Expression, Purification and Crystallization of the Xanthmonal Avirulence Protein, AvrBs1.1, and its Target in Capsicum annuum, the Transcription Factor WRKY‐1

Abstract

Brown spots and leaf loss of Capsicum annum, a pepper found in the Americas, are caused by prolonged activation of the plant's defense mechanisms to bacterial infection resulting in a phenomenon called the hypersensitive response. In the hypersensitive response, the plant “walls off” the infected tissue with lignin and then a variety of chemical processes occur within the lignified zone which results in the death of both bacterial and plant cells. Certain species‐specific bacterial pathogens inject effector proteins with different activities into the cytosol of the host to prevent activation of the plant's defense mechanisms. Our work focuses on the effector protein AvrBs1.1, a dual specificity protein tyrosine phosphatase produced by Xanthamonas euvesicatoria. Recent work has identified the transcription factor WRKY‐1 as a possible target for AvrBs1.1. We hypothesize that AvrBs1.1 dephosphorylates WRKY‐1 in the cytosol, thereby preventing it from entering the nucleus and activating the genes of the plant's defense response. In order to understand the molecular basis of the function of AvrBs1.1, we need to determine the x‐ray crystallographic structure of AvrBs1.1 and the AvrBs1.1/WRKY‐1 complex. We crystallized AvrBs1.1 in 2.0M (NH4)SO4, 10% PEG 400, 50mM HEPES pH 7.0, and 50mM ZnSO4. Using this condition as a base, we employed a small molecule additive screen to enhance crystal growth. Four different organic alcohols (MPD, tert‐butanol, PEG 400, and hexanediol) were found to induce the growth of single crystals of AvrBs1.1. In the future, we will screen the AvrBs1.1 crystals for x‐ray diffraction and optimize the expression of WRKY‐1 and attempt to crystallize the AvrBs1.1/WRKY‐1 complex.