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A unique genetic variation with respect to blast resistance was clarified in 201 rice accessions from Vietnam. These accessions were classified into three clusters-A, B1, and B2-based on their reactions to 26 standard differential blast isolates selected in Vietnam. Cluster A was the dominant cultivar group in Vietnam and the most susceptible of the three clusters. Cluster B1 was the smallest group and the most resistant. Cluster B2 was the second-most dominant group and of intermediate resistance between clusters A and B1. The percentages of accessions comprising each cluster varied by region and area. Accessions in cluster A were distributed widely throughout Vietnam and had the highest frequencies in both the Central and North regions. Accessions in cluster B2 were found with highest frequencies in the mountainous and intermediate areas of the North region. Accessions in cluster B1 were found with highest frequencies in the Central region and Red River Delta area (North region). These results suggest that rice accessions in Vietnam were basically susceptible (cluster A) or of intermediate resistance (cluster B2), and that high-resistance cultivars were mainly distributed in the low altitude areas, such as the Red River Delta area and Central region., (Copyright © 2023 by JAPANESE SOCIETY OF BREEDING.)

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.

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.

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., (Copyright © 2019 by JAPANESE SOCIETY OF BREEDING.)

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.

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., (© 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.)

Floods impede gas (O 2 and CO 2 ) exchange between plants and the environment. A mechanism to enhance plant gas exchange under water comprises gas films on hydrophobic leaves, but the genetic regulation of this mechanism is unknown. We used a rice mutant (dripping wet leaf 7, drp7) which does not retain gas films on leaves, and its wild-type (Kinmaze), in gene discovery for this trait. Gene complementation was tested in transgenic lines. Functional properties of leaves as related to gas film retention and underwater photosynthesis were evaluated. Leaf Gas Film 1 (LGF1) was identified as the gene determining leaf gas films. LGF1 regulates C30 primary alcohol synthesis, which is necessary for abundant epicuticular wax platelets, leaf hydrophobicity and gas films on submerged leaves. This trait enhanced underwater photosynthesis 8.2-fold and contributes to submergence tolerance. Gene function was verified by a complementation test of LGF1 expressed in the drp7 mutant background, which restored C30 primary alcohol synthesis, wax platelet abundance, leaf hydrophobicity, gas film retention, and underwater photosynthesis. The discovery of LGF1 provides an opportunity to better understand variation amongst rice genotypes for gas film retention ability and to target various alleles in breeding for improved submergence tolerance for yield stability in flood-prone areas., (© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.)

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.

Broad-spectrum disease resistance against two or more types of pathogen species is desirable for crop improvement. In rice, Xanthomonas oryzae pv. oryzae (Xoo), the causal bacteria of rice leaf blight, and Magnaporthe oryzae, the fungal pathogen causing rice blast, are two of the most devastating pathogens. We identified the rice BROAD-SPECTRUM RESISTANCE 1 (BSR1) gene for a BIK1-like receptor-like cytoplasmic kinase using the FOX hunting system, and demonstrated that BSR1-overexpressing (OX) rice showed strong resistance to the bacterial pathogen, Xoo and the fungal pathogen, M. oryzae. Here, we report that BSR1-OX rice showed extended resistance against two other different races of Xoo, and to at least one other race of M. oryzae. In addition, the rice showed resistance to another bacterial species, Burkholderia glumae, which causes bacterial seedling rot and bacterial grain rot, and to Cochliobolus miyabeanus, another fungal species causing brown spot. Furthermore, BSR1-OX rice showed slight resistance to rice stripe disease, a major viral disease caused by rice stripe virus. Thus, we demonstrated that BSR1-OX rice shows remarkable broad-spectrum resistance to at least two major bacterial species and two major fungal species, and slight resistance to one viral pathogen.

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., (© 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.)

The rice transcription factor WRKY45 plays a central role in the salicylic acid signalling pathway and mediates chemical-induced resistance to multiple pathogens, including Magnaporthe oryzae and Xanthomonas oryzae pv. oryzae. Previously, we reported that rice transformants overexpressing WRKY45 driven by the maize ubiquitin promoter were strongly resistant to both pathogens; however, their growth and yield were negatively affected because of the trade-off between the two conflicting traits. Also, some unknown environmental factor(s) exacerbated this problem. Here, we report the development of transgenic rice lines resistant to both pathogens and with agronomic traits almost comparable to those of wild-type rice. This was achieved by optimizing the promoter driving WRKY45 expression. We isolated 16 constitutive promoters from rice genomic DNA and tested their ability to drive WRKY45 expression. Comparisons among different transformant lines showed that, overall, the strength of WRKY45 expression was positively correlated with disease resistance and negatively correlated with agronomic traits. We conducted field trials to evaluate the growth of transgenic and control lines. The agronomic traits of two lines expressing WRKY45 driven by the OsUbi7 promoter (PO sUbi7 lines) were nearly comparable to those of untransformed rice, and both lines were pathogen resistant. Interestingly, excessive WRKY45 expression rendered rice plants sensitive to low temperature and salinity, and stress sensitivity was correlated with the induction of defence genes by these stresses. These negative effects were barely observed in the PO sUbi7 lines. Moreover, their patterns of defence gene expression were similar to those in plants primed by chemical defence inducers., (© 2014 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

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.

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.

CERK1 is a lysine motif-containing plant pattern recognition receptor for chitin and peptidoglycan. Chitin recognition by OsCERK1 triggers rapid engagement of a rice MAP kinase cascade, which leads to defense response activation. How the MAP kinase cascades are engaged downstream of OsCERK1 remains obscure. Searching for host proteins that interact with Xoo1488, an effector of the rice pathogen Xanthomonas oryzae, we identified the rice receptor-like cytoplasmic kinase, OsRLCK185. Silencing OsRLCK185 suppressed peptidoglycan- and chitin-induced immune responses, including MAP kinase activation and defense-gene expression. In response to chitin, OsRLCK185 associates with, and is directly phosphorylated by, OsCERK1 at the plasma membrane. Xoo1488 inhibits peptidoglycan- and chitin-induced immunity and pathogen resistance. Additionally, OsCERK1-mediated phosphorylation of OsRLCK185 is suppressed by Xoo1488, resulting in the inhibition of chitin-induced MAP kinase activation. These data support a role for OsRLCK185 as an essential immediate downstream signaling partner of OsCERK1 in mediating chitin- and peptidoglycan-induced plant immunity., (Copyright © 2013 Elsevier Inc. All rights reserved.)

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., (© 2012 The Authors The Plant Journal © 2012 Blackwell Publishing Ltd.)

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., (© 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.)

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 (H(2) O(2) ) 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 H(2) O(2) 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., (© 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.)

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 H(2)O(2) 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., (© 2011 THE AUTHORS. MOLECULAR PLANT PATHOLOGY © 2011 BSPP AND BLACKWELL PUBLISHING LTD.)

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., (© 2010 The Authors. Plant Biotechnology Journal © 2010 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.)

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., (© 2010 The Authors. Journal compilation © 2010 Blackwell Publishing Ltd.)

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.

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.

Blast disease is a devastating fungal disease of rice, one of the world's staple foods. Race-specific resistance to blast disease has usually not been durable. Here, we report the cloning of a previously unknown type of gene that confers non-race-specific resistance and its successful use in breeding. Pi21 encodes a proline-rich protein that includes a putative heavy metal-binding domain and putative protein-protein interaction motifs. Wild-type Pi21 appears to slow the plant's defense responses, which may support optimization of defense mechanisms. Deletions in its proline-rich motif inhibit this slowing. Pi21 is separable from a closely linked gene conferring poor flavor. The resistant pi21 allele, which is found in some strains of japonica rice, could improve blast resistance of rice worldwide.

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.

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 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.

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.

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.