Background: R gene-mediated resistance is one of the most effective mechanisms of immunity against pathogens in plants. To date some components that regulate the primary steps of plant immunity have been isolated, however, the molecular dissection of defense signaling downstream of the R proteins remains to be completed. In addition, R genes are known to be highly variable, however, the molecular mechanisms responsible for this variability remain obscure. Results: To identify novel factors required for R gene-mediated resistance in rice, we used rice insertional mutant lines, induced by the endogenous retrotransposon Tos17, in a genetic screening involving the rice blast fungus Magnaporthe oryzae. We inoculated 41,119 mutant lines with the fungus using a high throughput procedure, and identified 86 mutant lines with diminished resistance. A genome analysis revealed that 72 of the 86 lines contained mutations in a gene encoding a nucleotide binding site (NBS) and leucine rich repeat (LRR) domain-containing (NBS-LRR) protein. A genetic complementation analysis and a pathogenesis assay demonstrated that this NBS-LRR gene encodes Pish, which confers resistance against races of M. oryzae containing avrPish. The other 14 lines have intact copies of the Pish gene, suggesting that they may contain mutations in the signaling components downstream of Pish. The genome analysis indicated that Pish and its neighboring three NBS-LRR genes are high similar to one another and are tandemly located. An in silico analysis of a Tos17 flanking sequence database revealed that this region is a "hot spot" for insertion. Intriguingly, the insertion sites are not distributed evenly among these four NBS-LRR genes, despite their similarity at the sequence and expression levels. Conclusions: In this work we isolated the R gene Pish, and identified several other mutants involved in the signal transduction required for Pish-mediated resistance. These results indicate that our genetic approach is efficient and useful for unveiling novel aspects of defense signaling in rice. Furthermore, our data provide experimental evidence that R gene clusters have the potential to be highly preferred targets for transposable element insertions in plant genomes. Based on this finding, a possible mechanism underlying the high variability of R genes is discussed.

In total, 310 rice blast (Pyricularia oryzae Cavara) isolates from Japan showed wide variation in virulence. Virulence on rice (Oryza sativa L.) differential varieties (DV) harboring resistance genes Pish, Pia, Pii, Pi3, Pi5(t), Pik-s, and Pi19(t) ranged from 82.9 to 100.0%. In contrast, virulence on DV possessing Pib, Pit, Pik-m, Pi1, Pik-h, Pik, Pik-p, Pi7(t), Pi9(t), Piz, Piz-5, Piz-t, Pita-2, Pita, Pi12(t), and Pi20(t) ranged from 0 to 21.6%. Cluster analysis using the reaction patterns of the DV classified isolates into three groups: I, virulent to Pik, Pik-h, Pik-p, Pik-m, Pi1, and Pi7(t); IIa, avirulent to the preceding 6 genes and virulent to Pia, Pii, Pi3, and Pi5(t); and IIb, avirulent to all 10 genes. Group I was limited to northern Japan and group IIb to central Japan, while group IIa was distributed throughout Japan. We estimate that group IIa represents the original population and that groups I and IIb arose from it through minor changes in pathogenicity. We classified these isolates into 123 races by a new designation system and conclude that the rice blast races in Japan are less diverse than previously thought.

A total of 239 isolates of blast (Pyricularia oryzae Cavara) collected from northern and central Vietnam showed a wide variation in pathogenicity based on the reaction patterns to 25 differential varieties (DVs) harboring 23 resistance genes and susceptible cultivar Lijiangxintuanheigu (LTH). The frequencies of isolates virulent toward DVs for Pish, Pik-m, Pi1, Pik-h, Pik, Pik-p, Pi7(t), Pi9(t), Piz-5, Pita-2, and Pita were low, but they were high for DVs for Pib, Pit, Pia, Pii, Pi3, Pi5(t), Pik-s, Piz, Piz-t, Pi12(t), Pi19(t), and Pi20(t). Isolates were classified into three cluster groups Ia, Ib, and II based on reaction patterns to DVs and LTH. The frequencies of isolates virulent toward 11 DVs for Pik-m, Pi1, Pik-h, Pik, Pik-p, Pi7(t), Pi9(t), Piz, Piz-5, Pita-2, and Pita in cluster II and DV for Piz-t were higher and lower than those of Ia and Ib, respectively. The frequencies to DVs for Pii, Pi3, Pi5(t), and Piz-t were different between clusters Ia and Ib. Clusters Ia and Ib were distributed with similar frequencies in the northeast, north central, and south central coast regions, but the frequencies among three cluster groups in the Red River Delta and northwest regions were different. This means that the blast races in these two regions were different from the others. Overall, the blast isolates were categorized into 153 races. Among them, 26 were selected as a set of standard differential blast isolates for characterizing 23 resistance genes and developing a differential system in Vietnam.

Rice blast is the most serious disease afflicting rice and there is an urgent need for the use of disease resistance (R) genes in blast tolerance breeding programs. Pb1 is classified as a quantitative resistance gene and it does not have fungal specificity. Pb1-mediated resistance develops in the latter stages of growth. However, some cultivars, such as Kanto209 (K209), cultivar name Satojiman, despite possessing Pb1, do not exert resistance to rice blast during the reproductive stage. We found that the expression of WRKY45 gene downstream of Pb1 was weakly induced by rice blast inoculation at the full heading stage in K209. Genetic analysis using the SNP-based Golden Gate assay of K209 crossing with Koshihikari Aichi SBL (KASBL) found at least four regions related to the resistance in the rice genome (Chr8, Chr9, Chr7, Chr11). Mapping of QTL related to Chr7 confirmed the existence of factors that were required for the resistance of Pb1 in the 22 to 23 Mbp region of the rice genome. We clarified how the K209 cultivar is vulnerable to the blast disease despite possessing Pb1 and found the DNA marker responsible for the quantitative resistance of Pb1. We identified the QTL loci required for Pb1-mediated resistance to rice panicle blast. Pb1 was negatively dependent on at least three QTLs, 7, 9 and 11, and positively dependent on one, QTL 8, in the K209 genome. This finding paves the way for creating a line to select optimal QTLs in order to make use of Pb1-mediated resistance more effectively.

A total of 201 isolates of Pyricularia oryzae (the causal agent of rice blast) were collected from three rice ecosystems (upland, lowland, and swampy) in five regions of Indonesia (West Java, Lampung, South Sumatra, Kalimantan, and Bali). Their pathogenicities were characterized based on the patterns of reaction of 25 differential varieties (DVs) and the susceptible control Lijiangxintuanheigu (LTH), which was susceptible to all blast isolates. A high proportion of isolates (>80.0%) were virulent to DVs for resistance genes Pib, Pit, Pia, Pik-s, and Pi12(t), and a low proportion of isolates (<12.9%) were virulent to DVs for Pik-m, Pi1, Pik-h, Pik, Pik-p, and Pi7(t). Virulence to the other DVs for Pish, Pii, Pi3, Pi5(t), Pi9(t), Piz, Piz-5, Piz-t, Pita-2 (two lines), Pita (two lines), Pi19(t), and Pi20(t) showed intermediate frequencies from 20.0 to 80.0%. These isolates were classified into three cluster groups, Ia, Ib, and II, and the frequencies of cluster groups varied between the three ecosystems and the five regions. The frequencies of cluster groups varied between ecosystems and regions, and races varied according to the ecosystems. A total of 27 standard differential blast isolates (SDBIs) were selected from the 201 isolates collected. The set of 25 DVs and these 27 SDBIs will be used as a new differential system for analysis of the pathogenicity of blast isolates and analysis of resistance genes in rice cultivars, which will contribute to building a durable protection system against blast disease in Indonesia.