Browsing by Author "Wu, Shuchi"
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- AvrRxo1 Is a Bifunctional Type III Secreted Effector and Toxin-Antitoxin System Component with Homologs in Diverse Environmental ContextsTriplett, Lindsay R.; Shidore, Teja; Long, John J.; Miao, Jiamin; Wu, Shuchi; Han, Qian; Zhou, Changhe; Ishihara, Hiromichi; Li, Jianyong; Zhao, Bingyu Y.; Leach, Jan E. (PLOS, 2016-07-08)Toxin-antitoxin (TA) systems are ubiquitous bacterial systems that may function in genome maintenance and metabolic stress management, but are also thought to play a role in virulence by helping pathogens survive stress. We previously demonstrated that the Xanthomonas oryzae pv. oryzicola protein AvrRxo1 is a type III-secreted virulence factor that has structural similarities to the zeta family of TA toxins, and is toxic to plants and bacteria in the absence of its predicted chaperone Arc1. In this work, we confirm that AvrRxo1 and its binding partner Arc1 function as a TA system when expressed in Escherichia coli. Sequences of avrRxo1 homologs were culled from published and newly generated phytopathogen genomes, revealing that avrRxo1:arc1 modules are rare or frequently inactivated in some species and highly conserved in others. Cloning and functional analysis of avrRxo1 from Acidovorax avenae, A. citrulli, Burkholderia andropogonis, Xanthomonas translucens, and Xanthomonas euvesicatoria showed that some AvrRxo1 homologs share the bacteriostatic and Rxo1-mediated cell death triggering activities of AvrRxo1 from X. oryzae. Additional distant putative homologs of avrRxo1 and arc1 were identified in genomic or metagenomic sequence of environmental bacteria with no known pathogenic role. One of these distant homologs was cloned from the filamentous soil bacterium Cystobacter fuscus. avrRxo1 from C. fuscus caused watersoaking and triggered Rxo1-dependent cell collapse in Nicotiana benthamiana, but no growth suppression in E. coli was observed. This work confirms that a type III effector can function as a TA system toxin, and illustrates the potential of microbiome data to reveal new environmental origins or reservoirs of pathogen virulence factors.
- Structural and functional characterization of a Xanthomonas Type III effectorWu, Shuchi (Virginia Tech, 2015-04-23)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.
- The transcriptional network of WRKY53 in cereals links oxidative responses to biotic and abiotic stress inputsVan Eck, Leon; Davidson, Rebecca M.; Wu, Shuchi; Zhao, Bingyu Y.; Botha, Anna-Maria; Leach, Jan E.; Lapitan, Nora L. V. (Springer, 2014-01-01)The transcription factor WRKY53 is expressed during biotic and abiotic stress responses in cereals, but little is currently known about its regulation, structure and downstream targets. We sequenced the wheat ortholog TaWRKY53 and its promoter region, which revealed extensive similarity in gene architecture and cis-acting regulatory elements to the rice ortholog OsWRKY53, including the presence of stress-responsive abscisic acid-responsive elements (ABRE) motifs and GCC-boxes. Four proteins interacted with the WRKY53 promoter in yeast one-hybrid assays, suggesting that this gene can receive inputs from diverse stress-related pathways such as calcium signalling and senescence, and environmental cues such as drought and ultraviolet radiation. The Ser/Thr receptor kinase ORK10/LRK10 and the apoplastic peroxidase POC1 are two downstream targets for regulation by the WRKY53 transcription factor, predicted based on the presence of W-box motifs in their promoters and coregulation with WRKY53, and verified by electrophoretic mobility shift assay (EMSA). Both ORK10/LRK10 and POC1 are upregulated during cereal responses to pathogens and aphids and important components of the oxidative burst during the hypersensitive response. Taken with our yeast two-hybrid assay which identified a strong protein-protein interaction between microsomal glutathione S-transferase 3 and WRKY53, this implies that the WRKY53 transcriptional network regulates oxidative responses to a wide array of stresses. © 2014 The Author(s).