Zymoseptoria tritici Effectors Structurally Related to Killer Proteins UmV-KP4 and UmV-KP6 Inhibit Fungal Growth, and Define Extended Protein Families in Fungi.

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.