Structural Phylogenetics Identifies Conserved Effector Families and Cell Death-Inducing Proteins from Cercospora...

Cercospora zeina causes gray leaf spot disease on maize in Africa. As a foliar fungal pathogen, C. zeina relies on effectors to facilitate successful infection and colonization of its host. Predicting effector function is therefore essential for understanding the molecular mechanisms underlying this plant-pathogen interaction. Although effector functions are typically predicted using sequence-based analyses, many effectors are subject to strong selection pressure and exhibit high sequence diversity, reducing the accuracy of such predictions. In this study, a structural phylogenetics approach was employed to identify effector structural families in C. zeina and to assign putative functions to C. zeina candidate secreted effector proteins based on their structural clustering with functionally characterized effectors from related phytopathogens. Twenty-two effector structural families were identified, several of which contained effectors shown to induce cell death in Nicotiana benthamiana. It was therefore hypothesized that C. zeina effectors belonging to these structural families would exhibit similar functions. Three families containing cell death-inducing effectors ZtCDI1, MgNLP, VdPEVD1with high structural similarity and low sequence identity to C. zeina effectors were selected for biological validation. Transient expression of C. zeina effectors from these families induced cell death in N. benthamiana. These C. zeina structural analogs were expressed in either the latent (CDI1-like) or necrotrophic (NLP-like, PevD1-like) phase of GLS disease in maize. This work shows that structural phylogenetics can be employed to predict effector function and identify structurally related, cell death-inducing effectors with divergent amino acid sequences in C. zeina.