Epigenetic Control of Effector Gene Expression in the Plant Pathogenic Fungus Leptosphaeria maculans

Plant pathogens secrete an arsenal of small secreted proteins (SSPs) acting as effectors that modulate host immunity to facilitate infection. SSP-encoding genes are often located in particular genomic environments and show waves of concerted expression at diverse stages of plant infection. To date, little is known about the regulation of their expression. The genome of the Ascomycete Leptosphaeria maculans comprises alternating gene-rich GC-isochores and gene-poor AT-isochores. The AT-isochores harbor mosaics of transposable elements, encompassing one-third of the genome, and are enriched in putative effector genes that present similar expression patterns, namely no expression or low-level expression during axenic cultures compared to strong induction of expression during primary infection of oilseed rape (Brassica napus). Here, we investigated the involvement of one specific histone modification, histone H3 lysine 9 methylation (H3K9me3), in epigenetic regulation of concerted effector gene expression in L. maculans. For this purpose, we silenced the expression of two key players in heterochromatin assembly and maintenance, HP1 and DIM-5 by RNAi. By using HP1-GFP as a heterochromatin marker, we observed that almost no chromatin condensation is visible in strains in which LmDIM5 was silenced by RNAi. By whole genome oligoarrays we observed overexpression of 369 or 390 genes, respectively, in the silenced-LmHP1 and -LmDIM5 transformants during growth in axenic culture, clearly favouring expression of SSP-encoding genes within AT-isochores. The ectopic integration of four effector genes in GC-isochores led to their overexpression during growth in axenic culture. These data strongly suggest that epigenetic control, mediated by HP1 and DIM-5, represses the expression of at least part of the effector genes located in AT-isochores during growth in axenic culture. Our hypothesis is that changes of lifestyle and a switch toward pathogenesis lift chromatin-mediated repression, allowing a rapid response to new environmental conditions

L'expression des gènes codant des effecteurs nécessite une décondensation de la chromatine chez Leptosphaeria maculans, champignon pathogène du colza

absen

Analyse de l'interaction entre les membres d'un complexe d'espèces pathogènes du colza (Leptosphaeria maculans et L.biglobosa) par une approche transcriptomique

International audienc

The soil microbiota diversity influences clubroot disease by modulation of Brassica napus and Plasmodiophora brassicae transcriptomes

International audienc

Influence of soil microbial diversity and host plant genotype on the structure and diversity of root microbiota of Brassica napus infected with Plasmodiophora brassicae

International audienc

The soil microbiota diversity influences clubroot disease by modulation of host plant and pathogen transcriptomes

International audienc

Different waves of effector genes with contrasted genomic location are expressed by Leptosphaeria maculans during cotyledon and stem colonization of oilseed rape

Leptosphaeria maculans, the causal agent of stem canker disease, colonizes oilseed rape (Brassica napus) in two stages: a short and early colonization stage corresponding to cotyledon or leaf colonization, and a late colonization stage during which the fungus colonizes systemically and symptomlessly the plant during several months before stem canker appears. To date, the determinants of the late colonization stage are poorly understood; L. maculans may either successfully escape plant defences, leading to stem canker development, or the plant may develop an adult-stage' resistance reducing canker incidence. To obtain an insight into these determinants, we performed an RNA-sequencing (RNA-seq) pilot project comparing fungal gene expression in infected cotyledons and in symptomless or necrotic stems. Despite the low fraction of fungal material in infected stems, sufficient fungal transcripts were detected and a large number of fungal genes were expressed, thus validating the feasibility of the approach. Our analysis showed that all avirulence genes previously identified are under-expressed during stem colonization compared with cotyledon colonization. A validation RNA-seq experiment was then performed to investigate the expression of candidate effector genes during systemic colonization. Three hundred and seven late' effector candidates, under-expressed in the early colonization stage and over-expressed in the infected stems, were identified. Finally, our analysis revealed a link between the regulation of expression of effectors and their genomic location: the late' effector candidates, putatively involved in systemic colonization, are located in gene-rich genomic regions, whereas the early' effector genes, over-expressed in the early colonization stage, are located in gene-poor regions of the genome

Identification et analyse fonctionnelle des effecteurs tardifs impliqués dans la colonisation systémique du colza par Leptosphaeria maculans

National audienc

Influence de la diversité du microbiome du sol sur l'agent pathogène tellurique responsable de la hernie des crucifères

International audienc

Leptosphaeria maculans effectors involved in the oilseed rape systemic colonization.

The stem canker disease, caused by Leptosphaeria maculans, is one of the most devastating diseases of oilseed rape (canola). It colonizes the plant in two stages: a short and early colonisation stage corresponding to cotyledon or leaf colonisation, and a late colonisation stage during which the fungus colonises systemically and symptomlessly the plant during several months before stem canker appears. To date, determinants of the late colonisation stage remain poorly understood. By a transcriptomic approach, we previously identified two waves of effector candidate expression during the early and late colonisation stages (Gervais et al, 2016). The late effector candidates are located in gene-rich genomic regions, whereas the early effector genes are located in gene-poor regions of the genome. Among the late effector candidates identified, we selected 6 genes for further characterization. We created mutants silenced for these effector candidates. For one of these genes, its expression level correlated negatively with the size of the necrosis observed in the stem. The identification of new effector genes would contribute to the identification of new resistance genes specific to these effectors. To easily identify matching resistance genes in oilseed rape, we created transgenic isolates expressing these 6 late effectors at the early steps of infection to provide medium-throughput strategies to screen more efficiently different cultivars. Preliminary results indicate that some cultivars with adult resistance were more resistant to these transgenic isolates in cotyledon assays. With this approach, we also identified a cultivar carrying a specific resistance to one these 6 effector candidates. Reference Gervais, J., Plissonneau, C., Linglin, J., Meyer, M., Labadie, K., Cruaud, C., Fudal, I., Rouxel, T. and Balesdent, M.H. (2016) Different waves of effector genes with contrasted genomic location are expressed by Leptosphaeria maculans during cotyledon and stem colonization of oilseed rape. Mol. Plant Pathol