- Which domain is required for formation of heterodimers?
- Does heterodimerization alter DNA binding specificity?
- Do all IIa WRKYs interact with each other?
- Are all IIa WRKYs involved in the same pathways? (Arabidopsis?)
To identify other factors involved in Xb10 regulation of the XA21 defense response, Xb10 was used as a bait in the yeast two-hybrid system. Three proteins called Xb10 interactors (Xb10i-1, Xb10i-2, and Xb10i-3) that together with Xb10 comprise a family (referred to as IIa) of WRKY's that contain an N-terminal coiled-coil domain were identified. In arabidopsis at least one of the group IIa WRKY��s has already been shown to function in disease resistance. These results present another parallel with innate immunity in animals where the NF-kB/Rel family of transcriptional activator proteins exist as hetero- or homodimeric proteins. The proposed experiments will allow us to determine if XA21-mediated signaling occurs through a family of transcription factors by triggering the formation of Xb10/Xb10I heteromeric complexes in vivo.
It was first determine the role of the three XB10is in Xa21-mediated signal transduction as described in D2a for Xb10. RNAi constructs will be designed to specifically silence each WRKY so that one can assess the relative role for each WRKY in Xa21-mediated signaling. These transgenic lines will also be used in microarray experiments to identify individual defense related genes influenced by each WRKY.
The isolation of all four members of the rice IIa WRKY family allows us to design additional experiments to account for the possibility of functional redundancy. For this purpose dominant negative constructs will be created using truncated non-functional protein. The C-terminal domain of Xb10 will be overexpressed and each of the Xb10is in the XA21 background. This domain is required for interaction but lacks putative DNA binding and transcriptional activation activities. This approach may lead to the formation of non-functional complexes that would disrupt wild-type Xb10, Xb10i-1, Xb10i-2, and Xb10i-3 function, simultaneously.