Study Of Expression Regulation Of Endoxylanase Inhibitor RIXI In Rice

Xylanase inhibitor proteins (XIs) play a major role in plant defence. The transcriptomic changes between transgenic plants (overexpressing xylanase inhibitor RIXI gene) and wild-type plants showed that overexpression of RIXI induced changes in the expression of 12 WRKY transcription factors (TFs). WRKY family is one of the largest families of transcription factors in plants, responding to biotic and abiotic stresses. Bioinformatics analysis showed that RIXI promoter regions have several cis-elements of W-box which can bind to WRKY transcription factor. We speculated that WRKY may play a role in regulating the expression of RIXI. In this study, we selected two of the transcription factors OsWRKY46,OsWRKY6 to explore the relationships with xylanase inhibitor RIXI, investigating the regulation mode of RIXI. The main results are presented as following:1. OsWRKY and RIXI relative expression after the infection of Rice blast:Using qRT-PCR to analyse the relative expression of OsWRKY46、OsWRKY6 and RIXI after the infection of Rice blast. The result showed that the expression of OsWRKY46 was obviously decreased primitively then increased, whereas the expression of RIXI were progressively increased in wild type rice after the blast infection, indicating that OsWRKY46 negatively responds to Blast by reducing the expression of RIXI. While the expression of RIXI was positively correlated with OsWRKY6.OsWRKY6 may be the transcription activator of RIXI.2. OsWRKY combined with RIXI promoter:Yeast single-hybridization experiment showed that WRKY46 specifically binded to the region of the W-boxes in the RIXI promoter, not mutant W-boxes in the same region.We speculated that OsWRKY46 may play a regulatory role in the expression of RIXI through binding to specific elements in the RIXI promoter.3. Transcriptional activation of OsWRKY and RIXI promoter:We performed the transformation of tobacco (Nicotiana benthamiana) leaves by agro-infiltration, using the dual luciferases vector as a reporter system to analyze the transcriptional activation between RIXI promoters and OsWRKY. The result was clear that OsWRKY46 had the capacity of inhibiting reporter activity driven by the full length RIXI promoter, but not the 5’-deleted one, indicating that the full promoter region containing the certain enriched W-boxes is the essential part for the interaction between OsWRKY46 and the RIXI promoter. And OsWRKY46 may play a negative role in regulating the expression of RIXI. While OsWRKY6 had the capacity of activating reporter activity driven by the full length RIXI promoter, but not the 5’-deleted one, indicating OsWRKY46 may play a positive role in regulating the expression of RIXI through binding to the W-box.4. Obtained OsWRKY46-overexpression transgenic plants:To verify the resistance of OsWRKY46, we builded Ubi::OsWRKY46 over-expression vector, performed the transformation by agro-infiltration. qRT-PCR showed that the expression of OsWRKY46 was 3 times more in transgenice plant than in WT and the expression of RIXI was significantly reduced in OsWRKY46-overexpession rice comparing with WT. It indicated that OsWRKY46 inhibited the expression of RIXI. The OsWRKY46- overexpression rice can also be used in future study.