Two genes in a gene cluster encoding secreted proteins are required for appressorial penetration of the maize anthracnose fungus Colletotrichum graminicola

The contribution to virulence of 58 effector candidates of the hemibiotrophic maize pathogen Colletotrichum graminicola were analysed by gene deletion. In search of gene clusters encoding secreted proteins we identified a cluster of co-linear genes (CLU5a to CLU5e) in the genome of C. graminicola that is needed for pathogenicity. Analyses of the individual genes in that cluster showed that CLU5a deletion mutants are severely and CLU5e mutants are moderately impaired in virulence. Highly resolved TEM images and quantitative assessment of development of infection structures by LM showed that both mutants were impaired in appressorial penetration. Virulence deficiencies exist in the mutants independently from the host genotype and host organ inoculated. Papillae formed by CLU5a mutants are smaller and less structured than those elicited by the wild type, suggesting reduced PAMP exposure during arrested infection-related development. In contrast, CLU5e mutants elicited WT-like papillae, though at increased frequencies. Vegetative growth, sporulation and assays testing several types of stress conditions failed to exhibit significant differences between the mutants and the wild type reference highlighting the importance of the two proteins in the establishment of biotrophy. Possibly, defects of the appressorial cell wall may cause the phenotypes observed. Interestingly, genes in cluster 5 are highly syntenic only in the genomes of Colletotrichum spp. infecting grasses but not in Colletotrichum spp. infecting dicotyledonous plants.