Characterization of a Xanthomonas campestris pv. zinniae Oxidoreductase Involved in the Biodegradation of Cercosporin

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Title: Characterization of a Xanthomonas campestris pv. zinniae Oxidoreductase Involved in the Biodegradation of Cercosporin
Author: Taylor, Tanya Valera
Advisors: R. Gregory Upchurch, Committee Member
David F. Ritchie, Committee Member
Gary A. Payne, Committee Member
Margaret E. Daub, Committee Chair
Abstract: The fungal photoactivated toxin cercosporin plays a key role in pathogenesis by Cercospora spp. The bacterium Xanthomonas campestris pv. zinnia (XCZ) is able to rapidly degrade this toxin. Growth of XCZ strains in cercosporin–containing medium leads to the breakdown of cercosporin and to the formation of a non–toxic breakdown product called xanosporic acid. EMS mutagenesis was used to generate five non–degrading mutants of a rapid–degradation strain (XCZ&#8211h;3). Mutants were complemented using a wild–type genomic library. All five mutants were complemented with the same fragment, which encoded a putative transcriptional regulator and an oxidoreductase. Simultaneous expression of the two genes was necessary to complement the mutant phenotype. Sequence analysis of the mutants showed that all five had point mutations in the oxidoreductase sequence and no mutations in the regulator. Quantitative RT–PCR showed that expression of both of these genes in the wild–type strain is upregulated after exposure to cercosporin. Both the oxidoreductase and transcriptional regulator genes were transformed into three cercosporin non–degrading bacteria to determine if they are sufficient for cercosporin degradation. Quantitative RT–PCR analysis confirmed that the oxidoreductase was expressed in all transconjugants. However, none of the transconjugants were able to degrade cercosporin, suggesting that other factors are involved in this process. In an effort to determine if the oxidoreductase gene has a role in pathogenesis, quantitative RT–PCR was used to assay for oxidoreductase regulation in XCZ–3 grown in the presence of zinnia and tobacco extracts. The oxidoreductase gene was upregulated in the presence of both plant extracts, but the response was greater with the tobacco extract. The five XCZ–3 oxidoreductase mutants were assayed for pathogenicity on zinnia. The mutants yielded disease symptoms equal to those of the wild–type. Furthermore, previously identified bacteria (Xanthomonas axonopodis pv. pruni, strains XAP–50 and XAP–75, and Pseudomonas syringae pv. tomato, PST DC3000) with oxidoreductase homologues were not able to infect zinnia, suggesting that the presence of a functional oxidoreductase is not correlated with pathogenesis of zinnia.
Date: 2006-01-29
Degree: PhD
Discipline: Plant Pathology
URI: http://www.lib.ncsu.edu/resolver/1840.16/3992


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