Rice Defense Research Articles

Ascorbate oxidation activates systemic defence against root-knot nematode Meloidogyne graminicola in rice.

Ascorbic acid (AA) is the major antioxidant buffer produced in the shoot tissue of plants. Previous studies on root-knot nematode (RKN; Meloidogyne graminicola)-infected rice (Oryza sativa) plants showed differential expression of AA-recycling genes, although their functional role was unknown. Our results confirmed increased dehydroascorbate (DHA) levels in nematode-induced root galls, while AA mutants were significantly more susceptible to nematode infection. External applications of ascorbate oxidase (AO), DHA, or reduced AA, revealed systemic effects of ascorbate oxidation on rice defence versus RKN, associated with a primed accumulation of H2O2 upon nematode infection. To confirm and further investigate these systemic effects, a transcriptome analysis was done on roots of foliar AO-treated plants, revealing activation of the ethylene (ET) response and jasmonic acid (JA) biosynthesis pathways in roots, which was confirmed by hormone measurements. Activation of these pathways by methyl-JA, or ethephon treatment can complement the susceptibility phenotype of the rice Vitamin C (vtc1) mutant. Experiments on the jasmonate signalling (jar1) mutant or using chemical JA/ET inhibitors confirm that the effects of ascorbate oxidation are dependent on both the JA and ET pathways. Collectively, our data reveal a novel pathway in which ascorbate oxidation induces systemic defence against RKNs.

Indeterminate Domain Proteins Regulate Rice Defense to Sheath Blight Disease

BackgroundLoose Plant Architecture 1 (LPA1), an indeterminate domain (IDD) protein, exhibits almost no expression in the leaves, but the overexpression of LPA1 significantly increases the resistance of rice to sheath blight disease (ShB) via the activation of PIN-FORMED 1a (PIN1a).ResultsIn this study, we determined that Rhizoctonia solani infection significantly induced LPA1 expression in the leaves, and lpa1 was more susceptible to R. solani compared with the wild-type and revertant plants. In addition, infection with R. solani altered the expression of IDD3, IDD5, IDD10, and IDD13, and yeast two-hybrid, split-GFP, and coimmunoprecipitation assays showed that LPA1 interacts with IDD3 and IDD13. IDD13 RNAi plants were more susceptible, while IDD13 overexpressors were less susceptible to ShB compared with the wild-type. In parallel, idd3 exhibited no significant differences, while IDD3 overexpressors were more susceptible compared to the wild-type response to ShB. Additional chromatin-immunoprecipitation and electrophoretic mobility shift assay experiments indicated that IDD13 and IDD3 bound to the PIN1a promoter, and the transient assay indicated that IDD13 and IDD3 positively and negatively regulate PIN1a expression, respectively. Moreover, IDD13, IDD3, and LPA1 form a transcription factor complex that regulates PIN1a. A genetic study showed that the LPA1 repressor lines were similar to lpa1/IDD13 RNAi and were more susceptible than the lpa1 and IDD13 RNAi plants in response to ShB. The overexpression of IDD13 increased resistance to ShB in the lpa1 background.ConclusionsTaken together, our analyses established that IDD3, IDD13, and LPA1 form a transcription factor complex to regulate the defense of rice against ShB possibly via the regulation of PIN1a.

Isolation and Characterization of Root-Associated Bacterial Endophytes and Their Biocontrol Potential against Major Fungal Phytopathogens of Rice (Oryza sativa L.).

Rice (Oryza sativa L.) is a major cereal food crop worldwide, and its growth and yield are affected by several fungal phytopathogens, including Magnaporthe oryzae, Fusarium graminearum, F. moniliforme, and Rhizoctonia solani. In the present study, we have isolated and characterized root-associated bacterial endophytes that have antifungal activities against rice fungal phytopathogens. A total of 122 root-associated bacterial endophytes, belonging to six genera (Bacillus, Fictibacillus, Lysinibacillus, Paenibacillus, Cupriavidus, and Microbacterium) and 22 species were isolated from three rice cultivars. Furthermore, the 16S rRNA sequence-based phylogeny results revealed that Bacillus was the most dominant bacterial genera, and that there were 15 different species among the isolates. Moreover, 71 root-associated endophytes showed antagonistic effects against four major fungal phytopathogens, including M. oryzae, F. graminearum, F. moniliforme, and R. solani. Additionally, the biochemical, physiological, and PCR amplification results of the antibiotic-related genes further supported the endophytes as potential biocontrolling agents against the rice fungal pathogens. Consequently, the findings in this study suggested that the isolated bacterial endophytes might have beneficial roles in rice defense responses, including several bioactive compound syntheses. The outcomes of this study advocate the use of natural endophytes as an alternative strategy towards the rice resistance response.

Rice CaM-binding transcription factor (OsCBT) mediates defense signaling via transcriptional reprogramming

The mutant allele of rice calmodulin-binding transcription activator OsCBT, oscbt-1, exhibits broad-spectrum resistance against rice pathogens. Previously, we reported that the strong resistance of the oscbt-1 mutant to pathogens was conferred by a constitutive upregulation of defense-related genes even under pathogen-free conditions. We also found strong induction of the hypersensitive response as a reaction to pathogen invasion. The results suggest that OsCBT acts as a negative regulator of basal resistance to pathogen attack. To identify the transcriptional network regulated by OsCBT, we compared global gene expression profiles between wild-type (WT) and oscbt-1 rice plants grown under pathogen-free conditions. The results of a 3′ tiling microarray revealed that in oscbt-1 plants, 81 genes are upregulated and 200 genes are downregulated when compared with the WT. A gene ontology analysis showed that differentially regulated genes in oscbt-1 were very closely associated with “death” GO term in a biological process category, and “catalytic activity” and “binding” GO terms in a molecular function category. A MapMan analysis indicated that the functions of these genes were associated with plant responses to biotic stress. Moreover, the results from quantitative real-time PCR in the oscbt-1 mutant showed a significant effect on the gene expression patterns of the fungal pathogen response. Our results suggested that the OsCBT regulates a rice defense response by modulating the expressions of various defense-related genes.

Jasmonate Signaling Enhances RNA Silencing and Antiviral Defense in Rice

SUMMARY Small RNA-mediated RNA silencing is an important antiviral mechanism in higher plants. It has been shown that RNA silencing components can be upregulated by viral infection. However, the mechanisms underlying the upregulation remain largely unknown. Here, we show that JA signaling transcriptionally activates AGO18, a core RNA silencing component that promotes rice antiviral defense through sequestering miR168 and miR528. The JA-responsive transcription factor JAMYB directly binds the AGO18 promoter to activate AGO18 transcription. RSV coat protein (CP) is the elicitor that triggers JA accumulation and upregulates JAMYB. Our study reveals a novel crosstalk between the JA signaling and antiviral RNA silencing and elucidates a molecular mechanism for CP-mediated resistance in monocot crops.

Characteristic Dissection of Xanthomonas oryzae pv. oryzae Responsive MicroRNAs in Rice.

MicroRNAs (miRNAs) are crucial player in plant-pathogen interaction. While the evidence has demonstrated that rice miRNAs mediate immune response to pathogens invasion, the roles of miRNAs on Xanthomonas oryzae pv. oryzae (Xoo) attack remain be in place. Herein, we monitored the responsive changes of rice miRNAs at 0, 8, 24 h across Xoo strain PXO86 infection in its compatible rice variety IR24 and incompatible variety IRBB5 by small RNA sequencing, and the genes targeted by miRNAs were also detected via degradome technology. The faithfulness of sequencing data was validated through quantitative real-time stem-loop reverse transcription-polymerase chain reaction assay. Bioinformatic analysis showed that the differentially expressed miRNAs could be divided into three immunity-related clusters, and 80 regulatory units were emerged in infection process, which comprises 29 differentially expressed known miRNAs and 38 cleaved targets. Furthermore, the miRNA presumptive function of separate immunity cluster in rice-Xoo interplay was confirmed through overexpressing osa-miR164a, osa-miR167d and osa-miR159b, and the disruption of regulatory units, osa-miR164a/OsNAC60, osa-miR167d-5p/OsWD40-174 and osa-miR159b/OsMYBGA, OsLRR-RLK2, OsMPK20-4, may reset rice defense response to Xoo infestation in a controllable manner. These findings provide new insights into the complex roles of characteristic miRNAs and their targets in rice-Xoo interactions.

Identification and characterization of genes frequently responsive to Xanthomonas oryzae pv. oryzae and Magnaporthe oryzae infections in rice

BackgroundDisease resistance is an important factor that impacts rice production. However, the mechanisms underlying rice disease resistance remain to be elucidated.ResultsHere, we show that a robust set of genes has been defined in rice response to the infections of Xanthomonas oryzae pv. oryzae (Xoo) and Magnaporthe oryzae (Mor). We conducted a comprehensive analysis of the available microarray data from a variety of rice samples with inoculation of Xoo and Mor. A set of 12,932 genes was identified to be regulated by Xoo and another set of 2709 Mor-regulated genes was determined. GO enrichment analysis of the regulated genes by Xoo or Mor suggested mitochondrion may be an arena for the up-regulated genes and chloroplast be another for the down-regulated genes by Xoo or Mor. Cytokinin-related processes were most frequently repressed by Xoo, while processes relevant to jasmonic acid and abscisic acid were most frequently activated by Xoo and Mor. Among genes responsive to Xoo and Mor, defense responses and diverse signaling pathways were the most frequently enriched resistance mechanisms. InterPro annotation showed the zinc finger domain family, WRKY proteins, and Myb domain proteins were the most significant transcription factors regulated by Xoo and Mor. KEGG analysis demonstrated pathways including ‘phenylpropanoid biosynthesis’, ‘biosynthesis of antibiotics’, ‘phenylalanine metabolism’, and ‘biosynthesis of secondary metabolites’ were most frequently triggered by Xoo and Mor, whereas ‘circadian rhythm-plant’ was the most frequent pathway repressed by Xoo and Mor.ConclusionsThe genes identified here represent a robust set of genes responsive to the infections of Xoo and Mor, which provides an overview of transcriptional reprogramming during rice defense against Xoo and Mor infections. Our study would be helpful in understanding the mechanisms of rice disease resistance.

Black pepper (Piper nigrum L.) associated endophytic Pseudomonas putida BP25 alters root phenotype and induces defense in rice (Oryza sativa L.) against blast disease incited by Magnaporthe oryzae

Blast disease caused by Magnaporthe oryzae is an important production constraint in rice farming worldwide. Novel strategies are needed to combat blast disease as the current options like host resistance and fungicides are either unstable or not-acceptable. Black pepper associated Pseudomonas putida BP25 endogenously colonized rice -a monocot, in a density-dependent manner with altered root growth and defense activation, also showed biocontrol activity against blast caused by M. oryzae. Fluorescence imaging of P. putida BP25 primed seedlings revealed extensive bacterial colonization in roots that was further confirmed in plate assay and qPCR. Although initial counts could be correlated with bacterial dose used for priming, the bacterial population stabilized 14-days post priming suggesting internal regulation of endophyte colonization. Endophytic colonization of rice triggered alteration in root morphology, possibly, due trade-off between growth and defense. P. putida BP25 significantly inhibited the mycelium of M. oryzae by both volatile and non-volatile metabolites and also seed priming protected the rice from blast. Activities of defense related peroxidase and phenols were found increased in primed plants. qPCR assay of rice defense and developmental genes indicated increased expression of Systemic Acquired Resistance (SAR) related OsPR1-1 (Pathogenesis Related protein 1-1) and down-regulation of OsPR3. In addition, rice seedlings emerged from endophyte priming showed down-regulation of stress responsive OsACO4 and OsACS6 involved in inter-nodal elongation which plays an important role in growth and development.

Deciphering the molecular mechanism behind stimulated co-uptake of arsenic and fluoride from soil, associated toxicity, defence and glyoxalase machineries in arsenic-tolerant rice

The current study elucidates the uncharacterized biohazard associated with rice growth in arsenic and fluoride co-contaminated sites. Analysis of the arsenic-tolerant rice cultivar, Muktashri (known to restrict arsenic uptake) revealed that fluoride largely stimulated arsenic bioaccumulation in the stressed tissues and vice versa. Gene expression studies revealed that high arsenic uptake was facilitated by the fluoride-dependent up regulation of phosphate transporter2 (PT2), PT8 and low silicon rice1 (Lsi1), and elevated fluoride accumulation was stimulated by the arsenic-mediated induction of chloride channels (CLCs). The endogenous accumulation of fluoride and arsenic increased reactive oxygen species (ROS), O2−, membrane peroxidation and arsenic localization within tissues. This inhibited plant growth by triggering chlorosis, electrolyte leakage, malondialdehyde production (due to high lipoxygenase activity), protein carbonylation, protease activity and methylglyoxal accumulation due to inhibited glyoxylase activity. Metabolic analysis showed inhibited proline biosynthesis along with increased channelization of glutathione towards phytochelatin synthase and glutathione-S-tranferase-dependent pathways. Inhibition of the antioxidant enzymes like catalase, ascorbate peroxidase and guaiacol peroxidase validated the inefficient scavenging of H2O2 during combined stress. In silico analyses predicted the ecotoxicological risks of arsenic-fluoride complex formed during joint stress. Overall, our investigation illustrated the underlying mechanism of arsenic-fluoride co-uptake which resulted in complete suppression of the ‘tolerant’-phenotype in Muktashri seedlings.

Osa-miR7695 enhances transcriptional priming in defense responses against the rice blast fungus

BackgroundMicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression at the post-transcriptional level in eukaryotes. In rice, MIR7695 expression is regulated by infection with the rice blast fungus Magnaporthe oryzae with subsequent down-regulation of an alternatively spliced transcript of natural resistance-associated macrophage protein 6 (OsNramp6). NRAMP6 functions as an iron transporter in rice.ResultsRice plants grown under high iron supply showed blast resistance, which supports that iron is a factor in controlling blast resistance. During pathogen infection, iron accumulated in the vicinity of M. oryzae appressoria, the sites of pathogen entry, and in cells surrounding infected regions of the rice leaf. Activation-tagged MIR7695 rice plants (MIR7695-Ac) exhibited enhanced iron accumulation and resistance to M. oryzae infection. RNA-seq analysis revealed that blast resistance in MIR7695-Ac plants was associated with strong induction of defense-related genes, including pathogenesis-related and diterpenoid biosynthetic genes. Levels of phytoalexins during pathogen infection were higher in MIR7695-Ac than wild-type plants. Early phytoalexin biosynthetic genes, OsCPS2 and OsCPS4, were also highly upregulated in wild-type rice plants grown under high iron supply.ConclusionsOur data support a positive role of miR7695 in regulating rice immunity that further underpin links between defense and iron signaling in rice. These findings provides a basis to better understand regulatory mechanisms involved in rice immunity in which miR7695 participates which has a great potential for the development of strategies to improve blast resistance in rice.

Rice defense responses are induced upon leaf rolling by an insect herbivore

BackgroundPlant defense against herbivores begins with perception. The earlier plant detects the harm, the greater plant will benefit in its arm race with the herbivore. Before feeding, the larvae of the rice pest Cnaphalocrocis medinalis, initially spin silk and fold up a leaf. Rice can detect and protect itself against C. medinalis feeding. However, whether rice could perceive C. medinalis leaf rolling behavior is currently unknown. Here, we evaluated the role of leaf rolling by C. medinalis and artificial leaf rolling in rice plant defense and its indirect effect on two important C. medinalis parasitoids (Itoplectis naranyae and Apanteles sp.) through a combination of volatile profiling, gene-transcriptional and phytohormonal profiling.ResultsNatural leaf rolling by C. medinalis resulted in an increased attraction of I. naranyae when compared to the undamaged plant after 12 h. Volatile analysis revealed that six out of a total 22 components significantly increased in the headspace of C. medinalis rolled plant when compared to undamaged plant. Principal component analysis of these components revealed similarities in the headspace of undamaged plant and artificially rolled plant while the headspace volatiles of C. medinalis rolled plant deferred significantly. Leaf rolling and feeding by C. medinalis up-regulated the plant transcriptome and a series of jasmonic acid (JA) and salicylic acid (SA) related genes. While feeding significantly increased JA level after 12 to 36 h, rolling significantly increased SA level after 2 to 12 h. Compared to artificial rolling, natural rolling significantly increased JA level after 36 h and SA level after 2 and 12 h.ConclusionsOur findings suggest that natural leaf rolling by C. medinalis can be perceived by rice plant. The detection of this behavior may serve as an early warning signal in favor of the rice plant defenses against C. medinalis.

Comparative phosphoproteomic analysis of blast resistant and susceptible rice cultivars in response to salicylic acid

BackgroundSalicylic acid (SA) is a significant signaling molecule that induces rice resistance against pathogen invasion. Protein phosphorylation carries out an important regulatory function in plant defense responses, while the global phosphoproteome changes in rice response to SA-mediated defense response has not been reported. In this study, a comparative phosphoproteomic profiling was conducted by two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) analysis, with two near-isogenic rice cultivars after SA treatment.ResultsThirty-seven phosphoprotein spots were differentially expressed after SA treatment, twenty-nine of which were identified by MALDI-TOF/TOF MS, belonging to nine functional categories. Phosphoproteins involved in photosynthesis, antioxidative enzymes, molecular chaperones were similarly expressed in the two cultivars, suggesting SA might alleviate decreases in plant photosynthesis, regulate the antioxidant defense activities, thus improving basal resistance response in both cultivars. Meanwhile, phosphoproteins related to defense, carbohydrate metabolism, protein synthesis and degradation were differentially expressed, suggesting phosphorylation regulation mediated by SA may coordinate complex cellular activities in the two cultivars. Furthermore, the phosphorylation sites of four identified phosphoproteins were verified by NanoLC-MS/MS, and phosphorylated regulation of three enzymes (cinnamoyl-CoA reductase, phosphoglycerate mutase and ascorbate peroxidase) was validated by activity determination.ConclusionsOur study suggested that phosphorylation regulation mediated by SA may contribute to the different resistance response of the two cultivars. To our knowledge, this is the first report to measure rice phosphoproteomic changes in response to SA, which provides new insights into molecular mechanisms of SA-induced rice defense.

Jasmonates-the Master Regulator of Rice Development, Adaptation and Defense.

Rice is one of the most important food crops worldwide, as well as the model plant in molecular studies on the cereals group. Many different biotic and abiotic agents often limit rice production and threaten food security. Understanding the molecular mechanism, by which the rice plant reacts and resists these constraints, is the key to improving rice production to meet the demand of an increasing population. The phytohormone jasmonic acid (JA) and related compounds, collectively called jasmonates, are key regulators in plant growth and development. They are also one of the central players in plant immunity against biotic attacks and adaptation to unfavorable environmental conditions. Here, we review the most recent knowledge about jasmonates signaling in the rice crop model. We highlight the functions of jasmonates signaling in many adaptive responses, and also in rice growth and development processes. We also draw special attention to different signaling modules that are controlled by jasmonates in rice.

Modulation of defence and iron homeostasis genes in rice roots by the diazotrophic endophyte Herbaspirillum seropedicae

Rice is staple food of nearly half the world’s population. Rice yields must therefore increase to feed ever larger populations. By colonising rice and other plants, Herbaspirillum spp. stimulate plant growth and productivity. However the molecular factors involved are largely unknown. To further explore this interaction, the transcription profiles of Nipponbare rice roots inoculated with Herbaspirillum seropedicae were determined by RNA-seq. Mapping the 104 million reads against the Oryza sativa cv. Nipponbare genome produced 65 million unique mapped reads that represented 13,840 transcripts each with at least two-times coverage. About 7.4% (1,014) genes were differentially regulated and of these 255 changed expression levels more than two times. Several of the repressed genes encoded proteins related to plant defence (e.g. a putative probenazole inducible protein), plant disease resistance as well as enzymes involved in flavonoid and isoprenoid synthesis. Genes related to the synthesis and efflux of phytosiderophores (PS) and transport of PS-iron complexes were induced by the bacteria. These data suggest that the bacterium represses the rice defence system while concomitantly activating iron uptake. Transcripts of H. seropedicae were also detected amongst which transcripts of genes involved in nitrogen fixation, cell motility and cell wall synthesis were the most expressed.

Suppression of auxin signalling promotes rice susceptibility to Rice black streaked dwarf virus infection.

SummaryAuxin plays a fundamental role in plant growth and development, and also influences plant defence against various pathogens. Previous studies have examined the different roles of the auxin pathway during infection by biotrophic bacteria and necrotrophic fungi. We now show that the auxin signalling pathway was markedly down‐regulated following infection of rice by Rice black streaked dwarf virus (RBSDV), a dsRNA virus. Repression of the auxin receptor TIR1 by a mutant overexpressing miR393 increased rice susceptibility to RBSDV. Mutants overexpressing the auxin signalling repressors OsIAA20 and OsIAA31 were also more susceptible to RBSDV. The induction of jasmonic acid (JA) pathway genes in response to RBSDV was supressed in auxin signalling mutants, suggesting that activation of the JA pathway may be part of the auxin signalling‐mediated rice defence against RBSDV. More importantly, our results also revealed that OsRboh‐mediated reactive oxygen species levels played important roles in this defence. The results offer novel insights into the regulatory mechanisms of auxin signalling in the rice–RBSDV interaction.

Strigolactones positively regulate defense against Magnaporthe oryzae in rice (Oryza sativa)

This study presents evidence that strigolactones (SLs) promote defense against devastating rice blast fungal pathogen Magnaporthe oryzae. Impairment in either SL-biosynthetic dwarf17 (d17) or –signaling (d14) led to increased susceptibility towards M. oryzae. Comparative transcriptome profiling of the SL-signaling d14 mutant and WT plants revealed that a large number of defense-associated genes including hydrogen peroxide (H2O2)-, ethylene- and cell wall-synthesis-related genes were remarkably suppressed in d14 with respect to that of WT plants, during M. oryzae infection. In addition, various KEGG metabolic pathways related to sugar metabolism were significantly suppressed in the d14 plants compared to WT, during M. oryzae infection. Accordingly, WT plants accumulated increased levels of H2O2 and soluble sugar content compared to that of d17 and d14 in response to M. oryzae infection. Altogether, these results propose that SLs positively regulated rice defense against M. oryzae through involvement in the induction of various defense associated genes/pathways.

Current understanding of the molecular players involved in resistance to rice planthoppers.

Rice planthoppers are the most widespread and destructive pest of rice. Planthopper control depends greatly on the understanding of molecular players involved in resistance to planthoppers. This paper summarizes the recent progress in the understanding of some molecular players involved in resistance to planthoppers and the mechanisms involved. Recent researches showed that host-plant resistance is the most promising sustainable approach for controlling planthoppers. Planthopper-resistant varieties with a host-plant resistance gene have been released for rice products. Integrated planthopper management is a proposed strategy to prolong the durability of host-plant resistance. Bacillus spp. and their gene products or insect pathogenic fungi have great potential for application in the biological control of planthoppers. Enhancement of the activity of the natural enemies of planthoppers would be more cost-effective and environmentally friendly. Various molecular processes regulate rice-planthopper interactions. Rice encounters planthopper attacks via transcription factors, secondary metabolites, and signaling networks in which phytohormones have central roles. Maintenance of cell wall integrity and lignification act as physical barriers. Indirect defenses of rice are regulated via chemical elicitors, honeydew-associated elicitor, amendment with silicon and biochar, and salivary protein of BPH as elicitor or effector. Further research directions on planthopper control and rice defense against planthoppers are also put forward. © 2019 Society of Chemical Industry.

Phosphorylation of bip130 by OsMPK1 regulates abscisic acid-induced antioxidant defense in rice

In rice (Oryza sativa), the mitogen-activated protein kinase 1, OsMPK1, has been shown to play an important role in abscisic acid (ABA)-induced antioxidant defense and to enhance the tolerance of plants to drought, salinity and oxidative stress. However, its downstream molecular mechanisms are poorly understood. Here, we identified a BRI1-KD interacting protein 130, bip130, which interacts with OsMPK1 in vitro and in vivo. A transient expression analysis in combination with mutant analysis in rice protoplasts revealed that bip130 is required for ABA-induced antioxidant defense in an OsMPK1-dependent manner. Furthermore, bip130 can be phosphorylated by OsMPK1 at Thr-153 in vitro, and Thr-153 is essential for the ABA-induced antioxidant defense by OsMPK1. These results reveal that OsMPK1 phosphorylates bip130 at Thr-153 to regulate ABA-induced antioxidant defense.

Transcriptional Regulation of miR528 by OsSPL9 Orchestrates Antiviral Response in Rice

Many microRNAs (miRNAs) are critical regulators of plant antiviral defense. However, little is known about how these miRNAs respond to virus invasion at the transcriptional level. We previously show that defense against Rice stripe virus (RSV) invasion entailed a reduction of miR528 accumulation in rice, alleviating miR528-mediated degradation of L-Ascorbate Oxidase (AO) mRNA and bolstering the antiviral activity of AO. Here we show that the miR528-AO defense module is regulated by the transcription factor SPL9. SPL9 displayed high-affinity binding to specific motifs within the promoter region of miR528 and activated the expression of miR528 gene in vivo. Loss-of-function mutations in SPL9 caused a significant reduction in miR528 accumulation but a substantial increase of AO mRNA, resulting in enhanced plant resistance to RSV. Conversely, transgenic overexpression of SPL9 stimulated the expression of miR528 gene, hence lowering the level of AO mRNA and compromising rice defense against RSV. Importantly, gain in RSV susceptibility did not occur when SPL9 was overexpressed in mir528 loss-of-function mutants, or in transgenic rice expressing a miR528-resistant AO. Taken together, the finding of SPL9-mediated transcriptional activation of miR528 expression adds a new regulatory layer to the miR528-AO antiviral defense pathway.

Expression Analysis of Defense Related Genes in Rice Response to Bipolaris oryzae, the Causal Agent of Rice Brown Spot

The rice defense mechanism was studied against Bipolaris oryzae, the rice brown spot fungus, in two Thai rice varieties, Khao Dawk Mali 105 (KDML 105) and Jao Hom Nin (JHN) (showing highest and lowest susceptibility to B. oryzae, respectively). The expression was evaluated of eight genes through real-time quantitative reverse transcription polymerase chain reaction. The gene involved in the salicylic acid (SA) signaling pathway (OsPAL) and the pathogenesis related genes (OsPR1b and OsPBZ1) were upregulated in both varieties with no significant differences. Despite higher expression of the genes involved in the jasmonic acid (JA) signaling pathway (OsLOX and OsAOS2) in JHN, the expression of JiOsPR10 was not significantly different in both varieties. The genes involved in the ethylene (ET) signaling pathway (OsACS1 and OsEIN2) were expressed more highly and far more rapidly in KDML 105 than JHN. Overall, our results demonstrated that the investigated genes related to SA, JA and ET defense pathways may not play a major role in rice resistance against B. oryzae. Furthermore, the high level of transcript accumulation of genes related to the ET signaling pathway may interfere with the ability of rice to resist B. oryzae. The study provided information for a better understanding of rice defense mechanisms against B. oryzae.