Evolutionary History and Diversification of M35 Metalloproteases in Dothideomycetes: A Phylogenomic Overview and Case Study in Corynespora cassiicola

Deuterolysin metalloproteases (M35s), a family of zinc-dependent proteolytic enzymes, function as key virulence factors in bacteria and fungi. In plant-pathogenic fungi, these proteins induce host cell death and suppress plant chitinases, thereby protecting fungal cell walls from degradation and preventing the release of chitin oligomers that trigger plant immune responses. Here, we investigated the evolutionary history of the M35 gene family across Dothideomycetes fungi using genomic data from 79 Dothideomycetes species and 61 Corynespora cassiicola isolates. Predicted M35 proteins were classified as putative effectors or putative non-effectors based on bioinformatic criteria, with putative effectors defined as secreted proteins capable of manipulating host immunity. Across Dothideomycetes, we identified 146 M35 genes, of which 107 were putative effectors and 39 putative non-effectors. Family size varied widely (0-7 genes per genome), with the largest repertoires observed in the plant-pathogenic species Botryosphaeria dothidea and Diplodia seriata. In C. cassiicola, most isolates harbored three M35 genes per genome. Phylogenetic analysis revealed four distinct sub-clades within the C. cassiicola M35 family. Sub-clades Cc_M35_1.1 and Cc_M35_2.1 showed evidence of gene duplication followed by retention across most isolates, whereas Cc_M35_1.2 and Cc_M35_2.2 exhibited patterns consistent with gene loss. Notably, putative effector M35s in sub-clade Cc_M35_2.2 were exclusively recovered from isolates associated with cotton, suggesting a specialized role in C. cassiicola-cotton interactions. During soybean infection, all three M35 genes in C. cassiicola were expressed, but CC_29_g9669 (Cc_M35_1.1) displayed the highest relative expression, peaking at 8 days post-inoculation.