Responses to Hydric Stress in the Seed-Borne Necrotrophic Fungus Alternaria brassicicola

Abstract

Alternaria brassicicola is a necrotrophic fungus causing black spot disease and is an economically important seed-borne pathogen of cultivated brassicas. Seed transmission is a crucial component of its parasitic cycle as it promotes long-term survival and dispersal. Recent studies, conducted with the Arabidopsis thaliana/A. brassicicola pathosystem, showed that the level of susceptibility of the fungus to water stress strongly influenced its seed transmission ability. In this study, we gained further insights into the mechanisms involved in the seed infection process by analyzing the transcriptomic and metabolomic responses of germinated spores of A. brassicicola exposed to water stress. Then, the repertoire of putative hydrophilins, a group of proteins that are assumed to be involved in cellular dehydration tolerance, was established in A. brassicicola based on the expression data and additional structural and biochemical criteria. Phenotyping of single deletion mutants deficient for fungal hydrophilin-like proteins showed that they were affected in their transmission to A. thaliana seeds, although their aggressiveness on host vegetative tissues remained intact.

Keywords: Alternaria brassicicola; dehydration; hydrophilins; plant pathogenic fungus; seed transmission.

FIGURE 1

Global modulation of gene expression in Alternaria brassicicola in response to applied treatments. Sorbitol and desiccation treatments were compared to non-treated cultures at two time points 0.5 h, 2 h and 1 h, 4 h, respectively. Genes with a P-values ≤0.05 and a log2 ratio ≥0.7 or ≤–0.7 were considered as differentially expressed. Intersection with less than 10 genes were omitted. Colors displayed regulation compared to the control; red = up-regulated in all conditions, green = down-regulated in all conditions, orange = opposite regulation according to the considered condition.

FIGURE 2

Relevant Gene Ontology Biological Process (GO-BP) enrichments obtained from modulated gene sets after exposure to water stresses.

FIGURE 3

Impact of sorbitol and desiccation treatments on amino acid (A), saccharides and sugar alcohols (B) contents of fungal cultures. The heat map background displays the mean control contents. Internal colored boxes display the mean difference between treatments and controls. Quantifications were performed from three separate samples. Asterisks indicate a significant difference between the mutant and the parental isolate (Welch test, ^∗p ≤ 0.05).

FIGURE 4

Gene expression of Alternaria brassicicola hydrophilins in response to sorbitol and desiccation treatments. Sorbitol and desiccation treatments were compared to non-treated cultures at two time points 0.5 h, 2 h and 1 h, 4 h, respectively. (A) For each gene, numerical data were transformed into color-grid representations in which the fold gene expression induction (log2 values) is represented by a colored scale. In addition, hydrophilin gene expression were evaluated in mutant strains Δabnik1, Δabhog1 and Δabsch9 exposed to 1.2 M sorbitol for 0.5 h. The left part of the panel (A) highlights the possible regulation of hydrophilin gene expression by one or several of these protein kinases. (B) Volcano plot showing fold inductions of genes in the wild-type strain exposed to 1.2 M sorbitol for 0.5 h. The hydrophilin encoding genes are labeled and identified with red dots. The threshold values (P-values ≤0.05 and a log2 ratio ≥0.7 or ≤–0.7) are indicated by dash lines.

FIGURE 5

Growth inhibition rates of the wild-type strain (WT) and hydrophilin deficient mutants in the presence of H₂O₂ (10 mM) and menadione (20 mM). The results are expressed as the percentage of inhibition in treated samples compared to the control without additive. Conidia were used to inoculate microplate wells containing standard PDB medium that was supplemented with the appropriate test substance. Growth was automatically recorded for 30 h at 25°C using a nephelometric reader (see section “Materials and Methods”). Each genotype was analyzed in triplicate and the experiments were repeated at least three times per growth condition. Error bars indicate standard deviations. Asterisks indicate a significant difference between the mutant and the parental isolate (Dunn control test, ^∗p ≤ 0.01).

FIGURE 6

Complementation of yeast Δhsp12 mutants with AbSih3 coding sequences. (A) Growth curves of Saccharomyces cerevisiae (parental WT strain and Δhsp12 mutants) cells transformed with empty vector (pYES2CT) or recombinant vectors (pYAbsih3) exposed to 1 mM H₂O₂ or to water. (B) Comparisons of growth inhibition rates obtained for each yeast strain (Wilcoxon test, ^∗p < 0.001).

FIGURE 7

Representative symptoms at 5 dpi obtained by inoculation of wildtype, Δabsch9, Δabsih3, and Δabsih15 on cabbage leaves. Leaves were inoculated with 5 μL drops of conidia suspension (10⁵, 10⁴ or 10³ conidia/mL in water). As shown on the right side, mutants were inoculated on the right part of the central vein and compared on the same leaf with the parental strain (inoculated on the left part of the central vein). Bars = 2 cm.

FIGURE 8

Transmission capacity of Alternaria brassicicola wild-type (WT) and null strains to Arabidopsis thaliana seeds (Ler ecotype). The seed transmission capacity were measured as described by Pochon et al. (2012). The five youngest siliques of at least five plants were inoculated with each fungal genotype and the experiment was repeated twice. Contaminated siliques were harvested 10 dpi. After dissection, seeds were incubated separately on PDA medium for 2 days. A seed was considered contaminated when incubation resulted in typical Alternaria brassicicola colony development. For each inoculated fungal genotype, the seed infection probability was evaluated from at least 1000 seeds. Values represent infection probabilities with 95% confidence interval.