Functional Analysis Of AtWRKY8,28,48 And 71 And The Regulation Of SERRATE By WRKY Superfamily In Arabidopsis

The transcriptional factor WRKY protein superfamily, which exists exclusively in plants, can be classified into three groups on the basis of both the number of WRKY domains and the features of their zinc-finger-like motif. There exits at least 74 and 97 memberes in Arabidops and rice, respectively. WRKY proteins have been implicated in the regulation of various response porcesses, including pathogen infection, abiotic stress and plant development.Numerous in vitro experiments have demonstrated that WRKY proteins specifically bind to the W-box (T)TGAC(C/T), a cis-acting DNA element found frequently in the promoter of target genes. There are twelve TGAC core sequence in SERRATE promoter. As a single W box within a promoter is sometimes sufficient to mediate WRKY-dependent gene expression. SERRATE would be regulated by WRKY transcription factor for its numerous W box sequences in therotically. One part of the dissertation was on the regulation of SERRATE by WRKY transcription factors.Using CHIP analysis performed with anti-all-WRKY serum, we found that all the twelve W-boxes in the SERRATE promoter were bound by certain WRKY proteins. Using a report gene, we found that transgenic plants transformed with the SE:GUS construct produced significantly more GUS activity than those transformed with mSE:.GUSconstruct(3.78 fold); Consistent with the GUS activity, the transgenic plants transformed with the SE:GUS construct were stained blue as it is, while the transgenic plants transformed with the mSE-GUS construct can’t be stained blue in any part of the tissues just like WT. Using complementary experiment, we found that the transgenic plants transformed with the SE:SE successfully restore the se-1 mutants for phenotype change and miRNA accumulation, while the transgenic plants transformed with the mSE:SE not. In conlusion, the W box sequences in SERRATE promoter are necessary for the basal expression of SERRATE. These results provide strong evidence that certain WRKY genes act upstream of SERRATE and positively regulate its basal expression during plant developmental progress. Interestingly, mutation of SE enhanced the disease resistance toward PstDC3000.GroupⅠand groupⅢwere placed into two separate clusters whereas groupll were classified into several clusters based on the phylogenetic tree by Dong. AtWRKY8, 28,48, and 71 belongs to IId cluster on the tree. Another part of my research contents focus on functional analysis of AtWRKY8,28,48, and 71, but maily on At WRKY8.Based on the Northern bolt analysis, we showed that the expression of AtWRKY8, 28 and 71 mainly focus on root, and using the GUS reporter gene, we further showed that At WRKY8 expressed in root tip, lateral root initiations, the basis of lateral roots, root/hypocotyls transitions, shoot apex, vascular tissue and stigma, which imply that AtWRKY8 may play a role in development and certain stress signaling.Under abiotic stresses, Northern bolt analysis showed that AtWRKY8,28, and 71 was not induced by 4℃,42℃and PEG, but slightly induced by NaCl and strongly induced by wounding, similarly with them, AtWRKY48 was also induced by NaCl and wounding. Thus we thought that the function of AtWRKY8,28,48, and 71 probably involved in the NaCl stress and wounding signal. Because among these four genes we only got the AtWRKY8 and 48 mutants, so our futher work was maily foucs on AtWRKY8 and 48 mutants. Through treatment with NaCl toward 7 or 21d old seedlings, we found that mutation of AtWRKY8 decrease the ability of wrky8 to NaCl stress. Similarly, wrky48 has the same response to NaCl.Under biotic stresses, Northern bolt analysis and GUS staining showed that AtWRKY8 was induced by PstDC3000 or Botrytis infection, and also played roles in defence against PstDC3000 and Botrytis. Mutation of AtWRKY8 conferred enhanced tolerance taward PstDC3000 infection while decresased tolerance taward Botrytis infection; by contrast, over-expression of AtWRKY8 showed enhanced susceptibility toward PstDC3000 infection and enhanced resistance to Botrytis infectionAt the same time, based on Northern blot analysis and GUS staining, we demonstrated that AtWRKY8 played an important role in inhibiting the systemic transportation of TMV-Cg. The transportation of TMV-Cg in systemic leaves of Atwrky8 was clearly faster than in Wt’s. The existence of TMV-Cg can be detected in systemic leaves of Atwrky8 at 4dpi, but not in Wt’s;and the existence of TMV-Cg was abundant at 6dpi, but only few in Wt’s. Excitingly, the transcript level of PR2 in systemic leaves of Atwrky8 was undoubtedly higher than Wt’s. All of these showed that AtWRKY8 played an important role in inhibiting the transportation of TMV-Cg.Interestingly, by compare the microarray results between WT and wrky8 after inculation with TMV-Cg for 6 days, we found that a number of defence related genes (such as PR1 and PR2) and ET synthyized or responsive genes (such as ACS6, ACS11 and ETR6) were stronger induced in wrky8. Furthermore, by inculation the WT(treated with ACC or not) and acs6 with TMV-Cg, we found that ACC dramatically increased the accumulation of TMV-Cg while mutation of ACS6 greatly inhibited the TMV-Cg accumulation in systemic leaves. All these results showed that ET played a negative role in anti-virals which also validated the function of AtWRKY8 in anti- TMV-Cg.