Effect of overexpression of the rice WRKY transcription factor OsWRKY53 or its phospho-mimicking mutant on rice leaves 48 h after either mock treatment or the rice blast fungus Magnaporthe Oryzae infection
ABSTRACT: Analysis of transgenic rice plants overexpressing the rice WRKY transcription factor OsWRKY53 or its phospho-mimicking mutant (OsWRKY53SD). Results provide insight into the roles of OsWRKY53 and its phosphorylation in the basal defense signaling against the rice blast fungus. Expression profiling in wild-type, OsWRKY53- or its phospho-mimicking mutant-overexpressing rice leaves infected with or without Magnaporthe Oryzae was analyzed using one-color method with four biological replicates.
Project description:Analysis of transgenic rice overexpressing OsWRKY28, a WRKY type transcription factor. Results provide insight into the role of OsWRKY28 in the defense signaling against rice blast fungus. Expression profiling in wild-type and OsWRKY28 overexpressing rice leaves infected with or without Magnaporthe Oryzae was analyzed using one-color method with three biological replicates.
Project description:A novel rice NBS-LRR gene was cloned and overexpressed to explore its function in bacterial blight (BB) resistance. Due to its similarity to the Rp1 gene in maize (Zea mays), it was designated as OsRP1L1. We used microarrays to detail the global reprogram of gene expression in OsRP1L1-overexpressing plants. To find out pathways and genes related to this defense-related gene, microanalysis were carried out on OsPR1L1-overexpressing plants. Three independent replicates were perfomed for overexpressing plants and wild type plants.
Project description:The WRKY transcription factor family has 109 members in the rice genome, and has been reported to be involved in the regulation of biotic and abiotic stress in plants. Here, we demonstrated that a rice OsWRKY74 belonging to group III of the WRKY transcription factor family was involved in tolerance to phosphate (Pi) starvation. OsWRKY74 was localized in the nucleus and mainly expressed in roots and leaves. Overexpression of OsWRKY74 significantly enhanced tolerance to Pi starvation, whereas transgenic lines with down-regulation of OsWRKY74 were sensitive to Pi starvation. Root and shoot biomass, and phosphorus (P) concentration in rice OsWRKY74-overexpressing plants were ~16% higher than those of wild-type (WT) plants in Pi-deficient hydroponic solution. In soil pot experiments, >24% increases in tiller number, grain weight and P concentration were observed in rice OsWRKY74-overexpressing plants compared to WT plants when grown in P-deficient medium. Furthermore, Pi starvation-induced changes in root system architecture were more profound in OsWRKY74-overexpressing plants than in WT plants. Expression patterns of a number of Pi-responsive genes were altered in the OsWRKY74-overexpressing and RNA interference lines. In addition, OsWRKY74 may also be involved in the response to deficiencies in iron (Fe) and nitrogen (N) as well as cold stress in rice. In Pi-deficient conditions, OsWRKY74-overexpressing plants exhibited greater accumulation of Fe and up-regulation of the cold-responsive genes than WT plants. These findings highlight the role of OsWRKY74 in modulation of Pi homeostasis and potential crosstalk between P starvation and Fe starvation, and cold stress in rice.
Project description:WRKY transcription factors and mitogen-activated protein kinase (MAPK) cascades have been shown to play pivotal roles in the regulation of plant defense responses. We previously reported that OsWRKY53-overexpressing rice plants showed enhanced resistance to the rice blast fungus. In this study, we identified OsWRKY53 as a substrate of OsMPK3/OsMPK6, components of a fungal PAMP-responsive MAPK cascade in rice, and analyzed the effect of OsWRKY53 phosphorylation on the regulation of basal defense responses to a virulence race of rice blast fungus Magnaporthe oryzae strain Ina86-137. An in vitro phosphorylation assay revealed that the OsMPK3/OsMPK6 activated by OsMKK4 phosphorylated OsWRKY53 recombinant protein at its multiple clustered serine-proline residues (SP cluster). When OsWRKY53 was coexpressed with a constitutively active mutant of OsMKK4 in a transient reporter gene assay, the enhanced transactivation activity of OsWRKY53 was found to be dependent on phosphorylation of the SP cluster. Transgenic rice plants overexpressing a phospho-mimic mutant of OsWRKY53 (OsWRKY53SD) showed further-enhanced disease resistance to the blast fungus compared to native OsWRKY53-overexpressing rice plants, and a substantial number of defense-related genes, including pathogenesis-related protein genes, were more upregulated in the OsWRKY53SD-overexpressing plants compared to the OsWRKY53-overexpressing plants. These results strongly suggest that the OsMKK4-OsMPK3/OsMPK6 cascade regulates transactivation activity of OsWRKY53, and overexpression of the phospho-mimic mutant of OsWRKY53 results in a major change to the rice transcriptome at steady state that leads to activation of a defense response against the blast fungus in rice plants.
Project description:A transcription factor CYTOKININ-RESPONSIVE GATA FACTOR 1 (CGA1) regulates chloroplast development in rice (Oryza sativa) through modifying the expression of important nuclear expressed, chloroplast localized genes. A transcriptome analysis was done in wild type plants and transgenic rice over-expressing this OsCGA1 to identify the set of genes with altered expression. RNA was extracted from leaves of 4-wk old wild type and OsCGA1 overexpressing rice plants and hybridized to Affymetrix Rice Genome Array. Three biological replicates were sampled for wild type and OX plants.
Project description:The plant volatile linalool plays important roles in the regulation of defense responses in rice. To clarify the response to linalool in rice at gene expression level, we performed a microarray analysis using the Agilent Rice Oligo Microarray (44k, custom-made; Agilent Technologies, Redwood City, CA, USA). As a result, many defense-related genes including pathogenesis-related (PR) genes were upregulated in the linalool synthase-overexpressing transgenic rice. An Agilent Rice Oligo Microarray (44k, custom-made; Agilent Technologies, Redwood City, CA, USA) was used for the microarray analysis. Total RNA was extracted from leaf blades of four-leaf stage rice plants. Line 13 was used as the linalool synthase-overexpressing transgenic rice plant. For each biological replicate, material from at least three rice plants was pooled to provide a single sample for RNA extraction. We performed 3 biological replicates for each treatment. All microarray procedures and data analyses were performed according to the manufacturer’s instructions. RNA integrity was checked using an Agilent 2100 Bioanalyzer (Agilent Technologies, Redwood City, CA, USA). Total RNA (100 ng) was labeled with Cy-3 using an Agilent Low Input Quick Amp Labeling Kit, One-Color (Agilent Technologies). Fluorescently labeled targets were hybridized to Agilent Rice Oligo Microarrays for 17 h at 65°C. After hybridization, following wash processes were performed according to the manufacturer’s instructions, and hybridized microarrays were scanned using an Agilent Microarray Scanner (G2565CA; Agilent Technologies). Feature extraction software (Agilent Feature Extraction; Agilent Technologies) was used to delineate and measure the signal intensity of each spot in the array, and to normalize intensities. The background was measured around each spot as local background, calculated by the Feature Extraction software. Statistical data extraction processes were performed according to the manufacturer’s instructions.
Project description:Rice blast disease is a major threat to rice production worldwide, but the mechanisms underlying rice resistance to the causal agent Magnaporthe oryzae remain elusive. In this whole-genome transcriptome study of rice early defense response to M. oryzae, we applied Affymetrix Rice Genome Genechip to compare the compatible and incompatible rice-M. oryzae interactions in 24 hours post-inoculation. Leaf samples were harvested from three biological replicates of fungal- and mock-inoculated seedlings at 24 hours post-inoculation, from which RNA were extracted and analyzed with Genechip Rice Genome Array.
Project description:Accumulating information indicates that plant disease resistance signaling pathway frequently interact with other pathways regulating developmental processes or abiotic stress responses. However, the molecular mechanisms of these types of crosstalk remain poorly understood in most cases. Here we report that OsWRKY13, an activator of rice resistance to both bacterial and fungal pathogens, functions as a convergent point of the crosstalk between pathogen-induced salicylate-dependent defense pathway and at least five other physiological pathways. Genome-wide analysis of the expression profiling of OsWRKY13-overexpressing lines showed that OsWRKY13 directly or indirectly regulates the expression of more than 500 genes, which are potentially involved in different physiological processes according to the classification of Gene Ontology database. Comparing the expression patterns of genes functioning in known pathways or cellular processes upon pathogen infection and the phenotypes between OsWRKY13-overexpressing and susceptible wild-type plants, we find that OsWRKY13 is also regulator of other physiological processes during pathogen infection. OsWRKY13-involved disease resistance pathway synergistically interacts with glutathione/glutaredoxin system and flavonoid biosynthesis pathway via OsWRKY13 to monitor redox homeostasis and may enhance the biosynthesis of antimicrobial flavonoid phytoalexins. Meanwhile, OsWRKY13-invloved disease resistance pathway antagonistically interacts with SNAC1-mediated abiotic-stress defense pathway, JA signaling pathway, and terpenoid metabolism pathway via OsWRKY13 to suppress salt and cold defense responses as well as may retard rice growth and development. Experiment Overall Design: To determine the genes directly or indirectly regulated by OsWRKY13 genome-widely, we performed the microarray analysis using the Affymetrix Genechip Rice Genome Array containing total 57381 probe sets. Two independent OsWRKY13-overexpressing rice lines, D11UM 1-1 and D11UM 7-2 (Qiu et al., 2007), and wild-type Mudanjiang 8, which was susceptible to Xoo and M. grisea, were used for the analyses.The experiment was repeated three times.
Project description:To see the effect of overexpression of the bZIP transcription factor OsTGAP1, we compared the gene expression profiles in wild type rice cells and OsTGAP1-overexpressing rice cells. Expression profiling in the wild type rice cells and OsTGAP1-overexpressing rice cells treated with chitin oligosaccharide for 0, 6, and 24 h was compared using two-color method with four biological replicates.