ABSTRACT TAYLOR, TANYA VALERA. Characterization of a Xanthomonas campestris pv. zinniae Oxidoreductase Involved in the Biodegradation of Cercosporin. (Under the direction of Dr. Margaret E. Daub.) 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-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. Characterization of a Xanthomonas campestris pv. zinniae Oxidoreductase Involved in the Biodegradation of Cercosporin By TANYA VALERA TAYLOR A dissertation submitted to the Graduate Faculty of North Carolina State University In partial fulfillment of the Requirements for the degree of Doctor of Philosophy In PLANT PATHOLOGY Raleigh, NC 2005 Approved by: ______________________ ______________________ Margaret E. Daub Gary A. Payne Dissertation Committee Chair ______________________ ______________________ David F. Ritchie R. Gregory Upchurch PERSONAL BIOGRAPHY I was born to Thomas and Virginia Taylor in 1973 and raised in Hillsborough, NC along with my brother Harlan. After graduating from Orange High School in 1991, I enlisted in the Navy for four years. Through the Navy, I was able to travel to various parts of the United States, as well as to Spain. After completing my tour of duty, I attended the University of North Carolina at Chapel Hill and earned a Bachelors of Science degree in Biology in 2000. I began working on my Ph.D. at North Carolina State University in the Department of Plant Pathology in the fall of 2000. My project, which focused on characterization of bacterial genes responsible for degradation of the fungal toxin cercosporin, was completed under the direction of Dr. Margo Daub in the Department of Plant Pathology. ii TABLE OF CONTENTS Page LIST OF FIGURES ………………………………………………………………….…...... vi LIST OF TABLES …………………………………………………………………..……. viii Chapter 1: REVIEW OF RELEVANT LITERATURE .………………………………...…. 1 The fungal genus Cercospora …………………………………………………….…. 2 The toxin cercosporin …………………………………………………………..…….3 Cercospora resistance mechanisms ……………………………….……………...…..9 Control of Cercospora diseases ………………………………………………….….13 Cercosporin degradation ………………………………………………………….…16 Cytochrome P450s ………………………………………………….. ………….......19 Disease control through transgenic resistance to toxins ……….………………....…24 Summary …………………………………………………………………….…....…27 References cited ………………………………………………………………….….29 Chapter 2: A PUTATIVE OXIDOREDUCTASE IS INVOLVED IN CERCOSPORIN DEGRADATION BY THE BACTERIUM XANTHOMONAS CAMPESTRIS PV. ZINNIAE ..……….………………………………………………………………………….42 Abstract ………………………………………………………………………….…..43 Introduction ……………………………………………………………………….…44 Materials and Methods …………………………………………………………..…..48 Bacterial cultures …………………………………………………..………..48 Cercosporin stocks and chemicals ………………………………………..…48 Chemical mutagenesis of Xanthomonas campestris pv. zinniae and screening for mutants unable to degrade cercosporin ..………………….…..48 Box PCR …………………………………………………………………….49 Xanthomonas campestris pv. zinniae genomic library construction .…….…49 Complementation assays ……………………………………………………50 Subcloning of complementing library clone ………………………………...51 Sequencing of subclone …………………………………………………..…51 Sequencing of mutant genes ………………………………………………...51 Gene complementation ……………………………………………………...52 Quantitative RT-PCR of oxidoreductase and regulator in wild-type XCZ-3 …………………………………………………………….………....52 Southern analysis …………………………………………………………....53 Transformation of the regulator and oxidoreductase genes into non-degrading bacteria ………………………………………………………54 Time course of cercosporin degradation by transconjugants ……………......54 Results …………………………………………………………………………….…56 Isolation of Xanthomonas campestris pv. zinniae mutants unable to degrade cercosporin …………………………………………………………56 iii Identification of complementing genes ……………………………………...56 Search for additional genes ………………………………………………….58 Sequencing of mutants ……………………………………………………....59 Identification of sequences necessary for mutant complementation .……….59 Presence of cercosporin upregulates gene expression ………………………60 Southern analysis of oxidoreductase homologues in degrading and non-degrading species ……………………………………………………….60 The transcriptional regulator and oxidoreductase are not sufficient for cercosporin degradation in non-degrading species ………………………….61 Discussion …………………………………………………………………………...63 References cited ……………………………………………………………………..68 Chapter 3: THE XANTHOMONAS CAMPESTRIS PV. ZINNIAE OXIDOREDUCTASE GENE IS REGULATED BY PLANT EXTRACTS, BUT IS NOT NECESSARY FOR PATHOGENESIS .……………………………………………………………………....…..88 Abstract ……………………………………………………………………………...89 Introduction ………………………………………………………………………….90 Materials and Methods …………………………………………………………...….93 Bacterial Cultures ……………………………………………………………93 Plant Extract ……………………………………………………………..…..93 Quantitative RT-PCR of Oxidoreductase Expression in Wild-Type XCZ-3 ………………………………………………………………….…....94 Zinnia Inoculation …………………………………………………………...94 Results ……………………………………………………………………………….96 Presence of Plant Extract Upregulates Oxidoreductase Gene Expression …..96 Presence of the Oxidoreductase Not Correlated with Pathogenicity ………..96 Discussion …………………………………………………………………………...98 References cited ……………………………………………………………………100 APPENDIX - SEARCH FOR PROTEINS DIFFERENTIALLY EXPRESSED IN XCZ-3 UPON EXPOSURE TO CERCOSPORIN ………………………………….……..105 Goal …………………………………………………………………………….…..105 Methods ……………………………………………………………………….…....105 Results …………………………………………………………………………...…107 Conclusion …………………………………………………………………………107 References cited …………………………………………………………………....108 APPENDIX – XANTHOMONAS AXONOPODIS PV. CITRI DOES NOT DEGRADE CERCOSPORIN …………………………………………………………………………..110 Goal …………………………………………………………………………….….110 Methods …………………………………………………………………….….…..111 Results ………………………………………………………………………….….111 Conclusion …………………………………………………………………….…...112 References cited ………………………………………………………….…….…..113 iv APPENDIX – TRANSFORMATION OF TOBACCO WITH THE XANTHOMONAS CAMPESTRIS PV. ZINNIAE OXIDOREDUCTASE GENE INVOLVED IN CERCOSPORIN DEGRADATION ………………………………………………….…...117 Goal ………………………………………………………………………………..117 Methods ……………………………………………………………………………118 Cloning of Genes ………………………………………………….….……118 Plant Tissue Transformation ……………………………………….….…...118 Recovery of Plants and Callus ……………………………………...……...119 Screening of Callus ………………………………………………….……..120 Results ……………………………………………………………………….……..120 Conclusion …………………………………………………………………….…...121 References cited ……………………………………………………………..……..122 v LIST OF FIGURES Page A PUTATIVE OXIDOREDUCTASE IS INVOLVED IN CERCOSPORIN DEGRADATION BY THE BACTERIUM XANTHOMONAS CAMPESTRIS PV. ZINNIAE Figure 1 Chemical Structure of cercosporin and the breakdown product (xanosporic acid) produced by X. campestris pv. zinniae, isolate XCZ-3 ………………….…………………………………….76 Figure 2 Cercosporin non-degrading XCZ-3 mutants ..……………………….77 Figure 3 Agarose gel of Box PCR products ………………………………..…78 Figure 4 Complementing fragment derived from sequencing the complementing 7.5 kb subclone …………………………………….79 Figure 5 Sequence containing the putative transcriptional regulator and oxidoreductase genes of XCZ-3 (GenBank Accession #DQ087176) ………………………………….80 Figure 6 Alignment of amino acid sequences of oxidoreductase genes from Xanthomonas campestris pv. zinniae (XCZ-3) and Xanthomonas axonopodis pv. citri (XAC-306) ………………………………….…82 Figure 7 Alignment of amino acid sequences of transcriptional regulator genes from Xanthomonas campestris