Mitogen-activated protein kinase OsMPK6 negatively regulates rice disease resistance to bacterial pathogens

Mitogen-activated protein kinase (MAPK) cascades play important roles in diverse developmental and physiological processes of plants, including pathogen-induced defense responses. Although at least 17 rice MAPKs have been identified and more than half of these MAPK genes have been shown to be pathogen or elicitor responsive, the exact role of most of the MAPKs in host-pathogen interaction is unknown. Here we report that OsMPK6 is an important regulator in rice disease resistance. Suppressing OsMPK6 or knocking out of OsMPK6 enhanced rice resistance to different races of Xanthomonas oryzae pv. oryzae, causing bacterial blight, one of the most devastating diseases of rice worldwide. The resistant plants showed increased expression of a subset of defense-responsive genes functioning in the NH1 (an Arabidopsis NPR1 orthologue)-involved defense signal transduction pathway. These results suggest that OsMPK6 functions as a repressor to regulate rice defense responses upon bacterial invasion. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A Rice Gene of De Novo Origin Negatively Regulates Pathogen-Induced Defense Response

How defense genes originated with the evolution of their specific pathogen-responsive traits remains an important problem. It is generally known that a form of duplication can generate new genes, suggesting that a new gene usually evolves from an ancestral gene. However, we show that a new defense gene in plants may evolve by de novo origination, resulting in sophisticated disease-resistant functions in rice. Analyses of gene evolution showed that this new gene, OsDR10, had homologs only in the closest relative, Leersia genus, but not other subfamilies of the grass family; therefore, it is a rice tribe-specific gene that may have originated de novo in the tribe. We further show that this gene may evolve a highly conservative rice-specific function that contributes to the regulation difference between rice and other plant species in response to pathogen infections. Biologic analyses including gene silencing, pathologic analysis, and mutant characterization by transformation showed that the OsDR10-suppressed plants enhanced resistance to a broad spectrum of Xanthomonas oryzae pv. oryzae strains, which cause bacterial blight disease. This enhanced disease resistance was accompanied by increased accumulation of endogenous salicylic acid (SA) and suppressed accumulation of endogenous jasmonic acid (JA) as well as modified expression of a subset of defense-responsive genes functioning both upstream and downstream of SA and JA. These data and analyses provide fresh insights into the new biologic and evolutionary processes of a de novo gene recruited rapidly.     

Genome-wide analysis of defense-responsive genes in bacterial blight resistance of rice mediated by the recessive<Emphasis Type="Italic"> R</Emphasis> gene<Emphasis Type="Italic"> xa13</Emphasis>

Defense responses triggered by dominant and recessive disease resistance ( R) genes are presumed to be regulated by different molecular mechanisms. In order to characterize the genes activated in defense responses against bacterial blight mediated by the recessive R gene xa13, two pathogen-induced subtraction cDNA libraries were constructed using the resistant rice line IRBB13—which carries xa13 —and its susceptible, near-isogenic, parental line IR24. Clustering analysis of expressed sequence tags (ESTs) identified 702 unique expressed sequences as being involved in the defense responses triggered by xa13; 16% of these are new rice ESTs. These sequences define 702 genes, putatively encoding a wide range of products, including defense-responsive genes commonly involved in different host-pathogen interactions, genes that have not previously been reported to be associated with pathogen-induced defense responses, and genes (38%) with no homology to previously described functional genes. In addition, R -like genes putatively encoding nucleotide-binding site/leucine rich repeat (NBS-LRR) and LRR receptor kinase proteins were observed to be induced in the disease resistance activated by xa13. A total of 568 defense-responsive ESTs were mapped to 588 loci on the rice molecular linkage map through bioinformatic analysis. About 48% of the mapped ESTs co-localized with quantitative trait loci (QTLs) for resistance to various rice diseases, including bacterial blight, rice blast, sheath blight and yellow mottle virus. Furthermore, some defense-responsive sequences were conserved at similar locations on different chromosomes. These results reveal the complexity of xa13 -mediated resistance. The information obtained in this study provides a large source of candidate genes for understanding the molecular bases of defense responses activated by recessive R genes and of quantitative disease resistance.Electronic Supplementary Material Supplementary material is available in the online version of this article at The first two authors contributed equally to this workCommunicated by R. Hagemann     

Three types of defense-responsive genes are involved in resistance to bacterial blight and fungal blast diseases in rice

Bacterial blight and fungal blast diseases of rice, caused by Xanthomonas oryzae pv. oryzae and Pyricularia grisea Sacc., respectively, are two of the most devastating diseases in rice worldwide. To study the defense responses to infection with each of these pathogens, expression profiling of 12 defense-responsive genes was performed using near-isogenic rice lines that are resistant or susceptible to bacterial blight and fungal blast, respectively, and rice cultivars that are resistant or susceptible to both pathogens. All 12 genes showed constitutive expression, but expression levels increased in response to infection. Based on their expression patterns in 12 host-pathogen combinations, these genes could be classified into three types, pathogen non-specific (6), pathogen specific but race non-specific (4) and race specific (2). Most of the 12 genes were only responsive during incompatible interactions. These results suggest that bacterial blight and fungal blast resistances share common pathway(s), but are also regulated by different defense pathways in rice. Activation of the corresponding R gene is the key step that initiates the action of these genes in defense responses. The chromosomal locations and pathogen specificities of seven of the 12 genes were consistent with those of previously identified quantitative trait loci for rice disease resistance, which indicates that some of the 12 genes studied may have a phenotypic impact on disease resistance in rice.     

Effects of Colonization of a Bacterial Endophyte,Azospirillum sp. B510, on Disease Resistance in Rice

Agriculturally important grasses contain numerous diazotrophic bacteria, the interactions of which are speculated to have some other benefits to the host plants. In this study, we analyzed the effects of a bacterial endophyte, Azospirillum sp. B510, on disease resistance in host rice plants. Rice plants (Oryza sativa cv. Nipponbare) were inoculated with B510 exhibited enhanced resistance against diseases caused by the virulent rice blast fungus Magnaporthe oryzae and by the virulent bacterial pathogen Xanthomonas oryzae. In the rice plants, neither salicylic acid (SA) accumulation nor expression of pathogenesis-related (PR) genes was induced by interaction with this bacterium, except for slight induction of PBZ1. These results indicate the possibility that strain B510 is able to induce disease resistance in rice by activating a novel type of resistance mechanism independent of SA-mediated defense signaling.     

Perception of the chitin oligosaccharides contributes to disease resistance to blast fungus Magnaporthe oryzae in rice

Chitin is a component of fungal cell walls, and its fragments act as elicitors in many plants. The plasma membrane glycoprotein CEBiP, which possesses LysM domains, is a receptor for the chitin elicitor (CE) in rice. Here, we report that the perception of CE by CEBiP contributes to disease resistance against the rice blast fungus, Magnaporthe oryzae, and that enhanced responses to CE by engineering CEBiP increase disease tolerance. Knockdown of CEBiP expression allowed increased spread of the infection hyphae. To enhance defense responses to CE, we constructed chimeric genes composed of CEBiP and Xa21, which mediate resistance to rice bacterial leaf blight. The expression of either CRXa1 or CRXa3, each of which contains the whole extracellular portion of CEBiP, the whole intracellular domain of XA21, and the transmembrane domain from either CEBiP or XA21, induced cell death accompanied by an increased production of reactive oxygen and nitrogen species after treatment with CE. Rice plants expressing the chimeric receptor exhibited necrotic lesions in response to CE and became more resistant to M. oryzae. Deletion of the first LysM domain in CRXA1 abolished these cellular responses. These results suggest that CEs are produced and recognized through the LysM domain of CEBiP during the interaction between rice and M. oryzae and imply that engineering pattern recognition receptors represents a new strategy for crop protection against fungal diseases.     

Accumulation of salicylic acid,jasmonic acid and phytoalexins in rice, <Emphasis Type="Italic">Oryza sativa</Emphasis>, infested by the white-backed planthopper, <Emphasis Type="Italic">Sogatella furcifera</Emphasis> (Hemiptera: Delphacidae)

In order to clarify the mechanism of induced resistance to blast disease in rice, Oryza sativa, that had been previously infested by the white-backed planthopper, Sogatella furcifera Horváth, we first investigated the accumulation of salicylic acid (SA) and jasmonic acid (JA) in rice plants infested by the planthopper. The results confirmed that infestation of S. furcifera strongly stimulates the production of SA and JA in rice. These results indicate that both salicylate- and jasmonate-mediated pathways (SA and JA pathways), which are involved in the general defense system in plants, were activated in rice infested by S. furcifera. Further results confirmed that S. furcifera infestation induces accumulation of a major rice diterpenoid phytoalexin, momilactone A, and a flavonoid phytoalexin, sakuranetin, which are well known as antimicrobial chemicals, particularly in blast disease caused by the blast fungus, Magnaporthe oryzae B. Couch. All these results strongly suggest the following hypothetical mechanism of induced-resistance to M. oryzae in rice infested by S. furcifera. First, S. furcifera releases some elicitor-active compounds, which might be produced in the salivary glands, into the rice plant during feeding. Next, the defense signal systems, SA- and JA-mediated pathways, are activated by the elicitor. Finally, phytoalexins are induced in rice as antimicrobial compounds mainly through activation of the JA-mediated pathway.     

Analysis of the Relationship between Blast Resistance Genes and Disease Resistance of Rice Germplasm via Functional Molecular Markers

Rice blast disease is one of the most devastating diseases of rice (Oryza sativa L.) caused by the fungus Magnaporthe oryzae (M. oryzae), and neck blast is the most destructive phase of this illness. The underlying molecular mechanisms of rice blast resistance are not well known. Thus, we collected 150 rice varieties from different ecotypes in China and assessed the rice blast resistances under the natural conditions that favoured disease development in Jining, Shandong Province, China in 2017. Results showed that 92 (61.3%) and 58 (38.7%) rice varieties were resistant and susceptible to M. oryzae, respectively. Among the 150 rice varieties screened for the presence of 13 major blast resistance (R) genes against M. oryzae by using functional markers, 147 contained one to eight R genes. The relationship between R genes and disease response was discussed by analysing the phenotype and genotype of functional markers. The results showed that the rice blast resistance gene Pita was significantly correlated with rice blast resistance. Our results provided a basis for the further understanding of the distribution of 13 major R genes of rice blast in the germplasm resources of the tested rice varieties, and were meaningful for rice disease resistance breeding.     

The DnaJ protein OsDjA6 negatively regulates rice innate immunity to the blast fungus Magnaporthe oryzae

Rice blast, caused by Magnaporthe oryzae (synonym: Pyricularia oryzae), severely reduces rice production and grain quality. The molecular mechanism of rice resistance to M. oryzae is not fully understood. In this study, we identified a chaperone DnaJ protein, OsDjA6, which is involved in basal resistance to M. oryzae in rice. The OsDjA6 protein is distributed in the entire rice cell. The expression of OsDjA6 is significantly induced in rice after infection with a compatible isolate. Silencing of OsDjA6 in transgenic rice enhances resistance to M. oryzae and also results in an increased burst of reactive oxygen species after flg22 and chitin treatments. In addition, the expression levels of WRKY45, NPR1 and PR5 are increased in OsDjA6 RNAi plants, indicating that OsDjA6 may mediate resistance by affecting the salicylic acid pathway. Finally, we found that OsDjA6 interacts directly with the E3 ligase OsZFP1 in vitro and in vivo. These results suggest that the DnaJ protein OsDjA6 negatively regulates rice innate immunity, probably via the ubiquitination proteasome degradation pathway.     

Differential Gene Expression Reflects Morphological Characteristics and Physiological Processes in Rice Immunity against Blast Pathogen Magnaporthe oryzae

The rice blast fungus Magnaporthe oryzae is a serious pathogen that jeopardises the world’s most important food-security crop. Ten common Malaysian rice varieties were examined for their morphological, physiological and genomic responses to this rice blast pathogen. qPCR quantification was used to assess the growth of the pathogen population in resistant and susceptible rice varieties. The chlorophyll content and photosynthesis were also measured to further understand the disruptive effects that M. oryzae has on infected plants of these varieties. Real-time PCR was used to explore the differential expression of eight blast resistance genes among the ten local varieties. Blast disease has destructive effects on the growth of rice, and the findings of our study provide evidence that the Pikh, Pi9, Pi21, and Osw45 genes are involved in defence responses in the leaves of Malaysian rice at 31 h after inoculation with M. oryzae pathotype P7.2. Both the chlorophyll content and photosynthesis were reduced, but the levels of Pikh gene expression remained constant in susceptible varieties, with a developed pathogen population and mild or severe symptoms. The Pi9, Pi21, and Osw45 genes, however, were simultaneously upregulated in infected rice plants. Therefore, the presence of the Pikh, Pi9, Pi21, and Osw45 genes in the germplasm is useful for improving the resistance of rice varieties.     

Small RNAs and Gene Network in a Durable Disease Resistance Gene—Mediated Defense Responses in Rice

Accumulating data have suggested that small RNAs (sRNAs) have important functions in plant responses to pathogen invasion. However, it is largely unknown whether and how sRNAs are involved in the regulation of rice responses to the invasion of Xanthomonas oryzae pv. oryzae (Xoo), which causes bacterial blight, the most devastating bacterial disease of rice worldwide. We performed simultaneous genome-wide analyses of the expression of sRNAs and genes during early defense responses of rice to Xoo mediated by a major disease resistance gene, Xa3/Xa26, which confers durable and race-specific qualitative resistance. A large number of sRNAs and genes showed differential expression in Xa3/Xa26-mediated resistance. These differentially expressed sRNAs include known microRNAs (miRNAs), unreported miRNAs, and small interfering RNAs. The candidate genes, with expression that was negatively correlated with the expression of sRNAs, were identified, indicating that these genes may be regulated by sRNAs in disease resistance in rice. These results provide a new perspective regarding the putative roles of sRNA candidates and their putative target genes in durable disease resistance in rice.     

The RhoGAP SPIN6 Associates with SPL11 and OsRac1 and Negatively Regulates Programmed Cell Death and Innate Immunity in Rice

The ubiquitin proteasome system in plants plays important roles in plant-microbe interactions and in immune responses to pathogens. We previously demonstrated that the rice U-box E3 ligase SPL11 and its Arabidopsis ortholog PUB13 negatively regulate programmed cell death (PCD) and defense response. However, the components involved in the SPL11/PUB13-mediated PCD and immune signaling pathway remain unknown. In this study, we report that SPL11-interacting Protein 6 (SPIN6) is a Rho GTPase-activating protein (RhoGAP) that interacts with SPL11 in vitro and in vivo. SPL11 ubiquitinates SPIN6 in vitro and degrades SPIN6 in vivo via the 26S proteasome-dependent pathway. Both RNAi silencing in transgenic rice and knockout of Spin6 in a T-DNA insertion mutant lead to PCD and increased resistance to the rice blast pathogen Magnaporthe oryzae and the bacterial blight pathogen Xanthomonas oryzae pv. oryzae. The levels of reactive oxygen species and defense-related gene expression are significantly elevated in both the Spin6 RNAi and mutant plants. Strikingly, SPIN6 interacts with the small GTPase OsRac1, catalyze the GTP-bound OsRac1 into the GDP-bound state in vitro and has GAP activity towards OsRac1 in rice cells. Together, our results demonstrate that the RhoGAP SPIN6 acts as a linkage between a U-box E3 ligase-mediated ubiquitination pathway and a small GTPase-associated defensome system for plant immunity.