Functional Characterization Of A Pair Of WRKY Transcription Factors In Rice Defense Response

Rice disease is one of important factors resulting heavy yield loss, including fungal blast disease and bacterial blight and streak disease, which caused by Magnaporthe grisea, Xanthomonas oryza sative pv. Oryza and Xanthomonas campestris pv.Oryzicola respectively. Research on rice resistance to disease has important effects on rice genetic improvement. Characterization of rice defense-responsive genes enriched the genetic resource of rice resistance and conferred a new way to breed broad-spectrum and durable resistance varieties, also helped to elucidate the mechanism of rice resistance. Here we show that OsWRKY45 and its alleles function in the response to rice disease and abiotic stress through different methods in functional genomic research.OsWRKY13 is a positive regulator of rice defense response. Overexpression of OsWRKY13 enhance resistance to blight and blast disease.OsWRKY13 can bind to the promoter of OsWRKY45. Overexpression of OsWRKY13 suppressed the expression of OsWRKY45, and suppression of OsWRKY13 enhanced the expression of OsWRKY45. Previous report had suggested that OsWRKY45 played a important role in BTH-induced resistance to M.grisea. Overexpression of OsWRKY45 enhanced the resistance to M.grisea and knockdown of OsWRKY45 compromised the resistance. In our study, we isolated the OsWRKY45 from different rice genomes. The OsWRKY45 from Japonica varieties had identical sequence and encoded 326 amino acids, named as OsWRKY45-1; The OsWRKY45 from indica varieties had identical sequence and encoded 322 amino acids, named as OsWRKY45-2. The differences between two alleles were in presence of promoters, coding sequences and proteins. By Agrobacterium-mediated transformation, OsWRKY45-1- and OsWRKY45-2-overexpressing plants were obtained in susceptible Japonica variety mudanjiang 8, OsWRKY45-2-RNAi-suppressing plants were obtained in indica variety minghui63, a T-DNA inserted mutants were obtained which the expression of OsWRKY45-1 was knockout. After inoculation with Xoo and cosegregation analysis, the results showed that OsWRKY45-1 was a negative regulator of rice resistance to blight disease. Overexpression of OsWRKY45-1 enhanced susceptible to Xoo and knockout of OsWRKY45-1 conferred the resistance. In contrast, OsWRKY45-2 was a positive regulator. Overexpression of OsWRKY45-2 enhanced resistance to Xoo and suppressing OsWRKY45-2 compromised the resistance. There was also no race-specificity in resistance mediated by OsWRKY45-1 and OsWRKY45-2. The transgenic plants exhibit the similar resistance to another bacterial disease, caused by Xanthomonas campestris pv.Oryzicola. However, OsWRKY45-1 and OsWRKY45-2-overexpressing plant both confer the resistance to M.grisea. We detected the expression pattern of other defense-responsive genes in transgenic and wild-type plants after inoculated with Xoo by quantitative RT-PCR. The results indicated that OsWRKY45-1 negatively regulated the expression of PR1a, PR1b, AOS2, LOX, PALI, NH1 and OsWRKY13, OsWRKY45-2 positively regulated the expression of PR1a, PR1b, AOS2, LOX, PAL1 and OsWRKY13, no effect on NH1. We also detected the concentration of salicylic acid (SA) and jasmonic acid (JA) in experimented plants. Overexpression of OsWRKY45-1 accompanied with decreased levels of SA and JA, OsWRKY45-1 mutant accumulated higher SA and JA. In contrast overexpression of OsWRKY45-2 accompanied with increased JA and invarable SA. Thereafter, we suggest that the opposite roles in the resistance to Xoo mediated by OsWRKY45-1 and OsWRKY45-2 maybe result from differentially regulation these defense-responsive genes and SA and JA-dependent signal pathway.The allelic genes of OsWRKY45-1 and OsWRKY45-2 not only functioned in rice defense response, but also function in abiotic stress. During the tolerance to abscisic acid (ABA), drought, high salinity and cold, OsWRKY45-1 and OsWRKY45-2 exhibit opposite and similar response. Both OsWRKY45-1 and OsWRKY45-2 transgenic plant alter the sensitivity to ABA stress. Overexprssion of OsWRKY45-1 exhibit decreased sensitivity to ABA accompanied with elevated ABA levels while OsWRKY45-1 mutants increased sensitivity. In contrast, Overexprssion of OsWRKY45-2 exhibited hyper-sensitivity to ABA accompanied with decreased ABA. Both OsWRKY45-1 and OsWRKY45-2 functioned as negative regulator to cold stress. But the transgenic plants had no alteration after high salinity and drought stress when compared with their wild-type plants.In order to decipher the different function of OsWRKY45-1 and OsWRKY45-2, the whole-genome microarrays were performed with transgenic and wild-type plants.We observed there would be some types of different expressed genes behalf different regulation by two alleles. Some genes showed similar expression pattern in the OsWRKY45-1 and OsWRKY45-2 transgenic plants which would be co-regulated by two alleles. Some genes show differentially expression between the OsWRKY45-1 and OsWRKY45-2 which would be specifically regulated by OsWRKY45-1 or OsWRKY45-2. Some genes even show reverse expression pattern between OsWRKY45-1 and OsWRKY45-2 which would be differentially regulated by OsWRKY45-1 and OsWRKY45-2. We noticed some genes which exhibit down-regulated in both OsWRKY45-1 and OsWRKY45-2-overexpressing plants and also induced after cold stress to some degree. We validated the expression of these genes in the transgenic plants by qRT-PCR. The results showed that the expression level in the transgenic plants were in accordance with that in the microarays.In conclusion, OsWRKY45-1 and OsWRKY45-2 function differently in various physiological processes which would be resulted from the different regulation of some related genes or act in the different horomone-mediated signal pathways. But the factors which would be responsible for the different function of the two allelic genes should be investigated with further experiments.