Résumé
Fungal effectors play crucial roles in plant infection. Despite low sequence identity, they were recently discovered to belong to families with similar three-dimensional structures. In this study, we elucidated the structures of Zt-NIP1 and Mycgr3-91409-2 effectors of the wheat fungal pathogen Zymoseptoria tritici using X-ray crystallography and NMR. These effectors displayed a structural homology with, respectively, KP4 and KP6α killer toxins from UmV dsRNA viruses of the maize fungal pathogen Ustilago maydis. Consequently, Zt-NIP1 and Mycgr3-91409-2 were renamed Zt-KP4-1 and Zt-KP6-1. Orthologues and paralogues of Zt-KP4-1 and Zt-KP6-1 were identified in Zymoseptoria, but not in other fungi, except ECP2 effectors related to Zt-KP4-1. Assessment of the biological activities of Zt-KP6-1 and Zt-KP4-1 revealed their ability to inhibit fungal growth, but they were unable to induce wheat leaf necrosis. A novel pipeline relying on cysteine-pattern constrained HMM searches and Foldseek analysis of AlphaFold2 predicted structures from Uniprot generated a comprehensive inventory of KP4 and KP6 proteins in fungi and plants. Their structure-based classification revealed four KP4 and three KP6 structural superfamilies and provided far-reaching hypotheses on their biological function and evolution. This framework highlights the power of structure determination and modelling for the classification of effectors and their functional investigation.