Gene duplication drove functional divergence of two effectors in the maize anthracnose pathogen

Colletotrichum species rank among the most important fungal pathogens, threatening food security by infecting nearly all major crops worldwide. Colletotrichum graminicola, the causal agent of maize anthracnose, secretes effector proteins to manipulate host defences and promote colonization. Previously, we characterized the nuclear effector CgEP1 in C. graminicola and showed that it localizes to the host nucleus. Here, we conducted a phylogenomic analysis of CgEP1 and its homologs and identified its paralog, CgEP4, in C. graminicola. The phylogenomic analysis revealed that they originated from a gene duplication event approximately 28 to 18 million years ago, predating the diversification of the Graminicola species complex. This timing coincides with the ecological expansion of C₄ grasses, suggesting that the functional divergence of these effectors was an adaptive response to facilitate the colonization of emerging monocot hosts. Functional characterization using gene deletion mutants demonstrated that CgEP4 has a critical role in pathogenicity, characterized by a significant reduction in virulence, delayed penetration, enhanced papilla formation, and decreased fungal biomass. This virulence defect is associated with compromised host colonization and a failure to suppress basal host defences. In the absence of CgEP4, the pathogen also showed defects in general fungal physiology and stress tolerance. Overall, our findings establish CgEP4 as a new, essential nuclear-localized effector that promotes fungal entry and colonization by manipulating host responses. Our findings demonstrate that evolutionary analysis is a valuable tool for discovering new genes important for host adaptation and pathogen evolution.