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.

Keywords: Janthinobacterium agaricidamnosum; lytic enzymes; mushroom pathogens; secretome analysis; type III secretion.

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 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.

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]).

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 protein-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 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.

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.