Magnaporthe oryzae is a rice blast pathogen that seriously threatens rice yield. Benzovindiflupyr is a succinate dehydrogenase inhibitor (SDHI) fungicide that effectively controls many crop diseases. Benzovindiflupyr has a strong inhibitory effect on M. oryzae; however, control of rice blast by benzovindiflupyr and risk of resistance to benzovindiflupyr are not well studied in this pathogen. In this study, six benzovindiflupyr-resistant strains were obtained by domestication induced in the laboratory. The MoSdhBH245D mutation was the cause of M. oryzae resistance to benzovindiflupyr, which was verified through succinate dehydrogenase (SDH) activity assays, molecular docking, and site-specific mutations. Survival fitness analysis showed no significant difference between the benzovindiflupyr-resistant and parent strains. Positive cross-resistance to benzovindiflupyr and other SDHIs and negative cross-resistance to azoxystrobin were observed. Therefore, the risk of benzovindiflupyr resistance in M. oryzae might be medium to high. It should be combined with other classes of fungicides (tebuconazole and azoxystrobin) to slow the development of resistance.

Rice blast, caused by Magnaporthe oryzae, is a devastating fungal disease worldwide. Pydiflumetofen (Pyd) is a new succinate dehydrogenase inhibitor (SDHI) that exhibited anti-fungal activity against M. oryzae. However, control of rice blast by Pyd and risk of resistance to Pyd are not well studied in this pathogen. The baseline sensitivity of 109 M. oryzae strains to Pyd was determined using mycelial growth rate assay, with EC50 values ranging from 0.291 to 2.1313 μg/mL, and an average EC50 value of 1.1005 ± 0.3727 μg/mL. Totally 28 Pyd-resistant (PydR) mutants with 15 genotypes of point mutations in succinate dehydrogenase (SDH) complex were obtained, and the resistance level could be divided into three categories of very high resistance (VHR), high resistance (HR) and moderate resistance (MR) with the resistance factors (RFs) of >1000, 105.74-986.13 and 81.92-99.48, respectively. Molecular docking revealed that all 15 mutations decreased the binding-force score for the affinity between Pyd and target subunits, which further confirmed that these 15 genotypes of point mutations were responsible for the resistance to Pyd in M. oryzae. There was positive cross resistance between Pyd and other SDHIs, such as fluxapyroxad, penflufen or carboxin, while there was no cross-resistance between Pyd and carbendazim, prochloraz or azoxystrobin in M. oryzae, however, PydR mutants with SdhBP198Q, SdhCL66F or SdhCL66R genotype were still sensitive to the other 3 SDHIs, indicating lack of cross resistance. The results of fitness study revealed that the point mutations in MoSdhB/C/D genes might reduce the hyphae growth and sporulation, but could improve the pathogenicity in M. oryzae. Taken together, the risk of resistance to Pyd might be moderate to high, and it should be used as tank-mixtures with other classes of fungicides to delay resistance development when it is used for the control of rice blast in the field.

The cell wall is the first barrier against external adversity and plays roles in maintaining normal physiological functions of fungi. Previously, we reported a nucleosome assembly protein, MoNap1, in Magnaporthe oryzae that plays a role in cell wall integrity (CWI), stress response, and pathogenicity. Moreover, MoNap1 negatively regulates the expression of MoSMI1 encoded by MGG_03970. Here, we demonstrated that deletion of MoSMI1 resulted in a significant defect in appressorium function, CWI, cell morphology, and pathogenicity. Further investigation revealed that MoSmi1 interacted with MoOsm1 and MoMps1 and affected the phosphorylation levels of MoOsm1, MoMps1, and MoPmk1, suggesting that MoSmi1 regulates biological functions by mediating mitogen-activated protein kinase (MAPK) signalling pathway in M. oryzae. In addition, transcriptome data revealed that MoSmi1 regulates many infection-related processes in M. oryzae, such as membrane-related pathway and oxidation reduction process. In conclusion, our study demonstrated that MoSmi1 regulates CWI by mediating the MAPK pathway to affect development and pathogenicity of M. oryzae.

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.

Cell cycle progression, autophagic cell death during appressorium development, and ROS degradation at the infection site are important for the development of rice blast disease. However, the association of cell cycle, autophagy and ROS detoxification remains largely unknown in M. oryzae. Here, we identify the dual-specificity kinase MoLKH1, which serves as an important cell cycle regulator required for appressorium formation by regulating cytokinesis and cytoskeleton in M. oryzae. MoLKH1 is transcriptionally activated by H2O2 and required for H2O2-induced autophagic cell death and suppression of ROS-activated plant defense during plant invasion of M. oryzae. In addition, the Molkh1 mutant also showed several phenotypic defects, including delayed growth, abnormal conidiation, damaged cell wall integrity, impaired glycogen and lipid transport, reduced secretion of extracellular enzymes and effectors, and attenuated virulence of M. oryzae. Nuclear localization of MoLKH1 requires the nuclear localization sequence, Lammer motif, as well as the kinase active site and ATP-binding site in this protein. Site-directed mutagenesis showed that each of them plays crucial roles in fungal growth and pathogenicity of M. oryzae. In conclusion, our results demonstrate that MoLKH1-mediated cell cycle, autophagy, and suppression of plant immunity play crucial roles in development and pathogenicity of M. oryzae.

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.

Broad-spectrum disease resistance (BSR) is crucial for controlling plant diseases and relies on immune signals that are subject to transcriptional and post-translational regulation. How plants integrate and coordinate these signals remains unclear. We show here that the rice really interesting new gene (RING)-type E3 ubiquitin ligase OsRING113 targets APIP5, a negative regulator of plant immunity and programmed cell death (PCD), for 26S proteasomal degradation. The osring113 mutants in Nipponbare exhibited decreased BSR, while the overexpressing OsRING113 plants showed enhanced BSR against Magnaporthe oryzae (M. oryzae) and Xanthomonas oryzae pv. oryzae (Xoo). Furthermore, APIP5 directly suppressed the transcription of the Bowman-Birk trypsin inhibitor genes OsBBTI5 and AvrPiz-t-interacting protein 4 (APIP4). Overexpression of these two genes, which are partially required for APIP5-mediated PCD and disease resistance, conferred BSR. OsBBTI5 and APIP4 associated with and stabilized the pathogenesis-related protein OsPR1aL, which promotes M. oryzae resistance. Our results identify an immune module with integrated and coordinated hierarchical regulations that confer BSR in plants.

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.