Magnaporthe AVRs and ToxB-like (MAX) effectors constitute a family of secreted virulence proteins in the fungus Pyricularia oryzae (syn. Magnaporthe oryzae), which causes blast disease on numerous cereals and grasses. In spite of high sequence divergence, MAX effectors share a common fold characterized by a ß-sandwich core stabilized by a conserved disulfide bond. In this study, we investigated the structural landscape and diversity within the MAX effector repertoire of P. oryzae. Combining experimental protein structure determination and in silico structure modeling we validated the presence of the conserved MAX effector core domain in 77 out of 94 groups of orthologs (OG) identified in a previous population genomic study. Four novel MAX effector structures determined by NMR were in remarkably good agreement with AlphaFold2 (AF2) predictions. Based on the comparison of the AF2-generated 3D models we propose a classification of the MAX effectors superfamily in 20 structural groups that vary in the canonical MAX fold, disulfide bond patterns, and additional secondary structures in N- and C-terminal extensions. About one-third of the MAX family members remain singletons, without strong structural relationship to other MAX effectors. Analysis of the surface properties of the AF2 MAX models also highlights the high variability within the MAX family at the structural level, potentially reflecting the wide diversity of their virulence functions and host targets.

Summary: Phytopathogenic fungi cause enormous yield losses in many crops, threatening both agricultural production and global food security. To infect plants, they secrete effectors targeting various cellular processes in the host. Putative effector genes are numerous in fungal genomes, and they generally encode proteins with no sequence homology to each other or to other known proteins or domains. Recent studies have elucidated and predicted three‐dimensional structures of effectors from a wide diversity of plant pathogenic fungi, revealing a limited number of conserved folds. Effectors with very diverse amino acid sequences can thereby be grouped into families based on structural homology. Some structural families are conserved in many different fungi, and some are expanded in specific fungal taxa. Here, we describe the features of these structural families and discuss recent advances in predicting new structural families. We highlight the contribution of structural analyses to deepen our understanding of the function and evolution of fungal effectors. We also discuss prospects offered by advances in structural modeling for predicting and studying the virulence targets of fungal effectors in plants.

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.

Does a similar 3D structure mean a similar folding pathway? This question is particularly meaningful when it concerns proteins sharing a similar 3D structure, but low sequence identity or homology. MAX effectors secreted by the phytopathogenic fungus Magnaporthe oryzae present such characteristics. They share a common 3D structure, a ß-sandwich with the same topology for all the family members, but an extremely low sequence identity/homology. In a previous study, we have investigated the folding of two MAX effectors, AVR-Pia and AVR-Pib, using High-Hydrostatic-Pressure NMR and found that they display a similar folding pathway, with a common folding intermediate. In the present work, we used a similar strategy to investigate the folding conformational landscape of another MAX effector, MAX60, and found a very different folding intermediate. Our analysis strongly supports that the presence of a C-terminal $\mathsf{\alpha }$-helical extension in the 3D structure of MAX60 could be responsible for its different folding pathway.

Despite advances in experimental and computational methods, the mechanisms by which an unstructured polypeptide chain regains its unique three-dimensional structure remains one of the main puzzling questions in biology. Single-molecule techniques, ultra-fast perturbation and detection approaches and improvement in all-atom and coarse-grained simulation methods have greatly deepened our understanding of protein folding and the effects of environmental factors on folding landscape. However, a major challenge remains the detailed characterization of the protein folding landscape. Here, we used high hydrostatic pressure 2D NMR spectroscopy to obtain high-resolution experimental structural information in a site-specific manner across the polypeptide sequence and along the folding reaction coordinate. We used this residue-specific information to constrain Cyana3 calculations, in order to obtain a topological description of the entire folding landscape. This approach was used to describe the conformers populating the folding landscape of two small globular proteins, AVR-Pia and AVR-Pib, that belong to the structurally conserved but sequence-unrelated MAX effectors superfamily. Comparing the two folding landscapes, we found that, in spite of their divergent sequences, the folding pathway of these two proteins involves a similar, inescapable, folding intermediate, even if, statistically, the routes used are different.

Fungal effectors play crucial roles in plant infection. Despite low sequence identity, effectors were recently classified into structural families. In this study, we have elucidated the structures of Zt-NIP1 and Mycgr3-91409 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 infecting the corn fungal pathogen Ustilago maydis . Consequently, Zt-NIP1 and Mycgr3-91409 were renamed Zt-KP4-1 and Zt-KP6-1. Orthologs and paralogs 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 toxicity to fungi such as Botrytis cinerea and Z. tritici , but not to wheat. A novel pipeline relying on Foldseek and cysteine-pattern constrained HMM searches of AlphaFold2 predicted structures from Uniprot generated a comprehensive inventory of KP4 and KP6 proteins in fungi and plants. A structure-based classification of these proteins revealed four KP4 and three KP6 structural super families. This classification provided far-reaching hypotheses on their biological function and evolution. This unifying structural framework highlights the power of structure determination for the classification of effectors, and their functional investigation.