Bioengineering of plant immune receptors has emerged as a key strategy for generating novel disease resistance traits to counteract the expanding threat of plant pathogens to global food security. However, current approaches are limited by rapid evolution of plant pathogens in the field and may lack durability when deployed. Here, we show that the rice nucleotide-binding, leucine-rich repeat (NLR) immune receptor Pik-1 can be engineered to respond to a conserved family of effectors from the multihost blast fungus pathogen Magnaporthe oryzae. We switched the effector binding and response profile of the Pik NLR from its cognate rice blast effector AVR-Pik to the host-determining factor pathogenicity toward weeping lovegrass 2 (Pwl2) by installing a putative host target, OsHIPP43, in place of the native integrated heavy metal-associated domain (generating Pikm-1OsHIPP43). This chimeric receptor also responded to other PWL alleles from diverse blast isolates. The crystal structure of the Pwl2/OsHIPP43 complex revealed a multifaceted, robust interface that cannot be easily disrupted by mutagenesis, and may therefore provide durable, broad resistance to blast isolates carrying PWL effectors in the field. Our findings highlight how the host targets of pathogen effectors can be used to bioengineer recognition specificities that have more robust properties compared to naturally evolved disease resistance genes.

Filamentous plant pathogens deliver effector proteins into host cells to suppress host defence responses and manipulate metabolic processes to support colonization. Understanding the evolution and molecular function of these effectors provides knowledge about pathogenesis and can suggest novel strategies to reduce damage caused by pathogens. However, effector proteins are highly variable, share weak sequence similarity and, although they can be grouped according to their structure, only a few structurally conserved effector families have been functionally characterized to date. Here, we demonstrate that Zinc-finger fold (ZiF) secreted proteins form a functionally diverse effector family in the blast fungus Magnaporthe oryzae. This family relies on the Zinc-finger motif for protein stability and is ubiquitously present in blast fungus lineages infecting 13 different host species, forming different effector tribes. Homologs of the canonical ZiF effector, AVR-Pii, from rice infecting isolates are present in multiple M. oryzae lineages. Wheat infecting strains of the fungus also possess an AVR-Pii like allele that binds host Exo70 proteins and activates the immune receptor Pii. Furthermore, ZiF tribes may vary in the proteins they bind to, indicating functional diversification and an intricate effector/host interactome. Altogether, we uncovered a new effector family with a common protein fold that has functionally diversified in lineages of M. oryzae. This work expands our understanding of the diversity of M. oryzae effectors, the molecular basis of plant pathogenesis and may ultimately facilitate the development of new sources for pathogen resistance.

Blast disease caused by the fungus Magnaporthe oryzae is one of the most devastating rice diseases. Disease resistance genes such as Pi-ta or Pi-ta2 are critical in protecting rice production from blast. Published work reports that Pi-ta codes for a nucleotide-binding and leucine-rich repeat domain protein (NLR) that recognizes the fungal protease-like effector AVR-Pita by direct binding. However, this model was challenged by the recent discovery that Pi-ta2 resistance, which also relies on AVR-Pita detection, is conferred by the unconventional resistance gene Ptr, which codes for a membrane protein with a cytoplasmic armadillo repeat domain. Here, using NLR Pi-ta and Ptr RNAi knockdown and CRISPR/Cas9 knockout mutant rice lines, we found that AVR-Pita recognition relies solely on Ptr and that the NLR Pi-ta has no role in it, indicating that it is not the Pi-ta resistance gene. Different alleles of Ptr confer different recognition specificities. The A allele of Ptr (PtrA) detects all natural sequence variants of the effector and confers Pi-ta2 resistance, while the B allele of Ptr (PtrB) recognizes a restricted set of AVR-Pita alleles and, thereby, confers Pi-ta resistance. Analysis of the natural diversity in AVR-Pita and of mutant and transgenic strains identified one specific polymorphism in the effector sequence that controls escape from PtrB-mediated resistance. Taken together, our work establishes that the M. oryzae effector AVR-Pita is detected in an allele-specific manner by the unconventional rice resistance protein Ptr and that the NLR Pi-ta has no function in Pi-ta resistance and the recognition of AVR-Pita.

A novel negative-sense single-stranded RNA mycovirus, designated as \"Magnaporthe oryzae mymonavirus 1\" (MoMNV1), was identified in the rice blast fungus Magnaporthe oryzae isolate NJ39. MoMNV1 has a single genomic RNA segment consisting of 10,515 nucleotides, which contains six open reading frames. The largest open reading frame contains 5837 bases and encodes an RNA replicase. The six open reading frames have no overlap and are arranged linearly on the genome, but the spacing of the genes is small, with a maximum of 315 bases and a minimum of 80 bases. Genome comparison and phylogenetic analysis indicated that MoMNV1 is a new member of the genus Penicillimonavirus of the family Mymonaviridae.

Rice (Oryza sativa) is one of the most important staple foods worldwide. However, rice blast disease, caused by the ascomycete fungus Magnaporthe oryzae, seriously affects the yield and quality of rice. Calmodulin-binding transcriptional activators (CAMTAs) play vital roles in the response to biotic stresses. In this study, we showed that OsCAMTA3 and CAMTA PROTEIN LIKE (OsCAMTAPL), an OsCAMTA3 homolog that lacks the DNA-binding domain, functioned together in negatively regulating disease resistance in rice. OsCAMTA3 associated with OsCAMTAPL. The oscamta3 and oscamtapl mutants showed enhanced resistance compared to wild-type plants, and oscamta3/pl double mutants showed more robust resistance to M. oryzae than oscamta3 or oscamtapl. An RNA-Seq analysis revealed that 59 and 73 genes, respectively, were differentially expressed in wild-type plants and oscamta3 before and after inoculation with M. oryzae, including OsALDH2B1, an acetaldehyde dehydrogenase that negatively regulates plant immunity. OsCAMTA3 could directly bind to the promoter of OsALDH2B1, and OsALDH2B1 expression was decreased in oscamta3, oscamtapl, and oscamta3/pl mutants. In conclusion, OsCAMTA3 associates with OsCAMTAPL to regulate disease resistance by binding and activating the expression of OsALDH2B1 in rice, which reveals a strategy by which rice controls rice blast disease and provides important genes for resistance breeding holding a certain positive impact on ensuring food security.

CONCLUSIONS: The investigation is the first report on genome-wide identification and characterization of NBLRR genes in pearl millet. We have shown the role of gene loss and purifying selection in the divergence of NBLRRs in Poaceae lineage and candidate CaNBLRR genes for resistance to Magnaporthe grisea infection. Plants have evolved multiple integral mechanisms to counteract the pathogens\' infection, among which plant immunity through NBLRR (nucleotide-binding site, leucine-rich repeat) genes is at the forefront. The genome-wide mining in pearl millet (Cenchrus americanus (L.) Morrone) revealed 146 CaNBLRRs. The variation in the branch length of NBLRRs showed the dynamic nature of NBLRRs in response to evolving pathogen races. The orthology of NBLRRs showed a predominance of many-to-one orthologs, indicating the divergence of NBLRRs in the pearl millet lineage mainly through gene loss events followed by gene gain through single-copy duplications. Further, the purifying selection (Ka/Ks < 1) shaped the expansion of NBLRRs within the lineage of pear millet and other members of Poaceae. Presence of cis-acting elements, viz. TCA element, G-box, MYB, SARE, ABRE and conserved motifs annotated with P-loop, kinase 2, RNBS-A, RNBS-D, GLPL, MHD, Rx-CC and LRR suggests their putative role in disease resistance and stress regulation. The qRT-PCR analysis in pearl millet lines showing contrasting responses to Magnaporthe grisea infection identified CaNBLRR20, CaNBLRR33, CaNBLRR46 CaNBLRR51, CaNBLRR78 and CaNBLRR146 as putative candidates. Molecular docking showed the involvement of three and two amino acid residues of LRR domains forming hydrogen bonds (histidine, arginine and threonine) and salt bridges (arginine and lysine) with effectors. Whereas 14 and 20 amino acid residues of CaNBLRR78 and CaNBLRR20 showed hydrophobic interactions with 11 and 9 amino acid residues of effectors, Mg.00g064570.m01 and Mg.00g006570.m01, respectively. The present investigation gives a comprehensive overview of CaNBLRRs and paves the foundation for their utility in pearl millet resistance breeding through understanding of host-pathogen interactions.

Reverse genetic approaches are common tools in genomics for elucidating gene functions, involving techniques such as gene deletion followed by screening for aberrant phenotypes. If the generation of gene deletion mutants fails, the question arises whether the failure stems from technical issues or because the gene of interest (GOI) is essential, meaning that the deletion causes lethality. In this report, we introduce a novel method for assessing gene essentiality using the phytopathogenic ascomycete Magnaporthe oryzae. The method is based on the observation that telomere vectors are lost in transformants during cultivation without selection pressure. We tested the hypothesis that essential genes can be identified in deletion mutants co-transformed with a telomere vector. The M. oryzae gene MoPKC, described in literature as essential, was chosen as GOI. Using CRISPR/Cas9 technology transformants with deleted GOI were generated and backed up by a telomere vector carrying a copy of the GOI and conferring fenhexamid resistance. Transformants in which the GOI deletion in the genome was not successful lost the telomere vector on media without fenhexamid. In contrast, transformants with confirmed GOI deletion retained the telomere vector even in absence of fenhexamid selection. In the latter case, the maintenance of the telomere indicates that the GOI is essential for the surveillance of the fungi, as it would have been lost otherwise. The method presented here allows to test for essentiality of genes when no mutants can be obtained from gene deletion approaches, thereby expanding the toolbox for studying gene function in ascomycetes.

Polarized growth is critical for the development of filamentous phytopathogens, and the CHY-type zinc finger protein Chy1 regulates microtubule assembly to influence polarized growth and thereby affect plant infections. However, the biological role of a Chy1 homolog MoChy1 remains unknown in Magnaporthe oryzae. We found here that the MoChy1-GFP was distributed in the cytoplasm outside the vacuole in hyphae and localized mainly to the vacuole compartments as the appressorium matured. The Mochy1 mutants showed an extremely slow growth rate, curved and branched mycelium, reduced conidiation, and a smaller size in the appressorium. Meanwhile, the Mochy1 mutants showed increased sensitivity to benomyl, damaged microtubule cytoskeleton, and mislocalized polarisome protein MoSpa2 and chitin synthase MoChs6 in hyphae. Compared to Guy11, the Mochy1 mutants exhibited increased sensitivity to H2O2, impaired ability to eliminate host-derived ROS and reduced penetration into host plants, resulting in a strong reduction in pathogenicity of Mochy1 mutants. Furthermore, the Mochy1 mutants also exhibited defects in chitin distribution, osmotic stress tolerance, and septin ring organization during appressorium differentiation and fungal development. Nonselective autophagy was negatively regulated in Mochy1 mutants compared to Guy11. In summary, MoChy1 plays multiple roles in fungal polar growth and full virulence of M. oryzae.

The plant apoplast, which serves as the frontline battleground for long-term host-pathogen interactions, harbors a wealth of disease resistance resources. However, the identification of the disease resistance proteins in the apoplast is relatively lacking. In this study, we identified and characterized the rice secretory protein OsSSP1 (Oryza sativa secretory small protein 1). OsSSP1 can be secreted into the plant apoplast, and either in vitro treatment of recombinant OsSSP1 or overexpression of OsSSP1 in rice could trigger plant immune response. The expression of OsSSP1 is suppressed significantly during Magnaporthe oryzae infection in the susceptible rice variety Taibei 309, and OsSSP1-overexpressing lines all show strong resistance to M. oryzae. Combining the knockout and overexpression results, we found that OsSSP1 positively regulates plant immunity in response to fungal infection. Moreover, the recognition and immune response triggered by OsSSP1 depend on an uncharacterized transmembrane OsSSR1 (secretory small protein receptor 1) and the key co-receptor OsBAK1, since most of the induced immune response and resistance are lost in the absence of OsSSR1 or OsBAK1. Intriguingly, the OsSSP1 protein is relatively stable and can still induce plant resistance after 1 week of storage in the open environment, and exogenous OsSSP1 treatment for a 2-week period did not affect rice yield. Collectively, our study reveals that OsSSP1 can be secreted into the apoplast and percepted by OsSSR1 and OsBAK1 during fungal infection, thereby triggering the immune response to enhance plant resistance to M. oryzae. These findings provide novel resources and potential strategies for crop breeding and disease control.

Although the antagonistic effects of host resistance against biotrophic and necrotrophic pathogens have been documented in various plants, the underlying mechanisms are unknown. Here, we investigated the antagonistic resistance mediated by the transcription factor ETHYLENE-INSENSITIVE3-LIKE 3 (OsEIL3) in rice. The Oseil3 mutant confers enhanced resistance to the necrotroph Rhizoctonia solani but greater susceptibility to the hemibiotroph Magnaporthe oryzae and biotroph Xanthomonas oryzae pv. oryzae. OsEIL3 directly activates OsERF040 transcription while repressing OsWRKY28 transcription. The infection of R. solani and M. oryzae or Xoo influences the extent of binding of OsEIL3 to OsWRKY28 and OsERF040 promoters, resulting in the repression or activation of both salicylic acid (SA)- and jasmonic acid (JA)-dependent pathways and enhanced susceptibility or resistance, respectively. These results demonstrate that the distinct effects of plant immunity to different pathogen types are determined by two transcription factor modules that control transcriptional reprogramming and the SA and JA pathways.