Research in Microbiology 171 (2020) 64e73

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Research in Microbiology
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Original Article

Type VI secretion system is not required for virulence on rice but for
inter-bacterial competition in Xanthomonas oryzae pv. oryzicola
Ping-Chuan Zhu a, Yi-Ming Li a, Xia Yang a, Hai-Fan Zou a, Xiao-Lin Zhu a, Xiang-Na Niu a,
Ling-Hui Xu a, Wei Jiang a, Sheng Huang a, Ji-Liang Tang a, **, Yong-Qiang He a, b, *
a
State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, College of Life Science and Technology, Guangxi University, 100
Daxue Road, Nanning, Guangxi, 530004, China
b
National Demonstration Center for Experimental Plant Science Education, College of Agriculture, Guangxi University, 100 Daxue Road, Nanning, Guangxi,
530004, China

a r t i c l e i n f o a b s t r a c t

Article history: The type VI secretion system (T6SS), a multifunctional protein secretion device, plays very important
Received 23 June 2019 roles in bacterial killing and/or virulence to eukaryotic cells. Although T6SS genes have been found in
Accepted 17 October 2019 many Xanthomonas species, the biological function of T6SSs has not been elucidated in most xantho-
Available online 30 October 2019
monads. In this study, we identified two phylogenetically distinct T6SS clusters, T6SS1 and T6SS2, in a
newly sequenced Chinese strain GX01 of Xanthomonas oryzea pv. oryzicola (Xoc) which causes bacterial
Keywords:
leaf streak (BLS) of rice (Oryza sativa L.). Mutational assays demonstrated that T6SS1 and T6SS2 are not
Xanthomonas oryzea pv. oryzicola
required for the virulence of Xoc GX01 on rice. Nevertheless, we found that T6SS2, but not T6SS1, played
Type VI secretion system (T6SS)
Bacterial competition
an important role in bacterial killing. Transcription and secretion analysis revealed that hcp2 gene is
Hcp secretion actively expressed and that Hcp2 protein is secreted via T6SS. Moreover, several candidate T6SS effectors
T6SS effectors were predicted by bioinformatics analysis that might play a role in the antibacterial activity of Xoc. This is
the first report to investigate the type VI secretion system in Xanthomonas oryzae. We speculate that Xoc
T6SS2 might play an important role in inter-bacterial competition, allowing this plant pathogen to gain
niche advantage by killing other bacteria.
© 2019 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

1. Introduction
The type VI secretion system (T6SS), widely distributed in the
Gram-negative Proteobacteria, is a contact-dependent protein
secreting device that directly translocates effector proteins to
target other bacteria or eukaryotic cells, playing multiple roles
ranging from inter-bacterial relationships and biofilm formation,
to cytotoxicity and persistent survival [1e3]. Thirteen T6SS core
proteins (TssA-TssM) and a PAAR protein assemble into a bacte-
riophage tail-like structure, a dynamic protein injection machine
containing two distinct interacting subassemblies. Of the T6SS
core proteins, hemolysin-coregulated protein (Hcp), an important
* Corresponding author. National Demonstration Center for Experimental Plant
Science Education, College of Agriculture, Guangxi University, 100 Daxue Road,
Nanning, Guangxi, 530004. China.
** Corresponding author.
E-mail addresses: jltang@gxu.edu.cn (J.-L. Tang), yqhe@gxu.edu.cn (Y.-Q. He).
component of T6SS tube and the chaperone for T6SS effectors, is
considered as the hallmark of T6SS. TssB and TssC form a con-
tractile sheath, enclosing the Hcp hexamer ring tube. At the top of
the Hcp tube is a puncturing device consisting of trimeric VgrG
spikes, coated with PAAR protein [4]. The cytoplasmic portion of
T6SS may dock with a membrane complex (TssLMJ) by interacting
with a phage-like structure [5]. The ClpV ATPase binds and dis-
integrates the contracted sheath, thus resetting the system for
reassembly of an extended sheath that is ready to trigger again.
T6SSs are usually very modular and can accommodate different
combinations of VgrG/PAAR proteins to form tips. Effector pro-
teins, associated noncovalently with the Hcp, VgrG or PAAR sub-
units (cargo effectors) or occurring as additional domains on
these proteins (specialized effectors), are thus introduced and
delivered through the formation of tubular Hcp proteins into the
target cells they kill. These T6SS effectors (T6SEs) are all toxic to
bacteria; to prevent self-intoxication, bacteria also encode T6SS
effector immunity proteins (T6SIs) that make them immune to
T6SE toxins [6].

https://doi.org/10.1016/j.resmic.2019.10.004
0923-2508/© 2019 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
 P.-C. Zhu et al. / Research in Microbiology 171 (2020) 64e73
It has been estimated that about 25% of Proteobacteria encode
at least one T6SS system, including bacterial pathogens of humans,
animals and plants, as well as symbiotic, antagonistic and envi-
ronmental bacteria [2,7]. The functions of T6SSs have been char-
acterized in many bacteria, associated with virulence, host
immunity suppression, antibacterial and anti-eukaryotic activities
[8]. Different bacterial species and strains use their T6SS(s) for
specific roles according to the niche and strategy of the organism.
In general, T6SS coding genes are clustered in the genomes, with
the obvious features of horizontal gene transfer [9]. Genomic and
phylogenetic analysis showed that the existing T6SS clusters could
be classified into three types, type i, ii and iii [10]. Type i system in
turn contains six subtypes (i1, i2, i3, i4a, i4b and i5), while the type
ii class is uniquely populated by the Francisella pathogenicity
island-encoded system. The type iii class was identified in Bac-
teroidetes. In plant-associated bacteria, T6SS clusters were
phylogenetically grouped into five main clades, group 1e5, based
on the core component protein TssB [11], which is almost equal to
the corresponding relationship with type i system [10]. In many
cases, more than one T6SS gene clusters were found in a single
bacterial genome, and they often have different biological func-
tions [3]. In some bacteria, one T6SS is used for multiple roles, e.g.,
antibacterial and anti-eukaryotic, whereas in other cases different
T6SSs may play distinct roles, or only promote inter-bacterial
competition [12]. Like other bacterial secretion systems, the
expression and activation of T6SS is closely related to the niche
conditions and the competitors to which bacteria are confronted,
to maintain optimal performance in suppressing opponents and
energy consumption [13]. It was found that the T6SS systems
respond to various environmental conditions, e.g. pH, tempera-
ture, ion concentration and cell density, and the cellular extracts of
their hosts or opponents. The expression of T6SS genes might be
controlled by certain regulators in some bacterial species [14].
Recently, a variety of T6SS-dependent effectors and cognate im-
munity proteins have been described, including superfamilies of
antibacterial and anti-eukaryotic effectors [2], which further
expanded our understanding of the biological functions of T6SS
system.
Although the functions of T6SSs are widely characterized in
many animal pathogens, symbionts and probiotics, their roles still
remain unclear in many phytobacterial pathogens [3,11]. Xantho-
monas is a genus of Proteobacteria, most of which are important
plant pathogens. It has been calculated that about 80% of Xan-
thomonas species or strains with whole genome sequenced
possess one to three T6SS clusters, significantly higher than 25%,
the ratio of Gram-negative bacteria containing T6SS clusters [11].
However, with the exception of Xanthomonas citri [15], there are
no other reports about the functions of T6SS genes in Xanthomo-
nas genus.
Xanthomonas oryzae pv. oryzicola (Xoc) is the causal agent of rice
bacterial leaf streak (BLS), one of the most destructive diseases of
rice (Oryza sativa L.), the important staple food crop. Xoc can invade
host leaves via stomata and wounds, and colonizes intercellular
spaces in the mesophyll, causing water-soaked interveinal lesions
that develop into translucent streaks. A variety of virulence factors
or systems, such as adhesins, extracellular polysaccharides (EPS),
type II and III secretion systems, quorum sensing (QS) system, have
been identified successively [16,17]. Recently, we identified two
phylogenetically distinct T6SS gene clusters, T6SS1 and T6SS2, in a
newly sequenced Chinese strain GX01 of Xoc. To elucidate their
biological functions, we have constructed the single mutants of
each T6SS clusters and two hcp genes, and double mutants of two
clusters and two hcp genes, respectively. In this study, we describe
the functional characterization of T6SS systems in the important
bacterial phytopathogen.
65
2. Materials and methods
2.1. Bacterial strains, plasmids and growth conditions
Microbial strains, plasmids, and primers used in this study are
listed in Table 1. X. oryzae pv. oryzicola strains were grown at 28
C
with shaking at 200 rpm in nutrienterich medium NB (3 g/L beef
extract, 1 g/L yeast extract, 5 g/L polypeptone, and 10 g/L sucrose,
pH7.0) or on nutrient agar (NA), and minimal medium XOM3 (1.8 g/
L D-xylose, 670 mM D, L-methionine, 10 mM sodium L-glutamate,
240 mM NaFe2þ-EDTA, 5 mM MgCl2, 14.7 mM KH2PO4, 40 mM MnSO4,
pH7.0). Ochrobactrum oryzae strains were grown at 30
C with
shaking at 200 rpm in the medium (3 g/L beef extract, 5 g/L poly-
peptone, 5 g/L NaCl, pH7.0). Escherichia coli DH5a strains were
grown at 37
C with shaking at 200 rpm in this medium (5 g/L yeast
extract, 10 g/L polypeptone, 10 g/L NaCl, pH7.0). Yeast was cultured
in liquid PYG medium (20 g/L polypeptone, 10 g/L yeast extract,
20 g/L glucose). Antibiotics were used at the following final con-
centrations: Tetracycline (Tc), 15 mg/ml, and kanamycin (Km), 50 mg/
ml, for E. coli; rifampicin (Rif), 50 mg/ml, Tc, 5 mg/ml, and Km, 50 mg/
ml, for Xoc strains.
2.2. Construction of mutant strains
To construct the deletion mutant of T6SS1 (XOCgx_2341 to
XOCgx_2348) (Fig. 1), the region from 554 bp upstream to 666 bp
downstream was amplified by the primer pairs DMT6SS1-LF(E)/
DMT6SS1-LR(X) and DMT6SS1-RF(X)/DMT6SS1-RR(H)
(Supplementary Table S1), and fused into the suicide plasmid
pK18mobsacB to create the recombinant plasmid pKDT6SS1. The
plasmid pKDT6SS1 was transferred into the Xoc GX01 by triparental
conjugation, followed by the selection of colonies on NA plates
which have no sucrose containing Rif and Kan. The DT6SS1 deletion
mutant was obtained by further selection on NA supplemented
with Rif and 10% sucrose. The mutants were then checked for Kan
sensitivity and were further confirmed by PCR. The confirmed
mutant was named DT6SS1. DT6SS2 (XOCgx_3583 to XOCgx_3589)
mutants were constructed using the primer pairs DMT6SS2-LF(E)/
DMT6SS2-LR(X) and DMT6SS2-RF(X)/DMT6SS2-RR(H)
(Supplementary Table S1), in accordance with above method for
constructing DT6SS1. By introducing the recombinant plasmid
pKDT6SS1 into DT6SS2, the double mutant DT6SS1DT6SS2 was
generated and confirmed by PCR.
The deletion mutants Dhcp1 (XOCgx_2343) and Dhcp2
(XOCgx_3588) were constructed using the above-mentioned
method with DT6SS1 or DT6SS2, and the generation of double
mutant Dhcp1Dhcp2 (XOCgx_2343 and XOCgx_3588) also followed
the method for creating double mutant DT6SS1DT6SS2. The mu-
tants Dhcp1, Dhcp2 and Dhcp1Dhcp2 were confirmed by PCR using
primers listed in Supplementary Table S1.
For complementation of the hcp2 mutant, a 716-bp DNA frag-
ment containing the hcp2 coding region and extending from 200 bp
upstream of the ORF was amplified using the primer set
C3588eF(H)/C3588-R(X) (Supplementary Table S1), and the
amplified DNA fragment was cloned into the plasmid pXUK
(Table 1) to generate the recombinant plasmid pXUKhcp2 (Table 1).
The recombinant plasmid was transferred into the deletion mutant
of Dhcp2 by triparental conjugation, resulting in the complemented
strain CDhcp2, further confirmed by PCR (Table 1).
2.3. Phenotypic characterization of the T6SS mutant strains
In order to assess the T6SS mutational effects in Xoc GX01
mutants, different phenotypic assessments were determined and
compared to the wild-type strain. The growth of the strains was
66 P.-C. Zhu et al. / Research in Microbiology 171 (2020) 64e73

Table 1
Bacterial strains and plasmids used in this work.

Strains or plasmids Relevant characteristics Source

Strains
Xoc GX01 Xoc Chinese strain GX01, Rifr* [17]
Dhcp1 Deletion mutant of XOCgx_2343 (hcp1) gene in Xoc GX01, Rifr* This study
Dhcp2 Deletion mutant of XOCgx_3588 (hcp2) gene in Xoc GX01, Rifr* This study
Dhcp1D hcp2 Double deletion mutant of hcp1 and hcp2 in Xoc GX01, Rifr* This study
DT6SS1# Deletion mutant of cluster T6SS1 in Xoc GX01, Rifr* This study
DT6SS2# Deletion mutant of cluster T6SS2 in Xoc GX01, Rifr* This study
DT6SS1#DT6SS2# Double deletion mutant of T6SS1and T6SS2 in Xoc GX01, Rifr* This study
GX01-RPF GX01 carrying pKLacRed, Rifr*, TCr* This study
Dhcp1-RPF Dhcp1 carrying pKLacRed, Rifr*, TCr* This study
Dhcp2-RPF Dhcp2 carrying pKLacRed, Rifr*, TCr* This study
Dhcp1Dhcp2-RPF Dhcp1Dhcp2 carrying pKLacRed, Rifr*, TCr* This study
DT6SS1#-RPF DT6SS1 carrying pKLacRed, Rifr*, TCr* This study
DT6SS2#-RPF DT6SS2 carrying pKLacRed, Rifr*, TCr* This study
DT6SS1#DT6SS2#-RPF DT6SS1DT6SS2 carrying pKLacRed, Rifr*, TCr* This study
CDhcp2 Complement strain, Dhcp2 carrying pXUKhcp2, Rifr*, Kanr* This study
DhrpG Deletion mutant of hrpG gene in Xoc GX01, Rifr* Lab collection
DrpfC Deletion mutant of rpfC gene in Xoc GX01, Rifr* Lab collection
DrpfG Deletion mutant of rpfG gene in Xoc GX01, Rifr* Lab collection
E. coli DH5a Used for molecular cloning and competitive test Lab collection
DH5a-GFP DH5a carrying pVLacGreen, Kanr* This study
MTCC 4195T Ochrobactrum oryzae wild type strain, used for competitive test [18]
MTCC 4195T-GFP MTCC 4195T carrying pVLacGreen, Kanr* This study
NMY51 Yeast reporter strain, used for competitive test Lab collection
plasmids
pK18mobsacB Suicide vector to create mutant by double crossover recombination, Kanr* [19]
pKLacRed A derivative of broad host range plasmid pKEB31 carrying mCherry gene under LacZ promoter, TCr* Lab collection
pVLacGreen A derivative of broad host range plasmid carrying a gfp under LacZ promoter, Kanr* [20]
pRK2013 A help plasmid used in triparental conjugation, Kanr* Lab collection
pXUK A broad host range plasmid with LacZ promoter, Kanr* Lab collection
pXUKhcp2 pXUK carrying hcp2 gene, Kanr* This study
pLAFRJ A derivative of broad host range vector pLAFR3 containing the multiple cloning sites of pUC19, Tcr* Lab collection
pJXG pLAFRJ containing 3  Flag, Tcr* [21]
pJXGhcp2 pJXG containing ORF exclusive termination codon of hcp2, Tcr* This work

*Kanr, Rifr, and Tcr ¼ Kanamycin-, Rifampicin-, and Tetracycline-resistant, respectively.

#
DT6SS1, deletion from XOCgx_2341 to XOCgx_2348 genes; DT6SS2, deletion from XOCgx_3583 to XOCgx_3589 genes.

assayed both in rich and defined media. For the assessment of EPS
production, 2 ml of the cell suspension (OD600 ¼ 0.1) of each strain
was pipetted onto NA with 2% sucrose. The plates were kept for
72 h at 28
C [17]. To measure the activity of extracellular protease,
2 ml of bacterial culture (OD600 ¼ 1.0) was spotted on NA plates
containing 1% (m/v) skim milk powder. After incubation at 28
C
for 36 h, protease activity was measured and compared according
to the hydrolytic zones around bacterial colony [17]. The biofilm
formation assay was performed as follows. 100 ml of the cell sus-
pension (OD600 ¼ 1.0) of each strain was pipetted into 10 ml LB in
universal glass bottles. After 4 days incubation at 28
C steadily,
the liquid medium in each bottle was removed gently. The bottles
were rinsed with water and stained with 10 ml of 1‰ crystal vi-
olet for 5 min, rinsed with water until all unbound dye was
removed. The dye was solubilized with acetic acid solution, and
the absorbance of each sample was determined at 630 nm using a
spectrophotometer. All of the assays were performed at least in
triplicates.
2.4. Plant assays
Virulence tests were carried out in a glasshouse at about 28
C.
In brief, Xoc strains were cultivated in NB broth at 28
C with
appropriate antibiotics. Cell pellets were suspended in water,
adjusted to OD600 ¼ 0.5, and infiltrated into leaves of 2-month-old
rice, O. sativa L. ssp. japonica cv. Nipponbare, using needleless sy-
ringe. Disease lesion length was measured 14 days post-
inoculation, at least twenty leaves were inoculated for each Xoc
strain.
Hypersensitive response (HR) assays were performed by infil-
tration of bacterial suspension into intact tobacco (Nicotiana ben-
thamiana) leaves using a needleless syringe. The bacterial
suspensions of Xoc strains cultured in NB medium to the logarith-
mic growth phase were diluted in 10 mM MgCl2 at an approximate
OD600 of 0.5 and spot-infiltrated into intact tobacco. The water
soaking spots formed on the leaves inoculated, i.e., the symptoms of
HR elicited by Xoc strains, were observed at 48 and 72 h after
infiltration [17]. All trials were repeated at least three times.
2.5. Intercellular competition experiments
In order to clearly show the intercellular competitions between
Xoc strains and their potential rivals, two broad host range plasmids
carrying green fluorescence protein gene (gfp) and mCherry gene,
i.e., pVLacGreen and pKLacRed (Table 1), were introduced into Xoc
strains or their competitors by electroporation to construct fluo-
rescence labelled bacterial strains (Table 1).
Inter-bacterial competition assay. RFP-labelled Xoc strains
(GX01-RFP, Dhcp1-RFP, Dhcp2-RFP, Dhcp1Dhcp2-RFP, DT6SS1-RFP,
DT6SS2-RFP, DT6SS1DT6SS2-RFP) and E. coli DH5a-GFP (Table 1)
were cultivated to OD600 ¼ 1.0, centrifuged, washed and resus-
pended to OD600 ¼ 0.5. These Xoc cells were then mixed with E. coli
diluted at a 10:1 of Xac: E. coli ratio with 10 mM MgCl2. 4 ml mix-
tures were spotted on NB plates and allowed to grow together as a
co-culture at 30
C for 40 h. Fluorescence microscopy was carried
out using an Olympus SZX16 equipped with filters for GFP and RFP.
The results of competition experiments were quantified by deter-
mining the CFUs of Xoc and E. coli by dilutions on selective medium
 P.-C. Zhu et al. / Research in Microbiology 171 (2020) 64e73 67

Fig. 1. The locations of annotated T6SS genes and T6SS genomic islands on the genomic map of Xoc GX01. (A) Circular map of the chromosome of Xoc GX01. The internal circle shows
the scale in Kb, with 0 representing the origin of replication; the first ring from the interior denotes GC content; second circle denotes GC skew (þ) were green, skew () were
purple; third circle and fourth circle indicate forward genes and reverse genes, respectively; the outer circle shows the distribution of the T6SS islands (red), T6SS1 and T6SS2 cluster
(green) in the Xoc GX01 chromosome. (B) Circular map of plasmid pXOCgx01 in Xoc GX01. The circles from inside to outside denote the same details as the circular map of the
chromosome of Xoc GX01; the outer circle shows predicted T6SS genes (yellow) in the GX01 plasmid. (C) Genomic organization of the GX01 T6SS1 and T6SS2 clusters. Different
colors represent different genes; red indicates hcp, blue indicates vgrG, and blackish green stands for transposase gene; the gray indicates non-T6SS genes, like unknown or
hypothetal protein genes. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

plates, as the Xoc-RFP were resistant to rifampicin and tetracycline
but DH5a-GFP resistant to kanamycin. Competition experiments of
Xoc cells against O. oryzae MTCC 4195T (Table1) at 50:1 ratio with
10 mM MgCl2 were performed as above and analyzed after 40 h of
incubation. Finally, the CFUs of MTCC 4195T-GFP were calculated by
dilutions on kanamycin LA medium plates [18].
Yeast competition assay. Xoc GX01and its mutant derivatives
were cultivated in NB medium, and the NMY51 yeast was cultured
in liquid PYG medium. Xoc and yeast cells were suspended in
10 mM MgCl2 and diluted to an OD600 of 0.5. After 40 h of
incubation at 30
C, all of the cells in a well were suspended in
10 mM MgCl2 and the yeast cells were counted with Bürker cell
counting chamber under microscope using an Olympus SZX16. For
each sample, six 0.04-mm squares with a 0.1-mm depth were
counted at least three repeats [22].
2.6. Quantitative RT-PCR analysis
RNA was extracted from cells which in log-phase growth with the
PureLink™ RNA Mini Kit (12183018A, Thermo Fisher, USA). We
68 P.-C. Zhu et al. / Research in Microbiology 171 (2020) 64e73

added 500 ng RNA sample to produce cDNA with the reverse
transcription-based reaction (00567293, RevertAid First Strand
cDNA Synthesis Kit, Thermo Fisher). Quantitative PCR (qPCR) was
performed using Fast SYBR Green Chemistry (Q411-02, Vazyme,
China) according to the manufacturer’s instructions on an
qTOWER2.0 Real-Time PCR System (Analytik Jena AG, Germany). A
three-step RT-PCR program (95
C for 5 min, and then 41 cycles of
95
C for 15 s and 60
C for 60 s) was used for amplification with
specific primers (from BBI Life Sciences Corporation, China; listed in
Supplementary Table S1). Gene expression was analyzed according
to 2DDCt methods, and 16S rRNA was used as reference control [23].
2.7. Semi-quantitative RT-PCR
RNA was reverse transcribed by the reverse transcription-based
reaction (Thermo Fisher), according to the manufacturer’s in-
struction. The cDNA was diluted and used for semi-quantitative RT-
PCR with specific primers (Supplementary Table S1). Relative
quantification of gene expression was performed using gapA gene
as the control.
2.8. Detection of Xoc T6SS secretion by western blot
To assess the T6SS secretion of Xoc strains, a recombinant
plasmid, named pJXGhcp2 (Table 1), was constructed by cloning the
hcp2-encoding ORF (XOCgx_3588) without its stop codon into the
vector pJXG [21]. In pJXGhcp2, the 3-end of the hcp2 ORF was fused
to the 3  Flag-tag coding sequence to ensure the expression of an
Hcp2 protein with a 3  Flag-tag at its C-terminus. The recombinant
plasmid pJXGhcp2 was introduced into Xoc strains by triparental
conjugation. The recombinant strains obtained were grown to
0.8 at OD600, and harvested by centrifugation at 4000g for 30 min.
The proteins in the cells were dissolved in lysis buffer (8 M urea,
2 mM EDTA, 10 mM DTT, 1% cocktail proteinase inhibitor) on ice
with 30 min after washing twice with PBS, and ultrasonicating for
1 min, collecting supernatants after centrifugation with 13,000 rpm
for 10 min. The proteins secreted in the supernatant fraction were
filtered with 0.45 mm filter paper, precipitated with chilled 10%
trichloroacetic acid (TCA)/acetone at 20
C overnight, and washed
with acetone and resuspended in dissolution solution (8 M urea,
0.1 M triethylammonium bicarbonate). For western blot, proteins
were separated in 12% SDS-PAGE gel and transferred to a poly-
vinylidene difluoride membrane (Roche) by electrophoresis. Then
covered with 5% skimmed milk at room temperature for 2 h and
incubated with primary antibodies (Pierce) at room temperature
for 4 h. Finally, the membrane was rinsed with TBST buffer (150 mM
NaCl, 50 mM Tris, 0.05% Tween 20, pH7.6) three times and incu-
bated with a secondary antibody (Pierce) at room temperature for
2 h. Signals were detected using Chemiluminescence immunoassay
(Thermofisher) and collected by gel imaging system.
2.9. Bioinformatic and statistical analysis
T6SS Gene IDs of Xoc GX01 used in this study are from the new
annotation version of Xoc GX01 Genome (Table S2). Potential
protein-coding sequences were analyzed manually using BLAST
suite of programs, including BLASTN, BLASTP, BLASTX. The putative
T6SS gene clusters were identified and aligned by using the web-
based tool VR profile [24]. MEGA6.0 was used for phylogenetic
tree analysis, GraphPad prism software version 5 was used for all
sthe statistical analyses. The prediction of T6SEs and T6SIs was
conducted by using a combined method and homology search
(http://db-mml.sjtu.edu.cn/SecReT6/) [25]. Signal peptide was
detected by the SignalP program (http://www.cbs.dtu.dk/services/
SignalP/) [26]. Primers were designed using Vector NTI software.
Circular maps of the chromosome and plasmid were generated
with CGView Server. T-test was used to determine the statistical
significance of differences between the least three biological rep-
licates in an experiment. ANOVA with p  0.05 was considered.
3. Results
3.1. Xoc GX01 encodes two phylogenetically distinct type VI
secretion systems
Genome sequence analysis revealed two T6SS clusters encoding
T6SS apparatus components on the chromosome of Xanthomonas
oryzea pv. oryzicola GX01, named T6SS1 and T6SS2, respectively.
Table 2 lists the T6SS structural elements encoded in both clusters.
T6SS1 is 30.5 Kb containing 12 core T6SS genes, including hcp1 and
T6SS2 is 50.1 Kb containing 13 core genes, including hcp2. It was
found that the gene coding for the TssJ protein, which is part of the
membrane complex [5], is present only in T6SS2 cluster (Fig. 1,
Table 2). Genetic organizations of the two T6SS clusters of Xoc GX01
are not identical, and T6SS2 was interrupted by clusters of
conserved hypothetical genes and transposase genes, whereas
homologs of tssB, tssC, tssD (hcp), tssE, tssF, tssG and tssH are orga-
nized together in syntenic pattern between T6SS clusters (Fig. 1C).
Phylogenetic analysis showed that the T6SS1 has a long evolu-
tionary distance with T6SS2 (Fig. S1). The identities of the

Table 2
Lists of the T6SS structural genes in both clusters and comparison of the amino acid sequences of the components of both T6SS systems in Xoc GX01a

Unified T6SS nomenclature T6SS structural genes in T6SS1 T6SS structural genes inT6SS2 Identitiesb Function

TssA XOCgx_2363 XOCgx_3623 26.60% Baseplate

TssB XOCgx_2341 XOCgx_3590 40.60% contractile sheath
TssC XOCgx_2342 XOCgx_3589 49.00% contractile sheath
TssD XOCgx_2343 (hcp1) XOCgx_3588 (hcp2) 29.70% Hcp Tube
TssE XOCgx_2345 XOCgx_3586 27.70% Baseplate
TssF XOCgx_2346 XOCgx_3585 34.00% Baseplate
TssG XOCgx_2347 XOCgx_3584 33.90% Baseplate
TssH XOCgx_2349 XOCgx_3583 50.80% ATPase
TssI XOCgx_2353 XOCgx_3595 18.80% VgrG spike
XOCgx_2340 XOCgx_3581 49.80%
TssJ Nc XOCgx_3592 N/A Membrane complex
TssK XOCgx_2356 XOCgx_3593 31.30% Baseplate
TssL XOCgx_2357 XOCgx_3594 27.00% Membrane complex
TssM XOCgx_2358 XOCgx_3623 22.00% Membrane complex
a
T6SS Gene IDs of Xoc GX01 used in this study are from the new annotation version of Xoc GX01 Genome (Table S2).
b
The identities are the results of comparison of the amino acid sequences of the components of both T6SS systems in Xoc GX01.
c
N indicate no presence of the gene.
 P.-C. Zhu et al. / Research in Microbiology 171 (2020) 64e73 69

Fig. 2. Phenotypic characterization and virulence of the T6SS Mutant Strains. (A) The EPS assays for T6SS mutants of Xoc GX01 strain. Two microliters of each bacterial cell sus-
pension (OD600 ¼ 0.1) was pipetted onto NA with 2% sucrose in a Petri dish. The plates were kept for 72 h at 28
C. (B) Biofilm formation assays for T6SS mutants of Xoc GX01 strains.
100 mL of each bacterial cell suspension (OD600 ¼ 1.0) was pipetted into 10 ml LB, and incubated 4 days at room temperature. (C) Virulence assay of Xoc GX01 strains. The bacterial
suspensions concentration of each Xoc strain was adjusted to OD600 ¼ 0.5 by bacteria free water. Bacterial suspensions were infiltrated into 2-month-old rice leaves (Nipponbare),
using needleless syringe on the back of the main vein side, virulence length was measured 14 days after inoculation.

corresponding proteins encoded within two clusters were not high,
ranging from 13.1 to 50.8% (Table 2). These observations indicate
that the two clusters may have arisen from the different origin, and
not from a recent direct duplication.
Moreover, 17 T6SS genomic islands (GIs), were annotated on the
chromosome of Xoc GX01 and several vgrG orphan genes were
found scattered on the chromosome (Fig. 1, Table S2). Three genes
(XOCgx_0030p, XOCgx_0031p and XOCgx_0032p) on the indigenous
plasmid of Xoc GX01 might encode proteins for both T4SS and T6SS
[27]. Obviously, several transposase genes were linked or inserted
in both T6SS clusters and all of the T6SS GIs, strongly suggesting
that T6SS genes in Xoc GX01 might have originated from later
horizontal transfer events (Fig. 1).
3.2. Both T6SS1 and T6SS2 of GX01 are not required for virulence on
rice
To elucidate the biological function of T6SS systems in Xoc GX01,
we constructed single mutants of each T6SS cluster and each hcp
gene, and the double mutants of two clusters and two hcp genes,
using pK18mobsacB vector. The T6SS mutants constructed from Xoc
GX01 were confirmed by PCR, and named DT6SS1, DT6SS2, Dhcp1
and Dhcp2, representing the deletion mutants of each T6SS cluster or
each hcp gene, respectively. DT6SS1DT6SS2 and Dhcp1Dhcp2 refer to
double mutants of two clusters and two hcp genes, respectively
(Table 1). No growth difference was observed between the wild type
and mutant strains in both rich and minimal media (Fig. S3).
To analyze whether the T6SS system of Xoc GX01 plays a role in
the adaptation to adverse conditions, we have conducted a series of
plate assays, including morphologic, auxotrophic assays and toler-
ance tests [17]. There was no significant difference between the
wild type and any mutant strain of T6SS in growth, phenotypes and
tolerance to high osmotic pressure, heavy metal stress, and anti-
oxidant reactions and sodium dodecyl sulfate (SDS) lysis (data not
shown).
To determine whether the T6SS system is involved in the
pathogenicity of Xoc GX01, we have carried out the assays of the
production of major virulence factors of Xoc strains, including
extracellular protease ability, EPS production, motility and biofilm
formation, and the plant tests, including virulence test on rice host
(Nipponbare) and HR induction on tobacco (N. benthamiana). The
results showed that none of the virulence factors tested were
70 P.-C. Zhu et al. / Research in Microbiology 171 (2020) 64e73

influenced by mutations in any T6SS cluster or hcp gene (Fig. 2A, B).
Notably, after the mutants were inoculated into rice leaves, they
behaved as the wild type strain GX01, produced almost identical
disease symptoms during the experimental period (Fig. 2C). These
results suggest that T6SS system is not required for full virulence of
Xoc GX01 on rice.
3.3. T6SS2 but not T6SS1 plays an important role in bacterial
competition
To verify whether the T6SS confers a competitive advantage to
Xoc in intercellular competitions, a series of co-culture confronta-
tion trials between Xoc strains and other bacteria have been con-
ducted in this study. When Xoc cells carrying a plasmid for the
expression of red fluorescent protein (RFP), the RFP-labeled strains,
and E. coli DH5a cells expressing green fluorescent protein (GFP),
the GFP-labeled strains, were mixed and allowed to grow together
after 40 h of co-culture on agar plates, we observed a significant
increase in both the number and the size of the green E. coli col-
onies zones in the green background of the DT6SS2, DT6SS1DT6SS2,
Dhcp2 and Dhcp1Dhcp2 strains when compared with that of the Xoc
WT strain (Fig. 3A a, c, d, f, g), but there was no significant difference
in fluorescence between DT6SS1, Dhcp1 and the Xoc WT strain
(Fig. 3A. b, e). Xoc competitiveness is recovered when the Hcp2
protein is expressed in the Dhcp2 mutant (Fig. 3A. h).
Quantitatively, after 40 h of co-culture, the Xoc/E. coli ratio was four
orders of magnitude lower in the treatments of DT6SS2,
DT6SS1DT6SS2, Dhcp2 and Dhcp1Dhcp2 strains than those of the
wild type, DT6SS1, Dhcp1 and CDhcp2 strain with pXUKhcp2
(Fig. 3B).
Xoc competitiveness against O. oryzae, the endophytic bacte-
rium isolated from deep-water rice [18], was also observed to be
T6SS dependent in the experiments (Fig. 3C) after 40 h of co-
culture. We initially compared two groups, DT6SS2 and GX01, and
the DT6SS2 mutant showed a significantly reduced capacity for
bacterial competition. We then compared DT6SS1, DT6SS1DT6SS2,
Dhcp1, Dhcp2 and Dhcp1Dhcp2 strains (Fig. 3C). In the same way, we
observed a significant increase in the number of O. oryzae in the
presence of DT6SS1DT6SS2, Dhcp2 and Dhcp1Dhcp2 which showed
a marked decline in competitive capacity, and the defect was
restored by pXUK3888. The results demonstrated that T6SS2 in Xoc
GX01 was predominantly involved in competition with E. coli DH5a
and O. oryzae, the rice endophytic bacterium.
3.4. Genes in T6SS1 of Xoc GX01 were inactive under various
conditions
In our previous RNA-seq results of Xoc GX01 both in NB medium
and in planta [28], we found that most of the genes in T6SS1 cluster
showed very low expression level. In order to verify the expression

Fig. 3. Xoc cells competition with bacteria. (A) Xoc cells carrying a plasmid for the expression of RFP (red) and E. coli DH5a cells expressing GFP (green) were mixed at a 10:1 ratio,
spotted on agar plates and allowed to grow together as a co-culture 40 h. The images show fluorescence emitted from the resultant colonies. Xoc strains used were: a wild-type
(WT); b, DT6SS1; c, DT6SS2; d, DT6SS1DT6SS2; e, Dhcp1; f, D hcp2; g, Dhcp1D hcp2; h, CDhcp2. (B) Ratio of the number of viable Xoc and E. coli in experiments similar to those show
in a-h growth after 0 and 40 h of co-culture. Xoc WT, DT6SS1, CDT6SS2, DT6SS1DT6SS2, Dhcp1, D hcp2, Dhcp1D hcp2 and CDhcp2 were used. The error bars represent the standard
deviation of three experiments. (C) Competition assays of Xoc strains against Ochrobactrum oryzae. Xoc and Ochrobactrum oryzae cells expressing GFP (green) were mixed at a 50:1
ratio, spotted on agar plates and allowed to grow together as a co-culture 40 h. The graphs show the number of fluorescent colonies produced. Significance was tested by Student’s
T-test (*indicates significance at P < 0.05, **indicates significance at P < 0.01). The error bars represent the standard deviation of three experiments. (For interpretation of the
references to color in this figure legend, the reader is referred to the Web version of this article.)
 P.-C. Zhu et al. / Research in Microbiology 171 (2020) 64e73
Fig. 4. The expression of T6SS of Xoc GX01. Semi-quantitative PCR was used to detect
the expression of T6SS, with gapA as the reference gene, and samples were cultured
with NB medium. tssB, tssC, hcp and tssH genes code for contractile sheath, tube sheath,
landmark effector and ATPase respectively. (A) Samples were cultured in NB medium.
(B) Samples were cultured in XOM3 medium.
status of T6SS genes in Xoc GX01, four genes in each cluster
encoding T6SS core components were selected as targets and gapA
as the control. The semi-quantitative PCR tests showed that there
were detectable bands of the four marker genes in T6SS2, but no
detectable bands of the marker genes in T6SS1, when the Xoc
strains grew both in nutrient medium and minimal medium
(Fig. 4), suggesting that T6SS1 was inactive when Xoc cultured in
nutrient-rich and defined condition. In addition, although we tried
to use different temperature and pH conditions, genes in T6SS1
remained inactive (Fig. S6, Fig. S7).
3.5. Hcp2 protein secretion is dependent on T6SS2 in Xoc GX01
Hcp release is dependent on the T6SS and is a reliable marker for
assessing functionality of the system [1]. To confirm the secreting
function of T6SS in Xoc GX01, we engineered Xoc strains producing
a 3  Flag-tagged version of Hcp2 for tracing Hcp protein. Hcp2 was
readily detected in the supernatant of wild type cultures but not in
the T6SS2 mutant (Fig. 5), demonstrating that Hcp2 protein
secretion is dependent on T6SS2 in Xoc GX01, thus further estab-
lishing that the T6SS2 is a functional secretion machine in the
bacterium. We assume that the antibacterial activity of GX01 might
result from the secretion of T6SS effectors.
3.6. Bioinformatics analysis predicted several Xoc GX01 genes
encoding candidate T6SS effectors
To predict the candidate T6SS effectors in Xoc GX01, a genome-
wide searching was conducted based on combined methods and
sequence homology searching using BLASTP search (http://db-
mml.sjtu.edu.cn/SecReT6/) [25], with identity  35% and E-
value < 1010. The amino acid sequences of the collected proteins
were checked manually. Proteins with length less than 100 residues
or containing a signal peptide checked by the SignalP program were
removed. To avoid inaccuracy of the data, we also examined the
length deviation between all hit query sequences and identified
protein sequences [29]. A total of 23 T6SE candidates and 9 T6SI
candidates were predicted in Xoc GX01 and listed in Table S3, most
of which are Hcp, VgrG and Rhs-repeat proteins. Some effectors are
not only encoded in the T6SS main cluster but also found in the
T6SS GIs located outside the T6SS main clusters. Several vgrGs are
scattered on the T6SS gene cluster or orphan vgrG islands.
It has been proposed that effectors bind tightly to the VgrG
structural element [30]. Some genes linked with vgrG were also co-
regulated with some T6SS phylogenetic clusters, suggesting that
71
Fig. 5. Production and secretion of Hcp2 in the Xoc GX01 wild type and the DT6SS2
mutant strains. The Flag-tagged Hcp2 protein was detected by western blot analysis
using an anti-Flag antibody. Detection of the b-subunit of the RNA polymerase (b-
RNAP) was used as the control. The position of the molecular size marker (in kDa) is
indicated.
these genes are also related to T6SS. Interestingly, XOCgx_3596,
associated with an orphan vgrG XOCgx_3595, encodes a protein con-
taining no signal peptide and contains a glycosidase domain, making
it a good candidate T6SE, which might be secreted through the T6SS
and play a role in degrading bacterial cell wall peptidoglycan. It is
noteworthy that XOCgx_3597 was located near the predicated effector
XOCgx_3596 and they might encode toxin/immunity pairs.
4. Discussion
Type VI secretion system (T6SS) is a versatile bacterial weapon
which endows its owner with advantages for survival in a variety of
competitions, manifesting as bacterial killing or/and eukaryotic
toxicity [3]. A survey of available Xanthomonas genomes in Gen-
Bank indicated that the majority of the Xanthomonas species
encode at least one T6SS system, and T6SS-related genes account
for almost 1.20% of the total genome of a given Xanthomonas strain.
However, T6SS was only reported to be required for mediating
resistance of X. citri to the predation of a eukaryote Dictyostelium
discoideum so far [15]. In this study, we demonstrated that T6SS
play an anti-bacterial role in Xoc strain GX01. Considering the
relatively high frequency of T6SS genes in Xanthomonas, it is
reasonable to anticipate that the biological functions of T6SSs in
this genus could be much more complicated.
Phylogenetic analysis indicated that T6SS clusters in Xantho-
monads are grouped into two clades, e.g., clade 3 and clade 4b in
the T6SS classification of plant-associated bacteria based on tssB
nucleotide sequence [11]. T6SS1 and T6SS2 of Xoc GX01 belong to
clade 3 and clade 4b, respectively. Genome annotation predicted
that 17 genomic islands in Xoc GX01 might to be related to T6SS
system. These observations suggest that the occurrences of T6SS
systems in Xoc GX01 might have arisen from horizontal gene
transfer and not as the result of duplication, which is in conformity
with the inferences by Bernal et al. (2018). It was found that the
clade 3 T6SS system (T6SS1 in Xoc GX01) is commonly present in
Xanthomonas strains which have T6SS genes and the clade 4b is
only present in X. oryzae [11]. Our results support the previous
studies showing that T6SSs from different clades might have
diverse functions. Burkholderia thailandensis E264 contains six T6SS
clusters [12], only one cluster involved in host manipulation (T6SS-
5). Pseudomonas putida KT2440 is equipped with three T6SSs [31],
but only K1-T6SS is a potent antibacterial device. In our work, we
found that only T6SS2 has the anti-bacterial activity.
It is worth mentioning that clade 3 T6SS system, the homolog of
T6SS1 of Xoc GX01, is required for mediating resistance of X. citri to
the predation of D. discoideum [15]. However, although we tested
the mutant strains of T6SS1 or hcp1 in various conditions, we found
72 P.-C. Zhu et al. / Research in Microbiology 171 (2020) 64e73
no significant difference with the wild type strain in phenotypic
traits, tolerance to adverse conditions and virulence on host plants.
Genes in T6SS1 cluster always remained inactive both in tran-
scriptional tests and proteomic assays, under various experimental
conditions [28]. We performed semi-quantitative PCR to confirm
expression at the transcript level, and while hcp2 mRNA was clearly
visible, hcp1 mRNA could not be detected, suggesting that no hcp1
transcript was present in Xoc GX01. Previously, it was reported that
T6SS in many other bacteria could be induced under certain con-
ditions, such as temperature, pH, presence of chitin or antibiotics,
and their expression was controlled by certain regulators or
quorum sensing systems [32,33]. In this study, we did not find
suitable conditions for induction of T6SS1. Thus, hcp1 may be a
silent gene or it is expressed under as-yet-uncharacterized condi-
tions. Moreover, the expression of T6SS2, the functional T6SS in Xoc
GX01, was found to be slightly affected by these conditions.
T6SSs were initially reported as virulence factors and proven to
be involved in the pathogenesis in several pathogens [1]. Among
phytopathogenic bacteria, Pectobacterium atrosepticum, a pectolytic
bacterium that produces soft rot in plants, is one of the first plant
pathogens whose T6SS activity was linked with virulence [34]. In
Ralstonia solanacearum, a destructive plant spathogen of solanaceae
plants having a wide range of plant hosts, the virulence of a tssB
mutant is reduced when compared with the wild-type strain [35].
This mutation has a considerable effect on antibacterial activity and
biofilm formation, which may indirectly affect virulence. In order to
confirm the functions of T6SS with Xoc GX01, the T6SS strains were
inoculated into rice leaves to verify whether T6SS of Xoc GX01 plays
a role in plant virulence. The results revealed that T6SS plays no role
in Xoc GX01 virulence. This is consistent with the result in Pseu-
domonas syringae pv. tomato DC3000, a plant-pathogenic bacte-
rium found in a wide variety of agricultural environments, in which
T6SS does not significantly contribute to virulence in the plant host
[22]. Moreover, we found that there were no significant differences
in expression of major pathogenicity genes between T6SS mutants
and wild type strain (Fig. S6) or in hcp genes between pathogenicity
gene mutants and wild type strain (Fig. S7). Studies showed that
T6SS has been also related with biofilm formation in many other
plant pathogenic bacteria such as Acidovorax citrulli [36] and the
non-pathogenic Pseudomonas fluorescens MFE01 strain [37]. A
possible link between biofilm formation and T6SS has also been
found in animal pathogens such as Pseudomonas aeruginosa [38].
However, in our study, mutations in T6SS genes had no effect on
biofilm formation, EPS production and extracellular protease ac-
tivity of the pathogen.
Growing evidence revealed that the primary function of the
T6SS is as a device for inter-bacterial competition [3]. In this study,
we found that Xoc has a significant competitive effect not only on
the model organism E. coli, but also on a rice endophytic bacterium
O. oryzae [18]. Xoc mainly invades rice through the stomata and
wounds and colonizes intercellular space in the rice leaf mesophyll
[16]. Our preliminary investigations showed that the rice leaf sur-
face and interior inhabit the complex multi-microbial groups,
which maintain a fierce competitive environment in epiphytic and
endophytic growth of Xoc. We demonstrated that T6SS systems
might play important role in bacterial killing, but the competitive
advantage of Xoc with T6SS over the plant microbiota during
infecting plant hosts need to be verified further. Besides, the T6SSs
promotes bacterial competition with eukaryotic microbes, i.e.,
X. citri with D. discoideum [15], P. syringae pv. tomato DC3000 with
yeast [22]. In our work, although we tested various conditions, we
did not find any evidence that T6SS of Xoc GX01 plays a role in
competition with yeast.
Once the biological functions of T6SS are revealed, it is an
important work to discover and identify T6SS effectors (T6SEs) in
certain bacteria, which is an important basis to reveal the func-
tional mechanism of T6SSs. T6SEs have been identified by a variety
of approaches, including bioinformatics analysis, genetic analysis of
T6SS-associated genes, proteomics-based method, and mutant li-
brary screening [39]. Since T6SS effectors are all toxic to bacteria,
and they are all located near genes that code for proteins that give
bacteria immunity to toxins that prevent them from self-
intoxication, suggesting that effectors and their cognate immu-
nity proteins are encoded from their adjacent genes [6]. Based on
their enzymatic activity, the known effectors can be divided into
amidases (Tae), glycosyl hydrolases (Tge), lipolytic enzymes (Tle)
and nucleases (Tde) [40]. In this study, we predicted 21 candidates
of the T6SE in Xoc GX01 based on a homology search [25], most of
which are Hcp, VgrG and Rhs-repeat proteins. Effectors are not only
encoded in the T6SS main clusters but also found in the other T6SS
GIs, some of which were found near vgrG [6]. Genes encoding toxin/
immunity pairs were also found in this study, suggesting that they
are good candidates for T6SS effectors in our prediction.
In this study, we have demonstrated that a T6SS system plays an
important role in bacterial competition, allowing this plant path-
ogen to survive under conditions in which it has to compete with
other microorganisms for resources. This is the first study to
investigate the type VI secretion system in X. oryzae. Our results will
serve useful reference and guidance of T6SS studies in phytopath-
ogenic bacteria in future.
Declaration of Competing Interest
The authors declare that they have no any conflict of interests.
Acknowledgements
We thank Mr. Andrew Read (Plant Pathology and Plant-Microbe
Biology Section, School of Integrative Plant Science, Cornell Uni-
versity Ithaca, NY, USA) for providing the broad host range plas-
mids, pVLacGreen. The work is supported by the National Natural
Science Foundation of China (31270139 and 31660505), the Na-
tional Key R&D Program of China (2018YFD02003), the Ba Gui
Scholar Program of Guangxi Zhuang Autonomous Region of China
(2014A002), and the Natural Science Foundation of Guangxi
(2017GXNSFAA198310).
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.resmic.2019.10.004.
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