa
IFJ81 RS09090
JH293 RS08480
QMY63 RS11040
LOK39 RS12170
XTG RS15535
BJD11 RS05975
BHE83 RS09435
FPL05 RS13785
100.0
FPK90 RS14455
BGK55 RS25635
LPY96 RS09700
APY29 RS09475
APY30 RS13510
XapA RS09500
LCZ91 RS03705
FPL05 RS24570
FPK90 RS25030
BHE83 RS26150
BJD11 RS09260
Xanthomonas species
LPY96 RS06290
APY29 RS05890
APY30 RS17055
H8Z71 RS05205
AMD13 RS16920
IG630 RS16920
H8Z73 RS16735
B7L66 RS04365
AMD07 RS05675
EYR27 RS01770
CFBP8129 RS25130
KP727 RS13905
Escherichia coli
XACM RS08075
FPL05 RS10375
FPK90 RS10315
K4A87 RS04825
OZ429 RS14290
XCCCN15 RS09225
JH303 RS13035
XCCB100 RS24355
euvesicatoria
XCCCN03 RS08915
JH291 RS16890
NKJ47 RS02030
HEP75 RS10700
euroxanthea
NRY95 RS12615
KM539 RS10370
OCJ37 RS19440
axonopodis
HEP73 RS20695
HEP74 RS19970
albilineans
campestris
HEP75 RS20380
cucurbitae
arboricola
K6982 RS13340
K6979 RS05550
K6978 RS14005
LOK39 RS09320
fragariae
BJD10 RS15725
PML25 RS10040
NDY25 RS20705
DYQ48 RS09410
CFBP8129 RS16560
XHV734 RS12125
BI317 RS13745
G4Q83 RS22980
G4Q83 RS22720
XaFJ1 RS11545
92.0 NG825 RS12405
NG828 RS10195
NKJ47 RS07105
NUG21 RS06315
NUG21 RS11915
QBE03 RS00945
NG829 RS20780
NG824 RS20860
NG827 RS06235
citri
QBE03 RS07645
93.0 NG825 RS06490
NG828 RS21750
NG827 RS20835
NG831 RS06820
NG829 RS06270
NG824 RS14860
OCJ37 RS05755
OCJ37 RS05745
HEP73 RS22415
HEP73 RS22295
OCJ37 RS20860
NUG20 RS21735
A NUG20 RS15640
NRY95 RS05470
HEP73 RS22405
HEP75 RS06045
HEP74 RS05875
NRY95 RS06510
FZ025 RS15345
KFS85 RS14540
KCU58 RS15120
KM539 RS06285
NZ30 RS21745
KHF85 RS05665
KHA79 RS05675
ISN38 RS20150
ISN36 RS20150
K8O61 RS05725
MZO50 RS18285
ISN33 RS16900
ISN30 RS08370
LTC53 RS05350
A Presence and absence plot of Rhs C-terminal tips in Xanthomonas
1 0
Strain
CFBP6991
CFBP6990
T21
UnB−XtecTG02−2
YLX21
YT9−19−2
HR1−32
CN03
30−1
CN15
B100
29−5
jj2001
NCPPB 796
BAI23
NBC2815
1
YchA
IL_234
CPBF 1494
17
3
1311a
IVIA 1317
IVIA 3978
SL2098
8284
10103
ATCC 33913
CFBP1371
12112
5053
BJSJQ20200612
CFBP5824
CFBP6690
NCPPB 4379
CPBF 766
LH3
P3
UPB458
Xtu 4699
Xv1601
LM159
XtKm7
301
CFBP2286
MAFF106181
719
GZ09
CFBP7337
AXO1947
XtLr8
XtKm15
Xtu−UPB513
ART−Xtg2
CITA 33
15−088
R1
NCPPB 902
OH_261
CFBP 8443
YN01
YM15
0−9
CFBP7331
CPBF 765
CFBP7341
LMG26789
F1
CIX97
CFBP3836
MAI5069
GW
NEB122
LMG7439
CFBP7112
PD5205
Fap29
LMG 703
ICMP 16317
LMG 843
VT106
B07−007
CFBP 498
ICMP 7383
LM16734
JS749−3
OSU493
305
CFBP 8445
DD13
Xa−FJ1
LMG 728
CFBP 8444
DAR73886
DAR72029
NT17
WG16
AW15
MI_359
TX160149
CFBP7111
YNJC
NJ01
MAI134
LW16
CFBP 2055
LW16
NCPPB4346
HGA4
NCPPB 2649
FDC 1609
M12
CFBP6166
CFBP6975
ISO12C3
NCPPB 1060
X11−5A
CFBP 2541
CFBP7342
LT6−16−1
JR3−14
DJ16
LT6−2
NCPPB 3711
ICMP11055
HR3−46
Oregano 108
CPBF 424
45215
12049
576
GBBC 3077
XtKm33
MAI5034
M28
1314c
LMG930
85−10
SHQP01
XcmH1005
CFBP 2036
XcmN1003
MSCT
JS49−6
AUST2013
KXO85
PXO404
PXO211
PXO99A
ITCCBB0002
SS
SI
CIX383
CIX249
MAI5037
2
CFBP 4691
AM6
K2
8ra
CFBP7119
CFBP 1156
NJ611
BB156−2
BB151−3
rhs1
rhs2
rhs3
rhs4
rhs5
rhs6
rhs7
rhs8
rhs9
rhs10
rhs11
rhs12
rhs13
rhs14
rhs15
rhs16
rhs17
rhs18
rhs19
rhs20
rhs21
rhs22
rhs23
rhs24
rhs25
rhs26
rhs27
rhs28
rhs29
rhs30
rhs31
rhs32
rhs33
rhs34
rhs35
rhs36
rhs37
rhs38
rhs39
rhs40
rhs41
rhs42
rhs43
rhs44
rhs45
rhs46
rhs47
rhs48
rhs49
rhs50
rhs51
rhs52
rhs53
rhs54
rhs55
rhs56
rhs57
rhs58
rhs59
rhs60
rhs61
rhs62
rhs63
rhs64
rhs65
rhs66
rhs67
rhs68
rhs69
rhs70
rhs71
rhs72
rhs73
rhs74
rhs75
rhs76
rhs77
rhs78
rhs79
rhs80
rhs81
rhs82
rhs83
rhs84
rhs85
rhs86
rhs87
rhs88
rhs89
rhs90
rhs91
rhs92
rhs93
rhs94
rhs95
rhs96
rhs97
rhs98
rhs99
rhs100
rhs101
rhs102
rhs103
rhs104
rhs105
rhs106
rhs107
rhs108
rhs109
rhs110
rhs111
rhs112
rhs113
rhs114
rhs115
rhs116
rhs117
rhs118
rhs119
rhs120
rhs121
rhs122
rhs123
rhs124
rhs125
rhs126
rhs127
rhs128
rhs129
rhs130
rhs131
rhs132
rhs133
rhs134
rhs135
rhs136
rhs137
rhs138
rhs139
rhs140
rhs141
rhs142
rhs143
rhs144
rhs145
rhs146
rhs147
rhs148
rhs149
rhs150
rhs151
rhs152
rhs153
rhs154
rhs155
rhs156
rhs157
rhs158
rhs159
rhs160
rhs161
rhs162
rhs163
rhs164
rhs165
rhs166
rhs167
rhs168
rhs169
rhs170
rhs171
rhs172
rhs173
rhs174
rhs175
rhs176
rhs177
rhs178
rhs179
rhs180
rhs181
C-terminal tip identifiers
B 1
2 3 4
5 6 7 8 9 10 11
12 13 14 15 16 17 18 19 20
32
21 22 23 24 25 26 27 28 29 30 31 32
33 3441 35 36 37 38 39 40 41 42 43 44 48 45
46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61
62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77
78 90
91 96
Figure 2.4. A total of 181 unique Rhs C-Terminal tips in Xanthomonas species. (A) Presence
and absence plot of C-terminal toxin tip sequences which carry the active toxin domain. Unique
C-terminal tip identifiers are along the bottom, and on the right side showing Xanthomonas
strains. A blastP analysis was performed on 604 C-terminal tip sequences from 165 genomes to
search for homology; C-terminal tips were considered the same if homologous (>85% similarity).
(B) A similarity network analysis shows 96 clusters and 85 singleton C-terminal tip putative toxin
functions. Node names represent the Rhs C-terminal tip identifiers, are color-coded based on
species, and their size corresponds to the protein length. Small circles represent orphan C-
terminal tips and large circles represent full Rhs-toxins.
178
Figure 2.5. Unique, general, and large unknown functional diversity among Rhs-toxins in
Xanthomonas species. (A) A total of 33 distinct toxin domains from 173 Rhs C-terminal tips were
identified through a Pfam database analysis. Each ring is color coded to represent a different
Xanthomonas species and each source node represents a strain. Target node (gray) represents
identified toxin domain and shape represents putative predicted function: Dnases (circles); Rnases
(diamonds); proteases (squares); and deaminases (hexagon). Edges connecting source and target
nodes are also color-coded based on species. Of the 605 Rhs C-terminal tip sequences analyzed,
431 have undetermined toxin domains (octagon). (B) Alignment and percent identity of
representative protein sequences that are in different clusters but share the same toxin domain
(cluster 1, 27, and 37), supporting hypervariability of C-terminal tips outside of functional
domains.
179
Figure 3.1 Abundance and diversity of Rhs-toxin sequences in plant pathogenic Ralstonia. The x-axis show Ralstonia species,
the y-axis number of Rhs-toxin per genome, and each dot represent a genome color coded by species. The size of the circle
corresponds to the number of Rhs-toxins per genome. The dotted line intercepting the y-axis shows the genomes with the largest
number of Rhs-toxins. Short black lines, interspersed among dots, represent the median value of total Rhs-toxins found for each
species. Data represents 87 genomes across 6 species.
180
Protein length of Curated Rhs−toxins repertoires in Ralstonia
2000
1700
Species
1400 mannitolilytica sp.
Protein Length
pseudosolanacearum syzygii
solanacearum wenshanensis
<350aa
700
350−699aa
700−1886aa
350
90
RS −M
G 000
Po 2
M
AT 303 078
M CC 107 0
2
2
1
0
8
2
5
UWT98
40 4
SS R 1
IB F 2 4−1
a 3
CF Bs B2
T1 1
17
UW 63
UW386
UW551
UY763
W 31
_D 24
2
36 204
RS I07
D2
91 F8
LM I1 17
SL 340
24
RU SC 5
SE N24 9
PP 74
C −1
FJ JAT442 .F1
IB H F50
AF 1 09
AF 21 22
M FF 114 9
M FF 416 1
AF 30 48
31 0
M 93
Sw 31
PS 82
SL X05
SL 064
SL 729
SL 175
SL 300
T1 0
RS 2
BD RS6 7
BR 50
RS 9
N2 76
FJ AT 244 F1
45 45. 0
FJ 4.F5 50
RS 488
RS 9
56 1
PS 9
RU S 4 1
00
FJ T1 T_ 7
3
FJ T1 5244 F8
FJ JAT 52.F50
FJ T15 463. 0
FJ 153 4.F 1
FJ AT 40. F6
AT 53 F1
A 4 8
F1 1
FJ AT9 0−1
FJ C P29 5
FJ AT 04. 50
FJ 153 04. 0
FJ 153 53. 0
FJ 535 F50
K
K CC K6
T1
T4
T5
T6
T7
T8
T9
YC j64
−
Pe 967
0
20
Rs
7
A 2 7
A 2 9
F 156
64
64
LL 22
AT 4 .F5
48
b0
CQ3A3
R FJ
69
A IA 5
50
A 1 5
AT 524 .F
FJ AT 3.F
SB 57
B 71
AT 153 F5
AT 153 F5
0
G w7
RUN22
M F 07
M F 14
16
19
ol
37
AT 1−
F
22
F 14 .F
A 1 −
F T 42
SB A
2
2
3
3
.
1 .
R
M
8
SN
M
A
A
K
UG
Strain
Figure 3.2. Extensive variation in protein sequence length among Rhs-toxin repertoires within Ralstonia genomes. The x axis
represents individual strains, y axis represents the protein sequence length in amino acids (aa), and each dot represents an Rhs-toxin
sequence color coded by species. The size of the dot represents the amino acid sequence length that are divided into four categories: 1)
Any toxin <350 aa, this represents Rhs-toxin tips; 2) Any toxin between 350-699 aa, this represents Rhs-toxins tips and parts of the
core region; 3) Any toxin between 700-1900 aa, indicative of a full Rhs-toxin; and 4) Any toxin >1900 aa also containing full length
toxins. The dashed lines that intercept the y-axis at 350 and 1400 aa demarcate the orphan module C-terminal tip from full Rhs-toxins
and full Rhs-toxins from putative pseudogenes, respectively.
181
Figure 3.3. Three conserved Rhs-toxin system loci in genomes of Ralstonia species. (A) A phylogeny was constructed from 144
Rhs- toxin core region sequences representing 6 species. three distinct clades depicted in various shades of pink were identified, with
maximum likelihood. The white cluster correspond to outgroups from E. coli and P. aeruginosa. Branch labels indicate the Rhs-toxin
locus tag from which the core regions where extracted, and the outer strip represents their respective species. Branches are supported
by >90% with 1,000 bootstraps. (B) Representative structures of Rhs-toxin system loci from each of the nine different clades: Rhs-
toxin (light green); Coding sequence immediately upstream of the Rhs-toxin (purple); Immunity protein/hypothetical immunity
proteins (bright green); T6SS-linked proteins (light blue); T2SS-linked proteins (pink); and proteins not associated with secretion
(grey). Numbers to the left represent approximate full Rhs-toxin sequence length (aa).
182
A Presence and absence plot of Rhs C-terminal tips in Ralstonia
1 0 Strain
SL2064
KACC 10722
T51
T95
56D2
Rs5
UGMSS_Db01
BDBR229
SEPPX05
PSI07
FJAT15353.F1
FJAT1303.F50
FJAT15353.F50
FJAT15353.F8
UW386
KACC 10709
Sw698
Cw717
CQPS−1
202
204
Bs715
FJAT1463.F1
FJAT15304.F50
FJAT15304.F6
FJAT15340.F50
FJAT454.F50−1
FJAT91.F50
MAFF 211479
MAFF 211491
MAFF 241648
MAFF 311693
RSCM
SL2729
SL3300
SL3730
T117
T42
T60
T78
Wj644
B2
RS24
MAFF 301560
FJAT91−F8
SN83A39
362200
PeaFJ1
HA4−1
FJAT1452.F50
SL1931
FJAT15244−F8
FJAT15244.F1
FJAT15244.F50
FJAT442.F1
FJAT442.F50
FJAT445.F50
YC40−M
RS647
T12
T101
T82
R24
IBSBF 2571
CIAT_078
Po82
UW163
IBSBF1503
RUN2340
RUN2474
RUN2279
LMG 9673
UY031
RS 488
UW551
RS 489
MolK2
RS 476
GMI1000
UW763
K60
RS650
RS642
LLRS−1
CFBP2957
T98
SL3175
rhs1
rhs2
rhs3
rhs4
rhs5
rhs6
rhs7
rhs8
rhs9
rhs10
rhs11
rhs12
rhs13
rhs14
rhs15
rhs16
rhs17
rhs18
rhs19
rhs20
rhs21
rhs22
rhs23
rhs24
rhs25
rhs26
rhs27
rhs28
rhs29
rhs30
rhs31
rhs32
rhs33
rhs34
rhs35
rhs36
rhs37
rhs38
rhs39
rhs40
rhs41
rhs42
rhs43
rhs44
rhs45
rhs46
rhs47
rhs48
rhs49
rhs50
rhs51
rhs52
rhs53
rhs54
rhs55
rhs56
rhs57
rhs58
rhs59
rhs60
rhs61
rhs62
rhs63
rhs64
rhs65
rhs66
rhs67
rhs68
rhs69
rhs70
rhs71
rhs72
rhs73
rhs74
rhs75
rhs76
B 1 2
3 4 5 6 7 8
9 10 11 12 13 14 15 16 17 18
19 20 21 22 23 24 25 26 27 28 29 30 31
32 43
44
Figure 3.4. A total of 76 unique Rhs C-Terminal tips in Ralstonia species. (A) Presence and
absence plot of C-terminal toxin tip sequences which carry the active toxin domain. Unique C-
terminal tip identifiers are along the bottom, and on the right side showing Ralstonia strains. A
blastP analysis was performed on 294 C-terminal tip sequences from 87 genomes to search for
homology; C-terminal tips were considered the same if homologous (>85% similarity). (B) A
similarity network analysis shows 44 clusters and 32 singleton C-terminal tip putative toxin
183
Ralstonia species. A total of 18 distinct toxin domains from 94 Rhs C-terminal tips were identified
through a Pfam database analysis. Each ring is color coded to represent a different Ralstonia
species and each source node represents a strain. Target node (gray) represents identified toxin
domain and shape represents putative predicted function: Dnases (circles); Rnases (diamonds);
proteases (squares). Edges connecting source and target nodes are also color-coded based on
species. Of the 294 Rhs C-terminal tip sequences analyzed, 200 have undetermined toxin domains
(octagon).
184
Figure 4.1. Abundance and diversity of Rhs-toxin sequences in plant pathogenic Pectobacterium. The x-axis show Pectobacterium
species, the y-axis number of Rhs-toxin per genome, and each dot represent a genome color coded by species. The size of the circle
corresponds to the number of Rhs-toxins per genome. The dotted line intercepting the y-axis shows the genomes with the largest number
of Rhs-toxins. Short black lines, interspersed among dots, represent the median value of total Rhs-toxins found for each species. Data
represents 60 genomes across 14 species.
185
Protein length of Curated Rhs−toxins repertoires in Pectobacterium
Species
actinidiae odoriferum
aquaticum parmentieri
aroidearum parvum
2000 atrosepticum polaris
brasiliense quasiaquaticum
Protein Length
1700 cacticida sp.

carotovorum versatile
1400
<350aa
350−699aa
700
700−1886aa
350
>2000aa
90
10 5
8− ar
L6
G A
.1
2A
PC 1
BC 1
IF HC
1
C3 5
BI 06
A3 QK 392
2
:4 NA 12
64
5
19 a1
ZJ 3
−1
−2
2.1
XP 4
IF 08
IF 41
BI 21
31
1
1.1
11
21
13
43
AG 2
TS 309
M J1
RC 1
W 7
W 61
FN 163
12
S7
RI 1
YT 11
AK 37
39
W 193
03
CC HJ
15
SC K−
16
15
LC 7
LJ
13
BC
PC
ZM
SR
04
C1
−1
1
48
−S 13−
21
A_ 169
CS
14
SC en
RO
81
25
3− 2−M
77 −F1
21 J1
NI 10
P
PL
PP
NI 222
F1
F1
−4
J0
J0
J3
10
ZL 0H
52
91
SR
2
2N 2
:2
BS
JK
JR
O
−A
−O
1
AK
CF LS
B5
B5
B5
X−
SX
PP
re
20
:
IP 062 BZ
EC
Q
1−
O
O
K
Q
C1
4
2
1
18
G
−S
2
S1
−S
−S
98
PC
12
77
13
A4
A7
A2
A0
:4
21
O
IP
Strain
Figure 4.2. Extensive variation in protein sequence length among Rhs-toxin repertoires within Pectobacterium genomes. The x
axis represents individual strains, y axis represents the protein sequence length in amino acids (aa), and each dot represents an Rhs-toxin
186
Figure 4.3. Five conserved Rhs-toxin system loci in genomes of Pectobacterium species. (A) A phylogeny was constructed from 165
Rhs-toxin core region sequences representing 14 species. Five distinct clades depicted in various shades of orange were identified, with
maximum likelihood. The white cluster correspond to outgroups from E. coli and P. aeruginosa. Branch labels indicate the Rhs-toxin
locus tag from which the core regions where extracted, and the outer strip represents their respective species. Branches are supported by
>90% with 1,000 bootstraps. (B) Representative structures of Rhs-toxin system loci from each of the nine different clades: Rhs-toxin
(light green); Coding sequence immediately upstream of the Rhs-toxin (purple); Immunity protein/hypothetical immunity proteins
(bright green); T6SS-linked proteins (light blue). and proteins not associated with secretion (grey). Numbers to the left represent
approximate full Rhs-toxin sequence length (aa).
187
A Presence and absence plot of Rhs C-terminal tips in Pectobacterium
1 0
Strain
IFB5441
HC
WPP163
JK2.1
ZM1
NIBIO1392
BC1
21PCA_AGRO2
QJ021
LJ2
A212−S19−A16
NIBIO1006
QJ313
AK042
BZA12
GX−Pa1
IPO:4132 NAK:239
21A
25.1
XP−13
JR1.1
QK413−1
SR12
ECC15
130
QJ011
WPP14
IPO:4062 NAK:237
1692
CFCC10813
F131
PC1
44987
SR1
F1−1
FN20211
YT22221
BC S7
ZLMLSHJ5
TS20HJ1
SCRI1043
Green1
PL64
SX309
PCCS1
A077−S18−O15
2A
A477−S1−J17
A398−S21−F17
ZJ−4−2
L6
A73−S18−O15
14A
RC5297
21LCBS03
WC19161
IFB5408
QK−5
IFB5485
SCC3193
rhs1
rhs2
rhs3
rhs4
rhs5
rhs6
rhs7
rhs8
rhs9
rhs10
rhs11
rhs12
rhs13
rhs14
rhs15
rhs16
rhs17
rhs18
rhs19
rhs20
rhs21
rhs22
rhs23
rhs24
rhs25
rhs26
rhs27
rhs28
rhs29
rhs30
rhs31
rhs32
rhs33
rhs34
rhs35
rhs36
rhs37
rhs38
rhs39
rhs40
rhs41
rhs42
rhs43
rhs44
rhs45
rhs46
rhs47
rhs48
rhs49
rhs50
rhs51
rhs52
rhs53
rhs54
rhs55
rhs56
rhs57
rhs58
rhs59
rhs60
rhs61
rhs62
rhs63
rhs64
rhs65
rhs66
rhs67
rhs68
rhs69
rhs70
rhs71
rhs72
rhs73
rhs74
rhs75
rhs76
rhs77
rhs78
rhs79
rhs80
rhs81
rhs82
rhs83
rhs84
rhs85
rhs86
rhs87
rhs88
rhs89
rhs90
rhs91
rhs92
rhs93
rhs94
rhs95
rhs96
rhs97
rhs98
rhs99
rhs100
rhs101
rhs102
rhs103
rhs104
rhs105
rhs106
1 2 3 4 5 6
B
7 8 9 10 11 12 13
14 15 16 17 18 19 20 21 22
23 24 25 26 27 28 29 30 31
32
33 40
41 48
49 56
57 64
Figure 4.4. A total of 106 unique Rhs C-Terminal tips in Pectobacterium species. (A) Presence
and absence plot of C-terminal toxin tip sequences which carry the active toxin domain. Unique
C-terminal tip identifiers are along the bottom, and on the right side showing Pectobacterium
strains. A blastP analysis was performed on 255 C-terminal tip sequences from 60 genomes to
search for homology; C- terminal tips were considered the same if homologous (>85% similarity).
(B) A similarity network analysis shows 64 clusters and 42 singleton C-terminal tip putative
toxin functions. Node names represent the Rhs C-terminal tip identifiers, are color-coded based
on species, and their size corresponds to the protein length. Small circles represent orphan C-
188
Pectobacterium species. A total of 28 distinct toxin domains from 102 Rhs C-terminal tips were
identified through a Pfam database analysis. Each ring is color coded to represent a different
Ralstonia species and each source node represents a strain. Target node (gray) represents identified
toxin domain and shape represents putative predicted function: Dnases (circles); Rnases
(diamonds); proteases (squares); and deaminases (hexagon). Edges connecting source and target
nodes are also color-coded based on species. Of the 255 Rhs C-terminal tip sequences analyzed,
153 have undetermined toxin domains (octagon).
189
Figure 5.1. Abundance and diversity of Rhs-toxin sequences in plant pathogenic Dickeya. The x-axis show Dickeya species, the y-
axis number of Rhs-toxin per genome, and each dot represent a genome color coded by species. The size of the circle corresponds to
the number of Rhs-toxins per genome. The dotted line intercepting the y-axis shows the genomes with the largest number of Rhs-toxins.
Short black lines, interspersed among dots, represent the median value of total Rhs-toxins found for each species. Data represents 31
genomes across 8 species.
190
Protein length of Curated Rhs−toxins repertoires in Dickeya
2000
1700
Species
chrysanthemi parazeae
Protein Length
1400 dadantii solani

dianthicola sp.
fangzhongdai zeae

<350aa
700 350−699aa
700−1886aa
350
90
L7
3
6
2T
E1
S1
S2
ID
2
P6
65
b
37
3b
72
1
18
0
86
47
1
A
C
2−
17
0
EC
EC
A
J1
PA
00
14
nc
−
−1
02
59
FZ
PL
.2
ZX
JZ
.L
39
E2
52
C
64
20
S3
1.
h5
19
A
M
16
D
O
18
67
h1
.1
3.
03
8−
S_
10
Ec
M
05
3.
Ec
p
SM
6.
S1
16
M
cp
.2
SM
.1
08
6−
N
Se
D
R
LA
58
S
N
A
R
Strain
Figure 5.2. Extensive variation in protein sequence length among Rhs-toxin repertoires within Dickeya genomes. The x axis
represents individual strains, y axis represents the protein sequence length in amino acids (aa), and each dot represents an Rhs-toxin
191
Figure 5.3. Three conserved Rhs-toxin system loci in genomes of Dickeya species. (A) A phylogeny was constructed from 113 Rhs-
toxin core region sequences representing 8 species. Five distinct clades depicted in various shades of blue were identified, with maximum
likelihood. The white cluster correspond to outgroups from E. coli and P. aeruginosa. Branch labels indicate the Rhs-toxin locus tag
from which the core regions where extracted, and the outer strip represents their respective species. Branches are supported by >90%
with 1,000 bootstraps. (B) Representative structures of Rhs-toxin system loci from each of the nine different clades: Rhs-toxin (light
green); Coding sequence immediately upstream of the Rhs-toxin (purple); Immunity protein/hypothetical immunity proteins (bright
green); T6SS-linked proteins (light blue). and proteins not associated with secretion (grey). Numbers to the left represent approximate
full Rhs-toxin sequence length (aa).
192
A Presence and absence plot of Rhs C-terminal tips in Dickeya
1 0
Strain
MS_2018
RNS 05.1.2A
643b
PL65
DSM 101947
LAR.16.03.LID
16ME22T
JZL7
CE1
DSM 18020
67−19
RNS 08.23.3.1.A
Ech586
M2−3
XJ12
3937
FZ06
EC1
PA1
Onc5
ZXC1
MS2
EC2
A5272
S3−1
A586−S18−A17
ND14b
Secpp 1600
AP6
MS1
Ech1591
rhs1
rhs2
rhs3
rhs4
rhs5
rhs6
rhs7
rhs8
rhs9
rhs10
rhs11
rhs12
rhs13
rhs14
rhs15
rhs16
rhs17
rhs18
rhs19
rhs20
rhs21
rhs22
rhs23
rhs24
rhs25
rhs26
rhs27
rhs28
rhs29
rhs30
rhs31
rhs32
rhs33
rhs34
rhs35
rhs36
rhs37
rhs38
rhs39
rhs40
rhs41
rhs42
rhs43
rhs44
rhs45
rhs46
B 1 2 3 4
5 6 7 8 9 10
11 12 13 14 15 16
17 18 19 20 21
22 26
27 30
Figure 5.4. A total of 46 unique Rhs C-Terminal tips in Dickeya species. (A) Presence and
absence plot of C-terminal toxin tip sequences which carry the active toxin domain. Unique C-
terminal tip identifiers are along the bottom, and on the right side showing Dickeya strains. A
blastP analysis was performed on 113 C-terminal tip sequences from 31 genomes to search for
homology; C- terminal tips were considered the same if homologous (>85% similarity). (B) A
similarity network analysis shows 30 clusters and 16 singleton C-terminal tip putative toxin
193
Dickeya species. A total of 13 distinct toxin domains from 63 Rhs C-terminal tips were identified
through a Pfam database analysis. Each ring is color coded to represent a different Dickeya species
and each source node represents a strain. Target node (gray) represents identified toxin domain and
shape represents putative predicted function: Dnases (circles); Rnases (diamonds); proteases
(squares). Edges connecting source and target nodes are also color-coded based on species. Of the
113 Rhs C-terminal tip sequences analyzed, 50 have undetermined toxin domains (octagon).
194
Figure 6. Rhs-toxin abundance and diversity across species and genera of plant pathogenic
bacteria (A) A total of 343 genomes from Xanthomonas (green), Ralstonia (pink), Pectobacterium
(brown), and Dickeya (blue) were analyzed. This accounted for 19, 6, 14, and 8 species,
respectively. On the x-axis are different bacterial genera and, on the y-axis, Rhs-toxin count per
genome. Superimposed are median values (horizontal black lines) and total number of Rhs-toxin
(on top) per genera. Each dot represents a genome color coded by genera. (B) An all vs all BlastP
analysis of 1,267 C-terminal sequences tips from all bacterial genera were used to perform a
similarity network analysis. A total of 181, 44, 106, and 46 clusters and singletons C-terminal tip
putative toxin functions were identified for Xanthomonas, Ralstonia, Pectobacterium and Dickeya,
respectively. Node names represent the Rhs C-terminal tip identifiers, are color-coded based on
species, and their size corresponds to the protein length. Small circles represent orphan C-terminal
tips and large circles represent full Rhs-toxins. (C) Shared Rhs C-terminal tips between bacterial
genera. C-terminal tips were grouped if they showed >85% similarity (Ralstonia-Pectobacterium,
Ralstonia-Dickeya, and Pectobacterium-Dickeya) and 50% or more (Xanthomonas-Ralstonia).
195
Supplemental figures
Supplemental Figure 2.1. Abundance and diversity of Rhs-toxin sequences in plant pathogenic Xanthomonas. The x-axis show
xanthomonas species, the y-axis number of Rhs-toxin per genome, and each dot represent a genome color coded by species. The size
of the circle corresponds to the number of Rhs-toxins per genome. The dotted line intercepting the y-axis shows the genomes with the
largest number of Rhs-toxins. Short black lines, interspersed among dots, represent the median value of total Rhs-toxins found for
each species. Data represents 207 genomes across 21 species.
196
Protein Length
1400
1700
2000
350
700
90
Xa
−F
13 J1
1
13 1a
15 14c
−0
88
17
3
CP CIT 301
B A
CP F 14 33
9
CPBF 7 4
6
CPBF 7 5
BF 66
IV 7
I 6
IV A 13 7
IA 17
39
78
SL R1
20
Yc 98
LM hA
NC G2 F1
PP 678
B 9
7
10 96
1
12 03
0
12 49
16 112
−O
c
29 t
−
30 5
−
50 1
53
57
6
71
9
AT 8 8284
CC 5−
BJ 33 10
SJ 91
Q2
0 B 3
CF 2006100
B 1
CF P 8 2
4
CFBP1 44
3
CFBP5 71
BP 824
66
9
CN 0
GB C 03
BC N1
M 30 5
A 77
NC FF1 M2
PP 061 8
B 81
NE4379
B1
22
CF A 8ra
B W
CF P 2 15
B 0
CF P6 36
1
CFBP6 66
9
CFBP6 75
9
CFBP6 90
9
CFBP7 91
1
CFBP7 11
1
DABP7 12
1
DAR72 19
R7 029
FD 38
C 86
16
09
IS GZ
O1 09
2C
LM 3
G7 K2
43
M9
M 12
SC
Un NT T
B− T 17
Xte X1 T2
cT 601 1
Xc G0 49
m 2
Xc H1 −2
mN 00
1 5
IL 003
M _234
OHI_35
_2 9
CP 61
CF BF 1
BP 424
LM383
G9 6
3
LMFap2 0
NB G 7 9
C 03
PD2815
SH 520
QP 5
YL 01
X2
B0 3 1
CF 7−0 05
IC BP 07
M 49
P7 8
3
jj 8
JS 2003
7 1
Or LM 49−3
eg 16
an 73
o 4
OS 108
U4
CF VT 93
BP 10
11 6
56
Strain
AU
S 0−
AXT20 9
O1 13
94
BB BAI 7
1 2
B 51 3
CFB15 −3
6
CFBP2 −2
2
CFBP7 86
B
CF 7 1 P
33
3
CFBP7 37
BP 341
IT 73
CC HG42
BB A4
JS 0002
49
KX −6
NC M O8
PP AI1 5
B4 34
34
N 6
N J01
PX J61
1
PXO21
1
PX O40
O9 4
X1 9A
from full Rhs-toxins and full Rhs-toxins from putative pseudogenes, respectively.
1−
YM5A
YN 15
YN 01
JC
CI LH3
X
CI 249
X3
8
M CIX 3
AI 97
M 0 5
AI 37
50
DD 69
1 3
HRDJ1
6
HR1−3
2
J 3−
LT R3− 46
6− 14
16
YT LT −1
9− 6−2
19
−2
CF
A 2
CFBP 8 M6
BP 44
84 3
45
GW
SI
CF W SS
B G
Protein length of Non−curated Rhs−toxins repertoires in Xanthomonas
AR P 46 16
CF T− 91
X
CFBP 2 tg2
IC BP 055
M 2
IC P 1 541
M 63
P 1
LM110 7
5
LMG 7 5
G 28
84
3
NC MALW1
PP I5 6
B 034
37
11
UP P
3
XtKB458
XtKm1
5
XtKm33
m7
Xtu XtuXtLr
NC −UP 469 8
9
NCPPB B51
PP 10 3
NC B 60
PP 264
B 9
Xv 902
1
LM601
15
9
citri
fragariae
hortorum
cucurbitae
arboricola
campestris
albilineans
axonopodis
euroxanthea
euvesicatoria
Species
<350aa
>2000aa
sp.
350−699aa
700−1886aa
oryzae
theicola
vasicola
sacchari
hyacinthi
perforans
prunicola
vesicatoria
translucens
genomes. The x axis represents individual strains, y axis represents the protein sequence length in amino acids (aa), and each dot
197
containing full length toxins. The dashed lines that intercept the y-axis at 350 and 1400 aa demarcate the orphan module C-terminal tip
tips and parts of the core region; 3) Any toxin between 700-1900 aa, indicative of a full Rhs-toxin; and 4) Any toxin >1900 aa also
into four categories: 1) Any toxin <350 aa, this represents Rhs-toxin tips; 2) Any toxin between 350-699 aa, this represents Rhs-toxins
represents an Rhs-toxin sequence color coded by species. The size of the dot represents the amino acid sequence length that are divided
Supplemental Figure 2.2. Extensive variation in protein sequence length among Rhs-toxin repertoires within Xanthomonas
Supplemental Figure 3.1. Abundance and diversity of Rhs-toxin sequences in plant pathogenic Ralstonia. The x-axis show
Ralstonia species, the y-axis number of Rhs-toxin per genome, and each dot represent a genome color coded by species. The size of the
circle corresponds to the number of Rhs-toxins per genome. The dotted line intercepting the y-axis shows the genomes with the largest
number of Rhs-toxins. Short black lines, interspersed among dots, represent the median value of total Rhs-toxins found for each species.
Data represents 91 genomes across 8 species.
198
Protein length of Non−curated Rhs−toxins repertoires in Ralstonia
2000
1700
Species
1400 mannitolilytica sp.
Protein Length
pseudosolanacearum syzygii
solanacearum wenshanensis
<350aa
700 350−699aa
700−1886aa
350
90
RS −M
G 000
Po 2
M
AT 303 078
M CC 107 0
2
2
1
0
8
2
5
UW 98
40 4
SS R 1
IB F 2 4−1
a 3
CF Bs 2
T1 1
17
UW163
UW386
UW551
UY763
W 31
_D 24
2
36 204
D2
RS I07
91 F8
LM I1 17
SL 340
24
RU SC 5
SE N24 9
PP 74
C −1
FJ JAT442 .F1
IB H F50
AF 1 09
M FF 114 2
M FF 114 9
M FF 416 1
AF 30 48
31 0
M 93
Sw 31
PS 82
SL X05
SL 064
SL 729
SL 175
SL 300
T1 0
RS 42
BD RS6 7
BR 50
RS 9
N2 76
FJ AT 244 F1
45 45. 0
FJ 4.F5 50
RS 488
RS 9
56 1
PS 9
RU S 4 1
00
FJ T1 T_ 7
3
FJ T1 5244 F8
FJ JAT 52.F50
FJ T15 463. 0
FJ 153 4.F 1
FJ AT 40. F6
AT 53 F1
A 4 8
F1 1
FJ AT9 0−1
FJ CI P29 5
FJ AT 04. 50
FJ 153 04. 0
FJ 153 53. 0
FJ 535 F50
K
K CC K6
T1
T4
T5
T6
T7
T8
T9
YC j64
−
Pe 967
0
20
Rs
7
A 2 2
A 2 7
A 2 9
F 156
64
LL 22
AT 4 .F5
48
b0
CQ3A3
R FJ
69
A A 5
50
A 1 5
AT 524 .F
FJ AT 3.F
SB 57
B 71
AT 153 F5
AT 153 F5
0
G w7
RUN22
M F 07
16
19
ol
37
6
AT 1−
F
22
F 14 .F
A 1 −
F T 42
SB A
2
2
3
3
.
1 .
R
M
8
SN
M
A
A
K
UG
Strain
Supplemental Figure 3.2. Extensive variation in protein sequence length among Rhs-toxin repertoires within Ralstonia genomes.
The x axis represents individual strains, y axis represents the protein sequence length in amino acids (aa), and each dot represents an
Rhs-toxin sequence color coded by species. The size of the dot represents the amino acid sequence length that are divided into four
categories: 1) Any toxin <350 aa, this represents Rhs-toxin tips; 2) Any toxin between 350-699 aa, this represents Rhs-toxins tips and
parts of the core region; 3) Any toxin between 700-1900 aa, indicative of a full Rhs-toxin; and 4) Any toxin >1900 aa also containing
full length toxins. The dashed lines that intercept the y-axis at 350 and 1400 aa demarcate the orphan module C-terminal tip from full
Rhs-toxins and full Rhs-toxins from putative pseudogenes, respectively.
199
Supplemental Figure 4.1. Abundance and diversity of Rhs-toxin sequences in plant pathogenic Pectobacterium. The x-axis show
pectobacterium species, the y-axis number of Rhs-toxin per genome, and each dot represent a genome color coded by species. The size
of the circle corresponds to the number of Rhs-toxins per genome. The dotted line intercepting the y-axis shows the genomes with the
largest number of Rhs-toxins. Short black lines, interspersed among dots, represent the median value of total Rhs-toxins found for each
species. Data represents 62 genomes across 15 species.
200
Protein length of Non−curated Rhs−toxins repertoires in Pectobacterium
Species
actinidiae odoriferum
aquaticum parmentieri
aroidearum parvum
2000 atrosepticum polaris
brasiliense quasiaquaticum
1700
cacticida sp.
carotovorum versatile
Protein Length
1400
<350aa
700 350−699aa
700−1886aa
350
90 >2000aa
10 5
BC 1
IF HC
8− ar
L6
C3 5
QJ 2
2A
BI 06
A3 QK 392
2
Gr A
1
NA 12
PC 1
1
64
5
ZJ 3
−1
−2
2.1
0
XP 4
IF 08
IF 41
BI 21
31
1
1
.1
1.1
QJ 1
QJ 1
3
43
AG 2
TS 309
RC 1
W 7
W 61
FN 163
12
S7
1
YT 211
NA 37
W 193
03
CC HJ
ZM
5
−
16
15
LC 7
LJ
04
BC
PC
−S −Pa
C1
−1
13
48
SR
−S 13−
01
02
31
A_ 169
CS
9
SC en
RO
81
23
21
14
3− 2−M
25
O1
77 −F1
21 −J1
NI 10
QK
A
PL
PP
4
4
NI 222
F1
F1
−4
10
ZL H
52
91
SR
32 K:2
BS
JK
JR
−A
−O
O1
AK
CF LS
B5
B5
B5
SX
PP
e
20
K:
IP 062 BZ
EC
20
O
RI
C1
4
GX
1
1
19
18
M
SC
−S
2
S1
−S
98
PC
12
77
A4
A7
41
A2
A0
4
21
O:
O:
IP
Strain
Supplemental Figure 4.2. Extensive variation in protein sequence length among Rhs-toxin repertoires within Pectobacterium
genomes. The x axis represents individual strains, y axis represents the protein sequence length in amino acids (aa), and each dot
represents an Rhs-toxin sequence color coded by species. The size of the dot represents the amino acid sequence length that are divided
into four categories: 1) Any toxin <350 aa, this represents Rhs-toxin tips; 2) Any toxin between 350-699 aa, this represents Rhs-toxins
tips and parts of the core region; 3) Any toxin between 700-1900 aa, indicative of a full Rhs-toxin; and 4) Any toxin >1900 aa also
containing full length toxins. The dashed lines that intercept the y-axis at 350 and 1400 aa demarcate the orphan module C-terminal tip
from full Rhs-toxins and full Rhs-toxins from putative pseudogenes, respectively.
201
Supplemental Figure 5.1. Abundance and diversity of Rhs-toxin sequences in plant pathogenic Dickeya. The x-axis show Dickeya
species, the y-axis number of Rhs-toxin per genome, and each dot represent a genome color coded by species. The size of the circle
corresponds to the number of Rhs-toxins per genome. The dotted line intercepting the y-axis shows the genomes with the largest number
of Rhs-toxins. Short black lines, interspersed among dots, represent the median value of total Rhs-toxins found for each species. Data
represents 34 genomes across 11 species.
202
Protein length of Non−curated Rhs−toxins repertoires in Dickeya
2000
1700
Species
chrysanthemi parazeae
Protein Length
1400 dadantii solani

dianthicola sp.
fangzhongdai zeae

<350aa
700 350−699aa
700−1886aa
350
90
L7
3
06
2T
E1
S1
S2
ID
2
P6
65
b
37
3b
72
−1
18
0
86
47
1
A
C
2−
17
EC
EC
A
J1
PA
00
14
nc
−1
02
59
FZ
PL
.2
ZX
JZ
.L
39
E2
52
C
64
20
S3
1.
h5
19
A
M
16
D
O
18
67
h1
.1
3.
03
8−
S_
10
Ec
M
05
3.
Ec
p
SM
6.
S1
16
M
cp
.2
SM
.1
08
6−
N
Se
D
R
LA
58
S
N
A
R
Strain
Supplemental Figure 5.2. Extensive variation in protein sequence length among Rhs-toxin repertoires within Dickeya genomes.
The x axis represents individual strains, y axis represents the protein sequence length in amino acids (aa), and each dot represents an
Rhs-toxin sequence color coded by species. The size of the dot represents the amino acid sequence length that are divided into four
categories: 1) Any toxin <350 aa, this represents Rhs-toxin tips; 2) Any toxin between 350-699 aa, this represents Rhs-toxins tips and
parts of the core region; 3) Any toxin between 700-1900 aa, indicative of a full Rhs-toxin; and 4) Any toxin >1900 aa also containing
full length toxins. The dashed lines that intercept the y-axis at 350 and 1400 aa demarcate the orphan module C-terminal tip from full
Rhs-toxins and full Rhs-toxins from putative pseudogenes, respectively.
203
Supplemental Figure 6. Example of Rhs-toxin flanked by transposases. Closer examination of
Rhs-toxins bewteen 400-700 aminoacids long (light green) and its cognate immunity protein
(bright green) depicted the truncation of the N-terminal region flanked by two transposases, one in
the upstream and downstream region (yellow).
204
APPENDICES
205
Appendix A
Perl script to translate Whole genome sequence (WGS) to a CDS format.
#! /usr/bin/perl -w
use strict;
use warnings;
use Bio::SeqIO;
use Getopt::Long;
#2008-05-05
#- fork of gb2fasta_pep_loc.pl
#- modified to handle DNA and peptide export
#2008-04-30
#- if there is no locus_tag, look for a gene tag
my $genetcode = 11;
my $use_gene_tag;
my $new_loc;
my $dna;
# my $format = 'fasta';
GetOptions( "code=s" => \$genetcode,

"gene" => \$use_gene_tag,
"new=s" => \$new_loc,
"dna" => \$dna);
if ($use_gene_tag && $new_loc) {

print "Can't apply a new locus tag and use the gene tag in the same time!\n";
exit;
}
if($#ARGV<1){
print "Usage: $0 <gb file> <taxa name>\n";
print "This program export CDSs from a genbank file to a fasta file\n";
print "Options:\n\t-code <number>: NCBI genetic code [default=11]
\t-gene: Use the gene tag if the locus_tag is not available [default=OFF]
\t-new <tag>: Apply a new locus tag to each CDS starting form <tag>1
\t-dna: Export DNA sequences instead of proteins\n";
exit;
}
my $ext = '.pep.loc.fasta';
if($dna) { $ext = '.dna.loc.fasta' }
my $sp=$ARGV[1];
206
open OUT,">$ARGV[1]$ext";
my $seqio_object = Bio::SeqIO->new(-file => $ARGV[0], -format =>'genbank');
my $n = 0;
while (my $seq_object = $seqio_object->next_seq) {

for my $feat_object ($seq_object->get_SeqFeatures) {
if ($feat_object->primary_tag eq "CDS") {
if($new_loc) {
++$n;
print OUT ">${new_loc}${n}\n";
if($dna) { print OUT $feat_object->spliced_seq->seq, "\n"; }
else { print OUT $feat_object->spliced_seq->translate(-
codontable_id => $genetcode)->seq, "\n"; }
}
else {
if ($feat_object->has_tag('locus_tag')) {
for my $val ($feat_object->get_tag_values('locus_tag')){
print OUT ">$val\n";
if($dna) { print OUT $feat_object->spliced_seq-
>seq, "\n" }
else { print OUT $feat_object->spliced_seq-
>translate(-codontable_id => $genetcode)->seq, "\n" }
}
}
elsif ($use_gene_tag) {
if ($feat_object->has_tag('gene')) {
for my $val ($feat_object->get_tag_values('gene')){
# print OUT ">$sp","_$val\n";
print OUT ">$val\n";
if($dna) { print OUT $feat_object-
>spliced_seq->seq, "\n" }
else { print OUT $feat_object->spliced_seq-
>translate(-codontable_id => $genetcode)->seq, "\n" }
}
}
else {
print "Can't find a locus_tag or a gene name for this
CDS:\n";
for my $tag ($feat_object->get_all_tags) {
print " tag: ", $tag, "\n";
for my $value ($feat_object-
>get_tag_values($tag)) {
print " value: ", $value,
"\n";
207
}
}
}
}
else {
print "Can't find a locus_tag for this CDS:\n";
for my $tag ($feat_object->get_all_tags) {
print " tag: ", $tag, "\n";
for my $value ($feat_object->get_tag_values($tag))
{
print " value: ", $value, "\n";
}
}
}
}
}
}
}
Bash script to run the Rhs HMM
##This command forms a list

ls *.gbff > input_List
for x in `cat input_List`

do
##this command makes a protein CDS type version

perl gb2cds.pl $x"_genomic.gbff" $x
##This command searches the file for Rhs proteins using the model
hmmsearch 54_42_Model $x".pep.loc.fasta" > "results_"$x
grep ">>" "results_"$x > "RhsL_"$x
sed 's/>//g' "RhsL_"$x | sed 's/ //g' > "RhsList_"$x
#This command indexes the database (the whole protein genome file)
esl-sfetch --index $x".pep.loc.fasta"
##This command uses the list generated from the pipeline to fetch/present the proteins from the
database and save them into another file
esl-sfetch -f $x".pep.loc.fasta" "RhsList_"$x > $x"_X.fa"
done
208
#!/bin/bash
# Set the directory where your files are located

dir="Users/…..”
# Loop through each file in the directory

for file in "$dir"/*.fa; do
# Extract the names with ">"
names_with_arrow=$(grep "^>" "$file")
# If names were found, print them along with the filename

if [ -n "$names_with_arrow" ]; then
echo "$(basename "$file"):"
echo "$names_with_arrow"
fi
done > Output.txt
Protein length bash script
from Bio import SeqIO

import csv
# Define the input FASTA file and output CSV file names
input_fasta_file = "input.csv "
output_csv_file = "output.csv"
# Create a CSV file for writing

with open(output_csv_file, "w", newline="") as csv_file:
# Create a CSV writer
csv_writer = csv.writer(csv_file)
# Write the header row to the CSV file

csv_writer.writerow(["ID", "Protein Length"])
# Iterate through the sequences in the input FASTA file

for record in SeqIO.parse(input_fasta_file, "fasta"):
sequence_id = record.id
protein_length = len(record.seq)
# Write the ID and protein length to the CSV file

csv_writer.writerow([sequence_id, protein_length])
print(f"CSV file '{output_csv_file}' has been created.”
209

Oryzae, R. Solanacearum, and Pectobacterium Parmentieri That Carried Up To 13, 12, and 13

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Oryzae, R. Solanacearum, and Pectobacterium Parmentieri That Carried Up To 13, 12, and 13

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ABSTRACT

GÓMEZ ANDREA. Abundance Diversity and Function of Rhs Polymorphic Toxins in

proteinaceous multicomponent toxins produced by bacteria and used as weapons to kill or

are hypothesized to play an important role in bacterial communities dynamics across

plant pathogenic bacterial genera: Xanthomonas, Ralstonia, Pectobacterium, and Dickeya.

implications of Rhs- toxin medicated competition in phytopathogenic bacteria offers

All Rights Reserved

Andrea Gómez Cabrera

Thesis submitted to the Graduate

Raleigh, North Carolina

Dr. Alejandra Huerta Dr. David Ritchie

Dr. Ignazio Carbone

Bachelor of Science degree in Agricultural Engineering at Zamorano University, Honduras in

transmission of the protozoan, Histomonas meleagridis, the cause of histomoniasis (blackhead) in

many research projects to come.

LIST OF TABLES ......................................................................................................................... v

Chapter I: Review of the Literature ........................................................................................... 1

Appendices ................................................................................................................................. 205

Table 2 Bacterial genomes used in this study ........................................................................ 70

Figure 6 Example of Rhs-toxin flanked by transposases ...................................................... 204

bacterial evolution and fitness [11].

acquisition of fitness factors unique to a specific ecological niche [14,15].

As previously mentioned, bacteria can engage in competitive interactions with cells

positive selection of this toxin-immunity system is likely an outcome of cell-to-cell

bacteria in agricultural ecosystems [21,22,23,24].

Ecology of foliar bacterial plant pathogens

microenvironments ready to cause disease under favorable environmental conditions

Ecology of soilborne bacterial plant pathogens

nutrient cycling in the soil [49, 50, 51].

to changes in soil conditions or chemical applications [53,54]. Dissemination of soilborne

species complex into three different species: R. solanacearum, R. pseudosolanacearum, and R.

Asia, Phy III Africa and Phy IV Indonesia.

occurrence of soft rot on tubers [71,72].

types: exploitative and interference competition [73]. Exploitative competition is dependent on

“invader” cells. Interference competition is when a cell employs antagonistic tactics by

antibiotics. However, bacteriocins can be produced by both gram-positive and negative

Rhs-toxins and their delivery into target hosts

characterized by a three-component architecture. The initial part of the protein consists of an N-

major role in driving the diversity of Rhs-toxin in bacteria [101,102].

interactions, as they can be detected in the extracellular environment by bacteria in a microbiome

distribution of Rhs-toxins in plant pathogenic bacteria [22,23,26, 111, 112, 113,114,115].

Function of Rhs-toxins domains

peptidases cleave proteins and disrupt translocation mechanisms [21,92,120,121,122]. A study

abundance, diversity, and function of Rhs-toxins in gram-negative plant pathogenic bacteria,

diversity of Rhs-toxins in the genomes of economically significant bacterial plant pathogens. To

higher abundance and greater functional diversity of Rhs-toxins in their genomes

Xanthomonas, Ralstonia, Pectobacterium, and Dickeya.

foliar and soilborne bacterial plant pathogens.

Aim 3. Analyze Rhs-toxins repertoires across the previous mentioned bacterial

genera according to the pathogen lifestyle.

certain microbes from specific agricultural microbiomes.

Keywords: Soilborne, Foliar, Hidden Markov Model, Rhs-toxins, competition.

of action, including cell membrane disruption, degradation of key molecular components

toxin mechanism of action was described to be contact-dependent, meaning that inhibition of

bacteria, works on the basis of a two-partner secretion mechanism in which a subfamily of

bacterial genera [8].

region characterized by tyrosine/aspartate (YD) peptide repeats that mediate Rhs-toxin-protein

microbial structure in human, animal, and agricultural microbiomes remain a mystery.

scope of bioinformatics, empowering researchers to study complex organisms and microbial

genomes and 2) develop a comprehensive understanding of Rhs-toxins in bacteria. Through the

of destructive bacterial pathogens using Rhs-toxins.

Materials and Methods

(NCBI) public genome database (http://www.ncbi.nlm.nih.gov) in GenBank format