ABSTRACT Vélez, Heriberto. Alternaria Alternata Mannitol
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ABSTRACT Vélez, Heriberto. Alternaria alternata mannitol metabolism in plant-pathogen interactions. (Under the direction of Dr. Margaret E. Daub) Mannitol is purported to have role in fungi as a storage carbohydrate and has been shown to quench reactive oxygen species (ROS) both in vitro and in vivo. Mannitol metabolism in fungi is thought to occur through the mannitol cycle, which was proposed in the late 1970’s from studies of cell free extracts of the fungus Alternaria alternata. In this cycle, mannitol 1-phosphate 5-dehydrogenase (MPDH; EC 1.1.1.17) reduces fructose 6- phosphate into mannitol 1-phosphate, which is dephosphorylated by a mannitol 1- phosphatase (EC 3.1.3.22) resulting in mannitol and inorganic phosphate. Mannitol also can be made through the enzyme mannitol dehydrogenase (MtDH; EC 1.1.1.138), which reduces fructose to mannitol. Here we report confirmation of these enzymes in the fungus A. alternata, the isolation of the genes, and the generation of strains mutated in MPDH, MtDH, or both genes. PCR confirmed gene replacement and enzyme assays using these mutants showed no activity for MtDH or MPDH. GC-MS analysis showed that strains deficient in both enzymes did not produce mannitol, while strains deficient in either MPDH or MtDH had reduced mannitol production. Mannitol, as a quencher of ROS, may also have a role in host-pathogen interactions, by allowing the fungus to suppress ROS-mediated plant defense responses. To assess the contribution of mannitol in plant-pathogen interactions, wild type, single and double mutants were used in pathogenicity assays on tobacco plants. Severity of lesions caused by the MtDH disruptant was not significantly different from that of the wild type. By contrast, the MPDH disruptant and the double mutant caused significantly less disease. Microscopy analysis and histochemical staining for H2O2 showed that both the wild type strain and the double mutant were able to germinate, produced appressoria, and elicited a defense response from the host. Quantitative PCR studies showed that genes for both enzymes were up-regulated in the presence of tobacco extracts, with MPDH having a stronger response. We conclude that mannitol biosynthesis is required for pathogenesis of A. alternata on tobacco, but is not required for normal spore germination either in vitro or in planta or for initial infection. Alternaria alternata mannitol metabolism in plant-pathogen interactions by Heriberto Vélez 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: Dr. Gary A. Payne Dr. Marc A. Cubeta Dr. Margaret E Daub, Chair Dr. Greg G. Upchurch DEDICATION To Michael P. Meiners, for all the times we have shared, thank you for being you and for being there. ii BIOGRAPHY My name is Heriberto Vélez; however, I prefer my nick name “Hery” as my father would spell it or Eddie, as my English speaking friends call me. I was born in Puerto Rico and moved to Florida, with my siblings during the early 1980s. I attended Osceola High School, in Kissimmee, FL from which I graduated with honors. Prior to attending college, I decided to see the world and traveled to Boston, MA where I ended up working for Houghton Mifflin Co., a publishing company, for five years. However, having been interested in science since an early age, I decided to return to Florida where I began attending the University of Central Florida, while working for the Naval Air Warfare in Orlando. However, I transferred to North Carolina State University, where I got my degree in biochemistry. As an undergraduate working during a summer internship in the laboratory of Dr. Barry Goldfarb, I got interested in plants, so I decided to stay at NC State University and pursue a degree in Plant Pathology. And here I am….. iii ACKNOWLEDGMENTS I want to express my sincerest appreciation to Drs. Margo Daub, Gary Payne, Marc Cubeta, Greg Upchurch, and Scott Chilton….you are an inspiration to me. To Drs. Tanya Taylor, Sheri Denslow, and Sonia Herrero, for your encouragement…. and Beth Rueschhoff, is your turn now Beth! To Drs. Marilyn Ehrenshaft, John Williamson, and Mason Pharr… I hope that my findings are able to advance our knowledge… iv TABLE OF CONTENTS LIST OF FIGURES .......................................................................................................................vii LIST OF TABLES..........................................................................................................................ix Literature Review ............................................................................................................................1 Introduction .................................................................................................................................1 Mannitol metabolism in fungi .....................................................................................................2 Other polyols .............................................................................................................................10 Roles for mannitol and other polyols ........................................................................................15 Osmoregulation .........................................................................................................................15 Storage Carbohydrate ................................................................................................................18 Regulating cofactors..................................................................................................................20 Regulate pH...............................................................................................................................22 Quencher of reactive oxygen species ........................................................................................22 Other ways fungi defend themselves from ROS .......................................................................26 Plant defense responses .............................................................................................................30 Alternaria alternata – Brown spot ............................................................................................31 References .................................................................................................................................33 CHAPTER 2: Mannitol metabolism in the phytopathogenic fungus Alternaria alternata..........47 Abstract......................................................................................................................................48 Introduction ...............................................................................................................................50 Materials and Methods ..............................................................................................................52 Strain and media....................................................................................................................52 Genomic DNA isolation........................................................................................................53 Enzyme assays.......................................................................................................................53 Isolation of MtDH and MPDH genes ....................................................................................55 Gene disruption .....................................................................................................................57 Protoplast isolation and fungal transformation......................................................................58 Screening for MtDH and MPDH disruptants using PCR ......................................................59 Enzyme assays of disruption mutants ...................................................................................60 In vitro growth characteristics on different carbon sources ..................................................60 Mannitol analyses..................................................................................................................61 Results .......................................................................................................................................62 Enzyme Assays......................................................................................................................62 Gene isolation........................................................................................................................63 Gene disruption .....................................................................................................................63 Enzyme assays of disruption mutants ...................................................................................64 Mannitol analyses..................................................................................................................65 In vitro growth characteristics on different carbon sources ..................................................65 Discussion..................................................................................................................................65 References .................................................................................................................................70 v CHAPTER 3: Mannitol biosynthesis is required for pathogenicity of Alternaria alternata........94