Functional Characterization And Regulatory Analysis Of A Rice WRKY Transcription Factor, OsWRKY13

During the plant defense responses, the components involved in the cross-talkbetween salicylic acid (SA)- and jasmonic acid (JA)-dependent defense pathways againstpathogens and insects have been partially revealed in dicot plants. Many evidences haveshowed that the WRKY transcription factors as unique to plants are also involved inmodulation of SA- or JA-responsive gene expression. Bacterial blight and fungal blast aretwo of the most devastating diseases of rice worldwide. Although 109-112 WRKY geneshave been identified in the rice genome, the functions of most in rice disease resistanceare unknown, especially for their role in SA- and JA-dependent signaling transductionpathways. Here we show that OsWRKY13 plays a pivotal role in rice disease resistancethrough different methods in functional genomic research.Overexpression of OsWRKY13 in rice susceptible cultivar Mudanjiang 8 canenhance transgenic plants resistance to bacterial blight and fungal blast, two of the mostdevastating diseases of rice worldwide, at both the seedling and adult stages. Conversely,transgenic plants of OsWRKY13-antisense suppression appeared to be more sensitive tobacterial blight (Xoo strain PXO61). Furthermore, different pathogenesis-related genes,SA synthesis-related genes and JA synthesis-related genes were examined in transgenicplants by quantitative RT-PCR. The results showed that OsWRKY13-overexpressingplants accumulated significantly more PR1αtranscripts than wild-type plants in at leastone examine time-point, and OsWRKY13-suppressed plant complemented that expressionpattern. Moreover, the analysis of eleven genes involved SA synthesis, JA synthesis oroxylipin metabolism indicated that accumulation of OsWRKY13 transcripts significantlyinduced the expression of CHS, PAD4, and ICS1 in at least one time point examined, incontrast, partially suppressing OsWRKY13 expression showed no influence on theexpression of the three genes compared with wild-type plant. Overexpressing OsWRKY13significantly repressed the expression of AOS2 and POX, while suppressing OsWRKY13expression significantly increased the expression of the two genes compared withwild-type plants. Further analysis of yeast one hybrid suggested that OsWRKY13possessed specific and strongly DNA-binding ability to the promoters of PR1α, AOS1,AOS2 and LOX, not to the promoters of ICS1, NH1 and PAD4. And the gel mobility shiftassay showed that nuclear proteins from OsWRKY13-overexpressing plants andOsWRKY13-GST fusion protein had stronger binding signal to the probe, harboring theW-like box, than that from wild type or control. Notably, that DNA-binding ability wasinfluenced by challenged bacterial pathogen certified by a in vitro phosphorylationanalysis, which suggesting the phosphorylation or de-phosphorylation status ofOsWRKY13 play a crucial role in its regulation function. Considering the increasing ofendogenous SA concentration and decreasing of JA concentration inOsWRKY13-overexpressing plants, we concluded that OsWRKY13 participate in ricedefense response as an activator of the SA-dependent pathway and a repressor of JA-dependent pathways.To determine more downstream OsWRKY13-regulated genes and its regulationmechanism in OsWRKY13-overexpressing plants, we performed microarray analysisusing the Affymetrix oligonucletide rice chip array. Firstly, the analysis of geneontology(GO) classification in differential expressed genes showed that OsWRKY13 activelyregulated the flavonoids metabolism and negatively regulated terpene or lipid metabolism,those of which always related to the SA or JA signaling pathways, respectively. Secondly,the statistic analysis of conserved cis-regulatory element and discovering of putativeconserved motif in the promoters of OsWRKY13 downstream genes through performingan enumerative method showed that the promoters of most OsWRKY13 up-regulatedgenes over-represented a W-box element (GTTGAC), and Myb3 binding site enriched indown-regulated genes whereas the GCC-box depleted in down-regulated genes. Thoseresults are consistent with the previous results of differential expressed transcriptionfactors. Thus, OsWRKY13 regulated the metabolism of flavonoids and lipid by regulatedthe downstream genes through interaction with other MYB and AP2/ERF transcriptionfactors.Otherwise, we also explored the relationship between OsWRKY13 and other riceWRKY family members as well as Arabidopsis AtWRKY70. Interestingly, OsWRKY13negatively regulation the expression of most of other rice WRKY family members,especially for those related to ABA signaling pathway. Comparing analysis indicatedthere was a putative conservation of aging and anti-oxidation burst mechanism inAtWRKY70- and OsWRKY13-mediating defense responses.In summary, our results showed that OsWRKY13 participated in rice resistance tobacterial blight and fungal blast by regulating SA- and JA- related signaling pathways orflavonoids and lipid metabolism, through different regulation models.