Role of membrane compartment occupied by Can1 (MCC) and eisosome subdomains in plant pathogenicity of the necrotrophic fungus Alternaria brassicicola

Abstract

Background: MCC/eisosomes are membrane microdomains that have been proposed to participate in the plasma membrane function in particular by regulating the homeostasis of lipids, promoting the recruitment of specific proteins and acting as provider of membrane reservoirs.

Results: Here we showed that several potential MCC/eisosomal protein encoding genes in the necrotrophic fungus A. brassicicola were overexpressed when germinated spores were exposed to antimicrobial defence compounds, osmotic and hydric stresses, which are major constraints encountered by the fungus during the plant colonization process. Mutants deficient for key MCC/eisosome components did not exhibit any enhanced susceptibility to phytoalexins and to applied stress conditions compared to the reference strain, except for a slight hypersensitivity of the ∆∆abpil1a-abpil1b strain to 2 M sorbitol. Depending on the considered mutants, we showed that the leaf and silique colonization processes were impaired by comparison to the wild-type, and assumed that these defects in aggressiveness were probably caused by a reduced appressorium formation rate.

Conclusions: This is the first study on the role of MCC/eisosomes in the pathogenic process of a plant pathogenic fungus. A link between these membrane domains and the fungus ability to form functional penetration structures was shown, providing new potential directions for plant disease control strategies.

Keywords: Appressoria; Eisosome; Fungi; Plant pathogen; Plasma membrane; Seed.

Fig. 1

Bioinformatic features of PIL1A and PIL1B homologues in A. brassicicola. a Conserved domains predicted from the respective sequences of AbPIL1A and AbPIL1B using SMART software at http://smart.emblheidelberg.de/. b Phylogenetic tree for AbPIL1 and LSP1 homologs found in B. bassiana (Bba), Aspergillus species (Ate: A. terreus; Ano: A. nomius; and Ani: A. nidulans and yeasts (Cal: Candida albicans; Cor: C. orthopsilosis; and Sce: S. cerevisiae). The phylogenetic analysis was based on the neighbor-joining method in MEGA7 software at http://www.megasoftware.net/. Bootstrap values of 1000 replications are given at nodes

Fig. 2

Localizations in A. brassicicola germlings of AbPIL1A, AbSUR7 or AbNCE102 fused to GFP fusion proteins. Captures before and after a 90-min period of exposure to 1% DMSO are shown (scale bars = 50 μm)

Fig. 3

Ultrastructure of A. brassicicola conidial cells. Transmission electron micrographs were performed from 7-day-old wild-type (WT) and MCC/eisosome mutant conidia (scale bars = 2 μm). White arrows indicate autophagosomes and black arrows indicate abnormal tubular structures with cell wall material in the middle of the cell

Fig. 4

Disease symptoms of A. brassicicola on B. oleracea leaves. a Mean lesion diameter for all inoculation sites measured at 5 dpi. Values are means ± SEM for at least five replicate experiments. Stars indicate a significant difference between the wild-type and the mutant strains aggressiveness using the paired Wilcoxon test (P < 0.05) .b Representative symptoms obtained by inoculation of wild-type (WT) and respective MCC/eisosome mutants at 5 dpi. Leaves were inoculated with conidia suspensions of WT (right part of the central vein) and MCC/eisosome mutants (left part of the central vein) without artificial lesions

Fig. 5

Effects of targeted gene knockout in appressorium-like structure differentiation. a Percentages of germ tubes differentiating swollen tips. Values represent appressorium appearance probabilities with 95% confidence interval. b Microscopic observations of the infection structures on B. oleracea leaf surfaces. Leaf fragments inoculated with A. brassicicola wild-type strain or MCC/eisosome mutants were collected at 24 h dpi and directly imaged with an environmental scanning electron microscope in their natural state without modification or preparation. Appressoria-like structures are indicated by arrows. Scale bars = 10 μm

Fig. 6

Transmission capacity of A. brassicicola wild-type (WT) and MCC/eisosome mutants to Arabidopsis thaliana seeds (Ler ecotype). The seed transmission capacity according to the silique stage and global seed transmission capacity (strain model) were measured as described by [37]. The five oldest siliques of at least five plants were inoculated with each fungal genotype and the experiment was repeated . 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 A. 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