Bacterial secretion systems contribute to rapid tissue decay in button mushroom soft rot disease

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 contribution 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 secretion 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 progression.


Figures S1 to S16
Tables Table S1.Secretion associated genes and proposed function of encoded proteins Table S2.Potentially relevant T3 effector proteins encoded within the T3SS gene cluster Table S3.Proteins identified by bioactivity-guided fractionation Table S4.First ten results returned in a HHpred search of the RICIN domain-containing protein (CDG82784) Table S5.Strains used in this study Table S6.Oligonucleotide primers used in this study Table S7.Plasmids used in this study Table S8.Quantification of mushroom (A.bisporus) degradation caused by J. agaricidamnosum strains Table S9.Statistical analysis of mushroom degradation caused by J. agaricidamnosum strains References Fig. S1 Genetic organization of type III secretion systems (T3SSs) from Janthinobacterium species.In order to reveal the distribution of T3SS gene clusters within the genus Janthinobacterium, the proteins encoded by sctC (GJA_RS07790) and sctT (GJA_RS07915) from J. agaricidamnosum DSM 9628 were used in a protein BLAST search against the Janthinobacterium (taxid:29580) database.T3SS gene clusters were found in the genome of Janthinobacterium sp.B9-8 and Janthinobacterium sp.BJB412.In some cases, genes were reannotated/annotated with HHpred (6) and NCBI BLAST (7).Putative T3 effectors were predicted using the EFFECTIVE T3 software (8).Genes of T3SS core components are represented by arrows with letter designations.The predicted functions of other gene products are indicated in the key.Gene accession numbers are given for sctC and sctT genes of each cluster.Fig. S2 Evolutionary relationship between alanine-tryptophan-arginine triad (AWR) effectors.The result of the phylogenetic analysis displays the AWR effector protein from the button mushroom pathogen J. agaricidamnosum (purple) and previously described AWR effectors from Burkholderia spp., and plant pathogenic R. solanacearum and Xanthomonas spp.(9).The putative AWR effector from J. agaricidamnosum belongs neither to the AWR family (AWR 1-5) of R. solanacearum nor to the other known AWR members.The analysis was conducted in MEGA7 (2) using the Neighbor-Joining algorithm (3).NCBI accession numbers are displayed in brackets.Fig. S3 Multiple sequence alignment of predicted AWR effector proteins.The alignment depicted is an updated version of the alignment from Sole et al. (9) and comprises AWR proteins from the button mushroom pathogen J. agaricidamnosum, plant pathogenic R. solanacearum strains, plant pathogenic Xanthomonas spp., and Burkholderia spp.Bar charts above the alignment display the overall sequence identity from low (red) to high (green).NCBI accession numbers are shown in brackets.a Type III secretion signals were predicted using the Effective T3 prediction tool (8).
b Percent identities of similar protein sequences were obtained using NCBI BLAST (7). a Peptides were manually searched against proteins identified by LC-MS/MS secretome analysis and in silico cleaved putative exoproteins of J. agaricidamnosum DSM9628 using the PeptideMass tool on the ExPASy Server (10).
b Secretion signal-containing peptide (SP).SignalP 6.0 was used to predict whether the peptides would be secreted.

Tables
Figures Fig. S1 Genetic organization of type III secretion systems from Janthinobacterium species Fig. S2 Evolutionary relationship between alanine-tryptophan-arginine triad (AWR) effectors Fig. S3 Multiple sequence alignment of predicted AWR effector proteins Fig. S4 Sequence alignment of conserved regions in AWR proteins Fig. S5 Gene expression analyses of T2SS and T3SS related genes Fig. S6 Verification of the mutant gspE Fig. S7 Verification of the mutant sctC Fig. S8 Growth kinetics of J. agaricidamnosum wild type, gspE and sctC Fig. S9 Swarming assay of J. agaricidamnosum wild type and deletion mutants Fig. S10 Bioactivity-guided protein fractionation Fig. S11 SDS-PAGE analysis of protein fractions Fig. S12 MALDI-TOF-MS and domain analysis of protein 1 Fig.S13 MALDI-TOF-MS and domain analysis of protein 2 Fig. S14 MALDI-TOF-MS analysis of protein 3 Fig.S15 MALDI-TOF-MS analysis of protein 4 Fig.S16 MALDI-TOF-MS and domain analysis of protein 5 Fig. S4 Sequence alignment of conserved regions in AWR proteins.Shown are the distinctive alanine-tryptophan-arginine triads from the putative T3 AWR effector of J. agaricidamnosum and AWR effectors from Burkholderia spp., plant pathogenic R. solanacearum and Xanthomonas spp.The AWR effector of J. agaricidamnosum contains an AWR triad at position 321-323 and an aspartate-tryptophan-arginine triad (DWR) at position 454-456.The bar chart above the alignment indicates the overall sequence identity from low (red) to high (green).NCBI accession numbers are displayed in brackets.

Table S1 .
(8)7)tion associated genes and proposed function of encoded proteins 249 E-values of the proposed proteins were obtained using HHPred or NCBI BLAST(6,7).bTypeIII secretion signals were predicted using the Effective T3 prediction tool(8). a

Table S2 .
Potentially relevant T3 effector proteins encoded within the T3SS gene cluster

Table S3 .
Proteins identified by bioactivity-guided fractionation and MALDI-TOF-MS a

Table S5 .
Strains used in this study 267

Table S6 .
Oligonucleotide primers used in this study 269

Table S7 .
Plasmids used in this study 271

Table S8 .
Quantification of mushroom (A.bisporus) degradation caused by J. agaricidamnosum strains.The listed data derived from four independent experiments with six replicates each.Each replicate consisted of four sliced and weighed mushrooms (approx.0.5 cm thickness) that were treated with J. agaricidamnosum (wild type), J. agaricidamnosum gspE, or J. agaricidamnosum sctC.Slices were spotted with culture medium to give negative (untreated) controls.After inoculation the slices were incubated at room temperature for 6 days, then the mushroom tissue was re-weighed.The mushroom degradation is calculated in percent as follows: % weight loss = 100 − (final weight / starting weight × 100).Expt., Experiment.

Table S9 .
Statistical analysis of mushroom degradation caused by J. agaricidamnosum strains.(A) Two-way analysis of variance (ANOVA) and (B) Bonferroni's means comparison test for mushroom degradation induced by J. agaricidamnosum (wild type), J. agaricidamnosum gspE, or J. agaricidamnosum sctC.Uninoculated slices spotted with culture medium served as negative controls.P-values < 0.05 were considered statistically significant.DF, Degrees of freedom; Expt., Experiment; Mean Diff., Mean difference; MS, Mean square; SS, Sum of squares.