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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 (

The pathogenicity of 192 blast isolates collected from regions across the whole of Laos from 2007 to 2009 showed a wide variation in terms of the frequencies of virulence toward differential varieties (DVs) and the susceptible control cultivar Lijiangxintuanheigu. High frequencies of virulence (>50%) were found in the reactions of DVs for Pit, Pia, Pi3, Pi5(t), Pik-s, Piz-t, Pi19(t), and Pi20(t); intermediate values (from 10 to 50%) were found in DVs for Pib, Pii, Pik-m, Pi1, Pik-h, Pik, Pik-p, Pi7(t), Piz-5, Pita (two lines), Pita-2 (two lines), and Pi12(t); and low frequencies (

A total of 99 isolates of rice blast (Pyricularia oryzae Cavara) were collected from 2010 to 2015 from four regions in Kenya: Kirinyaga County and Embu County, Kisumu County, Tana River County, and Mombasa County. The pathogenicities of these isolates were clarified based on the reaction patterns of Lijiangxintuanheigu and differential varieties (DVs) targeting 23 resistance genes. The frequency of virulent isolates was high for DVs for Pib, Pia, Pii, Pi3, Pi5(t), Pik-s, Pik-m, Pi1, Pik-h, Pik, Pik-p, Pi7(t), Pi19(t), and Pi20(t); low for DVs for Pish, Pi9(t), Piz-5, and Piz-t; and intermediate for the remaining DVs for Pit, Piz, Pita-2, Pita, and Pi12(t). These blast isolates were classified into three cluster groups: Ia, Ib, and II. The frequencies of virulent isolates to DVs for Pit, Pii, Pik-m, Pi1, Pik-h, Pik, Pik-p, Pi7(t), Piz, and Pi12(t) differed markedly between clusters I and II, and those of DVs for Pib, Pit, Pia, Pi3, Pita-2, Pita, and Pi20(t) differed between Ia and Ib. The frequencies of cluster groups in the four geographical regions were different. A total of 62 races were found, with 19 blast isolates categorized into one race (U63-i7-k177-z00-ta003), whereas the other races included only some isolates in each.

A total of 47 rice accessions collected from Kenya were investigated the genetic variations and classified into two cluster groups, A and B, by polymorphism data of 65 simple sequence repeat (SSR) markers. Clusters A and B corresponded to Japonica and Indica Groups, respectively. The number of Japonica Group accessions was limited in comparison with those of the Indica Group. Based on their patterns of reaction to standard differential blast isolates (SDBIs), these accessions and 57 control cultivars including differential varieties and several accessions harboring partial resistance genes were classified again into three cluster groups: Ia (high resistance), Ib (intermediate resistance) and II (susceptible). The rice accessions from Kenya were classified only into groups Ia and Ib. The accessions from Kenya were finally classified into three categories, A-Ia, B-Ia and B-Ib, based on the two classifications of polymorphism of SSR markers and resistance. The Indica Group accessions had wider genetic variation for blast resistance than did the Japonica Group accessions. The three leading cultivars (Basmati 217, Basmati 370 and ITA 310) categorized into Cluster group Ia were susceptible to some SDBIs from Kenya. The genetic variation for blast resistance in Kenya was demonstrated as the first report using SDBIs.

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.

The fungal pathogen Pyricularia oryzae causes blast, a severe disease of rice (Oryza sativa L.). Improving blast resistance is important in rice breeding programs. Inoculation tests have been used to select for resistance genotypes, with DNA marker-based selection becoming an efficient alternative. No comprehensive DNA marker system for race-specific resistance alleles in the Japanese rice breeding program has been developed because some loci contain multiple resistance alleles. Here, we used the Fluidigm SNP genotyping platform to determine a set of 96 single nucleotide polymorphism (SNP) markers for 10 loci with race-specific resistance. The markers were then used to evaluate the presence or absence of 24 resistance alleles in 369 cultivars; results were 93.5% consistent with reported inoculation test-based genotypes in japonica varieties. The evaluation system was successfully applied to high-yield varieties with indica genetic backgrounds. The system includes polymorphisms that distinguish the resistant alleles at the tightly linked Pita and Pita-2 loci, thereby confirming that all the tested cultivars with Pita-2 allele carry Pita allele. We also developed and validated insertion/deletion (InDel) markers for ten resistance loci. Combining SNP and InDel markers is an accurate and efficient strategy for selection for race-specific resistance to blast in breeding programs.

Plants have evolved many receptor-like cytoplasmic kinases (RLCKs) to modulate their growth, development, and innate immunity. Broad-Spectrum Resistance 1 (BSR1) encodes a rice RLCK, whose overexpression confers resistance to multiple diseases, including fungal rice blast and bacterial leaf blight. However, the mechanisms underlying resistance remain largely unknown. In the present study, we report that BSR1 is a functional protein kinase that autophosphorylates and transphosphorylates an artificial substrate in vitro. Although BSR1 is classified as a serine/threonine kinase, it was shown to autophosphorylate on tyrosine as well as on serine/threonine residues when expressed in bacteria, demonstrating that it is a dual-specificity kinase. Protein kinase activity was found to be indispensable for resistance to rice blast and leaf blight in BSR1-overexpressing plants. Importantly, tyrosine phosphorylation of BSR1 was critical for proper localization of BSR1 in rice cells and played a crucial role in BSR1-mediated resistance to multiple diseases, as evidenced by compromised disease resistance in transgenic plants overexpressing a mutant BSR1 in which Tyr-63 was substituted with Ala. Overall, our data indicate that BSR1 is a non-receptor dual-specificity kinase and that both tyrosine and serine/threonine kinase activities are critical for the normal functioning of BSR1 in the resistance to multiple pathogens. Our results support the notion that tyrosine phosphorylation plays a major regulatory role in the transduction of defense signals from cell-surface receptor complexes to downstream signaling components in plants.

Membrane trafficking plays pivotal roles in many cellular processes including plant immunity. Here, we report the characterization of OsVAMP714, an intracellular SNARE protein, focusing on its role in resistance to rice blast disease caused by the fungal pathogen Magnaporthe oryzae. Disease resistance tests using OsVAMP714 knockdown and overexpressing rice plants demonstrated the involvement of OsVAMP714 in blast resistance. The overexpression of OsVAMP7111, whose product is highly homologous to OsVAMP714, did not enhance blast resistance to rice, implying a potential specificity of OsVAMP714 to blast resistance. OsVAMP714 was localized to the chloroplast in mesophyll cells and to the cellular periphery in epidermal cells of transgenic rice plant leaves. We showed that chloroplast localization is critical for the normal OsVAMP714 functioning in blast resistance by analyzing the rice plants overexpressing OsVAMP714 mutants whose products did not localize in the chloroplast. We also found that OsVAMP714 was located in the vacuolar membrane surrounding the invasive hyphae of M. oryzae. Furthermore, we showed that OsVAMP714 overexpression promotes leaf sheath elongation and that the first 19 amino acids, which are highly conserved between animal and plant VAMP7 proteins, are crucial for normal rice plant growths. Our studies imply that the OsVAMP714-mediated trafficking pathway plays an important role in rice blast resistance as well as in the vegetative growth of rice.

WRKY45 is an important transcription factor in the salicylic acid signalling pathway in rice that mediates chemical-induced resistance against multiple pathogens. Its constitutive overexpression confers extremely strong resistance against Magnaporthe oryzae and Xanthomonas oryzae pv. oryzae to rice, but has adverse effects on agronomic traits. Here, a new strategy to confer rice with strong disease resistance without any negative effects on agronomic traits was established by expressing WRKY45 under the control of pathogen-responsive promoters in combination with a translational enhancer derived from a 5'-untranslated region (UTR) of rice alcohol dehydrogenase (ADH). Rice promoters that responded to M. oryzae and X. oryzae pv. oryzae infections within 24 h were identified, and 2-kb upstream sequences from nine of them were isolated, fused to WRKY45 cDNA with or without the ADH 5'-UTR, and introduced into rice. Although pathogen-responsive promoters alone failed to confer effective disease resistance, the use of the ADH 5'-UTR in combination with them, in particular the PR1b and GST promoters, enhanced disease resistance. Field trials showed that overall, PR1b promoter-driven (with ADH 5'-UTR) lines performed the best and one had agronomic traits comparable to control untransformed rice. Thus, expressing WRKY45 under the control of the PR1b promoter with the ADH 5'-UTR is an excellent strategy to develop disease-resistant rice, and the line established could serve as a mother line for breeding disease-resistant rice.

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.

The evaluation of a total of 122 blast (Pyricularia oryzae Cavara) isolates collected from the Tonle Sap and Mekong river regions of Cambodia revealed wide variation. Using a new designation system, the blast isolates were categorized into 92 races based on the reaction patterns of rice (Oryza sativa L.) differential varieties (DVs) harboring 23 resistance genes and of 1 susceptible cultivar, Lijiangxintuanheigu (LTH). Cluster analysis was used to classify the blast isolates into 3 groups — I, IIa, and IIb — using data from these reaction patterns of the DVs and LTH. We used the classifications established under the new designation system, alongside cluster analysis and the geographical distribution of blast isolates, to investigate the diversity and differentiation of blast races in the Tonle Sap and Mekong river regions. The distributions of the blast races differed between the 2 regions, although blast isolates of group IIa were distributed commonly in both regions and groups I and IIb occurred at higher frequencies in the Tonle Sap region rather than the Mekong region. The blast isolates in groups I and IIb were also less diverse than those in group IIa. Accordingly, Group II blast isolates overall were distributed in both regions with high diversity, but some modified blast isolates were additionally distributed in the Tonle Sap region. We also investigated the pathogenicities of blast isolates from wild rice (Oryza rufipogon Griff) weeds neighboring the cultivated rice, and discuss the relationship between these isolates and those from cultivated rice. Discipline: Plant disease Additional key words: cluster analysis, differential variety, diversity, pathotype, resistance gene

Panicle blast 1 ( Pb1 ) is a panicle blast resistance gene derived from the indica rice cultivar “Modan.” Pb1 encodes a coiled-coil–nucleotide-binding site–leucine-rich repeat (CC-NB-LRR) protein and confers durable, broad-spectrum resistance to Magnaporthe oryzae races. Here, we investigated the molecular mechanisms underlying Pb1 -mediated blast resistance. The Pb1 protein interacted with WRKY45, a transcription factor involved in induced resistance via the salicylic acid signaling pathway that is regulated by the ubiquitin proteasome system. Pb1 -mediated panicle blast resistance was largely compromised when WRKY45 was knocked down in a Pb1 -containing rice cultivar. Leaf-blast resistance by Pb1 overexpression ( Pb1 -ox) was also compromised in WRKY45 knockdown /Pb1 -ox rice. Blast infection induced higher accumulation of WRKY45 in Pb1 -ox than in control Nipponbare rice. Overexpression of Pb1-Quad , a coiled-coil domain mutant that had weak interaction with WRKY45, resulted in significantly weaker blast resistance than that of wild-type Pb1 . Overexpression of Pb1 with a nuclear export sequence failed to confer blast resistance to rice. These results suggest that the blast resistance of Pb1 depends on its interaction with WRKY45 in the nucleus. In a transient system using rice protoplasts, coexpression of Pb1 enhanced WRKY45 accumulation and increased WRKY45-dependent transactivation activity, suggesting that protection of WRKY45 from ubiquitin proteasome system degradation is possibly involved in Pb1-dependent blast resistance.

Calcium-dependent protein kinases (CDPKs) constitute a large multigene family in various plant species. CDPKs have been shown to have important roles in various physiological processes, including plant growth and development and abiotic and biotic stress responses in plants. Functional analysis using gain-of-function and loss-of-function mutants has revealed the biological function of CDPKs in planta. Several CDPKs have been shown to be essential factors in abiotic stress tolerance, positively or negatively regulating stress tolerance by modulating ABA signaling and reducing the accumulation of reactive oxygen species (ROS). This review summarizes recent results describing the biological function of CDPKs that are involved in abiotic stress tolerance.

Rho family small GTPases are involved in diverse signaling processes including immunity, growth, and development. The activity of Rho GTPases is regulated by cycling between guanosine diphosphate (GDP)-bound inactive and guanosine triphosphate (GTP)-bound active forms, in which guanine nucleotide exchange factors (GEFs) predominantly function to promote activation of the GTPases. In animals, most Rho GEFs possess a Dbl (diffuse B-cell lymphoma) homology (DH) domain which functions as a GEF-catalytic domain. However, no proteins with the DH domain have been identified in plants so far. Instead, plant-specific Rho GEFs with the PRONE domain responsible for GEF activity have been found to constitute a large family in plants. In this study, we found rice homologs of human SWAP70, Oryza sativa (Os) SWAP70A and SWAP70B, containing the DH domain. OsSWAP70A interacted with rice Rho GTPase OsRac1, an important signaling factor for immune responses. The DH domain of OsSWAP70A exhibited the GEF-catalytic activity toward OsRac1 as found in animal Rho GEFs, indicating that plants have the functional DH domains. Transient expression of OsSWAP70A enhanced OsRac1-mediated production of reactive oxygen species in planta. Reduction of OsSWAP70A and OsSWAP70B mRNA levels by RNA interference resulted in the suppression of chitin elicitor-induced defense gene expression and ROS production. Thus, it is likely that OsSWAP70 regulates immune responses through activation of OsRac1.

*† ‡ SUMMARY Calcium-dependent protein kinases (CDPKs) regulate the downstream components in calcium signaling pathways. We investigated the effects of overexpression and disruption of an Oryza sativa (rice) CDPK (OsCPK12) on the plant’s response to abiotic and biotic stresses. OsCPK12-overexpressing (OsCPK12-OX) plants exhibited increased tolerance to salt stress. The accumulation of hydrogen peroxide (H2O2) in the leaves was less in OsCPK12-OX plants than in wild-type (WT) plants. Genes encoding reactive oxygen species (ROS) scavenging enzymes (OsAPx2 and OsAPx8) were more highly expressed in OsCPK12-OX plants than in WT plants, whereas the expression of the NADPH oxidase gene, OsrbohI, was decreased in OsCPK12-OX plants compared with WT plants. Conversely, a retrotransposon (Tos17) insertion mutant, oscpk12, and plants transformed with an OsCPK12 RNA interference (RNAi) construct were more sensitive to high salinity than were WT plants. The level of H2O2 accumulation was greater in oscpk12 and OsCPK12 RNAi plants than in the WT. These results suggest that OsCPK12 promotes tolerance to salt stress by reducing the accumulation of ROS. We also observed that OsCPK12-OX seedlings had increased sensitivity to abscisic acid (ABA) and increased susceptibility to blast fungus, probably resulting from the repression of ROS production and/or the involvement of OsCPK12 in the ABA signaling pathway. Collectively, our results suggest that OsCPK12 functions in multiple signaling pathways, positively regulating salt tolerance and negatively modulating blast resistance.

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.

SUMMARY Plant ‘activators’, such as benzothiadiazole (BTH), protect plants from various diseases by priming the plant salicylic acid (SA) signalling pathway. We have reported previously that a transcription factor identified in rice, WRKY45 (OsWRKY45), plays a pivotal role in BTH-induced disease resistance by mediating SA signalling. Here, we report further functional characterization of WRKY45. Different plant activators vary in their action points, either downstream (BTH and tiadinil) or upstream (probenazole) of SA. Rice resistance to Magnaporthe grisea, induced by both types of plant activator, was markedly reduced in WRKY45-knockdown (WRKY45-kd) rice, indicating a universal role for WRKY45 in chemical-induced resistance. Fungal invasion into rice cells was blocked at most attempted invasion sites (pre-invasive defence) in WRKY45-overexpressing (WRKY45-ox) rice. Hydrogen peroxide accumulated within the cell wall underneath invading fungus appressoria or between the cell wall and the cytoplasm, implying a possible role for H2O2 in pre-invasive defence. Moreover, a hypersensitive reaction-like reaction was observed in rice cells, in which fungal growth was inhibited after invasion (post-invasive defence). The two levels of defence mechanism appear to correspond to Type I and II nonhost resistances. The leaf blast resistance of WRKY45-ox rice plants was much higher than that of other known blast-resistant varieties. WRKY45-ox plants also showed strong panicle blast resistance. BTH-induced resistance to Xanthomonas oryzae pv. oryzae was compromised in WRKY45-kd rice, whereas WRKY45-ox plants were highly resistant to this pathogen. However, WRKY45-ox plants were susceptible to Rhizoctonia solani. These results indicate the versatility and limitations of the application of this gene.

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.

Approximately 20,000 of the rice-FOX Arabidopsis transgenic lines, which overexpress 13,000 rice full-length cDNAs at random in Arabidopsis, were screened for bacterial disease resistance by dip inoculation with Pseudomonas syringae pv. tomato DC3000 (Pst DC3000). The identities of the overexpressed genes were determined in 72 lines that showed consistent resistance after three independent screens. Pst DC3000 resistance was verified for 19 genes by characterizing other independent Arabidopsis lines for the same genes in the original rice-FOX hunting population or obtained by reintroducing the genes into ecotype Columbia by floral dip transformation. Thirteen lines of these 72 selections were also resistant to the fungal pathogen Colletotrichum higginsianum. Eight genes that conferred resistance to Pst DC3000 in Arabidopsis have been introduced into rice for overexpression, and transformants were evaluated for resistance to the rice bacterial pathogen, Xanthomonas oryzae pv. oryzae. One of the transgenic rice lines was highly resistant to Xanthomonas oryzae pv. oryzae. Interestingly, this line also showed remarkably high resistance to Magnaporthe grisea, the fungal pathogen causing rice blast, which is the most devastating rice disease in many countries. The causal rice gene, encoding a putative receptor-like cytoplasmic kinase, was therefore designated as BROAD-SPECTRUM RESISTANCE 1. Our results demonstrate the utility of the rice-FOX Arabidopsis lines as a tool for the identification of genes involved in plant defence and suggest the presence of a defence mechanism common between monocots and dicots.

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.

The APSES protein family includes important transcriptional regulators of morphological processes in ascomycetes. We identified a deletion mutant of the APSES protein Mstu1 in Magnaporthe grisea that showed reduced conidiation and mycelial growth. Mstu1 formed a number of appressoria comparable to the wild type, although appressorium formation was delayed. In M. grisea, rapid transfer of conidial glycogen and lipid droplets to incipient appressoria is required for appressorial turgor generation, which the fungus uses to penetrate plant cuticles. Appressorial turgor was low in mstu1 and the mutant was deficient in appressorium-mediated invasion of rice leaves. The transfer of conidial glycogen and lipid droplets was remarkably delayed in mstu1, and a consequent delay in degradation of these conidial reserves was observed. Our results indicate that Mstu1 is required for appressorium-mediated infection due to its involvement in the mobilization of lipids and glycogen.

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.

A conidia suspension of Magnaporthe grisea carried elicitor activity that induced the expression of defense-related genes and the production of H(2)O(2) in suspension-cultured rice cells. The levels of H(2)O(2) produced were dependent on fungal isolates and were correlated with the catalase activity in the supernatant fraction of each conidia suspension, not with gene-for-gene interactions.

The OsNAC6 gene is a member of the NAC transcription factor gene family in rice. Expression of OsNAC6 is induced by abiotic stresses, including cold, drought and high salinity. OsNAC6 gene expression is also induced by wounding and blast disease. A transactivation assay using a yeast system demonstrated that OsNAC6 functions as a transcriptional activator, and transient localization studies with OsNAC6-sGFP fusion protein revealed its nuclear localization. Transgenic rice plants over-expressing OsNAC6 constitutively exhibited growth retardation and low reproductive yields. These transgenic rice plants showed an improved tolerance to dehydration and high-salt stresses, and also exhibited increased tolerance to blast disease. By utilizing stress-inducible promoters, such as the OsNAC6 promoter, it is hoped that stress-inducible over-expression of OsNAC6 in rice can improve stress tolerance by suppressing the negative effects of OsNAC6 on growth under normal growth conditions. The results of microarray analysis revealed that many genes that are inducible by abiotic and biotic stresses were upregulated in rice plants over-expressing OsNAC6. A transient transactivation assay showed that OsNAC6 activates the expression of at least two genes, including a gene encoding peroxidase. Collectively, these results indicate that OsNAC6 functions as a transcriptional activator in response to abiotic and biotic stresses in plants. We conclude that OsNAC6 may serve as a useful biotechnological tool for the improvement of stress tolerance in various kinds of plants.

To clarify the geographic variation and isolate specificity in blast resistance, 20 cultivars of foxtail millet (Setaria italica (L.) P. Beauv.) originating from Eurasia were examined for their resistance using 11 Japanese Setaria isolates of blast fungus. Cultivars from the countries east of Pakistan generally showed resistance to most of the 11 fungus isolates, whereas those from the countries west of Afghanistan indicated higher susceptibility. The origin of this geographical regularity was discussed in relation to the deficiency or specificity of relevant resistance gene(s). No identical reaction pattern was observed among cultivars, indicating their highly distinctive isolate specificity. The virulence of isolates was also diverse, since only 2 out of 10 virulent isolates were identical for their reaction patterns. Preliminary genetic analysis of blast resistance to four fungus isolates suggests that they are governed by more than two dominant genes.

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.

現在,防除を要する水稲の主要病害は紋枯病を除いて,いもち病,ごま葉枯病,ばか苗病,もみ枯細菌病,苗立枯細菌病等種子伝染性病害が大部分を占める.これらの種子伝染性病害の根本的な防除を目的として無病種子の作成を試みた.通常種子として用いるもみでの種子伝染性病害病原菌の存在部位は主にもみ殻で,玄米はほぼ無病原菌であることから,玄米に保護膜として水溶性ポリアクリル樹脂と粉末活性炭で二重被膜した水稲の無病化種子“玄米種子”を開発した.本玄米種子を育苗箱で通常に育苗することにより育苗期の病害であるばか苗病,もみ枯細菌病,苗立枯細菌病,本田移植後ではいもち病が種子消毒剤による薬剤防除とほぼ同等に防除可能であることが明らかになった.本種子を用いることにより種子もみの長期間の種子浸漬や種子消毒の削除,種子消毒剤の廃液処理問題解消,本田期での種子伝染性病害防除作業の削減が可能となり,種子伝染性病害の根本的防除および,環境と人間に優しい農業確立に大きく貢献するものである.

We precisely mapped the Pb1 locus for the panicle blast resistence of a quantitative nature on rice chromosome 11 using RFLP markers. Based on the cosegregation of the resistant genotypes of Pb1 and Stvb-i, a rice stripe virus (RSV) resistance gene derived from an Indica cultivar Modan, we examined the linkage relationships between Pb1 and 13 Stvb-i-linked RFLP markers located on the long arm of chromosome 11. The Pb1 locus was mapped in the Modan-derived chromosomal region in the middle part of the long arm of chromosome 11. Pb1 was located on the telomere side in relation to the Stvb-i locus. The Pb1 gene was closely located at 1.2cM from three RFLP markers: S723, CDO226, C189. Then, we examined the graphical genotypes of 34 Modan-derived RSV-resistant cultivars with or without panicle blast resistance, and 12 susceptible Japonica cultivars, using 21 RFLP markers. Among them, cultivars with panicle blast resistance were classified into four types: A, B, C and D, and those without it into six types: E, F, G, H, I and J. In all of the panicle blast-resistant cultivars, the Modan-type bands were observed in S723, CDO226 and C189. On the other hand, in all the panicle blast-susceptible cultivars, the Japonica-type band was observed in S723, whereas the Modan-type bands were noticed in CDO226 and C189 in Type E cultivars. Consequently, only the genotypes of the S723 locus completely coincided with the genotypes of the Pb1 locus. Although, it was determined, based on linkage analysis, that S723, CDO226 and C189 were located at the same locus, graphical genotyping analysis, using many progeny cultivars derived from Modan, revealed that S723 was the closest marker to Pb1 among the three.

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.

Genetic analysis of the rice cultivar Aichi Asahi and some other Japanese cultivars for the high resistance to the blast fungus isolate CHNOS58-3-1 from China was performed. All the Japanese differential cultivars were resistant to the isolate except for ‘Pi No. 4’, which showed moderate resistance. Analysis of the F2 population of a cross of the susceptible cultivar Reiho and the resistant cultivar Aichi Asahi indicated that the resistance of ‘Aichi Asahi’ to the isolate was conferred by one dominant gene. To identify the gene in other Japanese differential cultivars, AK lines, which were derived from a cross of ‘Aichi Asahi’ × ‘K59’ and assumed to harbor no known genes except for the new one, were used for the allelism tests. The new, completely dominant resistance gene was detected in 14 differential cultivars, but not in ‘Pi No. 4’, ‘Yashiro-mochi’, and ‘K1’, and was designated as Pi19(t). Pi19(t) was allelic or closely linked to Pita2 on chromosome 12. Pi19(t) was extensively distributed among Japanese traditional local cultivars.

We have constructed a BAC library of the rice blast fungus Magnaporthe grisea consisting of 5760 clones. The insert size ranged from 35 to 175 kbp, with an average of 120 kbp. The library is about 18 genomes equivalent, therefore covering more than 99.999% of the genome. This library is the first to be constructed using a rice pathogenic wild type isolate. Improved high molecular weight DNA size fractionating helped to construct the library with high efficiency. Total library clones were arranged onto two nylon membranes for efficient screening. Test hybridization with a single-copy RFLP marker showed ten positive clones, of which restriction patterns indicated no chimerality or deletions. As a model case of application of this library, the distribution of the well-studied fungal retrotransposons MGSR1, MGR583, and MAGGY and DNA transposons MGR586 and Pot2 was analyzed. Of all the BAC clones, 10%, 13%, 18%, 12%, and 23% contained MGSR1, MGR583, MAGGY, MGR586 and Pot2, respectively. The percentage of clones possessing more than five kinds of transposons was 1.4%, 215 times greater than the expected number. The results show that these transposons were distributed in clusters in the M. grisea genome.

Three hundred eight isolates of Magnaporthe grisea were collected from rice plants in 14 counties of Yunnan Province, China in 1988-89. Each isolate was tested for mating type and fertility with Mat1-1 and Mat1-2 female fertile tester strains. Three percent of these isolates consisted of Mat1-1, and 23% of Mat1-2. The remaining 74% isolates did not produce perithecia in crosses with both Mat1-1 and Mat1-2 fertile testers. The percentage of fertile isolates in each county ranged from 90% in Lincang to 0% in Lijiang, Dali, and Honghe. Almost all the isolates were differentiated into races based on the reaction of Japanese differential cultivars. Sixteen races were identified within the fertile isolates. Isolates of both mating types were found in two counties, Qujing and Xishuangbanna, and those in Xishuangbanna were slightly interfertile. In contrast, crosses between the hermaphroditic isolate CHNOS37-1-1 from Xishuangbanna and three male isolates from other counties led to the successful production of large numbers of perithecia containing viable ascospores. The crosses were fertile enough to permit the isolation of germinating ascospores. The southwestern area where fertile blast fungi were isolated with a considerable frequency, coincided with the area with a high diversity of rice cultivars.

The transcriptional activator WRKY45 plays a major role in the salicylic acid/benzothiadiazole-induced defense program in rice. Here, we show that the nuclear ubiquitin-proteasome system (UPS) plays a role in regulating the function of WRKY45. Proteasome inhibitors induced accumulation of polyubiquitinated WRKY45 and transient up-regulation of WRKY45 target genes in rice cells, suggesting that WRKY45 is constantly degraded by the UPS to suppress defense responses in the absence of defense signals. Mutational analysis of the nuclear localization signal indicated that UPS-dependent WRKY45 degradation occurs in the nuclei. Interestingly, the transcriptional activity of WRKY45 after salicylic acid treatment was impaired by proteasome inhibition. The same C-terminal region in WRKY45 was essential for both transcriptional activity and UPS-dependent degradation. These results suggest that UPS regulation also plays a role in the transcriptional activity of WRKY45. It has been reported that AtNPR1, the central regulator of the salicylic acid pathway in Arabidopsis, is regulated by the UPS. We found that OsNPR1/NH1, the rice counterpart of NPR1, was not stabilized by proteasome inhibition under uninfected conditions. We discuss the differences in post-translational regulation of salicylic acid pathway components between rice and Arabidopsis.

Plant hormones play pivotal signaling roles in plant–pathogen interactions. Here, we report characterization of an antagonistic interaction of abscisic acid (ABA) with salicylic acid (SA) signaling pathways in the rice–Magnaporthe grisea interaction. Exogenous application of ABA drastically compromised the rice resistance to both compatible and incompatible M. grisea strains, indicating that ABA negatively regulates both basal and resistance gene–mediated blast resistance. ABA markedly suppressed the transcriptional upregulation of WRKY45 and OsNPR1, the two key components of the SA signaling pathway in rice, induced by SA or benzothiadiazole or by blast infection. Overexpression of OsNPR1 or WRKY45 largely negated the enhancement of blast susceptibility by ABA, suggesting that ABA acts upstream of WRKY45 and OsNPR1 in the rice SA pathway. ABA-responsive genes were induced during blast infection in a pattern reciprocal to those of WRKY45 and OsPR1b in the compatible rice–blast interaction but only marginally in the incompatible one. These results suggest that the balance of SA and ABA signaling is an important determinant for the outcome of the rice–M. grisea interaction. ABA was detected in hyphae and conidia of M. grisea as well as in culture media, implying that blast-fungus-derived ABA could play a role in triggering ABA signaling at host infection sites.

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.