Browsing by Author "Leach, Jan E."

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    AvrRxo1 Is a Bifunctional Type III Secreted Effector and Toxin-Antitoxin System Component with Homologs in Diverse Environmental Contexts
    Triplett, 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.
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    Community-Driven Metadata Standards for Agricultural Microbiome Research
    Dundore-Arias, Jose Pablo; Eloe-Fadrosh, Emiley A.; Schriml, Lynn M.; Beattie, Gwyn A.; Brennan, Fiona P.; Busby, Posy E.; Calderon, Rosalie B.; Castle, Sarah C.; Emerson, Joanne B.; Everhart, Sydney E.; Eversole, Kellye; Frost, Kenneth E.; Herr, Joshua R.; Huerta, Alejandra I.; Iyer-Pascuzzi, Anjali S.; Kalil, Audrey K.; Leach, Jan E.; Leonard, J.; Maul, Jude E.; Prithiviraj, Bharath; Potrykus, Marta; Redekar, Neelam R.; Rojas, J. Alejandro; Silverstein, Kevin A. T.; Tomso, Daniel J.; Tringe, Susannah G.; Vinatzer, Boris A.; Kinkel, Linda L. (2020-02-20)
    Accelerating the pace of microbiome science to enhance crop productivity and agroecosystem health will require transdisciplinary studies, comparisons among datasets, and synthetic analyses of research from diverse crop management contexts. However, despite the widespread availability of crop-associated microbiome data, variation in field sampling and laboratory processing methodologies, as well as metadata collection and reporting, significantly constrains the potential for integrative and comparative analyses. Here we discuss the need for agriculture-specific metadata standards for microbiome research, and propose a list of "required" and "desirable" metadata categories and ontologies essential to be included in a future minimum information metadata standards checklist for describing agricultural microbiome studies. We begin by briefly reviewing existing metadata standards relevant to agricultural microbiome research, and describe ongoing efforts to enhance the potential for integration of data across research studies. Our goal is not to delineate a fixed list of metadata requirements. Instead, we hope to advance the field by providing a starting point for discussion, and inspire researchers to adopt standardized procedures for collecting and reporting consistent and well-annotated metadata for agricultural microbiome research.
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    The effector AvrRxo1 phosphorylates NAD in planta
    Shidore, Teja; Broeckling, Corey D.; Kirkwood, Jay S.; Long, John J.; Miao, Jiamin; Zhao, Bingyu Y.; Leach, Jan E.; Triplett, Lindsay R. (PLOS, 2017-06-19)
    Gram-negative bacterial pathogens of plants and animals employ type III secreted effectors to suppress innate immunity. Most characterized effectors work through modification of host proteins or transcriptional regulators, although a few are known to modify small molecule targets. The Xanthomonas type III secreted avirulence factor AvrRxo1 is a structural homolog of the zeta toxin family of sugar-nucleotide kinases that suppresses bacterial growth. AvrRxo1 was recently reported to phosphorylate the central metabolite and signaling molecule NAD in vitro, suggesting that the effector might enhance bacterial virulence on plants through manipulation of primary metabolic pathways. In this study, we determine that AvrRxo1 phosphorylates NAD in planta, and that its kinase catalytic sites are necessary for its toxic and resistance-triggering phenotypes. A global metabolomics approach was used to independently identify 3'-NADP as the sole detectable product of AvrRxo1 expression in yeast and bacteria, and NAD kinase activity was confirmed in vitro. 3'-NADP accumulated upon transient expression of AvrRxo1 in Nicotiana benthamiana and in rice leaves infected with avrRxo1-expressing strains of X. oryzae. Mutation of the catalytic aspartic acid residue D193 abolished AvrRxo1 kinase activity and several phenotypes of AvrRxo1, including toxicity in yeast, bacteria, and plants, suppression of the flg22-triggered ROS burst, and ability to trigger an R gene-mediated hypersensitive response. A mutation in the Walker A ATPbinding motif abolished the toxicity of AvrRxo1, but did not abolish the 3'-NADP production, virulence enhancement, ROS suppression, or HR-triggering phenotypes of AvrRxo1. These results demonstrate that a type III effector targets the central metabolite and redox carrier NAD in planta, and that this catalytic activity is required for toxicity and suppression of the ROS burst.
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    The transcriptional network of WRKY53 in cereals links oxidative responses to biotic and abiotic stress inputs
    Van 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.