| Environmental Microbiology | Research Article

Bacterial secretion systems contribute to rapid tissue decay in

button mushroom soft rot disease
Philipp Wein,1 Katharina Dornblut,1 Sebastian Herkersdorf,2,3 Thomas Krüger,4 Evelyn M. Molloy,1 Axel A. Brakhage,3,4 Dirk
Hoffmeister,2,3 Christian Hertweck1,3

AUTHOR AFFILIATIONS See affiliation list on p. 14.

ABSTRACT The soft rot pathogen Janthinobacterium agaricidamnosum causes

devastating damage to button mushrooms (Agaricus bisporus), one of the most
cultivated and commercially relevant mushrooms. We previously discovered that this
pathogen releases the membrane-disrupting lipopeptide jagaricin. This bacterial toxin,
however, could not solely explain the rapid decay of mushroom fruiting bodies,
indicating that J. agaricidamnosum implements a more sophisticated infection strategy.
In this study, we show that secretion systems play a crucial role in soft rot disease. By
mining the genome of J. agaricidamnosum, we identified gene clusters encoding a type
I (T1SS), a type II (T2SS), a type III (T3SS), and two type VI secretion systems (T6SSs).
We targeted the T2SS and T3SS for gene inactivation studies, and subsequent bioassays
implicated both in soft rot disease. Furthermore, through a combination of comparative
secretome analysis and activity-guided fractionation, we identified a number of secreted
lytic enzymes responsible for mushroom damage. Our findings regarding the contribu­
tion of secretion systems to the disease process expand the current knowledge of
bacterial soft rot pathogens and represent a significant stride toward identifying targets
for their disarmament with secretion system inhibitors.

IMPORTANCE The button mushroom (Agaricus bisporus) is the most popular edible
mushroom in the Western world. However, mushroom crops can fall victim to serious
bacterial diseases that are a major threat to the mushroom industry, among them
being soft rot disease caused by Janthinobacterium agaricidamnosum. Here, we show
that the rapid dissolution of mushroom fruiting bodies after bacterial invasion is due
to degradative enzymes and putative effector proteins secreted via the type II secre­
tion system (T2SS) and the type III secretion system (T3SS), respectively. The ability
to degrade mushroom tissue is significantly attenuated in secretion-deficient mutants,
which establishes that secretion systems are key factors in mushroom soft rot disease.
This insight is of both ecological and agricultural relevance by shedding light on the
disease processes behind a pathogenic bacterial-fungal interaction which, in turn, serves
as a starting point for the development of secretion system inhibitors to control disease Editor Reinhard Fischer, Karlsruhe Institute of
progression. Technology, Karlsruhe, Germany

Address correspondence to Christian Hertweck,

KEYWORDS Janthinobacterium agaricidamnosum, lytic enzymes, mushroom patho­ Christian.hertweck@leibniz-hki.de.
gens, secretome analysis, type III secretion The authors declare no conflict of interest.

T
See the funding table on p. 15.
he agricultural sector continually suffers severe economic losses due to the adverse
effect of Gram-negative bacterial soft rot pathogens on the yields of vegetable, fruit, Received 28 March 2023
and mushroom farming (1–4). These bacteria exert their devastating effects by releasing Accepted 8 June 2023
Published 24 July 2023
multiple virulence factors, lytic enzymes, and toxins to exploit eukaryotic host cells,
thereby degrading the crop to mush (5–7). While the disease processes of plant-infecting Copyright © 2023 Wein et al. This is an open-access
article distributed under the terms of the Creative
soft rot pathogens are well studied, with many using dedicated secretion systems to
Commons Attribution 4.0 International license.

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deliver disease mediators, their mushroom-infecting counterparts are poorly understood

(8, 9). Indeed, Janthinobacterium agaricidamnosum DSM 9628 is a causative agent of
mushroom soft rot disease, but the associated virulence determinants remain obscure.
J. agaricidamnosum infection of the button mushroom Agaricus bisporus is characterized
by pitting, sticky blotches, and rapid dissolution of fruiting bodies (10). Awareness of
this pathogen is of particular importance to mushroom farming since A. bisporus is
one of the most cultivated and widely consumed mushrooms, constituting around
15% of the world’s supply (11). Intriguingly, the Janthinobacterium genus is considered
non-pathogenic, with members being commonly isolated from water, forest soils, Arctic,
and Antarctic habitats (12–16). Besides J. agaricidamnosum, only very few representatives
of the genus engage in a pathogenic lifestyle (17, 18), among them Janthinobacterium
lividum has been associated with mortal disease of rainbow trouts and human sepsis
(19–21). Conversely, J. lividum is also known as cutaneous mutualistic bacterium that
protects amphibians from the lethal skin disease chytridiomycosis (22, 23). The protective
effect has been traced to the antifungal secondary metabolite violacein, whose intense
purple color is eponymous for the genus Janthinobacterium (23, 24). We previously
implicated another antifungal secondary metabolite, the cyclic lipopeptide jagaricin,
in the mushroom soft rot disease caused by J. agaricidamnosum (25). This toxin acts
by impairing the membrane integrity of host cells, resulting in a rapid influx of ions,
including Ca2+ (26). However, the striking soft rot symptoms could not be explained
solely by the lipopeptide (25). Thus, the molecular basis of this mushroom soft rot
disease and the effectors involved remained elusive. Here, we show that the type II
secretion system (T2SS) and type III secretion system (T3SS) of J. agaricidamnosum are
central to its infection strategy and implicate a number of T2SS-secreted lytic enzymes
in the deleterious effects on A. bisporus. Providing detailed knowledge on the pathobi­
ology of soft rot pathogens may aid the development of protective measures against
devastating losses in mushroom agriculture.

RESULTS AND DISCUSSION

J. agaricidamnosum harbors four different secretion systems
Given that some of the most virulent Gram-negative bacteria use secretion systems
to release or inject toxic proteins into eukaryotic host cells (27, 28), we browsed
the whole-genome sequence of J. agaricidamnosum (GenBank accession number:
HG322949) for potential secretion system gene clusters. In doing so, we uncovered four
different types that were scattered separately throughout the genome (Fig. 1A and B).
The products of unannotated genes encoded within the putative clusters were further
inspected for their similarity to characterized proteins using the basic local alignment
search tool (BLAST) (29) and HHpred (30) (see Table S1 in the supplemental material).
We found three genes that together potentially encode a type I secretion system
(T1SS). In pathogenic bacteria, these systems often secrete protein substrates implicated
in virulence and nutrient acquisition (31–34). Furthermore, we identified a gene cluster
coding for 12 proteins of the general secretion pathway (GspC–N), suggesting that the
bacterium has a functional T2SS. We also discovered a large T3SS gene cluster that spans
about 42,000 bp and contains 39 open reading frames (ORFs). A protein BLAST search
using the well-conserved SctC (secretin) and SctT (export apparatus) revealed that
among the 106 publicly available Janthinobacterium whole-genome sequences, only the
environmental isolates Janthinobacterium sp. B9-8 and the red-pigmented Janthinobac­
terium sp. BJB412 also harbor genes for a functional T3SS (see Fig. S1 in the supplemental
material). Six ORFs (ORF 20, 21, 23, and 25–27) within the T3SS cluster of J. agaricidamno­
sum encode proteins with unknown functions that possess N-terminal T3SS signals as
predicted by the EFFECTIVE T3 tool (35), indicating that these proteins might be T3
effectors (Table S2). ORF 20 (GJA_RS07825) codes for a protein that exhibits similarity
(24%) to the alanine-tryptophan-arginine triad (AWR, also named RipA) effector family of
the plant pathogen Ralstonia solanacearum (36). Although their putative function cannot
be deduced from the protein sequence information alone, it has been shown that AWR

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FIG 1 The Janthinobacterium agaricidamnosum genome encodes four different secretion systems. (A) Schematic representation of the type 1, 2, 3, and 6
secretion systems (T1SS, T2SS, T3SS, and T6SS) of J. agaricidamnosum. (B) Genetic organization of the T1SS, T2SS (gsp), T3SS (sct), and T6SS (tss) gene clusters
within the genome of J. agaricidamnosum. Arrows display protein-coding regions. Conserved genes encoding secretion system core proteins with similarity to
(Continued on next page)

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FIG 1 (Continued)
well-characterized secretion system components are represented by arrows with letter designations (color code continues from panel A). The predicted functions
of other gene products are indicated in the key. T3 secretion signals of open reading frames (ORFs) 20, 21, 23, and 25–27 were predicted with EFFECTIVE T3
software.

T3 effectors contribute to R. solanacearum virulence in numerous ways (37). A multiple

sequence alignment and phylogenetic analyses demonstrated that the AWR effector of J.
agaricidamnosum deviates from the AWR members of R. solanacearum in regard to the
conserved regions (data set Fig. S2 to S4). The putative effector encoded by ORF 21
(GJA_RS07835) does not share similarity with any known protein. The ORFs 23
(GJA_RS27170) and 25–27 (GJA_RS07855, GJA_RS07860, and GJA_RS07865) encode
proteins that exhibit sequence similarities (26%–37%) to putative T3 effectors with
unknown function from various plant pathogens, including Pseudomonas spp., Xantho­
monas spp., and R. solanacearum. In addition, we encountered two complete gene
clusters (tss-1 and tss-3) and an incomplete cluster (tss-2) that are reminiscent of type VI
secretion system (T6SS) clusters. The effectors delivered by this type of contractile
nanomachine usually have a pivotal role not only in interbacterial and interkingdom
competition (38–40) but also in bacterial-host interactions and nutrient acquisition (41–
44).
Any of the detected putative secretion systems (T1SS, T2SS, T3SS, and T6SSs), or a
combination thereof, may be involved in the interaction of J. agaricidamnosum with A.
bisporus. Because of the known degradative nature of T2SS-dependent proteins (45), we
deemed the bioinformatically identified T2SS a likely candidate to promote the damage
of mushroom tissue. In addition, considering the role of T3SS-associated effectors in
human pathogens like Salmonella or Yersinia (46), and plant pathogens like Pseudomonas
syringae and R. solanacearum (47, 48), we wondered whether the predicted T3SS might
be also involved in mushroom soft rot.

Deficiency in T2SS or T3SS attenuates the capacity of J. agaricidamnosum to

cause button mushroom soft rot
In order to investigate whether the bioinformatically identified T2SS and T3SS of J.
agaricidamnosum play a role in pathogenesis, we first verified by reverse-transcriptase
PCR that the associated genes (gspD, gspE, sctC, and sctT) are expressed under laboratory
conditions (Fig. S5). Subsequently, we inactivated the relevant genetic loci. To this end,
we targeted the secretin encoding gene gspD as well as gspE, which encodes an ATPase,
the driving force of the T2SS machinery. In case of the T3SS, the secretin-encoding gene
sctC and the inner membrane export apparatus protein encoded by sctT were targeted.
Due to the lack of genetic tools and standard cloning procedures for J. agaricidamnosum,
we adapted a double-crossover strategy, previously applied to identify the biosynthetic
gene cluster of jagaricin (25), to disrupt the corresponding genes with a kanamycin
resistance cassette. We succeeded in generating one of the two desired mutations
to each secretion system, resulting in the T2SS-deficient strain J. agaricidamnosum
ΔgspE and the T3SS-deficient strain J. agaricidamnosum ΔsctC (Fig. S6 and S7). Both
mutants do not show morphological differences or defects in growth and motility when
compared to the wild type under standard laboratory conditions (Fig. S8 and S9). We
proceeded to examine the effect of the mutations on pathogenesis by inoculating the
trama (inner flesh of the fruiting body) of separate button mushroom slices with J.
agaricidamnosum, J. agaricidamnosum ΔgspE, and J. agaricidamnosum ΔsctC (Fig. 2A). We
also included the jagaricin-deficient strain J. agaricidamnosum ΔjagA (25) for compari­
son. First, a visual inspection of severity of soft rot symptoms was performed (Fig. 2B).
Subsequently, we also quantified the level of mushroom degradation by comparing
the weight of starting mushroom tissue with that of degraded tissue after 6 days in
four independent experiments (Fig. 2C). We found that the T2SS- and T3SS-deficient
mutants cause significantly reduced trama degradation. While J. agaricidamnosum lysed
the mushroom tissue almost completely, the infection with J. agaricidamnosum ΔgspE

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and J. agaricidamnosum ΔsctC led to less severe symptoms such as browning and
sticky blotches. We noted that after 3 days of infection, J. agaricidamnosum wild type,
and to a lesser extent J. agaricidamnosum ΔgspE, cause characteristic cavities on the
mushroom slices. In contrast, these cavities are absent on mushroom slices infected with
J. agaricidamnosum ΔsctC. Furthermore, the antifungal effect of jagaricin is not apparent
in this mushroom infection assay since there was no visible difference in symptoms
caused by J. agaricidamnosum ΔjagA compared to the J. agaricidamnosum wild type,
corroborating that additional factors contribute to the infection strategy (25).
This finding that secretion systems are involved in mushroom soft rot disease is
interesting in light of known pathomechanisms of soft rot pathogens. For instance, the
pathogenic strategies of fruit- and vegetable-infecting Dickeya spp. and Pectobacterium
spp. require a T2SS and its secreted proteins (5, 49). While T3SS-secreted effector proteins
make only a small contribution to the pathogenicity of Pectobacterium spp. (50), they
play a key role in the initial infection process of Dickeya dadantii (51). However, little is
known about the role of T3SS in bacterial-fungal interactions, whether they be mutualis­
tic or pathogenic in nature (52, 53). Only a few studies have been reported showing that
T3SSs contribute to pathogenic bacterial-fungal interactions (54, 55). For example,
Salmonella enterica serovar Typhimurium competes with the pathogen Candida albicans
by releasing an inositol phosphatase (SopB) via a T3SS that negatively affects the viability
of the fungus (55). The bioinformatically identified T3 effector proteins of J. agaricidam­
nosum exhibit low similarity to phytopathogenic effectors, some of which are known to
alter plant physiology and interfere with the plant immune response (47). However,
similar functions cannot simply be predicted since those processes are unknown in
higher fungi like A. bisporus. Nonetheless, the mushroom infection assays indicated that
both T2SS- and T3SS-secreted proteins are involved in mushroom trama damage.

The T2SS releases tissue-degrading and chitinolytic proteins

Based on the observation that both the T2SS- and the T3SS-deficient mutants are
impaired in their ability to cause soft rot disease symptoms, we assumed the lack of
degradative proteins may explain this phenotype. Whereas putative T3 effector proteins
are encoded within the T3SS gene cluster, the genomic neighborhood of the T2SS gene
cluster does not reveal candidates that could contribute to mushroom soft rot disease.
However, T2SSs are typically involved in secreting toxins and lytic enzymes (45). An
extensive in silico secretome analysis confirmed that the J. agaricidamnosum genome
contains a number of genes that encode putative exoproteins with N-terminal secretion
signals such as lytic enzymes (data not shown). We monitored J. agaricidamnosum and J.
agaricidamnosum ΔgspE for differences in chitinolytic and proteolytic activity by
culturing them on split plates containing standard growth agar on one half of the petri
dish and chitin or milk agar on the other. Proteolytic activity was not visibly affected by
the inactivation of the T2SS; both strains showed strong proteolytic activity on milk agar.
While J. agaricidamnosum displayed chitinolytic activity, visible by a halo on the chitin
agar, chitin degradation was not detected for J. agaricidamnosum ΔgspE (Fig. 3A).
Intrigued by the observation that the secretion of chitinases is impaired in the
absence of a functional T2SS, we set out to identify the T2SS-secreted suite of chitinases
and other proteins. Therefore, we performed a comparative analysis of the total secre­
tomes of J. agaricidamnosum and J. agaricidamnosum ΔgspE. Both strains were cultured
in medium containing lyophilized, ground button mushrooms as the sole nutrient source
(Fig. 3B). Subsequently, the secretomes were analyzed by means of LC-MS/MS, demon­
strating that two proteases and one glucanase are less abundant in the secretome of J.
agaricidamnosum ΔgspE compared to that of J. agaricidamnosum wild type (Fig. 3C).
Surprisingly, three proteases and another glucanase are more abundant in the J.
agaricidamnosum ΔgspE secretome. This finding is consistent with the observation that
both J. agaricidamnosum and J. agaricidamnosum ΔgspE show protease activity on milk
agar, indicating that certain exoproteins can be secreted independently of the T2SS. The
production of exoproteins, such as extracellular proteases, is known to be regulated by

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FIG 2 Contribution of T2SS and T3SS to soft rot disease. (A) Schematic illustration of the button mushroom infection assay. Mushroom slices were placed in a
petri dish and weighed (i), then inoculated with 2.7 × 107 cells of J. agaricidamnosum or mutants onto two different spots on each trama (ii). A wet sterilized tissue
was placed in the center of the petri dish to prevent the slices from drying out. After 6 days of incubation, non-degraded mushroom tissue was collected and
re-weighed to determine the level of degradation (iii). RT, room temperature. (B) Representative images of mushroom slices infected with J. agaricidamnosum, J.
agaricidamnosum Δjag, J. agaricidamnosum ΔgspE, or J. agaricidamnosum ΔsctC from day 0 to day 6. (C) Statistical evaluation of infected mushroom slices after
6 days of inoculation. Mushrooms slices inoculated with J. agaricidamnosum ΔgspE, J. agaricidamnosum ΔsctC, or culture medium (control) show significantly
less (P < 0.05) degrees of degradation when compared to mushrooms slices infected with J. agaricidamnosum wild type. The results depicted are derived from
four independent experiments. The values displayed for each experiment are the mean of six replicates consisting of four mushroom slices each, with error bars
showing the standard error of the mean. ***P < 0.001 (Bonferroni’s means comparison test, two-way analysis of variance [ANOVA]).

the repression of carbon, nitrogen, and sulfur in bacteria (56). Considering that the T2SS-
deficient mutant has diminished capacity to secrete various hydrolyzing enzymes that
provide utilizable breakdown products needed for growth and survival, it is conceivable
that the bacterium responds with increased exoprotein production. Thus, it could be that
proteins that are secreted independently of the T2SS appear more abundant in the
supernatant of the T2SS-deficient mutant.
Notably, the triplicate experiments demonstrated that chitinases and chitosanases are
substantially less abundant in the secretome of the T2SS-deficient mutant in agreement
with the phenotypes observed in the chitinase activity assay (Fig. 3A and C). Moreover,
while we did not detect T3 effector proteins in the supernatants of J. agaricidamnosum
and J. agaricidamnosum ΔgspE, the secretion of proteins related to glucanases and ricin-
type β-trefoil lectin (RICIN) domain-containing proteins appears to be impaired in the
T2SS-deficient mutant (Fig. 3C; Table 1).
To identify the exoproteins responsible for soft rot symptoms, we again cultured J.
agaricidamnosum and J. agaricidamnosum ΔgspE in button mushroom media. The
secretomes were fractionated by ammonium sulfate precipitation of filtered superna­
tants and, after dialysis, tested in a mushroom assay and on chitin agar. Whereas protein
fractions of J. agaricidamnosum ΔgspE sporadically elicited mushroom browning where

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FIG 3 Bioactivity-guided secretome analysis. (A) Split-plate assay with chitin (top row) or milk agar (bottom row) to screen for secreted chitin- and pro­
tein-degrading enzymes of J. agaricidamnosum (wild type) and T2SS-deficient J. agaricidamnosum ΔgspE. Halo formation indicates protease activity from J.
agaricidamnosum and J. agaricidamnosum ΔgspE. Chitinase activity was only observed from J. agaricidamnosum as indicated by a halo. Arrows point to visible
(Continued on next page)

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FIG 3 (Continued)
halos caused by enzymatic activity. (B) Workflow for the secretome analysis of J. agaricidamnosum or J. agaricidamnosum ΔgspE cultivated in media containing
button mushrooms as the sole nutrient source. (C) Volcano-plot visualization of protein abundance changes [threshold of log2(ΔgspE/wt) < −1 and >1 and the
ratio adjusted P-value < 0.05] obtained from comparative LC-MS/MS secretome analysis of J. agaricidamnosum and J. agaricidamnosum ΔgspE. The significantly
lower abundance of lytic enzymes (chitinases, chitosanases, proteases, glucanases, and ricin B lectins) in the J. agaricidamnosumΔgspE secretome indicates their
impaired secretion in the absence of a functional T2SS. (D) Bioactivity-guided secretome analysis of J. agaricidamnosum and J. agaricidamnosum ΔgspE reveals
that a single protein fraction [precipitated in 30% (NH4)2SO4 saturation] from J. agaricidamnosum causes lesions on mushroom slices and halo formation on
chitin agar. Arrows point to visible lesions. (E) MALDI-TOF-MS of SDS-PAGE-separated proteins unveil two chitinases of the glycoside hydrolase family 18 and 19
and a ricin-type beta-trefoil lectin domain protein in the bioactive fraction.

spotted, one particular fraction of J. agaricidamnosum [30% (NH4)2SO4 saturation]

repeatedly showed chitinolytic activity and caused superficial lesions on mushroom
tissue (Fig. 3D; Fig. S10). Importantly, the equivalent fractions from the T2SS-deficient
mutant were inactive. When heat-treated aliquots of the J. agaricidamnosum active
fraction were tested in the mushroom assay and on chitin agar, neither activity was
detected. Protein separation of all fractions by SDS-PAGE revealed five visible protein
bands between 30 and 70 kDa in the active fraction that were not present in any
fractions of J. agaricidamnosum ΔgspE (Fig. 3E; Fig. S11). Due to low abundance, the two
proteins of approximately 40 and 50 kDa in size could not be identified. However, an
analysis of the remaining proteins by MALDI-time of flight mass spectrometry (MALDI-
TOF-MS) led to the identification of three proteins, each possessing signal sequences for
secretion (Table S3), that are encoded in the J. agaricidamnosum genome (Fig. S12 to
S16). The largest protein (approximately 70 kDa) is predicted to comprise an N-terminal
RICIN domain and a C-terminal right-handed β-helix structure that is common to
proteins belonging to the pectate lyase superfamily (Fig. S12A through C) (57). Pectolytic
enzymes like pectin lyases and pectate lyases cleave pectin, a major component of the
cell wall of higher plants, and play important roles as virulence factors of plant patho­
gens such as D. dadantii and Pectobacterium carotovorum (49, 58). Several pectate lyases
of these bacteria are known to be secreted by the T2SS. However, these enzymes feature
an N-terminal fibronectin type III fold (Fn3) domain (59), rather than the RICIN domain

TABLE 1 Proteins identified by LC-MS/MS that are predicted as exoproteinsa

NCBI accession no. Protein identification Pfam Mass (kDa) Log2 ratio ΔgspE/wild type Signal peptide
CDG84650 Chitinase (GH 18)b PF00704 47.2 −8.419 Yes
CDG84085 Chitinase (GH 18) PF00704 59.8 −7.942 Yes
CDG85585 Glucanase (GH 8) PF01270 46.4 −7.028 Yes
CDG86073 Ricin B lectin PF14200 12.7 −6.902 Yes
CDG82113 Chitodextrinase (GH 18) PF00704 83.8 −6.675 Yes
CDG81136 Chitinase (GH 19) PF00182 29.0 −5.700 Noc
CDG82784 Ricin B lectin PF14200 77.8 −4.904 Yes
CDG84529 Leucyl aminopeptidase PF04389 55.6 −4.982 Yes
CDG83702 Ricin B lectin (GH 71/99) PF14200 51.9 −4.568 Noc
CDG86074 Ricin B lectin (GH 71/99) PF14200 51.8 −4.528 Noc
CDG81767 Chitosanase (GH 46) PF01374 30.9 −4.356 Yes
CDG85621 Peptidase S41 family protein PF00595 53.5 −3.958 Yes
CDG81764 Chitosanase (GH 46) PF01374 28.7 −2.490 Yes
CDG84341 Endo-1,3(4)-β-glucanase (GH 81) PF00754 136.0 −1.208 Yes
CDG84587 Subtilisin-like protease PF00082 106.3 −0.662 Yes
CDG82666 Peptidase M28 family protein PF04389 50.7 3.629 Yes
CDG82433 Endoglucanase (GH 9) PF00759 64.9 3.978 Yes
CDG85746 Peptidase M13 family protein PF01431, PF05649 76.0 4.223 Yes
CDG85353 Peptidase M28 family protein PF04389 58.8 5.978 Yes
a
Secretion signal peptide (SP) was predicted with SignalP 6.
b
GH, glycoside hydrolase family protein.
c
Non-classical secretion was predicted with SecretomeP 2.0.

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observed here. Notably, the N-terminal domain determines the mode of secretion.
Nevertheless, since the fungal cell wall does not contain pectin or pectate, the mecha­
nism by which the predicted pectin lyase could contribute to J. agaricidamnosum
pathogenicity is not obvious (60). An analysis of structural homology using HHpred
indicates that the RICIN domain-containing protein shares more similarity with character­
ized exo-1,3-β-glucanases (E-value 3.4E−91, 54% identity) than with pectate lyases (E-value
1.6E−5, 11% identity) (Table S4). The two proteins of approximate sizes 60 and 30 kDa
could be assigned as chitinases that belong to the glycoside hydrolase families 18 and
19, respectively (Fig. 3E, and data set Fig. S13 and S16). One could envision that glucana­
ses and chitinases degrade mushroom tissue since glucan and chitin are major compo­
nents of the A. bisporus cell wall, along with other polysaccharides such as mannan and
chitosan (60). This enzymatic activity could potentially facilitate access of jagaricin to its
target, the cell membrane. An analogous case in which both T2SS-secreted chitinases
and secondary metabolites support the infection process has been described for the
mushroom pathogen Burkholderia gladioli pv. agaricicola (7, 61, 62). Considering the
variety of lytic enzymes identified in the secretome of J. agaricidamnosum, we assume
that J. agaricidamnosum releases an armory of lytic enzymes to decompose mushroom
tissue.
In conclusion, we demonstrated that various factors contribute to the pathogenicity
of J. agaricidamnosum. We successfully constructed mutant strains that enabled the
role of the T2SS and T3SS in pathogenesis to be dissected. Given the stark differences
between the phenotypes of J. agaricidamnosum and the secretion system deficient
mutants J. agaricidamnosum ΔgspE and J. agaricidamnosum ΔsctC in the mushroom
infection assay, we implicate T2SS- and T3SS-secreted proteins of J. agaricidamnosum in
the damage of its fungal host. The collective results of bioassays, comparative LC-MS/MS
secretome analysis, and activity-guided fractionation point to a blend of T2SS-secreted
lytic enzymes (including chitinases, chitosanases, glucanases, and proteases) that causes
mushroom degradation. In light of our results, we propose an updated model of soft rot
disease in which degradation of the compact mushroom cell wall by these T2SS-secre­
ted proteins likely facilitates access of jagaricin to the membrane (Fig. 4). In this way,
J. agaricidamnosum joins the ranks of mushroom-, fruit-, and vegetable-infecting soft

FIG 4 Model of the pathogenic mechanism of J. agaricidamnosum involved in button mushroom soft rot disease. Lytic enzymes secreted via a bacterial T2SS
induce the decay of the A. bisporus cell wall. This enables virulence factors such as jagaricin to attack the fungal membrane. T3 effector proteins are secreted
directly into the host cell and promote the infection process by a so far unknown mechanism.

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rot pathogens that follow a common strategy to evoke symptoms, i.e., secretion of
hydrolytic enzymes by a T2SS (5, 49, 61).
Moreover, we show that the T3SS contributes significantly to mushroom degradation
and present the first example of a T3SS involved in mushroom soft rot disease. We used
bioinformatic analysis to uncover six potential T3 effectors with five of them resembling
known effectors of phytopathogenic bacteria. However, the molecular details of how
the T3SS-secreted proteins of J. agaricidamnosum drive disease progression remain to
be deciphered. Since T3 effector proteins have long been recognized as important
virulence factors of various plant-pathogenic bacteria (51, 63, 64), our discovery reveals
a common principle of the pathobiology of mushroom- and plant-pathogenic bacte­
ria. Remarkably, while secretion systems of plant-pathogenic Gram-negative bacteria
are quite well studied, the role of secretion systems in mycopathogenic bacteria has
remained underexplored. We addressed this knowledge gap and unveiled the previously
elusive roles of J. agaricidamnosum secretion systems in mushroom soft rot disease.
This finding opens up a new avenue of research into the development of secretion
system inhibitors to disarm J. agaricidamnosum. We expect our work will eventually help
mitigate the severe economic losses caused by mushroom soft rot disease.

MATERIALS AND METHODS

Bacterial strains and growth conditions
Strains used in this study are listed in Table S5 in the supplemental material. Janthino­
bacterium agaricidamnosum DSM 9628 was procured from the German Collection of
Microorganisms (DSMZ). Routine cultivation was performed in modified nutrient media
(DSMZ Medium 605; 1 g L−1 beef extract, 2 g L−1 yeast extract, 5 g L−1 peptone, and 15
g L−1 agar, pH 6.5) and CYM (2 g L−1 yeast extract, 2 g L−1 polypeptone, 20 g L−1 glucose,
15 g L−1 agar, pH 6.5). Solid and liquid cultures (with orbital shaking in baffled flasks)
were incubated at room temperature and 26°C, respectively. Secretion system-deficient
mutants were selected on modified nutrient media supplemented with 50 µg mL−1
kanamycin. Escherichia coli HST08 Stellar (Takara Bio, Saint-Germain-en-Laye, France) and
E. coli ER2925 (New England Biolabs GmbH, Frankfurt am Main, Germany) were used to
construct plasmids for gene disruption. E. coli strains were cultured at 37°C in lysogeny
broth (LB; 10 g L−1 tryptone, 5 g L−1 yeast extract, 5 g L−1 NaCl, and 1 g L−1 glucose),
supplemented with 5 µg mL−1 kanamycin for selection, whenever appropriate.

Construction of plasmids for T2SS and T3SS gene disruption

Oligonucleotide primers and plasmids are listed in Tables S6 and S7, respectively. The
mutation strategy was based on target gene disruption via homologous recombination
inspired by previous studies (25, 52). Genomic DNA of J. agaricidamnosum was used as
template to amplify a portion of the region encoding the targeted secretion system.
In order to construct the vector pKOgspE, homologous regions (1,000 bp) downstream
(hr1) and upstream (hr2) of the target gene gspE were amplified by PCR using the
primer pair gspE_hr1_fw/gspE_hr1_rv and gspE_hr2_fw/gspE_hr2_rv, respectively, with
15bp extensions complementary to the linearized vector and 8 bp extensions comple­
mentary to the kanamycin-resistance gene kanr. Additionally, kanr was amplified from the
commercially available vector pK19 (65) with the primer pair gspE_kanr_fw/gspE_ kanr_rv
that incorporates 8 bp extensions, homologous to hr1 and hr2 of the target region. All
amplifications were carried out using Phusion High-Fidelity DNA Polymerase. The three
resulting PCR fragments (hr1, hr2, and kanr) were assembled with SmaI-linearized pGL42a
by using the In-Fusion HD Cloning Kit (Takara Bio), yielding the plasmid pKOgspE. The
reaction mixture was subsequently used to transform chemically competent E. coli HST
08 Stellar cells. The plasmid was verified by restriction digest and Sanger sequencing of
the cloned region using the primers M13_fw and M13_rv. Finally, the methylase-deficient
E. coli strain ER2925 was transformed with pKOgspE by electroporation (2 mm cuvette,

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2,500 V, 25 µF, and 200 Ω). The unmethylated plasmid pKOgspE was purified from
overnight cultures using the Monarch Plasmid Miniprep Kit (New England Biolabs) and
verified by restriction digest. The vector pKOsctC was analogously constructed. Briefly,
homologous regions (1,000 bp) upstream and downstream of the target gene sctC
were amplified by PCR using the primer pairs sctC_hr1_fw/sctC_hr1_rv and sctC_hr2_fw/
sctC_hr2_rv, respectively. The resistance gene kanr was amplified with the primers
sctC_kanr_fw and sctC_kanr_rv. Assembly and cloning were performed as described
above.

Transformation of J. agaricidamnosum
Twenty milliliters of modified nutrient medium was inoculated with 1 mL of bacterial
overnight preculture to give an optical density at 600 nm (OD600) of 0.1. The culture was
incubated at 23°C with orbital shaking until an OD600 of 0.4 was reached. All subsequent
steps were performed on ice. Cells were harvested by centrifuging at 4,000 × g for
5 minutes at 4°C. The pellet was washed twice with 300 mM sucrose and dissolved
in 0.5 mL ice-cold sterile water. An aliquot of 60 µL competent cells was mixed with
20–200 ng of unmethylated pKOgspE or pKOsctC and electroporation was performed
(2 mm cuvette, 2,500 V, 25 µF, and 200 Ω). Cells were incubated in CYM at 23°C with
orbital shaking for 3 hours to recover, then spread onto modified nutrient agar plates
supplemented with 50 µg mL−1 kanamycin. Colonies were screened for mutations by PCR
of the relevant genomic regions and compared to the equivalent region amplified from
the wild type by agarose gel electrophoresis.

Button mushroom infection assay

J. agaricidamnosum, J. agaricidamnosum ΔgspE (deficient in T2SS), and J. agaricidamno­
sum ΔsctC (deficient in T3SS) were grown overnight in 20 mL modified nutrient medium
in baffled flasks at 26°C with orbital shaking. Prior to inoculation, button mushrooms
were cut into 0.5 cm thick slices, weighed, and placed in sterile petri dishes. To prevent
the slices from drying out, a wet sterilized tissue was placed in the center of the plates.
Inocula of 2.7 × 107 cells were spotted onto two different areas of each trama. Slices
inoculated with medium served as negative (untreated) controls. Plates were sealed and
mushroom slices were incubated for 6 days at room temperature, then re-weighed. The
experiments were done in quadruplicate and each experiment consisted of six biological
replicates with four mushroom slices each. The disease symptoms were quantified by
measuring the degradation of mushroom slices; specifically, the differences between the
weights of mushroom tissue at the beginning versus at the end of the experiment were
statistically analyzed using two-way analysis of variance (ANOVA) followed by Bonferro­
ni’s mean comparison test as implemented in the GraphPad Prism 5 software. Raw data
are listed in Tables S8 and S9.

Preparation of colloidal chitin

One gram of practical grade shrimp shell chitin (Sigma C9213) was added to 40 mL of
37% HCl at room temperature and stirred for 2 hours. This solution was poured into 1 L
of cold distilled water (dH2O) and washed several times until the pH was neutral. After
filtration (Miracloth), the chitin pellet was lyophilized, autoclaved, and stored at 4°C until
use.

In vivo activity assay

In order to determine if J. agaricidamnosum and J. agaricidamnosum ΔgspE have
proteolytic or chitosan-/chitin-degrading activity, the strains were individually grown
overnight in 2 mL modified nutrient medium, then centrifuged for 2 minutes at 4,000
× g. The cultures were washed and diluted in fresh nutrient medium. A loopful of J.
agaricidamnosum or J. agaricidamnosum ΔgspE was streaked onto agar plates where
half the area consisted of nutrient agar while the other was filled with either milk agar

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(milk powder 10 g L−1 and 1% agarose) or chitin agar [10 g L−1 acid-swollen chitin,
3 g L−1 (NH4)2SO4, 2 g L−1 KH2PO4, 0.3 g L−1 MgSO4 × 7 H2O, Calcofluor White MR2
(Sigma-Aldrich Chemie GmbH, Schnelldorf, Germany) 1% (vol/vol), pH 6.5]. The plates
were incubated at room temperature for 3 days.

Bioinformatic analyses
Geneious Prime 2022 was used to visualize the annotated Janthinobacterium agarici­
damnosum genome, which was manually browsed for secretion system gene clusters.
Obscure gene products encoded adjacent and within the secretion system gene clusters
were analyzed for their similarity to characterized proteins using both HHpred (30) and
NCBI BLAST (29) and re-annotated. Putative effector proteins were predicted using the
EFFECTIVE T3 prediction tool (35). Potential exoproteins encoded in the J. agaricidam­
nosum genome were manually screened using a set of computational methods for
prediction. Secretion signal peptides (SPs) and twin-arginine translocation (tat) signal
peptides were predicted with SignalP 6.0 (66). Alternative secretion was predicted with
SecretomeP 2.0 (67). Putative exoproteins were characterized using InterPro (68) and
HHpred (30).

Bioactivity-guided protein fractionation

In order to find proteins that degrade mushroom tissue, J. agaricidamnosum and J.
agaricidamnosum ΔgspE were cultivated in mushroom medium (0.5 g L−1 lyophilized,
ground brown button mushrooms, pH 6.5) at 26°C with orbital shaking overnight. The
cultures were centrifuged at 6,000 × g for 10 minutes and the supernatants were filtered
through a 0.22 µm filter. Ground (NH4)2SO4 was added stepwise to the supernatant
(20%–80%). The precipitated proteins were collected by centrifugation at 12,000 × g for
10 minutes and dissolved in 200 µL water. After dialysis, 10 µL of the resolubilized protein
fractions were spotted on two different areas of button mushroom slices and incubated
for 1 day at room temperature. Additionally, the fractions were tested in a calcofluor
chitinase assay. Heat-treated fractions were used as negative controls.
All protein fractions were further analyzed by SDS-PAGE and tryptic digestion as
previously described (69). The proteins were digested with 3 µL Trypsin/Lys-C (Promega
GmbH, Walldorf, Germany) solution (25 ng µL−1 in 10 mM NH4HCO3) and incubated at
4°C for 45 minutes. Excess solution was removed and 3 µL 10 mM NH4HCO3 were added.
The gel particles were incubated at 37°C for 18 hours. The peptides were extracted
stepwise with 50 µL of 1/49/50 trifluoroacetic acid (TFA)/H2O/ACN (vol/vol/vol), 50 µL of
1/29/70 TFA/H2O/ACN (vol/vol/vol), and 50 µL of 1/9/90 TFA/H2O/ACN (vol/vol/vol). After
each step, the gels were sonicated in the water bath for 15 minutes and the supernatants
were collected and pooled. The proteins were dried in a SpeedVac and stored at −80°C.
For MALDI-TOF-MS measurements, samples were solubilized in 4 µL of 0.05% TFA
in 50% (vol/vol) ACN, then mixed with 52 mM α-cyano-4-hydroxycinnamate (HCCA)
at a ratio of 1/1 and 2 µL of this mixture was spotted onto a MALDI plate. Samples
were analyzed using a Bruker Daltonics UltrafleXtreme MALDI-TOF mass spectrometer.
Spectra were obtained in reflector positive mode and the instrument was calibrated to a
commercially available standard (Protein Calibration Standard II, Bruker Daltonics GmbH
& Co. KG, Bremen, Germany) prior to each measurement. Data analysis was performed
with flexAnalysis 3.3 (Bruker). Mass spectra were manually searched against in silico
cleaved putative exoproteins of J. agaricidamnosum DSM 9628 using the PeptideMass
tool on the ExPASy Server (70). Up to two missed cleavages were allowed for the tryptic
digestion. Modifications were defined as methionine oxidation and cysteine carbamido­
methylation.

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Purification of secreted proteins from J. agaricidamnosum and J. agaricidam­

nosum ΔgspE for LC-MS/MS
In order to identify secreted proteins, 100 mL mushroom medium (0.5 g L−1 lyophilized,
ground brown button mushrooms, pH 6.5) was inoculated with 0.5 mL overnight cultures
of J. agaricidamnosum or J. agaricidamnosum ΔgspE and incubated overnight. Cultures
were pelleted by centrifugation at 6,000 × g for 10 minutes at 4°C and the supernatants
were filtered through a 0.22 µm filter. Subsequently, TFA was added to a final concen­
tration of 0.1% (vol/vol). Proteins were purified using a Supelco Supelclean LC-4 SPE
Cartridge (Supelco, Schnelldorf, Germany) that had been prepared by washing with
4 mL ACN and reconstituting with 4 mL 0.1% (vol/vol) TFA. The supernatants were
aspirated through the column, then the column was washed with 4 mL of 5% (vol/vol)
MeOH in 0.1% (vol/vol) TFA. The samples were eluted with 4 mL 0.1% (vol/vol) TFA
in 80% (vol/vol) ACN, then the eluates were evaporated in a vacuum centrifuge to
near-dryness and stored overnight at −80°C. Afterward, the samples were resolubilized
in 100 µL denaturation buffer (50 mM triethylammonium bicarbonate (TEAB) in 50% (vol/
vol) 2,2,2-trifluoroethanol (TFE) and subjected to ultrasonication in a water bath for 15
minutes at room temperature. The samples were reduced and alkylated simultaneously
at 70°C with orbital shaking in the dark for 30 minutes after adding 2 µL each of
500 mM tris(2-carboxyethyl)phosphine and 625 mM 2-chloroacetamide. Subsequently,
proteins were precipitated as previously described (71). The resulting protein precipitate
was resolubilized in 100 µL 100 mM TEAB in 5% (vol/vol) TFE and sonicated for 15
minutes. The total protein concentration was determined by the Merck Millipore Direct
Detect System. The protein samples were diluted with 100 mM TEAB in 5% (vol/vol)
TFE to give a final protein concentration of approximately 1 µg µL−1, then treated with
2 µg Trypsin/Lys-C protease mix per 50 µg protein sample for 18 hours at 37°C. The
reaction mixture was evaporated in a vacuum centrifuge to dryness and resolubilized
in 30 µL of 0.05% (vol/vol) TFA in 2% (vol/vol) ACN, followed by sonication in the water
bath for 15 minutes. Peptides were transferred to a 10 kDa molecular weight cut off
filter (VWR, Darmstadt, Germany) and centrifuged at 18,000 × g for 15 minutes at 4°C.
The experiments were performed with three biological replicates and the filtrates were
transferred to HPLC vials and stored at −20°C until LC-MS/MS analysis.

LC-MS/MS secretome analysis

LC-MS/MS analysis was performed using an Ultimate 3000 Nano RSLC system connected
to a Q Exactive HF mass spectrometer (both Thermo Fisher Scientific, Waltham, MA, USA).
Peptide trapping for 5 minutes on an Acclaim Pep Map 100 Column (2 cm × 75 µm, 3
µm particle size) at 5 µL minutes−1 was followed by separation on an analytical Acclaim
Pep Map RSLC Cano Column (50 cm × 75 µm, 2 µm). Eluent A was 0.1% (vol/vol) formic
acid, eluent B was 0.1% (vol/vol) formic acid in 90% (vol/vol) ACN. Mobile phase gradient
elution was performed as follows: 0–5 minutes at 4% B, 30 minutes at 7% B, 60 minutes at
10% B, 100 minutes at 15% B, 140 minutes at 25% B, 180 minutes at 45% B, 200 minutes
at 65% B, 210–215 minutes at 96% B, and 215.1–240 minutes at 4% B.
Positively charged ions were generated at a spray voltage of 2.2 kV using a stainless-
steel emitter attached to the Nanospray Flex Ion Source (Thermo Fisher Scientific). The
quadrupole/orbitrap instrument was operated in Full MS/data-dependent MS2 Top 15
mode. Precursor ions were monitored at m/z 300–1500 at a resolution of 120,000 FWHM
(full width at half maximum) using a maximum injection time (ITmax) of 140 ms and an
AGC (automatic gain control) target of 3 × 106. Precursor ions with a charge state of z =
2–5 were filtered at an isolation width of m/z 2.0 amu for further HCD fragmentation at
30% normalized collision energy (NCE). MS2 ions were scanned at 15,000 FWHM, ITmax of
140 ms and an AGC target of 2 × 105. Dynamic exclusion of precursor ions was set to 30
seconds.

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Protein database search and data analysis

Tandem mass spectra were searched against the UniProt databases of Janthinobacterium
agaricidamnosum DSM 9628 (https://www.uniprot.org/proteomes/UP000027604) and
Agaricus bisporus (https://www.uniprot.org/proteomes/UP000629468) from 22/08/11
(YYMMDD) using Thermo Scientific Proteome Discoverer (PD) 2.4 and the algorithms
of Mascot 2.8, Sequest HT, MS Amanda 2.0, and MS Fragger 3.4. Two missed cleavages
were allowed for the tryptic digestion. The precursor mass tolerance was set to 10 ppm
and the fragment mass tolerance was set to 0.02 Da. Modifications were defined as
dynamic methionine (Met) oxidation and protein N-terminal Met-loss and/or acetylation
as well as static cysteine carbamidomethylation. A strict false discovery rate (FDR) <1%
(peptide and protein level) and search engine thresholds of >30 (Mascot), >4 (Sequest
HT), >300 (MS Amanda), and >8 (MS Fragger) were required for positive protein hits.
The Percolator node and a reverse decoy database was used for q-value validation of
spectral matches. Only rank 1 proteins and peptides of the top scored proteins were
counted. Label-free protein quantification was based on the Minora algorithm of PD2.4
using a signal-to-noise ratio of >5. The following requirements needed to be met to
qualify as a significant change in protein abundance between J. agaricidamnosum and
J. agaricidamnosum ΔgspE: a fold change greater than 2 or less than −2, a P-value from
two-sided t-test divided by the absolute value of the log4 ratio [P-value/ABS(log4 ratio)]
<0.05. Furthermore, the protein should be identified in at least two of three biological
replicates of the sample group with the highest protein abundance.

ACKNOWLEDGMENTS

We thank Finn Gude for MALDI-TOF-MS measurements, Markus Greßler for valuable
discussions and comments, and Ingrid Richter for proofreading. We are grateful for
financial support by the Deutsche Forschungsgemeinschaft (DFG, German Research
Foundation), Project-ID 239748522, Sonderforschungsbereich (SFB) 1127 ChemBioSys—
239748522, and CRC/TR 124 FungiNet under project ID 210879364 (Project Z2).
C.H., K.D., and P.W. designed the research. K.D. and P.W. performed bioinformatic
analyses. P.W. performed gene disruption experiments and in vivo studies. P.W. and S.H.
performed in vitro assays and P.W. interpreted the data (genetic, biochemical, analytical).
P.W. and T.K. performed LS-MS/MS analyses. C.H., A.A.B., and D.H. supervised experimen­
tal work. P.W., E.M.M., and C.H. wrote the manuscript with contributions from all authors.
The authors declare no conflict of interest.

AUTHOR AFFILIATIONS
1
Department of Biomolecular Chemistry, Leibniz Institute for Natural Product Research
and Infection Biology (HKI), Jena, Germany
2
Department of Pharmaceutical Microbiology, Leibniz Institute for Natural Product
Research and Infection Biology, Friedrich Schiller University Jena, Jena, Germany
3
Faculty of Biological Sciences, Friedrich Schiller University Jena, Jena, Germany
4
Department of Molecular and Applied Microbiology, Leibniz Institute for Natural
Product Research and Infection Biology (HKI), Jena, Germany

AUTHOR ORCIDs

Philipp Wein http://orcid.org/0000-0002-4340-9899

Thomas Krüger http://orcid.org/0000-0001-8984-3853
Evelyn M. Molloy http://orcid.org/0000-0001-8822-7207
Axel A. Brakhage http://orcid.org/0000-0002-8814-4193
Dirk Hoffmeister http://orcid.org/0000-0002-5302-6461
Christian Hertweck http://orcid.org/0000-0002-0367-337X

Month XXXX Volume 0 Issue 0 10.1128/mbio.00787-23 14

Research Article mBio

FUNDING

Funder Grant(s) Author(s)

Deutsche Forschungsgemeinschaft (DFG) 239748522 Christian Hertweck
Deutsche Forschungsgemeinschaft (DFG) 210879364 Axel A Brakhage

AUTHOR CONTRIBUTIONS

Philipp Wein, Conceptualization, Formal analysis, Investigation, Writing – original draft,

Writing – review and editing | Katharina Dornblut, Conceptualization, Formal analysis,
Investigation | Sebastian Herkersdorf, Formal analysis, Investigation | Thomas Krüger,
Formal analysis, Investigation, Data curation | Evelyn M. Molloy, Writing – original draft,
Writing – review and editing | Axel A. Brakhage, Supervision | Dirk Hoffmeister, Writing –
review and editing, Supervision | Christian Hertweck, Conceptualization, Writing – review
and editing, Supervision, Funding acquisition, Project administration

DATA AVAILABILITY

The mass spectrometry proteomics data have been deposited to the ProteomeXchange
Consortium via the PRIDE (72) partner repository with the data set identifier PXD040852.

ADDITIONAL FILES

The following material is available online.

Supplemental Material

Supporting Information (mBio00787-23-S0001.pdf). Supplemental figures, tables, and

procedures.

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