Structural and functional characterization of a Xanthomonas Type III effector
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Rice bacterial leaf streak disease caused by Xanthomonas oryzae pv. oryzicola (Xoc) is one of the most important rice bacterial diseases. Xanthomonas type III effector gene avrRxo1 is conserved in diverse Xoc strains and its homologues have been identified from several other gram-negative bacteria species such as Burkholderia and Acidovorax. In this research, we studied the protein structure of AvrRxo1 and illustrated its virulence mechanism.We determined the three-dimensional structure of the complex of AvrRxo1 and its cognate chaperone Arc1 (AvrRxo1 required chaperone 1). The AvrRxo1: Arc1 complex is structurally similar to the Zeta-epsilon family of toxin: antitoxin systems from the human bacterial pathogen Streptococcus pyogenes. AvrRxo1 and Arc1 have toxin: antitoxin-like activity in bacteria, and the toxin activity of AvrRxo1 is required for its virulence function in planta. These findings suggest that AvrRxo1 evolved from an endogenous bacterial toxin-antitoxin system.Furthermore, AvrRxo1 was shown to have virulence functions in diverse host plants including Arabidopsis thaliana. The ectopic expression of wild type avrRxo1 in Arabidopsis suppresses plant basal defense. AtVOZ (Arabidopsis vascular one zinc-finger transcription factor), which has two homologues in the Arabidopsis genome, VOZ1 and VOZ2, was identified as one of AvrRxo1 candidate interactor. The knockout of voz1/voz2 renders the plants more susceptible to the virulent pathogen Pseudomonas syringae pv. tomato (Pst) DC3000, but compromises the virulence function of AvrRxo1. The expression profiling of transgenic Arabidopsis plants expressing the avrRxo1 gene allowed us to identify Arabidopsis genes regulated by AvrRxo1 and VOZ1/2. AvrRxo1 interacts with and stabilizes VOZ2 in vivo and directly binds to the promoter region of AtCYS2 (Arabidopsis phytoCYStatin 2) to induce its expression. The overexpression of CYS2 in increased stomatal aperture size, and enhanced plant susceptibility to Pst. Therefore one of AvrRxo1 virulent functions is to regulate the expression of CYS2 by manipulating VOZ2, resulting in increased stomatal aperture. Presumably, this renders the host leaf more susceptible to colonization via the stomata. Another component of my dissertation was based on a genome-wide survey of Arabidopsis papin-like cysteine protease genes (PLCPs). The Arabidopsis genome has 31 PLCP and 7 cystatin genes, and they often worked in pairs to regulate signaling pathways in response to biotic and abiotic stress. The coordinated transcriptional regulation of all Arabidopsis PLCP and cystatin genes has never been systematically investigated. In order to unveil the mechanism of stomata-related plant immunity regulated by CYS2, we analyzed the expression patterns of 28 PLCPs and 7 cystatins in Arabidopsis in response to biotic or abiotic stress, by reprocessing and integrating microarray data from the AtGenExpress database. We also performed enzyme assays and evaluated the inhibition specificity of seven cystatins to the five most abundant PLCPs in Arabidopsis. Finally, we utilized the SVMs (support vector machines) package in R software to predict a functional network of PLCP-cystatin interplay in Arabidopsis. We identified the PLCP protein PAP4 as one of the putative targets of CYS2. The co-expression profiling indicated that the expression patterns of PAP4 and CYS2 were strongly correlated during virulent bacterial infection, and weakly correlated under drought stress. Therefore, PAP4 was determined to be a promising gene in regulating stomatal aperture size. Further research on the interplay of PAP4-CYS2 could be important for understanding AvrRxo1's virulence mechanism and regulation of plant stomatal immunity.
- Doctoral Dissertations