André Padilla

Phytopathogenic fungi secrete effector proteins to promote virulence. The MAX ( Magnaporthe Avrs and ToxB-like) effectors form a structurally conserved family despite significant sequence diversity. AVR-Pia, a MAX effector from the rice blast fungus Magnaporthe oryzae, is recognised by the model rice nucleotide-binding leucine-rich repeat (NLR) receptor pair OsRGA4/OsRGA5 via direct binding to a heavy metal-associated (HMA) integrated domain (ID) in OsRGA5. While the structural basis of AVR-Pia recognition is well defined, the role of this effector in promoting virulence has remained elusive. Here, we reveal that AVR-Pia specifically interacts with four previously uncharacterised rice HMA domain-containing proteins, three HMA Plant Proteins (OsHPP09, OsHPP10 and OsHPP11), and one HMA Isoprenylated Plant Protein (OsHIPP21). AVR-Pia binds these proteins in vitro and in planta, engaging their HMA domains with differential affinities. Notably, AVR-Pia binds OsHPP09-HMA with considerably higher affinity than the HMA-ID of OsRGA5. By solving the crystal structure of the AVR-Pia/OsHPP09-HMA complex, we identified additional molecular contacts at the interface which underpin high affinity binding. Importantly, the H(I)PPs identified as AVR-Pia interactors are distinct from those bound by the MAX effectors AVR-Pik and Pwl2, underscoring target specialisation within the MAX effector family. Further, structural analyses of the AVR-Pia/OsHPP09-HMA complex revealed a markedly different interface compared to other MAX effector/H(I)PP complexes. Finally, structure-guided mutagenesis of OsHPP09 identified a single residue that is critical for AVR-Pia binding. This work provides structural insight into how distinct MAX effectors exploit HMA domain-containing proteins and offers a foundation towards targeted modification of HMA domains to disrupt effector binding and enhance cereal resistance to blast disease.

Phytopathogenic fungi secrete effector proteins to promote virulence. MAX ( M agnaporthe A vrs and T oxB-like) effectors form a sequence-diverse family sharing a conserved protein structure. AVR-Pia, a MAX effector from the rice blast fungus Magnaporthe oryzae, is recognised by the paired rice nucleotide-binding leucine-rich repeat immune receptors OsRGA4/OsRGA5 through direct binding to an integrated heavy metal-associated (HMA) domain in OsRGA5. Here, we identify previously unknown host targets of AVR-Pia: four HMA domain-containing rice proteins, belonging to the H MA Isoprenylated P lant P roteins (HIPPs) and H MA P lant P roteins (HPPs). AVR-Pia interacted with all four proteins, both in vitro and in planta, and bound their HMA domains with varying affinities. The crystal structure of AVR-Pia in complex with the HMA domain of OsHPP09 revealed the molecular details of the binding interface. Structure-guided mutagenesis of OsHPP09 identified a single point mutation which prevents AVR-Pia binding, providing a foundation for targeted engineering of HMA domains to evade effector binding.

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, 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.

A critical step to maximize the usefulness of genome-wide association studies (GWAS) in plant breeding is the identification and validation of candidate genes underlying genetic associations. This is of particular importance in disease resistance breeding where allelic variants of resistance genes often confer resistance to distinct populations, or races, of a pathogen. Here, we perform a genome-wide association analysis of rice blast resistance in 500 genetically diverse rice accessions. To facilitate candidate gene identification, we produce de-novo genome assemblies of ten rice accessions with various rice blast resistance associations. These genome assemblies facilitate the identification and functional validation of novel alleles of the rice blast resistance genes Ptr and Pia. We uncover an allelic series for the unusual Ptr rice blast resistance gene, and additional alleles of the Pia resistance genes RGA4 and RGA5. By linking these associations to three thousand rice genomes we provide a useful tool to inform future rice blast breeding efforts. Our work shows that GWAS in combination with whole-genome sequencing is a powerful tool for gene cloning and to facilitate selection of specific resistance alleles for plant breeding.

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.

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. Author summary: MAX effectors are a family of virulence proteins from the plant pathogenic fungus Pyricularia (syn. Magnaporthe) oryzae that share a similar 3D structure despite very low amino-acid sequence identity. Characterizing the function and evolution of these proteins requires a detailed understanding of their structural diversity. With this in mind, we have determined the NMR structures of four new MAX effectors and shown a near-perfect match with the corresponding AlphaFold2 (AF2) models. We then applied a prediction pipeline based on similarity searches with structural modeling using the AF2 software to predict MAX effectors in a collection of 120 P. oryzae genomes. The resulting models and experimental structures revealed that the MAX core while preserved is highly permissive to secondary structure variations and may coexists with extensive structural diversity in terms of structured N- or C-terminal extensions permitting their classification. For a subset of AF2 models, we have also analyzed the physico-chemical properties of the core domain surfaces, adding another, more functional perspective, notably surface electrostatics and stickiness. This work constitutes a major step in understanding the relationships among MAX effectors by analyzing their structural landscape and cataloguing specific physico-chemical properties. It also provides valuable insights for guiding research into the putative targets of these effectors in infected plant hosts.

Plant pathogens secrete proteins called effectors that target host cellular processes to promote disease. Recently, structural genomics has identified several families of fungal effectors that share a similar three-dimensional structure despite remarkably variable amino-acid sequences and surface properties. To explore the selective forces that underlie the sequence variability of structurally-analogous effectors, we focused on MAX effectors, a structural family of effectors that are major determinants of virulence in the rice blast fungus Pyricularia oryzae . Using structure-informed gene annotation, we identified 58 to 78 MAX effector genes per genome in a set of 120 isolates representing seven host-associated lineages. The expression of MAX effector genes was primarily restricted to the early biotrophic phase of infection and strongly influenced by the host plant. Pangenome analyses of MAX effectors demonstrated extensive presence/absence polymorphism and identified gene loss events possibly involved in host range adaptation. However, gene knock-in experiments did not reveal a strong effect on virulence phenotypes suggesting that other evolutionary mechanisms are the main drivers of MAX effector losses. MAX effectors displayed high levels of standing variation and high rates of non-synonymous substitutions, pointing to widespread positive selection shaping the molecular diversity of MAX effectors. The combination of these analyses with structural data revealed that positive selection acts mostly on residues located in particular structural elements and at specific positions. By providing a comprehensive catalog of amino acid polymorphism, and by identifying the structural determinants of the sequence diversity, our work will inform future studies aimed at elucidating the function and mode of action of MAX effectors. AUTHOR SUMMARY: Fungal plant pathogens use small secreted proteins, called effectors, to manipulate to their own advantage their host’s physiology and immunity. The evolution of these effectors, whether spontaneously or in response to human actions, can lead to epidemics or the emergence of new diseases. It is therefore crucial to understand the mechanisms underlying this evolution. In this article, we report on the evolution of effectors in one of the prime experimental model systems of plant pathology, Pyricularia oryzae , the fungus causing blast diseases in rice, wheat, and other cereals or grasses. We further characterize in this fungus a particular class of effectors, called MAX effectors, using structural models based on experimental protein structures of effectors. We show that this class of effector is produced by the pathogen during the early stages of infection, when plant cells are still alive. By comparing the gene content of isolates infecting different plant species, we show that the MAX effector arsenal is highly variable from one isolate to another. Finally, using the inferential framework of population genetics, we demonstrate that MAX effectors exhibit very high genetic variability and that this results from the action of natural selection.

One of the most critical steps following genome-wide association studies (GWAS) is the identification and validation of candidate genes underlying genetic associations. Gene presence-absence and copy number variations can significantly hamper candidate gene discovery if high-quality genomic resources from specific crop cultivars are unavailable. This is particularly true for disease resistance genes, which are often located in highly dynamic genomic regions. Here, we perform a genome-wide association analysis of rice blast resistance in 500 genetically diverse rice accessions. To facilitate candidate gene identification, we produced de-novo genome assemblies of ten rice accessions that showed rice blast resistance associations. These assemblies facilitated the identification and validation of novel alleles of the rice blast resistance genes Ptr and Pia. Our work shows that GWAS in combination with whole-genome sequencing are powerful tools for gene cloning and uncovers an allelic series for the unusual Ptr rice blast resistance gene.