Plant Stress Signaling Pathways. WRKY Family Regulation Of The Preliminary Study Of The Molecular Mechanisms

The WRKY proteins mainly present in higher plants, and are extensive involved in defense responsive, stress signal, senescence and some development processes.OsWRKY30 is a member of WRKY transcription factor family in rice. It has two WRKY domains and belongs to Group I of WRKY family. GFP fusion protein of OsWRKY30 was mainly expressed in the nucleus of rice cells; OsWRKY30 protein can bind W-box in vitro, W-box is a common cis element binded by WRKY transcription factors; in yeast cells, OsWRKY30 had transcription activation, the ability of transcription activation of different truncated mutant of OsWRKY30 was also detected in yeast cells and transcription activation domain of OsWRKY30 was found in its N-terminal region. These results indicated that OsWRKY30 had the typical characters of WRKY transcription factors.The expression profiles of OsWRKY30 under ABA treatment and drought stress were analyzed by RT-PCR and Real-time PCR. The expression levels of OsWRKY30 were up-regulated by ABA treatment and drought stress, suggesting that OsWRKY30 was involved in ABA-related drought signal pathway in rice. It was also found that over-expression transgenic lines of OsWRKY30 had more drought tolerance than wild types under drought stress.It was reported that OsWRKY30 was also induced by SA, JA treatments and pathogens infection, suggesting the functional relationship with MAPK which is a kinase family involved in biotic and abiotic signal pathways. Meanwhile, the structure of OsWRKY30 was analyzed and a conserved domain including several two linked animo acids: Serine-Proline (SP site) in the N-terminal of OsWRKY30 was found. SP site is candidate phosphated site by MAPK. By yeast two-hybrid assay, it was found that OsMAPK5a which is a member of MAPK A group and OsWJUMK1 which is a member of MAPK D group interacted with OsWRKY30, OsBWMK1 which is also a member of MAPK D group did not interact with OsWRKY30.Interactions between different truncated versions of OsWRKY30 and two MAPKs were analyzed. It was found that OsWRKY30 interacted with two MAPKs by its SP sites. WRKY domain was important in the process of interaction between OsWRKY30 and two MAPKs , the regions including SP1 to SP6 or SP2 to SP5 could interact with OsMAPK5a, but strength was very poor. WRKY domain could strengthen the interaction between OsWRKY30 and OsMAPK5a. Two WRKY domains were necessary when OsWRKY30 interacted with OsWJUMK1, this was different from OsMAPK5a. by BiFC visualization, OsWRKY30 interacting with OsMAPK5a in rice cells was confirmed. It was the first report that WRKY transcription factors interacted with MAPK in vivo. OsWRKY30 was phosphorated by OsMAPK5a in vitro. All these results verified OsWRKY30 was a substrate of MAPK.After using the sequence of OsWRKY30 N-terminal conserved domain including multi SP sites to blast in Genebank database, 28 members of WRKY proteins which also have this conserved domain were discovered. According five continued amino acids residues, this conserved domain was named as SPTTG domain. 4 more WRKY proteins which included SPTTG domain were found after searching Arabidopsis and rice transcription factors database, so all of 32 members of WRKY proteins were found finally. All of them belong to Ia group of plant WRKY family, which was the most conserved group of WRKY family.5 members in Arabidopsis and 2 members in rice which have SPTTG domain were cloned. The interactions between these proteins and two MAPKs: OsMAPK5a and OsWJUMK1 were analyzed by yeast two-hybrid assays. The strengths of interactions between these WRKY proteins and two MAPKs were analyzed by detectingβ-gal activity and growth in SD/-Trp-Leu-His-Ade plate. The strengths of interactions between OsMAPK5a and AtWRKY2, AtWRKY20, OsWRKY30, OsWRKY53 and OsWRKY80 were higher than that between OsMAPK5a and AtWRKY25, AtWRKY26 and AtWRKY33. OsWJUMK1 had some different from OsMAPK5a. it did not interact with OsWRKY53, but with other members OsWJUMK1 was similar with OsMAPK5a. AtMAPK3, a member of MAPK family of Arabidopsis and a homologue of OsMAPK5a, was also cloned. The strengths of interactions between AtMAPK3 and these cloned WRKY proteins which have SPTTG domain were also analyzed, the results were similar with OsWJUMK1. These results proved WRKY proteins including SPTTG domain interacted with MAPKs. By BiFC visualization, it was found that OsWRKY80 interacted with OsMAPK5a in rice cells.Finally, we discussed the key role of SPTTG domain in related with WRKY family and MAPK family, the importance of WRKY protein which containing SPTTG domain in biotic and abiotic tress signal pathway.