The structural landscape and diversity of Pyricularia oryzae MAX effectors revisited.

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.

1H, 13C, 15 N backbone and side-chain NMR assignments for three MAX effectors from Magnaporthe oryzae.

Effectors are small and very diverse proteins secreted by fungi and translocated in plant cells during infection. Among them, MAX effectors (for Magnaporthe Avrs and ToxB) were identified as a family of effectors that share an identical fold topology despite having highly divergent sequences. They are mostly secreted by ascomycetes from the Magnaporthe genus, a fungus that causes the rice blast, a plant disease leading to huge crop losses. As rice is the first source of calories in many countries, especially in Asia and Africa, this constitutes a threat for world food security. Hence, a better understanding of these effectors, including structural and functional characterization, constitutes a strategic milestone in the fight against phytopathogen fungi and may give clues for the development of resistant varieties of rice. We report here the near complete 1H, 15 N and 13C NMR resonance assignment of three new putative MAX effectors (MAX47, MAX60 and MAX67). Secondary structure determination using TALOS-N and CSI.3 demonstrates a high content of ß-strands in all the three proteins, in agreement with the canonic ß-sandwich structure of MAX effectors. This preliminary study provides foundations for further structural characterization, that will help in turn to improve sequence predictions of other MAX effectors through data mining.