An Agrobacterium-Mediated Transient Expression Method for Functional Assay of Genes Promoting Disease in Monocots.

Agrobacterium-mediated transient expression (AMTE) has been widely used for high-throughput assays of gene function in diverse plant species. However, its application in monocots is still limited due to low expression efficiency. Here, by using histochemical staining and a quantitative fluorescence assay of ß-glucuronidase (GUS) gene expression, we investigated factors affecting the efficiency of AMTE on intact barley plants. We found prominent variation in GUS expression levels across diverse vectors commonly used for stable transformation and that the vector pCBEP produced the highest expression. Additionally, concurrent treatments of plants with one day of high humidity and two days of darkness following agro-infiltration also significantly increased GUS expression efficiency. We thus established an optimized method for efficient AMTE on barley and further demonstrated its efficiency on wheat and rice plants. We showed that this approach could produce enough proteins suitable for split-luciferase assays of protein-protein interactions on barley leaves. Moreover, we incorporated the AMTE protocol into the functional dissection of a complex biological process such as plant disease. Based on our previous research, we used the pCBEP vector to construct a full-length cDNA library of genes upregulated during the early stage of rice blast disease. A subsequent screen of the library by AMTE identified 15 candidate genes (out of ~2000 clones) promoting blast disease on barley plants. Four identified genes encode chloroplast-related proteins: OsNYC3, OsNUDX21, OsMRS2-9, and OsAk2. These genes were induced during rice blast disease; however, constitutive overexpression of these genes conferred enhanced disease susceptibility to Colletotrichum higginsianum in Arabidopsis. These observations highlight the power of the optimized AMTE approach on monocots as an effective tool for facilitating functional assays of genes mediating complex processes such as plant-microbe interactions.

Phosphorylation and ubiquitination of OsWRKY31 are integral to OsMKK10-2-mediated defense responses in rice.

Mitogen-activated protein kinase (MPK) cascades play vital roles in plant innate immunity, growth, and development. Here, we report that the rice (Oryza sativa) transcription factor gene OsWRKY31 is a key component in a MPK signaling pathway involved in plant disease resistance in rice. We found that the activation of OsMKK10-2 enhances resistance against the rice blast pathogen Magnaporthe oryzae and suppresses growth through an increase in jasmonic acid and salicylic acid accumulation and a decrease of indole-3-acetic acid levels. Knockout of OsWRKY31 compromises the defense responses mediated by OsMKK10-2. OsMKK10-2 and OsWRKY31 physically interact, and OsWRKY31 is phosphorylated by OsMPK3, OsMPK4, and OsMPK6. Phosphomimetic OsWRKY31 has elevated DNA-binding activity and confers enhanced resistance to M. oryzae. In addition, OsWRKY31 stability is regulated by phosphorylation and ubiquitination via RING-finger E3 ubiquitin ligases interacting with WRKY 1 (OsREIW1). Taken together, our findings indicate that modification of OsWRKY31 by phosphorylation and ubiquitination functions in the OsMKK10-2-mediated defense signaling pathway.

OsWRKY62 and OsWRKY76 Interact with Importin α1s for Negative Regulation of Defensive Responses in Rice Nucleus.

Background OsWRKY62 and OsWRKY76, two close members of WRKY transcription factors, function together as transcriptional repressors. OsWRKY62 is predominantly localized in the cytosol. What are the regulatory factors for OsWRKY62 nuclear translocation? Results In this study, we characterized the interaction of OsWRKY62 and OsWRKY76 with rice importin, OsIMα1a and OsIMα1b, for nuclear translocation. Chimeric OsWRKY62.1-GFP, which is predominantly localized in the cytoplasm, was translocated to the nucleus of Nicotiana benthamiana leaf cells in the presence of OsIMα1a or OsIMαΔIBB1a lacking the auto-inhibitory importin ß-binding domain. OsIMαΔIBB1a interacted with the WRKY domain of OsWRKY62.1, which has specific bipartite positively charged concatenated amino acids functioning as a nuclear localization signal (NLS). Similarly, we found that OsIMαΔIBB1a interacted with the AvrPib effector of rice blast fungus Magnaporthe oryzae, which contains a scattered distribution of positively charged amino acids. Furthermore, we identified a nuclear export signal (NES) in OsWRKY62.1 that inhibited nuclear transportation. Overexpression of OsIMα1a or OsIMα1b enhanced resistance to M. oryzae, whereas knockout mutants decreased resistance to the pathogen. However, overexpressing both OsIMα1a and OsWRKY62.1 were slightly more susceptible to M. oryzae than OsWRKY62.1 alone. Ectopic overexpression of OsWRKY62.1-NES fused gene compromised the enhanced susceptibility of OsWRKY62.1 to M. oryzae. Conclusion These results revealed the existence of NLS and NES in OsWRKY62. OsWRKY62, OsWRKY76, and AvrPib effector translocate to nucleus in association with importin α1s through new types of nuclear localization signals for negatively regulating defense responses.

Salicylic Acid Is Required for Broad-Spectrum Disease Resistance in Rice.

Rice plants contain high basal levels of salicylic acid (SA), but some of their functions remain elusive. To elucidate the importance of SA homeostasis in rice immunity, we characterized four rice SA hydroxylase genes (OsSAHs) and verified their roles in SA metabolism and disease resistance. Recombinant OsSAH proteins catalyzed SA in vitro, while OsSAH3 protein showed only SA 5-hydroxylase (SA5H) activity, which was remarkably higher than that of other OsSAHs that presented both SA3H and SA5H activities. Amino acid substitutions revealed that three amino acids in the binding pocket affected SAH enzyme activity and/or specificity. Knockout OsSAH2 and OsSAH3 (sahKO) genes conferred enhanced resistance to both hemibiotrophic and necrotrophic pathogens, whereas overexpression of each OsSAH gene increased susceptibility to the pathogens. sahKO mutants showed increased SA and jasmonate levels compared to those of the wild type and OsSAH-overexpressing plants. Analysis of the OsSAH3 promoter indicated that its induction was mainly restricted around Magnaporthe oryzae infection sites. Taken together, our findings indicate that SA plays a vital role in immune signaling. Moreover, fine-tuning SA homeostasis through suppression of SA metabolism is an effective approach in studying broad-spectrum disease resistance in rice.

Comparison of mitochondrial genomes from multi-, Bi-, and uninucleate Rhizoctonia.

Six circular mitochondrial genomes of multi-, bi-, and uninucleate Rhizoctonia isolates were assembled and found that all the genomes contain 14 conserved protein-coding genes, one ribosomal protein (rps3), and 23 tRNA in the same order. The mitogenome sizes of uninucleate isolates were relatively smaller than binucleate and multinucleate stains. The size variations between uninucleate and multinucleate isolates were from both intergenic and intronic regions, whereas the differences between uninucleate and binucleate isolates were predominantly from intergenic regions. The phylogenetic analysis revealed that Rhizoctonia strains of the same nucleate types had a closer relationship.

Evolutionary and genomic comparisons of hybrid uninucleate and nonhybrid Rhizoctonia fungi.

The basidiomycetous fungal genus, Rhizoctonia, can cause severe damage to many plants and is composed of multinucleate, binucleate, and uninucleate species differing in pathogenicity. Here we generated chromosome-scale genome assemblies of the three nuclear types of Rhizoctonia isolates. The genomic comparisons revealed that the uninucleate JN strain likely arose by somatic hybridization of two binucleate isolates, and maintained a diploid nucleus. Homeolog gene pairs in the JN genome have experienced both decelerated or accelerated evolution. Homeolog expression dominance occurred between JN subgenomes, in which differentially expressed genes show potentially less evolutionary constraint than the genes without. Analysis of mating-type genes suggested that Rhizoctonia maintains the ancestral tetrapolarity of the Basidiomycota. Long terminal repeat-retrotransposons displayed a reciprocal correlation with the chromosomal GC content in the three chromosome-scale genomes. The more aggressive multinucleate XN strain had more genes encoding enzymes for host cell wall decomposition. These findings demonstrate some evolutionary changes of a recently derived hybrid and in multiple nuclear types of Rhizoctonia.

A fungal effector targets a heat shock-dynamin protein complex to modulate mitochondrial dynamics and reduce plant immunity.

Mitochondria are essential for animal and plant immunity. Here, we report that the effector MoCDIP4 of the fungal pathogen Magnaporthe oryzae targets the mitochondria-associated OsDjA9-OsDRP1E protein complex to reduce rice immunity. The DnaJ protein OsDjA9 interacts with the dynamin-related protein OsDRP1E and promotes the degradation of OsDRP1E, which functions in mitochondrial fission. By contrast, MoCDIP4 binds OsDjA9 to compete with OsDRP1E, resulting in OsDRP1E accumulation. Knockout of OsDjA9 or overexpression of OsDRP1E or MoCDIP4 in transgenic rice results in shortened mitochondria and enhanced susceptibility to M. oryzae Overexpression of OsDjA9 or knockout of OsDRP1E in transgenic rice, in contrast, leads to elongated mitochondria and enhanced resistance to M. oryzae Our study therefore reveals a previously unidentified pathogen-infection strategy in which the pathogen delivers an effector into plant cells to target an HSP40-DRP complex; the targeting leads to the perturbation of mitochondrial dynamics, thereby inhibiting mitochondria-mediated plant immunity.

The rice terpene synthase gene OsTPS19 functions as an (S)-limonene synthase in planta, and its overexpression leads to enhanced resistance to the blast fungus Magnaporthe oryzae.

Rice blast disease, caused by the fungus Magnaporthe oryzae, is the most devastating disease of rice. In our ongoing characterization of the defence mechanisms of rice plants against M. oryzae, a terpene synthase gene OsTPS19 was identified as a candidate defence gene. Here, we report the functional characterization of OsTPS19, which is up-regulated by M. oryzae infection. Overexpression of OsTPS19 in rice plants enhanced resistance against M. oryzae, while OsTPS19 RNAi lines were more susceptible to the pathogen. Metabolic analysis revealed that the production of a monoterpene (S)-limonene was increased and decreased in OsTPS19 overexpression and RNAi lines, respectively, suggesting that OsTPS19 functions as a limonene synthase in planta. This notion was further supported by in vitro enzyme assays with recombinant OsTPS19, in which OsTPS19 had both sesquiterpene activity and monoterpene synthase activity, with limonene as a major product. Furthermore, in a subcellular localization experiment, OsTPS19 was localized in plastids. OsTPS19 has a highly homologous paralog, OsTPS20, which likely resulted from a recent gene duplication event. We found that the variation in OsTPS19 and OsTPS20 enzyme activities was determined by a single amino acid in the active site cavity. The expression of OsTPS20 was not affected by M. oryzae infection. This indicates functional divergence of OsTPS19 and OsTPS20. Lastly, (S)-limonene inhibited the germination of M. oryzae spores in vitro. OsTPS19 was determined to function as an (S)-limonene synthase in rice and plays a role in defence against M. oryzae, at least partly, by inhibiting spore germination.

Metabolic and transcriptional alternations for defense by interfering OsWRKY62 and OsWRKY76 transcriptions in rice.

Metabolomic and transcriptomic approaches were used to dissect the enhanced disease resistance in the plants harbouring a RNA interfering construct of OsWRKY62 and OsWRKY76 (dsOW62/76) genes. The primary metabolic pathways were activated in dsOW62/76 compared with wild-type (ZH17) plants, revealed by increased accumulation of amino acids and constituents of citric acid cycle etc. Contents of phenolic acids derived from phenylpropanoid pathway were elevated in dsOW62/76 plants. Importantly, phenolamides, conjugates of the phenolic acids with amines, were detected in large number and mostly at higher levels in dsOW62/76 compared with ZH17 plants; however, the free pools of flavonoids were mostly decreased in dsOW62/76. Salicylic acid (SA) and jasmonic acid (JA)/JA-Ile contents were increased in dsOW62/76 and knockout lines of individual OsWRKY62 and OsWRKY76 genes. Transcription of isochorismate synthase (OsICS1) gene was suppressed in dsOW62/76 and in MeJA-treated rice plants, whereas the transcription level of cinnamoyl-CoA hydratase-dehydrogenase (OsCHD) gene for ß-oxidation in peroxisome was increased. The calli with OsCHD mutation showed markedly decreased SA accumulation. These results indicate that OsWRKY62 and OsWRKY76 function as negative regulators of biosynthetic defense-related metabolites and provide evidence for an important role of phenylpropanoid pathway in SA production in rice.

Rice black streaked dwarf virus P7-2 forms a SCF complex through binding to Oryza sativa SKP1-like proteins, and interacts with GID2 involved in the gibberellin pathway.

As a core subunit of the SCF complex that promotes protein degradation through the 26S proteasome, S-phase kinase-associated protein 1 (SKP1) plays important roles in multiple cellular processes in eukaryotes, including gibberellin (GA), jasmonate, ethylene, auxin and light responses. P7-2 encoded by Rice black streaked dwarf virus (RBSDV), a devastating viral pathogen that causes severe symptoms in infected plants, interacts with SKP1 from different plants. However, whether RBSDV P7-2 forms a SCF complex and targets host proteins is poorly understood. In this study, we conducted yeast two-hybrid assays to further explore the interactions between P7-2 and 25 type I Oryza sativa SKP1-like (OSK) proteins, and found that P7-2 interacted with eight OSK members with different binding affinity. Co-immunoprecipitation assay further confirmed the interaction of P7-2 with OSK1, OSK5 and OSK20. It was also shown that P7-2, together with OSK1 and O. sativa Cullin-1, was able to form the SCF complex. Moreover, yeast two-hybrid assays revealed that P7-2 interacted with gibberellin insensitive dwarf2 (GID2) from rice and maize plants, which is essential for regulating the GA signaling pathway. It was further demonstrated that the N-terminal region of P7-2 was necessary for the interaction with GID2. Overall, these results indicated that P7-2 functioned as a component of the SCF complex in rice, and interaction of P7-2 with GID2 implied possible roles of the GA signaling pathway during RBSDV infection.

Alternative Splicing of Rice WRKY62 and WRKY76 Transcription Factor Genes in Pathogen Defense.

The WRKY family of transcription factors (TFs) functions as transcriptional activators or repressors in various signaling pathways. In this study, we discovered that OsWRKY62 and OsWRKY76, two genes of the WRKY IIa subfamily, undergo constitutive and inducible alternative splicing. The full-length OsWRKY62.1 and OsWRKY76.1 proteins formed homocomplexes and heterocomplexes, and the heterocomplex dominates in the nuclei when analyzed in Nicotiana benthamiana leaves. Transgenic overexpression of OsWRKY62.1 and OsWRKY76.1 in rice (Oryza sativa) enhanced plant susceptibility to the blast fungus Magnaporthe oryzae and the leaf blight bacterium Xanthomonas oryzae pv oryzae, whereas RNA interference and loss-of-function knockout plants exhibited elevated resistance. The dsOW62/76 and knockout lines of OsWRKY62 and OsWRKY76 also showed greatly increased expression of defense-related genes and the accumulation of phytoalexins. The ratio of full-length versus truncated transcripts changed in dsOW62/76 plants as well as in response to pathogen infection. The short alternative OsWRKY62.2 and OsWRKY76.2 isoforms could interact with each other and with full-length proteins. OsWRKY62.2 showed a reduced repressor activity in planta, and two sequence determinants required for the repressor activity were identified in the amino terminus of OsWRKY62.1. The amino termini of OsWRKY62 and OsWRKY76 splice variants also showed reduced binding to the canonical W box motif. These results not only enhance our understanding of the DNA-binding property, the repressor sequence motifs, and the negative feedback regulation of the IIa subfamily of WRKYs but also provide evidence for alternative splicing of WRKY TFs during the plant defense response.

Rice OsFLS2-Mediated Perception of Bacterial Flagellins Is Evaded by Xanthomonas oryzae pvs. oryzae and oryzicola.

Bacterial flagellins are often recognized by the receptor kinase FLAGELLIN SENSITIVE2 (FLS2) and activate MAMP-triggered immunity in dicotyledonous plants. However, the capacity of monocotyledonous rice to recognize flagellins of key rice pathogens and its biological relevance remain poorly understood. We demonstrate that ectopically expressed OsFLS2 in Arabidopsis senses the eliciting flg22 peptide and in vitro purified Acidovorax avenae (Aa) flagellin in an expression level-dependent manner, but does not recognize purified flagellins or derivative flg22(Xo) peptides of Xanthomonas oryzae pvs. oryzae (Xoo) and oryzicola (Xoc). Consistently, the flg22 peptide and purified Aa flagellin, but not Xoo/Xoc flagellins, induce various immune responses such as defense gene induction and MAPK activation in rice. Perception of flagellin by rice does induce strong resistance to Xoo infection, as shown after pre-treatment of rice leaves with Aa flagellin. OsFLS2 was found to differ from AtFLS2 in its perception specificities or sensitivities to different flg22 sequences. In addition, post-translational modification of Xoc flagellin was altered by deletion of glycosyltransferase-encoding rbfC, but this had little effect on Xoc motility and rpfC mutation did not detectably reduce Xoc virulence on rice. Deletion of flagellin-encoding fliC from Xoo/Xoc blocked swimming motility but also did not significantly alter Xoo/Xoc virulence. These results suggest that Xoo/Xoc carry flg22-region amino acid changes that allow motility while evading the ancient flagellin detection system in rice, which retains recognition capacity for other bacterial pathogens.

Dlf1, a WRKY transcription factor, is involved in the control of flowering time and plant height in rice.

Flowering time and plant height are important agronomic traits for crop production. In this study, we characterized a semi-dwarf and late flowering (dlf1) mutation of rice that has pleiotropic effects on these traits. The dlf1 mutation was caused by a T-DNA insertion and the cloned Dlf1 gene was found to encode a WRKY transcription factor (OsWRKY11). The dlf1 mutant contains a T-DNA insertion at the promoter region, leading to enhanced accumulation of Dlf1 transcripts, resulting in a semidominant mutation. The dlf1 mutation suppressed the transcription of Ehd2/RID1/OsId1 and its downstream flowering-time genes including Hd1, Ehd1 and Hd3a under both long-day (LD) and short-day (SD) conditions. Knock-down of Dlf1 expression exhibited early flowering at LD condition related to the wild-type plants. Accumulation of Dlf1 mRNA was observed in most tissues, and two splicing forms of Dlf1 cDNAs were obtained (OsWRKY11.1 and OsWRKY11.2). These two proteins showed transactivation activity in yeast cells. Dlf1 protein was found to be localized in the nucleus. Enhanced expression of OsWRKY11.2 or its 5' truncated gene showed similar phenotypes to the dlf1 mutant, suggesting that it might function as a negative regulator. We conclude that Dlf1 acts as a transactivator to downregulate Ehd2/RID1/OsId1 in the signal transduction pathway of flowering and plays an important role in the regulation of plant height in rice.

RBS1, an RNA binding protein, interacts with SPIN1 and is involved in flowering time control in rice.

The rice U-box/ARM E3 ubiquitin ligase SPL11 negatively regulates programmed cell death (PCD) and disease resistance, and controls flowering time through interacting with the novel RNA/DNA binding KH domain protein SPIN1. Overexpression of Spin1 causes late flowering in transgenic rice under short-day (SD) and long-day (LD) conditions. In this study, we characterized the function of the RNA-binding and SPIN1-interacting 1 (RBS1) protein in flowering time regulation. Rbs1 was identified in a yeast-two-hybrid screen using the full-length Spin1 cDNA as a bait and encodes an RNA binding protein with three RNA recognition motifs. The protein binds RNA in vitro and interacts with SPIN1 in the nucleus. Rbs1 overexpression causes delayed flowering under SD and LD conditions in rice. Expression analyses of flowering marker genes show that Rbs1 overexpression represses the expression of Hd3a under SD and LD conditions. Rbs1 is upregulated in both Spin1 overexpression plants and in the spl11 mutant. Interestingly, Spin1 expression is increased but Spl11 expression is repressed in the Rbs1 overexpression plants. Western blot analysis revealed that the SPIN1 protein level is increased in the Rbs1 overexpression plants and that the RBS1 protein level is also up-regulated in the Spin1 overexpression plants. These results suggest that RBS1 is a new negative regulator of flowering time that itself is positively regulated by SPIN1 but negatively regulated by SPL11 in rice.

Constitutive expression of rice WRKY30 gene increases the endogenous jasmonic acid accumulation, PR gene expression and resistance to fungal pathogens in rice.

WRKY transcription factors are crucial regulatory components of plant responses to pathogen infection. In the present study, we report isolation and functional characterization of the pathogen-responsive rice WRKY30 gene, whose transcripts accumulate rapidly in response to salicylic acid (SA) and jasmonic acid (JA) treatment. Overexpression of WRKY30 in rice enhanced resistance to rice sheath blight fungus Rhizoctonia solani and blast fungus Magnaporthe grisea. The enhanced resistance in the transgenic lines overexpressing WRKY30 was associated with activated expression of JA synthesis-related genes LOX, AOS2 and pathogenesis-related (PR)3 and PR10, and increased endogenous JA accumulation under the challenge of fungal pathogens. WRKY30 was nuclear-localized and had transcriptional activation ability in yeast cells, supporting that it functions as a transcription factor. Together, our findings indicate that JA plays a crucial role in the WRKY30-mediated defense responses to fungal pathogens, and that the rice WRKY30 seems promising as an important candidate gene to improve disease resistance in rice.

The rice ERF transcription factor OsERF922 negatively regulates resistance to Magnaporthe oryzae and salt tolerance.

Rice OsERF922, encoding an APETELA2/ethylene response factor (AP2/ERF) type transcription factor, is rapidly and strongly induced by abscisic acid (ABA) and salt treatments, as well as by both virulent and avirulent pathovars of Magnaporthe oryzae, the causal agent of rice blast disease. OsERF922 is localized to the nucleus, binds specifically to the GCC box sequence, and acts as a transcriptional activator in plant cells. Knockdown of OsERF922 by means of RNAi enhanced resistance against M. oryzae. The elevated disease resistance of the RNAi plants was associated with increased expression of PR, PAL, and the other genes encoding phytoalexin biosynthetic enzymes and without M. oryzae infection. In contrast, OsERF922-overexpressing plants showed reduced expression of these defence-related genes and enhanced susceptibility to M. oryzae. In addition, the OsERF922-overexpressing lines exhibited decreased tolerance to salt stress with an increased Na(+)/K(+) ratio in the shoots. The ABA levels were found increased in the overexpressing lines and decreased in the RNAi plants. Expression of the ABA biosynthesis-related genes, 9-cis-epoxycarotenoid dioxygenase (NCED) 3 and 4, was upregulated in the OsERF922-overexpressing plants, and NCED4 was downregulated in the RNAi lines. These results suggest that OsERF922 is integrated into the cross-talk between biotic and abiotic stress-signalling networks perhaps through modulation of the ABA levels.

Antifungal activity of the essential oil of Illicium verum fruit and its main component trans-anethole.

In order to identify natural products for plant disease control, the essential oil of star anise (Illicium verum Hook. f.) fruit was investigated for its antifungal activity on plant pathogenic fungi. The fruit essential oil obtained by hydro-distillation was analyzed for its chemical composition by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). trans-Anethole (89.5%), 2-(1-cyclopentenyl)-furan (0.9%) and cis-anethole (0.7%) were found to be the main components among 22 identified compounds, which accounted for 94.6% of the total oil. The antifungal activity of the oil and its main component trans-anethole against plant pathogenic fungi were determined. Both the essential oil and trans-anethole exhibited strong inhibitory effect against all test fungi indicating that most of the observed antifungal properties was due to the presence of trans-anethole in the oil, which could be developed as natural fungicides for plant disease control in fruit and vegetable preservation.

Constitutive expression of pathogen-inducible OsWRKY31 enhances disease resistance and affects root growth and auxin response in transgenic rice plants.

WRKY transcription factors have many regulatory roles in response to biotic and abiotic stresses. In this study, we isolated a rice WRKY gene (OsWRKY31) that is induced by the rice blast fungus Magnaporthe grisea and auxin. This gene encodes a polypeptide of 211 amino-acid residues and belongs to a subgroup of the rice WRKY gene family that probably originated after the divergence of monocot and dicot plants. OsWRKY31 was found to be localized to the nucleus of onion epidermis cells to transiently express OsWRKY31-eGFP fusion protein. Analysis of OsWRKY31 and its mutants fused with a Gal4 DNA-binding domain indicated that OsWRKY31 has transactivation activity in yeast. Overexpression of the OsWRKY31 gene was found to enhance resistance against infection with M. grisea, and the transgenic lines exhibited reduced lateral root formation and elongation compared with wild-type and RNAi plants. The lines with overexpression showed constitutive expression of many defense-related genes, such as PBZ1 and OsSci2, as well as early auxin-response genes, such as OsIAA4 and OsCrl1 genes. Furthermore, the plants with overexpression were less sensitive to exogenously supplied IBA, NAA and 2,4-D at high concentrations, suggesting that overexpression of the OsWRKY31 gene might alter the auxin response or transport. These results also suggest that OsWRKY31 might be a common component in the signal transduction pathways of the auxin response and the defense response in rice.

Tobacco OPBP1 enhances salt tolerance and disease resistance of transgenic rice.

Osmotin promoter binding protein 1 (OPBP1), an AP2/ERF transcription factor of tobacco, has been demonstrated to function in disease resistance and salt tolerance in tobacco. To increase stress tolerant capability of rice, we generated rice plants with an OPBP1 overexpressing construct. Salinity shock treatment with 250 mM NaCl indicated that most of the OPBP1 transgenic plants can survive, whereas the control seedlings cannot. Similar recovery was found by using the seedlings grown in 200 mM NaCl for two weeks. The OPBP1 transgenic and control plants were also studied for oxidative stress tolerance by treatment with paraquat, showing the transgenic lines were damaged less in comparison with the control plants. Further, the OPBP1 overexpression lines exhibited enhanced resistance to infections of Magnaporthe oryzae and Rhizoctonia solani pathogens. Gene expressing analysis showed increase in mRNA accumulation of several stress related genes. These results suggest that expression of OPBP1 gene increase the detoxification capability of rice.

Overexpression of rice WRKY89 enhances ultraviolet B tolerance and disease resistance in rice plants.

WRKY proteins are a large family of transcriptional regulators involved in a variety of biological processes in plants. Here we report functional characterization of a rice WRKY gene, OsWRKY89. RNA gel blot analysis indicated that OsWRKY89 was strongly induced by treatments of methyl jasmonate and UV-B radiation. The transient expression analysis of the OsWRKY89-eGFP reporter in onion epidermal cells revealed that OsWRKY89 was targeted to nuclei. Transcriptional activity assays of OsWRKY89 and its mutants fused with a GAL4 DNA binding domain indicated that the 67 C-terminal amino acids were required for the transcriptional activation and that the leucine zipper region at the N-terminus enhanced its transcriptional activity. Overexpression of OsWRKY89 led to growth retardation at the early stage and reduction of internode length. Scanning electron microscopy revealed an increase in wax deposition on leaf surfaces of the OsWRKY89 overexpression lines and a decrease in wax loading in the RNAi-mediated OsWRKY89 suppression lines. Moreover, extractable and cell-wall-bound phenolic compounds were decreased in the overexpressor lines, but its SA levels were increased. Lignin staining showed an increase in lignification in culms of the overexpressor lines. Interestingly, overexpression of the OsWRKY89 gene enhanced resistance to the rice blast fungus and white-backed planthopper as well as tolerance to UV-B irradiation. These results suggest that OsWRKY89 plays an important role in response to biotic and abiotic stresses.