Abstract
Abstract Fungal effectors play crucial roles in plant infection. Despite low sequence identity, their classification into families is feasible using structural homology. In this study, we have elucidated the structures of the two effectors Mycgr3-91409 and Zt-NIP1 from the wheat fungal pathogen Zymoseptoria tritici using X-ray crystallography and NMR. These proteins shared structural homology with, respectively, KP6α and KP4 killer toxins, encoded by UmV dsRNA viruses infecting the corn fungal pathogen Ustilago maydis . Consequently, Zt-Mycgr3-91409 and Zt-NIP1 were renamed Zt-KP6-1 and Zt- KP4-1. Orthologs and paralogs of Zt-KP6-1 and Zt-KP4-1 were identified in Zymoseptoria species, but not in other fungi, with the exception of ECP2 effectors related to Zt-KP4-1. A novel pipeline relying on Foldseek and cysteine-pattern constrained HMM searches identified a large number of proteins structurally related to Zt-KP6-1 and Zt-KP4-1 in fungi, and to Zt-KP4-1 in plants. These proteins were classified into distinct structural super families (4 KP4 and 3 KP6) including effectors not known as structurally related to KP4 (ECP2) or KP6 (BAS4). The biological activities of Zt-KP6-1 and Zt-KP4-1 were assessed on plants and fungi. They did not induce symptoms on wheat leaves. However, they inhibited the growth of Botrytis cinerea and Z. tritici , demonstrating their toxicity to fungi. These results highlight the importance of structure determination of effectors for their classification and the prediction of their function.