Phytopathogenic ascomycete fungi possess huge effector repertoires that are dominated by hundreds of sequence-unrelated small secreted proteins. The molecular function of these effectors and the evolutionary mechanisms that generate this tremendous number of singleton genes are largely unknown. To get a deeper understanding of fungal effectors, we determined by NMR spectroscopy the 3-dimensional structures of the Magnaporthe oryzae effectors AVR1-CO39 and AVR-Pia. Despite a lack of sequence similarity, both proteins have very similar 6 β-sandwich structures that are stabilized in both cases by a disulfide bridge between 2 conserved cysteins located in similar positions of the proteins. Structural similarity searches revealed that AvrPiz-t, another effector from M. oryzae, and ToxB, an effector of the wheat tan spot pathogen Pyrenophora tritici-repentis have the same structures suggesting the existence of a family of sequence-unrelated but structurally conserved fungal effectors that we named MAX-effectors (Magnaporthe Avrs and ToxB like). Structure-informed pattern searches strengthened this hypothesis by identifying MAX-effector candidates in a broad range of ascomycete phytopathogens. Strong expansion of the MAX-effector family was detected in M. oryzae and M. grisea where they seem to be particularly important since they account for 5–10% of the effector repertoire and 50% of the cloned avirulence effectors. Expression analysis indicated that the majority of M. oryzae MAX-effectors are expressed specifically during early infection suggesting important functions during biotrophic host colonization. We hypothesize that the scenario observed for MAX-effectors can serve as a paradigm for ascomycete effector diversity and that the enormous number of sequence-unrelated ascomycete effectors may in fact belong to a restricted set of structurally conserved effector families. Fungal plant pathogens are of outstanding economic and ecological importance and cause destructive diseases on many cultivated and wild plants. Effector proteins that are secreted during infection to manipulate the host and to promote disease are a key element in fungal virulence. Phytopathogenic fungi possess huge effector repertoires that are dominated by hundreds of sequence-unrelated small secreted proteins. The molecular functions of this most important class of fungal effectors and the evolutionary mechanisms that generate this tremendous numbers of apparently unrelated proteins are largely unknown. By investigating the 3-dimensional structures of effectors from the rice blast fungus M. oryzae, we discovered an effector family comprising structurally conserved but sequence-unrelated effectors from M. oryzae and the phylogenetically distant wheat pathogen Pyrenophora tritici-repentis that we named MAX-effectors (M. oryzae Avrs and ToxB). Structure-informed searches of whole genome sequence databases suggest that MAX-effectors are present at low frequencies and with a patchy phylogenetic distribution in many ascomycete phytopathogens. They underwent strong lineage-specific expansion in fungi of the Pyriculariae family that contains M. oryzae where they seem particularly important during biotrophic plant colonization and account for 50% of the cloned Avr effectors and 5–10% of the effector repertoire. Based on our results on the MAX-effectors and the widely accepted concept that fungal effectors evolve according to a birth-and-death model we propose the hypothesis that the majority of the immense numbers of different ascomycete effectors could in fact belong to a limited set of structurally defined families whose members are phylogenetically related.

The Magnaporthe oryzae effector AVR-Pia interacts with the decoy domain of the rice NLR immune receptor RGA5 through a defined surface area, and this interactio

The structurally conserved but sequence-unrelated MAX ( Magnaporthe oryzae avirulence and ToxB-like) effectors AVR1-CO39 and AVR-PikD from the blast fungus M. oryzae are recognized by the rice nucleotide-binding domain and leucine-rich repeat proteins (NLRs) RGA5 and Pikp-1, respectively. This involves, in both cases, direct interaction of the effector with a heavy metal-associated (HMA) integrated domain (ID) in the NLR. Here, we solved the crystal structures of a C-terminal fragment of RGA5 carrying the HMA ID (RGA5_S), alone, and in complex with AVR1-CO39 and compared it to the structure of the Pikp1 _HMA /AVR-PikD complex. In both complexes, HMA ID/MAX effector interactions involve antiparallel alignment of β-sheets from each partner. However, effector-binding occurs at different surfaces in Pikp1 _HMA and RGA5 _HMA , indicating that these interactions evolved independently by convergence of these two MAX effectors to the same type of plant target proteins. Interestingly, the effector-binding surface in RGA5 _HMA overlaps with the surface that mediates RGA5 _HMA self-interaction. Mutations in the HMA-binding interface of AVR1-CO39 perturb RGA5 _HMA -binding, in vitro and in vivo, and affect the recognition of M. oryzae in a rice cultivar containing Pi-CO39 . Our study provides detailed insight into the mechanisms of effector recognition by NLRs, which has substantial implications for future engineering of NLRs to expand their recognition specificities. In addition, we propose, as a hypothesis for the understanding of effector diversity, that in the structurally conserved MAX effectors the molecular mechanism of host target protein-binding is conserved rather than the host target proteins themselves.

Plant nucleotide-binding and leucine-rich repeat domain proteins (NLRs) are immune sensors that recognize pathogen effectors. Here, we show that molecular engineering of the integrated decoy domain (ID) of an NLR can extend its recognition spectrum to a new effector. We relied for this on detailed knowledge on the recognition of the Magnaporthe oryzae effectors AVR-PikD, AVR-Pia, and AVR1-CO39 by, respectively, the rice NLRs Pikp-1 and RGA5. Both receptors detect their effectors through physical binding to their HMA (Heavy Metal-Associated) IDs. By introducing into RGA5_HMA the AVR-PikD binding residues of Pikp-1_HMA, we create a high-affinity binding surface for this effector. RGA5 variants carrying this engineered binding surface perceive the new ligand, AVR-PikD, and still recognize AVR-Pia and AVR1-CO39 in the model plant N. benthamiana. However, they do not confer extended disease resistance specificity against M. oryzae in transgenic rice plants. Altogether, our study provides a proof of concept for the design of new effector recognition specificities in NLRs through molecular engineering of IDs.

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.