Isolation of Pikps, an allele of Pik, from the aus rice cultivar Shoni.

Blast disease caused by the filamentous fungus Pyricularia oryzae (syn. Magnaporthe oryzae) is one of the most destructive diseases of rice (Oryza sativa L.) around the globe. An aus cultivar, Shoni, showed resistance against at least four Japanese P. oryzae isolates. To understand Shoni's resistance against the P. oryzae isolate Naga69-150, genetic analysis was carried out using recombinant inbred lines developed by a cross between Shoni and the japonica cultivar Hitomebore, which is susceptible to Naga69-150. The result indicated that the resistance was controlled by a single locus, which was named Pi-Shoni. A QTL analysis identified Pi-Shoni as being located in the telomeric region of chromosome 11. A candidate gene approach in the region indicated that Pi-Shoni corresponds to the previously cloned Pik locus, and we named this allele Pikps. Loss of gene function mediated by RNA interference demonstrated that a head-to-head-orientated pair of NBS-LRR receptor genes (Pikps-1 and Pikps-2) are required for the Pikps-mediated resistance. Amino acid sequence comparison showed that Pikps-1 is 99% identical to Pikp-1, while Pikps-2 is identical to Pikp-2. Pikps-1 had one amino acid substitution (Pro351Ser) in the NBS domain as compared to Pikp-1. The recognition specificity of Pikps against known AVR-Pik alleles is identical to that of Pikp.

Disentangling the complex gene interaction networks between rice and the blast fungus identifies a new pathogen effector.

Studies focused solely on single organisms can fail to identify the networks underlying host-pathogen gene-for-gene interactions. Here, we integrate genetic analyses of rice (Oryza sativa, host) and rice blast fungus (Magnaporthe oryzae, pathogen) and uncover a new pathogen recognition specificity of the rice nucleotide-binding domain and leucine-rich repeat protein (NLR) immune receptor Pik, which mediates resistance to M. oryzae expressing the avirulence effector gene AVR-Pik. Rice Piks-1, encoded by an allele of Pik-1, recognizes a previously unidentified effector encoded by the M. oryzae avirulence gene AVR-Mgk1, which is found on a mini-chromosome. AVR-Mgk1 has no sequence similarity to known AVR-Pik effectors and is prone to deletion from the mini-chromosome mediated by repeated Inago2 retrotransposon sequences. AVR-Mgk1 is detected by Piks-1 and by other Pik-1 alleles known to recognize AVR-Pik effectors; recognition is mediated by AVR-Mgk1 binding to the integrated heavy metal-associated (HMA) domain of Piks-1 and other Pik-1 alleles. Our findings highlight how complex gene-for-gene interaction networks can be disentangled by applying forward genetics approaches simultaneously to the host and pathogen. We demonstrate dynamic coevolution between an NLR integrated domain and multiple families of effector proteins.

Rice apoplastic CBM1-interacting protein counters blast pathogen invasion by binding conserved carbohydrate binding module 1 motif of fungal proteins.

When infecting plants, fungal pathogens secrete cell wall-degrading enzymes (CWDEs) that break down cellulose and hemicellulose, the primary components of plant cell walls. Some fungal CWDEs contain a unique domain, named the carbohydrate binding module (CBM), that facilitates their access to polysaccharides. However, little is known about how plants counteract pathogen degradation of their cell walls. Here, we show that the rice cysteine-rich repeat secretion protein OsRMC binds to and inhibits xylanase MoCel10A of the blast fungus pathogen Magnaporthe oryzae, interfering with its access to the rice cell wall and degradation of rice xylan. We found binding of OsRMC to various CBM1-containing enzymes, suggesting that it has a general role in inhibiting the action of CBM1. OsRMC is localized to the apoplast, and its expression is strongly induced in leaves infected with M. oryzae. Remarkably, knockdown and overexpression of OsRMC reduced and enhanced rice defense against M. oryzae, respectively, demonstrating that inhibition of CBM1-containing fungal enzymes by OsRMC is crucial for rice defense. We also identified additional CBM-interacting proteins (CBMIPs) from Arabidopsis thaliana and Setaria italica, indicating that a wide range of plants counteract pathogens through this mechanism.

A genetically linked pair of NLR immune receptors shows contrasting patterns of evolution.

Throughout their evolution, plant nucleotide-binding leucine-rich-repeat receptors (NLRs) have acquired widely divergent unconventional integrated domains that enhance their ability to detect pathogen effectors. However, the functional dynamics that drive the evolution of NLRs with integrated domains (NLR-IDs) remain poorly understood. Here, we reconstructed the evolutionary history of an NLR locus prone to unconventional domain integration and experimentally tested hypotheses about the evolution of NLR-IDs. We show that the rice (Oryza sativa) NLR Pias recognizes the effector AVR-Pias of the blast fungal pathogen Magnaporthe oryzae. Pias consists of a functionally specialized NLR pair, the helper Pias-1 and the sensor Pias-2, that is allelic to the previously characterized Pia pair of NLRs: the helper RGA4 and the sensor RGA5. Remarkably, Pias-2 carries a C-terminal DUF761 domain at a similar position to the heavy metal-associated (HMA) domain of RGA5. Phylogenomic analysis showed that Pias-2/RGA5 sensor NLRs have undergone recurrent genomic recombination within the genus Oryza, resulting in up to six sequence-divergent domain integrations. Allelic NLRs with divergent functions have been maintained transspecies in different Oryza lineages to detect sequence-divergent pathogen effectors. By contrast, Pias-1 has retained its NLR helper activity throughout evolution and is capable of functioning together with the divergent sensor-NLR RGA5 to respond to AVR-Pia. These results suggest that opposite selective forces have driven the evolution of paired NLRs: highly dynamic domain integration events maintained by balancing selection for sensor NLRs, in sharp contrast to purifying selection and functional conservation of immune signaling for helper NLRs.

Rice Exo70 interacts with a fungal effector, AVR-Pii, and is required for AVR-Pii-triggered immunity.

Vesicle trafficking including the exocytosis pathway is intimately associated with host immunity against pathogens. However, we still have insufficient knowledge about how it contributes to immunity, and how pathogen factors affect it. In this study, we explore host factors that interact with the Magnaporthe oryzae effector AVR-Pii. Gel filtration chromatography and co-immunoprecipitation assays identified a 150 kDa complex of proteins in the soluble fraction comprising AVR-Pii and OsExo70-F2 and OsExo70-F3, two rice Exo70 proteins presumably involved in exocytosis. Simultaneous knockdown of OsExo70-F2 and F3 totally abrogated Pii immune receptor-dependent resistance, but had no effect on Pia- and Pik-dependent resistance. Knockdown levels of OsExo70-F3 but not OsExo70-F2 correlated with reduction of Pii function, suggesting that OsExo70-F3 is specifically involved in Pii-dependent resistance. Under our current experimental conditions, over-expression of AVR-Pii or knockdown of OsExo70-F2 and -F3 genes in rice did not affect the virulence of compatible isolates of M. oryzae. AVR-Pii interaction with OsExo70-F3 appears to play a crucial role in immunity triggered by Pii, suggesting a role for OsExo70 as a decoy or helper in Pii/AVR-Pii interactions.

The rice (Oryza sativa L.) LESION MIMIC RESEMBLING, which encodes an AAA-type ATPase, is implicated in defense response.

Lesion mimic mutants (LMMs) provide a useful tool to study defense-related programmed cell death (PCD) in plants. Although a number of LMMs have been identified in multiple species, most of the candidate genes are yet to be isolated. Here, we report the identification and characterization of a novel rice (Oryza sativa L.) lesion mimic resembling (lmr) mutant, and cloning of the corresponding LMR gene. The LMR locus was initially delineated to 1.2 Mb region on chromosome 6, which was further narrowed down to 155-kb using insertions/deletions (INDELs) and cleavage amplified polymorphic sequence markers developed in this study. We sequenced the open reading frames predicted within the candidate genomic region, and identified a G-A base substitution causing a premature translation termination in a gene that encodes an ATPase associated with various cellular activities type (AAA-type) protein. RNA interference transgenic lines with reduced LMR transcripts exhibited the lesion mimic phenotype similar to that of lmr plants. Furthermore, expression of the wild-type LMR in the mutant background complemented the lesion phenotype, confirming that the mutation identified in LMR is responsible for the mutant phenotype. The pathogenesis-related (PR) genes PBZ1 and PR1 were induced in lmr, which also showed enhanced resistance to rice blast (Magnaporthe oryzae) and bacterial blight (Xanthomonas oryzae pv. oryzae), suggesting LMR is a negative regulator of cell death in rice. The identification of lmr and cloning of the corresponding LMR gene provide an additional resource for the study of PCD in plants.

MutMap+: genetic mapping and mutant identification without crossing in rice.

Advances in genome sequencing technologies have enabled researchers and breeders to rapidly associate phenotypic variation to genome sequence differences. We recently took advantage of next-generation sequencing technology to develop MutMap, a method that allows rapid identification of causal nucleotide changes of rice mutants by whole genome resequencing of pooled DNA of mutant F2 progeny derived from crosses made between candidate mutants and the parental line. Here we describe MutMap+, a versatile extension of MutMap, that identifies causal mutations by comparing SNP frequencies of bulked DNA of mutant and wild-type progeny of M3 generation derived from selfing of an M2 heterozygous individual. Notably, MutMap+ does not necessitate artificial crossing between mutants and the wild-type parental line. This method is therefore suitable for identifying mutations that cause early development lethality, sterility, or generally hamper crossing. Furthermore, MutMap+ is potentially useful for gene isolation in crops that are recalcitrant to artificial crosses.