I I the EVOLUTION of FUNGAL PECTINASES in GLYCOSYL
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i THE EVOLUTION OF FUNGAL PECTINASES IN GLYCOSYL HYDROLASE FAMILY 28 AND THEIR ASSOCIATION WITH ECOLOGICAL STRATEGY A thesis submitted to Kent State University in partial fulfillment of the requirements for the degree of Master of Sciences By Daniel David Sprockett December 2009 i ii Thesis written by Daniel David Sprockett B.S., Kent State University, 2006 M.S., Kent State University, 2009 Approved by ___________________________________, Advisor Christopher B. Blackwood ___________________________________, Advisor Helen Piontkivska ___________________________________, Member, Masters Thesis Committee Walter R. Hoeh Accepted by ___________________________________, Chair, Department of Biology James L. Blank ___________________________________, Dean, College of Arts and Sciences John R.D. Stalvey ii iii TABLE OF CONTENTS LIST OF FIGURES.......................................................................................................v LIST OF TABLES........................................................................................................vii ACKNOWLEDGEMENTS…………………………………………….……………viii CHAPTER 1: I. General Introduction………………………………….…………………….1 Pectin Structure ………………………………………………………1 Pectin Distribution and Environmental Role…………………………2 Pectin Industrial and Biomedical Uses…………………….…………3 Pectinase Structure and Biochemistry………………………..………4 Pectinase Distribution and Environmental Role……..…….............…5 Pectinase Industrial and Biomedical Uses………………….…...……6 Thesis Overview………………………………………………...……7 CHAPTER 2: II. The Distribution, Functional Diversity, and Evolution of Glycosyl Hydrolase Family 28 in Fungi……………………………………………………….…12 Introduction…………………………………………………………12 Methods…………………………………………………………..…14 Results and Discussion…………………………………...…………17 Conclusion………………………………………………………..…24 CHAPTER 3: III. Fungal Glycosyl Hydrolase Family 28 Repertoire is Significantly Influenced by Ecological Strategy………………………………………….......………30 Introduction…………………………………………….…...…....…30 Methods……………………………………………………..........…33 Results……………………………………………………...…….…36 Discussion………………………………………………….…….…38 Conclusion……………………………………………...….....…….44 LITERATURE CITED………………………………………………..…..….….…49 APPENDIX 1: Summary of Organisms Used in this Study …………................…65 APPENDIX 2: Aspergillus niger GH28 BLAST Query Sequences……………….84 iii iv APPENDIX 3: Neighbor-Joining Gene Tree Clades………………………………86 APPENDIX 4: Maximum-Likelihood Gene Tree Clades………………….………95 iv v List of Figures Fig. 1.1 This diagram shows the structure of the pectin chain and sites of GH28 cleavage. Modified from Willats et al. 2006……………………………………………...….10 Fig. 2.1 Fungal Species Tree and Maximum Parsimony Reconstruction of Ancestral GH8 Copy Number……………………………………………………………....………25 Fig 2.2.a GH28 Neighbor-Joining Phylogenetic Reconstruction. ………….......…26 Fig 2.2.b GH28 Maximum-Likelihood Phylogenetic Reconstruction. …….......…27 Fig 2.3 This scatterplot shows the relationship between GH28 copy number and the number of clades those copies occur. ……………………………….…...........…28 Fig 2.4 This scatterplot shows the relationship between GH28 copy number and the number of different functional groups in which those GH28 gene copies are categorized, including endo-PG, exo-PG, endo-RG, and exo-RG. …………………………..…29 Fig 3.1.a Bar graph showing the differences in the genome sizes (Mb) in biotrophic, necrotrophic, and non-pathogenic fungi. ………………………………….…...…45 Fig 3.1.b Bar graph showing the differences in GH28 copy number in biotrophic, necrotrophic, and non-pathogenic fungi. ……………………………….……..…46 Fig 3.2 Scatterplot showing the relationship between genome size (Mb) and GH28 gene copy number. Biotrophs, necrotrophs, and non-pathogens are labeled, and groups of biotrophs and necrotrophs identified with colored ovals. ……………………....…47 v vi Fig 3.3 Mirror Tree of fungal species. Genome size and GH28 copy number has been reconstructed using parsimony analysis. Biotrophs and necrotrophs are identified. Felsenstein’s Contrast Correlation P < 0.0001, r 2 = 0.19………………………..…48 vi vii LIST OF TABLES Table 1.1 A summary of GH28 functional types, E.C. numbers, and a brief description of each mode of enzymatic action. ……………………………………………………11 vii viii ACKNOWLEDGEMENTS It is a pleasure for me to acknowledge the many people who made this thesis research possible. I would first like to sincerely thank my graduate advisors, Christopher B. Blackwood and Helen Piontkivska, for their careful guidance during this research project, as well as my thesis committee member, Walter Hoeh. I would also like to thank other biology faculty members that have significantly impacted my development as a research scientist over my tenure at Kent State. These faculty members include John Stalvey, Andrea Case, Oscar Rocha, Pat Lorch, Ferenc de Szalay, and Mark Kershner. I also need to thank Justin Reeves, Justin Montemarano and Sinu Paul for their help analyzing data and building figures, as well as Mikayel Hovhannisyan for writing me a valuable sequence re-naming program. I owe a large debt of gratitude to Chris Blackwood and Bess Heidenreich for their help in completing an extensive literature search of fungal pathogens. I additionally need to acknowledge my many lab mates, including Stephanie Hovatter, Doug Antibus, Larry Feinstein, and Oscar Valverde, as well as fellow graduate students Eric Floro, Julie Proell, Julie Morris, and Jenn Clark. Their enthusiasm, encouragement and advice has not only helped make this a successful project, but has also aided in my growth as a person and as a scientist. Finally, I am grateful for my wonderful friends and family. Their endless love and support have made me the person that I am today. I wish to dedicate this thesis to my parents, Larry and Barbara Sprockett, and my fiancé, Andrea Loomis. I could have not made it this far without them. viii 1 Chapter 1: General Introduction Pectin Structure One major distinguishing characteristic of plant cells is their rigid cell wall. This specialized structure gives plant tissue rigidity and protection, while its semi-permeable nature also precludes the uptake of large molecules. Plant cell walls have three layers, the inner lamella, the primary cell wall, and the secondary cell wall, and are primarily composed of cellulose, hemicellulose, various soluble proteins, and pectin. Pectin, a diverse family of polysaccharides, is a major structural component of both the primary cell wall and the inner lamella. The cell wall can be conceptualized as a complex cellulose-glycan network surrounded by a soluble matrix of polysaccharides, glycoproteins, proteoglycans, and ions. Pectin is the most abundant class of macromolecule in this matrix, accounting for up to one third of all primary cell wall macromolecules (Willats et al. 2001). Biochemically, the pectin family of carbohydrates is comprised of members dominated by a chain of 1,4-linked alpha-D-galactosyluronic acid residues (GalA). There are three types of pectic domains, which include homogalacturonan, rhamnogalacturonan-I, and rhamnogalacturonan-II (Fig 1.1). Homogalacturonan (HG) is composed of a linear chain of 1,4-linked alpha-D-galactosyluronic acid residues. Demethyl-esterification at the C-6 carboxyl allows calcium cross-linkages to form, resulting in large assemblies of homogalacuronan within the cell wall matrix. Rhamnogalacturonan-I (RG-I) occurs when regions of galacturonic acid are replaced with 1 2 the disaccharide repeat [(1 →2)-α-L-rhamnose-(1 →4)- α-D-galacturonic acid]n. This replacement causes steric hindrance or a “kink” in the linear backbone and allows the bonding of various sugar side chains, including D-galactose, L-arabinose, and D-xylose. Pectic regions with high densities of rhamnose are considered “hairy” regions due to their highly branched configuration, while those with less branching are termed “smooth” regions (Pérez et al. 2000). Rhamnogalacturonan-II (RG-II) is a branched pectic domain containing a homogalacturonan backbone with various complex side chains bonded to the GalA residues (O’Neill et al. 2004). These three polysaccharide domains form covalent linkages throughout the primary cell wall matrix and middle lamellae, and provide considerable potential for structural modulation by a wide range of pectinase enzymes. This matrix forms a crystalline structure that allows it to trap water and other molecules, giving pectin its widely used gelling properties. It has been traditionally assumed that the structure of pectin was primarily composed of a HG backbone interspersed with RG-I and RG-II regions (Fig 1.1). However, recent alternative structures have been proposed, placing RG-I at the backbone with long side chains of HG, further branching into RG-II (Willats et al. 2006). Pectin Distribution and Environmental Role Pectin is found in both angiosperms and gymnosperms, along with pteridophytes, bryophytes, lycophytes and carophytes, an algal clade that is believed to be the closest extant relative of land plants (Matsunaga et al. 2004, O’Neill et al. 2004). The occurrence of pectin in lignified cell walls is thought to be a key plant adaptation that allowed plants to colonize land by way of upright growth (Matsunaga et al. 2004). This carbohydrate 2 3 family makes up ~35% of primary walls in dicots and non-graminaceous monocots and anywhere between 2 and 10% of grass and other commelinoid primary walls (Mohnen 2008). It is synthesized in the Golgi apparatus and transported throughout the cell in membrane-bound vesicles. Pectic chains are largely deposited in