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The segregation of avirulence/virulence was studied in 115 F1 progeny isolates of Magnaporthe oryzae from a cross of two field isolates on three Japanese race-differential rice cultivars Kanto 51, Fukunishiki, and Toride 1. The χ2 tests of goodness-of-fit for a 1:1 ratio indicated that avirulence on cvs. Kanto 51, Fukunishiki, and Toride 1 was under monogenic control. The relationship between the avirulence (Avr) gene in the parental isolate and the Avr gene in the standard isolate was investigated by using 100 lines each of three F3 families from the crosses of the rice cultivars Norin 3/Kanto 51, AK61/Fukunishiki, and Norin 3/Toride 1, respectively. Based on the resistant reactions of the F3 rice lines to the parental isolates and the standard isolates harboring three known Avr genes, three genetically independent Avr genes, AvrPik, AvrPiz, and AvrPiz-t, were identified. The three identified Avr genes were mapped using random amplified polymorphic DNA (RAPD) analysis, and a partial linkage map was constructed with 17 RAPD markers closely linked to the Avr genes. Twelve markers and AvrPik, three markers and AvrPiz, and two markers and AvrPiz-t, as well as mating locus MAT1, constructed linkage groups A, B, and C, respectively.

The pathogenicity of 331 blast isolates (Pyricularia oryzae Cavara) collected from different regions and ecosystems for rice cultivation in Bangladesh was evaluated by compatibility on 23 differential varieties (DV), each harboring a single blast resistance gene, and susceptible ‘Lijiangxintuanheigu’ (LTH). A wide variation in virulence was found among the isolates, and 267 races were classified using a new designation system. Virulence of blast isolates against DV carrying the resistance genes Pia, Pib, Pit, Pik-s, Piz-t, Pi12(t), Pi19(t), and Pi20(t), as well as avirulence against those carrying Pish, Pi9, Pita-2, and Pita, was distributed widely in Bangladesh. Cluster analysis of the compatibility data on the DV initially classified the isolates into groups I and II. The virulence spectra of the two groups differed mainly according to the reactions of the DV to Pii, Pi3, Pi5(t), Pik-m, Pi1, Pik-h, Pik, Pik-p, and Pi7(t). Group I isolates were distributed mainly in rainfed lowlands, whereas group II isolates were found mainly in irrigated lowlands; however, there were no critical differences in geographic distribution of the blast isolates. In total, 26 isolates, which could be used to identify the 23 resistance genes of the DV on the basis of their reaction patterns, were selected as a set of standard differential blast isolates. To our knowledge, this is the first clear demonstration of the diversity and differentiation of blast races in Bangladesh. This information will be used to develop a durable blast protection system in that country.

Rice blast, which is caused by the fungus Magnaporthe oryzae, is a widespread and destructive plant disease that greatly reduces yield and grain quality worldwide. The use of host resistance is the most effective and economic way to control plant diseases. Many studies on blast resistance genes have focused on the nucleotide-binding site leucine-rich repeat (NB–LRR) resistance (called typical R) genes that mediate effector-triggered immunity. However, incorporating these R genes into rice varieties has not achieved durable blast resistance, because R-gene-mediated resistance is race-specific and often breaks down owing to the appearance of fungal variants that escape host recognition.

Rice blast resistance caused by Resistance (R) genes, most of which encode coiled-coil (CC), nucleotide-binding (NB), and leucine-rich-repeat (LRR) domains, is race-specific. Panicle blast 1 (Pb1) is a panicle blast resistance gene that confers a durable non-race-specific blast resistance to rice. Despite being non-R, Pb1 also encodes a CC-NB-LRR protein. Pb1 specifically interacts with rice WRKY45, a transcription factor (TF) in the rice salicylic acid signaling pathway, through its CC domain, and that this interaction is important for the blast resistance conferred by Pb1. Additionally, barley MLA10, an R protein for Blumeria graminis, specifically interacts with HvWRKY1/2. Here, we investigated whether the blast resistance between conferred by R genes and non-R Pb1 genes is mediated by their interaction with WRKY TFs. We performed targeted yeast two-hybrid and glutathione S-transferase (GST)-pull-down assays, which showed that all the five rice R proteins of the CC-NB-LRR type tested, Pi36, Pib, Pita, Pit, and Piz-t, interacted with WRKY45 and WRKY66 through their CC domains. However, elicitor-triggered immunity due to these R genes was not affected by the knockdown of these WRKY genes. The results suggest that R proteins can potentially transduce defense signals by interacting with WRKY TFs but the potentials are masked by HR-mediated elicitor-triggered immunity responses.

Background 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. Results 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. Conclusion 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. Electronic supplementary material The online version of this article (doi:10.1186/s12284-017-0175-0) contains supplementary material, which is available to authorized users.

Pikahei-1(t) is the strongest quantitative trait locus (QTL) for blast resistance in upland rice cv. Kahei, which has strong field resistance to the rice blast disease. A high-quality bacterial artificial chromosome library was used to fine-map Pikahei-1(t) within ~300 kb on the 31-Mb region on rice chromosome 4. Of the 42 predicted open reading frames, seven resistance gene analogs (RGAs) with the nucleotide-binding site and leucine-rich repeat (NBS-LRR) domain were identified. Among these, RGA1, 2, 3, 5, and 7, but not RGA4 and 6, were found to be expressed in Kahei and monogenic lines containing Pikahei-1(t). Blast inoculation of transgenic rice lines carrying the genomic fragment of each RGA revealed that only RGA3 was associated with blast resistance. On the basis of these results, we concluded that RGA3 is the Pikahei-1(t) and named it Pi63. Pi63 encoded a typical coiled-coil-NBS-LRR protein and showed isolate-specificity. These results suggest that Pi63 behaves like a typical Resistance (R) gene, and the strong and broad-spectrum resistance of Kahei is dependent on natural pyramiding of multiple QTLs. The blast resistance levels of Pi63 were closely correlated with its gene expression levels, indicating a dose-dependent response of Pi63 function in rice resistance. Pi63 is the first cloned R gene in the R gene cluster on rice chromosome 4, and its cloning might facilitate genomic dissection of this cluster region.

To study similar, but distinct, plant disease resistance (R) specificities exhibited by allelic genes at the rice blast resistance locus Pik/Pikm, we cloned the Pik gene from rice cultivar Kanto51 and compared its molecular features with those of Pikm and of another Pik gene cloned from cv. Kusabue. Like Pikm, Pik is composed of two adjacent NBS-LRR (nucleotide-binding site, leucine-rich repeat) genes: the first gene, Pik1-KA, and the second gene, Pik2-KA. Pik from Kanto51 and Pik from Kusabue were not identical; although the predicted protein sequences of the second genes were identical, the sequences differed by three amino acids within the NBS domain of the first genes. The Pik proteins from Kanto51 and Kusabue differed from Pikm in eight and seven amino acids, respectively. Most of these substituted amino acids were within the coiled-coil (CC) and NBS domains encoded by the first gene. Of these substitutions, all within the CC domain were conserved between the two Pik proteins, whereas all within the NBS domain differed between them. Comparison of the two Pik proteins and Pikm suggests the importance of the CC domain in determining the resistance specificities of Pik and Pikm. This feature contrasts with that of most allelic or homologous NBS-LRR genes characterized to date, in which the major specificity determinant is believed to lie in the highly diverged LRR domain. In addition, our study revealed high evolutionary flexibility in the genome at the Pik locus, which may be relevant to the generation of new R specificities at this locus.

Characterization of resistance genes is essential for the development of a new variety resistant to blast (Magnaporthe grisea) in rice (Oryza sativa L.). In this study, we found that an indica variety, Kasalath, harbored a resistance gene that showed a broad spectrum of resistance reactions against standard differential blast isolates from the Philippines and Japan. Quantitative trait locus (QTL) analysis was conducted using backcross inbred lines derived from crosses between Kasalath and the japonica cultivar, Koshihikari, to detect the QTL for blast resistance. QTLs were detected on chromosomes 6 and 8. Further, QTL analysis of chromosome segment substitution lines of Kasalath in the Koshihikari genetic background revealed that the Kasalath allele at the QTL on chromosome 6 strongly contributed to blast resistance. These results suggested that the gene locus might be identical to Pi13(t) that had already been reported. By marker-assisted breeding, a near-isogenic line, Koshihikari Toyama BL7, with a Kasalath chromosome segment containing Pi13(t) introduced into Koshihikari genetic background, was developed. The reaction of Koshihikari Toyama BL7 to the differential blast isolates indicated that Pi13(t) was a broad-spectrum and moderate resistance gene. Since Pi13(t) showed single-gene segregation and was characterized, the gene was re-designated as Pi13.

NPR1 is a central regulator of salicylic-acid (SA)-mediated defense signaling in Arabidopsis. Here, we report the characterization of OsNPR1, an Oryzae sativa (rice) ortholog of NPR1, focusing on its role in blast disease resistance and identification of OsNPR1-regulated genes. Blast resistance tests using OsNPR1 knockdown and overexpressing rice lines demonstrated the essential role of OsNPR1 in benzothiadiazole (BTH)-induced blast resistance. Genome-wide transcript profiling using OsNPR1-knockdown lines revealed that 358 genes out of 1,228 BTH-upregulated genes and 724 genes out of 1,069 BTH-downregulated genes were OsNPR1-dependent with respect to BTH responsiveness, thereby indicating that OsNPR1 plays a more vital role in gene downregulation. The OsNPR1-dependently downregulated genes included many of those involved in photosynthesis and in chloroplast translation and transcription. Reduction of photosynthetic activity after BTH treatment and its negation by OsNPR1 knockdown were indeed reflected in the changes in Fv/Fm values in leaves. These results imply the role of OsNPR1 in the reallocation of energy and resources during defense responses. We also examined the OsNPR1-dependence of SA-mediated suppression of ABA-induced genes.

Rice blast is one of the most widespread and destructive plant diseases worldwide. Breeders have used disease resistance (R) genes that mediate fungal race-specific 'gene-for-gene' resistance to manage rice blast, but the resistance is prone to breakdown due to high pathogenic variability of blast fungus. Panicle blast 1 (Pb1) is a blast-resistance gene derived from the indica cultivar 'Modan'. Pb1-mediated resistance, which is characterized by durability of resistance and adult/panicle blast resistance, has been introduced into elite varieties for commercial cultivation. We isolated the Pb1 gene by map-based cloning. It encoded a coiled-coil-nucleotide-binding-site-leucine-rich repeat (CC-NBS-LRR) protein. The Pb1 protein sequence differed from previously reported R-proteins, particularly in the NBS domain, in which the P-loop was apparently absent and some other motifs were degenerated. Pb1 was located within one of tandemly repeated 60-kb units, which presumably arose through local genome duplication. Pb1 transcript levels increased during the development of Pb1+ cultivars; this expression pattern accounts for their adult/panicle resistance. Promoter:GUS analysis indicated that genome duplication played a crucial role in the generation of Pb1 by placing a promoter sequence upstream of its coding sequence, thereby conferring a Pb1-characteristic expression pattern to a transcriptionally inactive 'sleeping' resistance gene. We discuss possible determinants for the durability of Pb1-mediated blast resistance.

Blast-Resistant Rice The durability of disease resistance for an agricultural crop reflects the extent to which the defense stands up to evolutionary dodges on the part of the pathogen. Pi21 , which is a quantitative trait locus (QTL) of rice, contributes to a particularly durable resistance to a fungal rice blast disease: Rice plants carrying the resistant allele have been in cultivation for more than a century, and yet the pathogen has been unable to find a way through the defense. Fukuoka et al. (p. 998 ; see the news story by Normile ) have now cloned the responsible Pi21 QTL allele and were able to separate Pi21 resistance from tightly linked reductions in grain quality, paving the way for more widespread use of this allele in rice breeding.

The Pik-h gene in rice confers resistance to several races of rice blast fungus (Magnaporthe oryzae), and has been classified as a member of the Pik cluster, one of the most resistance (R) gene-dense regions in the rice genome. However, the loss of a key mutant isolate has long made it difficult to differentiate Pik-h from other Pik group genes especially from Pik-m. We identified new natural isolates enabling the differentiation between Pik-h and Pik-m genes, and first confirmed the authenticity of the International Rice Research Institute (IRRI) “monogenic” line IRBLkh-K3, and then fine-mapped the Pik-h gene in the Pik cluster. Using 701 susceptible individuals among 3,060 siblings from a cross of IRBLkh-K3×CO39, the Pik-h region was delimited to 270 kb, the narrowest interval among the Pik group genes reported to date, in the cv. Nipponbare genome. Annotation of this genome region first revealed 6 NBS-LRR type R-gene analogs (RGAs), clustered within the central 120 kb, as possible counterparts of Pik-h and 6 other Pik group R genes. Interestingly, the Pik-h region and the cluster of RGAs were shown to be located 130 kb and 230 kb apart from Xa4 and Xa2 bacterial blight resistance genes, respectively, once classified as belonging to the Pik cluster. The closest recombination events were limited to the margins of the Pik-h region, and recombination was suppressed in the core interval with the RGA cluster. This fine-mapping, performed in a short time using an HEGS system, will facilitate utilization of the cluster’s genetic resources and help to elucidate the mechanism of evolution of R-genes. The presence of natural isolates also confirmed that evolution of Pik-h corresponds to pathogen evolution.

In plants, calcium acts as a universal second messenger in various signal transduction pathways. The plant-specific calcium-dependent protein kinases (CDPKs) play important roles regulating downstream components of calcium signaling. We conducted a genome-wide analysis of rice CDPKs and identified 29 CDPK genes and eight closely related kinase genes, including five CDPK-related kinases (CRKs), one calcium and calmodulin-dependent protein kinase (CCaMK) and two phosphoenolpyruvate (PEP) carboxylase kinase-related kinases (PEPRKs). The mRNA splicing sites of the rice CDPKs, CRKs and PEPRKs (but not OsCCaMK) are highly conserved, suggesting that these kinases are derived from a common ancestor. RNA gel blot analyses revealed that the majority of rice CDPK genes exhibited tissue-specific expression. Expression of OsCPK9 was elevated in seedlings infected by rice blast, indicating that this gene plays an important role in signaling in response to rice blast treatment. Our genomic and bioinformatic analyses will provide an important foundation for further functional dissection of the rice CDPK gene family.

インド型イネ「Modan」に由来する穂いもち圃場抵抗性遺伝子Pb1の発病抑制効果を定量的に評価する目的で，「黄金晴」（Pb1-+） ／ 「月の光」（Pb1）の交配後代に由来する約2,000のF7組換え型近交系（RILs）から作出したPb1座に関する3種のF8準同質遺伝子系統（NIL）ペアと， Pb1座およびイネ縞葉枯病抵抗性遺伝子Stvb-i座に関する「コシヒカリ」のNILとを用いて2ヶ所の検定地で2ヶ年にわたりいもち病抵抗性検定を行った．Pb1遺伝子の発病抑制効果は，葉いもち＜止葉葉いもち＜穂いもちの順に高く，イネの発育ステージの進展に応じてより強い圃場抵抗性を発現した． Pb1の罹病籾率低減効果を示す「防除価」は，2 ヶ年ともに平均93で極めて高い値を示し， Pb1は，幅広い穂いもち発生条件下において高い発病抑制効果を発現した．Pb1座に関する3種のNILペア内で Pb1を持つNILと持たないNILとの精玄米収量比は，穂いもち少発生条件では0.78 ～ 1.29（1.07±0.18）と1に近かったが，多発生条件では2.40～16.22と大きく，穂いもち多発条件ほど減収軽減効果は高かった．また，穂いもち多発生条件下で， Pb1を持つNILは，持たないNILより精玄米歩合が有意に高く，千粒重が有意に重く，玄米蛋白質含量が有意に低く， Pb1の穂いもち発病抑制効果により，品質および食味低下を軽減する二次的効果が認められた．

Effective control of blast, a devastating fungal disease of rice, would increase and stabilize worldwide food production. Resistance mediated by quantitative trait loci (QTLs), which usually have smaller individual effects than R-genes but confer broad-spectrum or non-race-specific resistance, is a promising alternative to less durable race-specific resistance for crop improvement, yet evidence that validates the impact of QTL combinations (pyramids) on the durability of plant disease resistance has been lacking. Here, we developed near-isogenic experimental lines representing all possible combinations of four QTL alleles from a durably resistant cultivar. These lines enabled us to evaluate the QTLs singly and in combination in a homogeneous genetic background. We present evidence that pyramiding QTL alleles, each controlling a different response to M. oryzae, confers strong, non-race-specific, environmentally stable resistance to blast disease. Our results suggest that this robust defence system provides durable resistance, thus avoiding an evolutionary "arms race" between a crop and its pathogen.

One hundred and twenty-nine monoconidial isolates of Pyricularia grisea from rice were collected in Vietnam, mainly in the Mekong River Delta (MRD), in 1995-1996 to characterize their virulence. These isolates were classified into 12 pathogenic groups (races) based on their virulence to 12 Japanese differential rice varieties (modified Kiyosawa's differentials). Race 002.4 was predominant and was found in 10 provinces of MRD, followed by races 106.4, 006.4, 102.4 and 002.0. Differential varieties Aichi Asahi (which carries resistance gene Pia) and K59 (Pit) were susceptible to 93.8% and 86.0% of the isolates, respectively. On the other hand, none of the isolates were virulent to differentials Shin 2 (Pik-s, Pish), Kusabue (Pik, Pish), Fukunishiki (Piz, Pish), Pi No.4 (Pita-2, Pish), Toride 1 (Piz-t), K60 (Pik-p) and BL1 (Pib, Pish). The reference rice variety AA/S2-3, which carries only one resistance gene Pish, was also resistant to all the isolates, but the variety AA/S2-75, carrying only Pik-s, was susceptible to 95.3% of the isolates. Therefore, the resistance in Shin 2, with Pik-s and Pish, might be due to Pish or an unknown gene(s). Reaction patterns of 45 Vietnamese rice varieties to four isolates belonging to different races, based on their virulence to the Japanese differential varieties, could be classified into eight groups. These results suggested that the pathogenicity of the Vietnamese isolates was complex and that the pathogenic specialization of Vietnamese isolates may be difficult to classify in detail using the Japanese differential varieties.

Probenazole (3-allyloxy-1,2-benzisothiazole-1,1-dioxide) is an agricultural chemical primarily used to prevent rice blast disease. Probenazole-treated rice acquires resistance to blast fungus irrespective of the rice variety. The chemical is applied prophylactically, and is thought to induce or bolster endogenous plant defenses. However, the mechanisms underlying this effect have not been established. To understand the mode of the chemical's action, we screened for novel probenazole-responsive genes in rice by means of differential display and identified a candidate gene, RPR1. RPR1 contains a nucleotide binding site and leucine-rich repeats, thus sharing structural similarity with known disease resistance genes. The expression of RPR1 in rice can be up-regulated by treatment with chemical inducers of systemic acquired resistance (SAR) and by inoculation with pathogens. RPR1-related sequences in rice varieties seem to be varied in sequence and/or expression, indicating that RPR1 itself is not a crucial factor for induced resistance in rice. However, Southern blot analysis revealed the existence of homologous sequences in all varieties examined. While the role of RPR1 has yet to be clarified, this is the first report of the identification of a member of this gene class and its induction during the systemic expression of induced disease resistance.

イネ縞葉枯ウイルス (RSV) 抵抗性の日本型水稲品種「月の光」および同じくRSV抵抗性をもつその姉妹品種「朝の光」が有する穂いもち抵抗性の遺伝子を同定するため, これら品種と感受性品種との交雑F1, F3およびF4を用いて穂いもち抵抗性に関する遺伝子分析を行った.「月の光」と感受性の「あ系他494」とのF1における穂いもち罹病籾率の逆正弦変換値は, 「月の光」のそれと有意差がなく, 穂いもち抵抗性が優性形質であることが明らかになった. 一方, 「朝の光」と感受性品種の「コシヒカリ」とのF3119系統は, 朝の光型固定系統, 分離型系統およびコシヒカリ型固定系統の明瞭な3群に分類され, その比は1遺伝子分離の期待比1: 2: 1に適合した.また, 「月の光」と感受性品種の「黄金晴」とのF460系統は, 月の光型固定系統, 分離型系統および黄金晴型固定系統に分類され, その比は1遺伝子分離の期待比3: 2: 3に適合した. これらの結果と, 「月の光」と「朝の光」の育成歴から, これら2品種の穂いもち抵抗性は同一の優性主働遺伝子に支配されていると考えられた. この穂いもち抵抗性遺伝子は, その作用力からみて新規の遺伝子とみなせることから, Pb1と命名した. Pb1とRSV抵抗性遺伝子Stvbiとの連鎖分析を, 両抵抗性を欠くコシヒカリと両抵抗性を持つ朝の光とのF3119系統を用いて行ったところ, 両抵抗性遺伝子は組換価5.2±1.5%で連鎖していることが明らかとなった. また, 月の光/黄金晴のF460系統, 葵の風の育成過程で得られたRSV抵抗性と感受性の姉妹系統6種を用いた分析でも, 両抵抗性遺伝子間の連鎖が確認された.

The IRRI-Japan Collaborative Research Project has developed four universal differential variety sets, monogenic lines (MLs) with a Japonica-type variety Lijianxintuanheigu (LTH) genetic background, and near-isogenic lines (NILs) with three different genetic backgrounds, LTH, an Indica type variety CO39, and a universal susceptible line, US-2. These lines targeted 24 resistance genes:Pia, Pib, Pii, Pik, Pik-h, Pik-m, Pik-p, Pik-s, Pish, Pita(Pi4), Pita-2, Pit, Piz, Piz-5(Pi-2), Piz-t, Pi1, Pi3, Pi5(t), Pi7(t), Pi9, Pi11(t), Pi12(t), Pi19 and Pi20(t). The MLs have been distributed to more than 15 countries. To characterize these genetic components and confirm the introgression of chromosome segments harboring resistance genes, the graphical genotypes were determined using 162 simple sequence repeat (SSR) markers distributed throughout the rice genome. The genotypes of the three sets of NILs were more uniform than those of MLs. Several introgression segments, which corresponded to the locations of blast resistance genes, were also confirmed. The genome restoration rates of 31 MLs to LTH ranged from 50 to 90.0%, averaging 77.3%. All LTH, CO39, and US-2 NILs were developed by backcrossing six times with each recurrent parent. The genome restoration rates of the parent in 34 LTH NILs ranged from 75.6 to 96.9% with an average of 90.6%, lower than the theoretical value of 99%. The 31 CO39 NILs showed greater than 90% genome restoration rate compared to the recurrent parent with an average of 97.3%. The genomic restoration rate of the 16 US-2 NILs closely resembled those of the recurrent parent, with frequencies ranging from 88.9 to 98.8% with an average of 94.6%. Genetic characterization of four universal differential variety sets was carried out using DNA markers. This information will be linked with resistance genes that are potentially very useful for marker-assisted selection (MAS) in rice breeding programs, since the differential varieties can be applied as genetic sources.

The rice blast resistance gene Pikm was cloned by a map-based cloning strategy. High-resolution genetic mapping and sequencing of the gene region in the Pikm-containing cultivar Tsuyuake narrowed down the candidate region to a 131-kb genomic interval. Sequence analysis predicted two adjacently arranged resistance-like genes, Pikm1-TS and Pikm2-TS, within this candidate region. These genes encoded proteins with a nucleotide-binding site (NBS) and leucine-rich repeats (LRRs) and were considered the most probable candidates for Pikm. However, genetic complementation analysis of transgenic lines individually carrying these two genes negated the possibility that either Pikm1-TS or Pikm2-TS alone was Pikm. Instead, it was revealed that transgenic lines carrying both of these genes expressed blast resistance. The results of the complementation analysis and an evaluation of the resistance specificity of the transgenic lines to blast isolates demonstrated that Pikm-specific resistance is conferred by cooperation of Pikm1-TS and Pikm2-TS. Although these two genes are not homologous with each other, they both contain all the conserved motifs necessary for an NBS–LRR class gene to function independently as a resistance gene.

We developed an efficient method to analyze gene function and expression of the rice blast fungus. We constructed a GATEWAY binary vector, which generates a gene-targeted disruptant carrying a green fluorescent protein gene under the native promoter of the target gene. Using this method, the knockout efficiency and expression patterns of two hypothetical genes were determined.

A lesion mimic mutant that we designated Spotted leaf 18 (Spl18) was isolated from 13,000 activation-tagging lines of rice produced by our modified activation-tagging vector and further characterized. Spl18 was dominant and its phenotype was linked to the T-DNA insertion. An ORF was located about 500 bp downstream of the inserted T-DNA, and the deduced protein, designated OsAT1, showed sequence similarity to an acyltransferase whose expression is induced by hypersensitive reaction in tobacco. The transcriptional level of OsAT1 was very low in the WT leaf blade but high in Spl18 leaf blade. In wild-type rice, OsAT1 was transcribed mainly in the young panicle, in the panicle just after heading, and in the leaf sheath. In addition, transcription of the genes for PR protein was upregulated in Spl18, accumulation of phytoalexins (both momilactone A and sakuranetin) was increased, and resistance to blast disease was improved. We then combined OsAT1 genomic DNA downstream of the modified 35S promoter and re-transformed it into rice. Lesion mimic and blast resistance phenotypes were detected in the transgenic lines produced, clearly indicating that overexpression of OsAT1 caused the Spl18 phenotypes. In addition, plants overexpressing OsAT1 showed resistance to bacterial blight.

Race-cultivar specificity of the rice blast fungus complies with the ‘genefor-gene’ system. Several avirulence genes, which determine cultivar specificity have been identified and characterized (Valent and Chumley, 1994, Notteghem et al., 1994, Mandel et al., 1997, Dioh et al., 2000, Kang et al., 2001, Fukiya et al., 2001, Farman et al., 2002). However, no common features of these genes other than recognition factors have been found. The molecular mechanisms behind race-cultivar interactions might vary with the host-pathogen combinations, while the mode and frequency of the avirulence gene mutations resulting in virulent races might vary with the genes. Studies on the molecular background and evolution of race-cultivar interactions will lead to the effective use of resistance genes. In this paper, we describe and identify new avirulence genes and corresponding resistance genes in the rice blast system.

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.

The complete nucleotide sequence of segment 5 (S5) of the rice dwarf virus (RDV) genome was determined. RDV S5 is 2571 bp in length and has a single long open reading frame which encodes a polypeptide of 801 amino acids (M r 90495). When compared to the wound tumor virus genome S5, there was 56.9% and 52.8% similarity in the nucleotide and amino acid sequences, respectively. This high similarity suggests that the S5 proteins of these viruses are functionally similar.