Function Study Of Defense-related WRKY Gene In Soybean

The WRKY tamcription factor family is only presented in plant genomes,and is one of the largest transcription factor family in plants.The WRKY gene family is involved in numerous biological processes in plants,including plant growth and development,abiotic and biotic stress responses.The previous results in model plants showed that Arabidopsis WRKY33 plays a pivotal role in the MAPK3/6-mediated camalexin biosynthesis and reactive oxygen species(ROS)burst,indicating that WRKY33 positively regulates plant disease responses.Even though the WRKY33 is extensively studied in the model plant species,the function of WRKYs in soybean,the most important source of plant protein and edible oil,is still poorly understood.Our previous results showed that silencing GmWRKY33 homologs in soybean attenuated the activation of GmMPK6 and compromised the resistance against multiple bacterial pathogens,suggesting that Gm WRKY33s play a positive role in disease resistance.In this study,we determined to provide additional evidence that GmWRKY33s play a critical role in disease resistance through gain-of-function studies.Our bioinformatics analysis identified six soybean GmWRKY33 homologs sharing the highest homology with AtWRKY33.The WRKY33s across different species contain a highly conserved SP cluster(4-6 SPs)at their N-termini and the S is the phosphorylation target sites of MAPK3/6.To investigate the functional importance of the SP clusters,each individual phosphorylation target site within the SP clusters of these 6 GmWRKY33s was sequentially mutated to either a D,which mimics a constitutive phosphorylation state,or to an A,which abolishes the phosphorylation site.At present,we have obtained 32 various phospho-mimicking mutants(with all 4 or 6 Ss mutated to either Ds or As)and 18 non-phospho mutants.All these mutant GmWRKY33s together with the wild-type GmWRKY33s have been cloned into binary vecters.The transgenic lines that over-express some of these mutant Gm WRKY33s have been generated successfully and used for pathogen infection assays.our preliminary results showed that over-expressing one of the phospho-mimic GmWRKY33s(Gm09g2802004D)resulted in enhanced resistnce to Potato virus X(PVX)infections,further comfirming that GmWRKY33s play a positive role in disease resistance.Together,our results indicate that the GmWRKY33s could be potentially used for improving soybean resistance against multiple pathogens.The importance of the phosphorylation sites presented in the SP clustered will be further investigated after obtaining the transgenic lines that overexpress all the combinations of both phospo-mimic and non-phospo SP mutants.