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Genetic and Transcriptomic Analyses of the Rice Pathogenic Bacterium, Burkholderia Glumae, Reveal the Important Roles of The Louisiana State University LSU Digital Commons LSU Master's Theses Graduate School 2015 Genetic and Transcriptomic Analyses of the Rice Pathogenic Bacterium, Burkholderia glumae, Reveal the Important Roles of the Regulatory Gene, tepR, for Bacterial Survival in Environmental Stresses Jingyu Peng Louisiana State University and Agricultural and Mechanical College, [email protected] Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_theses Part of the Plant Sciences Commons Recommended Citation Peng, Jingyu, "Genetic and Transcriptomic Analyses of the Rice Pathogenic Bacterium, Burkholderia glumae, Reveal the Important Roles of the Regulatory Gene, tepR, for Bacterial Survival in Environmental Stresses" (2015). LSU Master's Theses. 2722. https://digitalcommons.lsu.edu/gradschool_theses/2722 This Thesis is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Master's Theses by an authorized graduate school editor of LSU Digital Commons. For more information, please contact [email protected]. GENETIC AND TRANSCRIPTOMIC ANALYSES OF THE RICE PATHOGENIC BACTERIUM, BURKHOLDERIA GLUMAE, REVEAL THE IMPORTANT ROLES OF THE REGULATORY GENE, TEPR, FOR BACTERIAL SURVIVAL IN ENVIRONMENTAL STRESSES A Thesis Submitted to the Graduate Faculty of the Louisiana State University and Agriculture and Mechanical College in partial fulfillment of the requirements for the degree of Master of Science in The Department of Plant Pathology and Crop Physiology by Jingyu Peng B.S., Shandong Agricultural University, 2013 December 2015 ACKNOWLEDGEMENTS It has never been easy for me to gain such a great opportunity to study in Louisiana State University, one of the best public university in America. I have been so lucky to study Plant Pathology in such a harmonious and efficient department, the Department of Plant Pathology & Crop Physiology. It is my great honor to work with a group of graduate students and plant pathologists who are so passionate about research and science. I want to take this opportunity to give my greatest appreciation to all the person who have accompanied me, helped me, appraised and criticized me. I want to give my greatest thanks to my parents, Bo Sun and Liqian Peng. They have never been hesitant to support me to study in America even at the cost that I could not see them for more than two years. Words can hardly explain how much they love me and I love them just as much. I want to give my great appreciation to my advisor Dr. Jong Hyun Ham, who is always the brain of our lab. He is very enthusiastic about our research and has provided me with the best resource and environment for conducting researches. I feel so glad to work on the research project I am very interested in, and I am always so excited to discuss with him about our research progress and spark new ideas. I want to thank Dr. Eric C. Achberger for his great help throughout the Microbial Genetics class in my first semester in America. He was so nice and patient to me, no matter what simple and silly questions I asked. His class opened me the door for molecular biology. ii I want to thank Inderjit Barphagha, the research associate of our lab. She was the person who taught me all the laboratory techniques from the basic PCR to cloning techniques, hand by hand, step by step. She was never reserved in teaching me whatever she knew. I want to thank my committee members Dr. Christopher A. Clark and Dr. J. Cameron Thrash. They both are the professors I respect most in LSU. I really appreciate them for their acceptance to advise my Master’s research. It has always been great time to talk to them about my research progress and listen to their suggestions. I appreciate all the time they have taken, all the encouragement they have given, and all the advices they have provided! Finally, I want to thank myself for never giving up! Thank myself for being the proudness of my parents! And one day, I wish to be the proudness of all the people who have helped me! iii TABLE OF CONTENTS ACKNOWLEDGEMENTS ................................................................................................ii ABSTRACT .....................................................................................................................vi CHAPTER 1: INTRODUCTION ....................................................................................... 1 Bacterial Panicle Blight (BPB) of Rice caused by Burkholderia glumae ....................... 1 Virulence factors and their regulation in B. glumae ...................................................... 5 Significance and Rationale ......................................................................................... 12 References ................................................................................................................. 14 CHAPTER 2: GLOBAL REGULATORY ROLE OF TEPR IN BURKHOLDERIA GLUMAE AS REVEALED BY WHOLE TRANSCRIPTOMIC ANALYSES .................... 18 Introduction ................................................................................................................ 18 Materials and Methods ............................................................................................... 21 Results ....................................................................................................................... 31 Discussion .................................................................................................................. 48 References ................................................................................................................. 55 CHAPTER 3: VALIDATION OF THE PHENOTYPES REGULATED BY TEPR AS REVEALED BY RNA-SEQ ............................................................................................ 61 Introduction ................................................................................................................ 61 Materials and Methods ............................................................................................... 68 Results ....................................................................................................................... 70 Discussion .................................................................................................................. 75 References ................................................................................................................. 79 CHAPTER 4: REGULATION OF TOXOFLAVIN AND PROTEASE BY TEPR IS QSMR-DEPENDENT .................................................................................................... 85 Introduction ................................................................................................................ 85 Materials and Methods ............................................................................................... 86 Results ....................................................................................................................... 93 Discussion .................................................................................................................. 95 References ................................................................................................................. 96 CHAPTER 5: CONCLUSIONS ...................................................................................... 99 APPENDIX A: PER BASE QUALITY OF RNA-SEQ RAW DATA ............................... 102 APPENDIX B: IDENTIFIED DIFFERENTIALLY EXPRESSED GENES FROM RNA-SEQ .................................................................................................................... 104 APPENDIX C: STATISTICS ANALYSES OF WHOLE TRANSCRIPTOME GENE ONTOLOGY ANNOTATION ....................................................................................... 114 iv VITA ............................................................................................................................ 115 v ABSTRACT Burkholderia glumae causes bacterial panicle blight in rice and leads to severe yield losses. B. glumae causes disease by producing multiple virulence factors including toxoflavin, lipase, flagella, type III secretion system, catalase, and exopolysaccharides (EPS). The production of toxoflavin, the most important virulence factor, is tightly regulated by TofI/TofR-mediated quorum sensing (QS) and the transcriptional activators ToxJ and ToxR. However, other virulence regulatory systems are poorly studied. Recently, tepR, encoding a sigma 54-dependent response regulator, was characterized to negatively regulate toxoflavin and extracellular protease production. In order to gain insights into the comprehensive biological functions of tepR in B. glumae, the genome- wide transcriptional profile dependent on tepR was investigated through comparative RNA sequencing (RNA-seq) analyses between the wild-type B. glumae strain 336gr-1 and its tepR knockout derivative, LSUPB401. The RNA-seq analyses yielded the identification of 238 differentially expressed genes (DEGs). Gene Ontology enrichment analysis of the DEGs indicated that genes involved in flagella assembly and stress response were up-regulated in LSUPB401. Kyoto Encyclopaedia of Genes and Genomes pathway enrichment further revealed that tepR positively regulated the gene cluster encoding a putative type VI secretion system (T6SS) and the genes responsible for a branched-chain amino acid ABC Transporter system but negatively regulated the genes involved in thiamine, cysteine and methionine metabolism. Phenotypic tests
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