UNIVERSITY OF OKLAHOMA GRADUATE COLLEGE SEQUENCE AND ANALYSIS OF THE PHYMATOTRICHOPSIS OMNIVORA GENOME AND EXPRESSED SEQUENCE TAGS A DISSERTATION SUBMITTED TO THE GRADUATE FACULTY in partial fulfillment of the requirements for the Degree of DOCTOR OF PHILOSOPHY BY SIMONE MACMIL Norman, Oklahoma 2009 SEQUENCE AND ANALYSIS OF PHYMATOTRICHOPSIS OMNIVORA GENOME AND EXPRESSED SEQUENCE TAGS A DISSERTATION APPROVED FOR THE DEPARTMENT OF CHEMISTRY AND BIOCHEMISTRY By Dr. Bruce A. Roe, Chair Dr. Paul F. Cook Dr. Ann H. West Dr. Kathleen E. Duncan Dr. Charles V. Rice ©Copyright by SIMONE LYNETTE MACMIL 2009 All Rights Reserved. Acknowledgements I wish to express my sincere gratitude to my major advisor Dr. Bruce Roe for accepting me to his lab at the brink of his retirement, giving me the opportunity to work on this challenging yet rewarding project and for cheering me on through the many roadblocks and bumps that I encountered during the course of this study. His scientific accomplishments and discussions have always inspired me and have spurred me to reach higher in my goals and delve deeper in my understanding and it would be impossible to complete this project sans his patience, guidance and encouragement. I am extremely grateful to my advisory committee, Dr. Paul Cook, Dr. Kathleen Duncan, and Dr. Charles Rice for their advice, encouragement and participation in my research and dissertation. I would specially like to thank Dr. Ann West for chairing my committee during my candidacy exam and for her willingness to be present on my doctoral defense committee as well. My special thanks also goes to Dr. Carolyn Young at the Noble Foundation for providing us with chromosomal bands, informative discussions and giving me the opportunity to learn at her lab. I am extremely grateful to Steve Kenton and Dr. Fares Najar for imparting to me some of their bioinformatic skills and for their assistance with the assembly and annotation. I would also like to extend my appreciation to all the members of Dr. Roe’s lab; Graham Wiley, Doug White, Jim White, Yanbo Xing, Chunmei Qu, Ping Wang, Baifang Qin, Keqin Wang, Liping Zhou who worked together with me in processing pyrosequencing experiments on the 454 Genome Sequencer; Jiaxi Quan, Jing Yi, Ruihua Shi, Shaoping Lin, Fu iv Ying, Axin Hua, Ziyun Yao, Limei Yang, Hongshing Lai, Dixie Wishnuck, Jennifer Lewis, Mounir Elharam and the many lab members who have now moved on for collectively making my experience at the lab memorable. I wish to thank my brothers Llewellyn, Lyon, Warren, my friend Sini and my sister Sharon and her family for their love and support. I dedicate this dissertation to my mum Patricia Rowe and dad Albert Macmil for having faith in my abilities and instilling in me a love for learning. Above all, I am eternally grateful to my heavenly Father, who daily loads me with His goodness, mercies and grace. v Table of Contents List of Tables xii List of Figures xiii Abstract xvi Chapter1 Introduction 1 1.1 Fungi and their role in agriculture 1 1.1.1 Introduction to Fungi 1 1.2 Phymatotrichopsis omnivora – the “cotton root rot fungus” 3 1.2.1 Life cycle of Phymatotrichopsis omnivora 4 1.2.2 Habitat of Phymatotrichopsis omnivora 6 1.3 Fungal Genomics 7 1.3.1 Fungal genomes used in this study 8 1.4 The discovery of nucleic acids and elucidation of the Central Dogma 10 1.5 Fungal Expressed Sequence Tags 13 1.6 DNA Sequencing Strategies 16 1.7 Bioinformatic Tools 19 1.7.1 BLAST 19 1.7.2 FgenesH 21 1.7.3 tRNA scan-SE 21 v i 1.7.4 Databases 22 Chapter2 Material and Methods 25 2.1 Construction of Shotgun Library for Sanger Sequencing and Paired-end Pyrosequencing 25 2.1.1 Fragmentation of Genomic DNA for both Sanger and Paired-end Pyrosequencing 26 2.2 Sanger Sequencing 26 2.2.1 End Repair and Size Selection for Sanger sequencing 26 2.2.2 Ligation of DNA fragements with pUC 18 27 2.2.3 Transformation 28 2.2.4 Automatic Isolation of Subclone DNA 29 2.2.5 Reaction and Clean Up 30 2.2.6 Sequencing on the ABI 3730 31 2.3 Pyrosequencing on the 454 31 2.3.1 DNA fragmentation, SPRI bead purification and size selection 33 2.3.2 Methylation of the EcoRI site, shearing and blunt-ending 34 2.3.3 Hairpin adaptor ligation, exonuclease and EcoR1 digestion 35 2.3.4 DNA circularization, DNA Nebulization and Concentration 36 2.3.5 Adaptor Ligation and Quantitation 37 2.4 Emulsion PCR 38 vi i 2.4.1 Bead recovery, enrichment and sequencing primer annealing 39 2.4.2 Loading the PicoTiterPlate for Sequencing 40 2.4.3 Sequencing and signal processing on the 454 Genome Sequencer. 41 2.5 Assembling the massively parallel pyrosequencing data for cDNA and genomic DNA 42 2.6 Isolation of Individual P. omnivora Chromosomes 43 2.7 Analysis of cDNAs 44 2.7.1 Biological function assignments 44 2.7.2 Determining EST abundance in each library 45 2.8 Analysis of the P. omnivora genome 46 2.8.1 Gene prediction and annotation 46 2.8.2 Comparison of P. omnivora predicted proteins with that of other fungi 47 Chapter3 Results and Discussion 48 3.1 Sequencing and analyzing the ESTs from three different life stages of P. omnivora and after exposure to different environmental conditions. 48 3.1.1.1 Assembly of ESTs 49 3.1.1.2 The possible role of Tar 1p ESTs in P. omnivora cDNA libraries 52 3.1.2 EST Sequencing of mycelia grown on M1078 medium 53 3.1.3 EST Sequencing of Sclerotia 57 3.1.4 EST Sequencing of Conidial Spore mat 61 vii i 3.1.5 EST Sequencing of Carbon and Nitrogen Starved mycelia 65 3.1.6 EST Sequencing of mycelia exposed to host root exudates 68 3.1.7 EST Sequencing of mycelia exposed to non-host root exudates 71 3.2 Comparative analysis of P. omnivora EST libraries 75 3.2.1 Comparison of metabolic profile of ESTs derived from different life stages and environments 75 3.2.2 Does oxidative stress trigger metamorphosis in P. omnivora? 80 3.3 The P. omnivora genome and assembly 82 3.3.1 Sequencing individual P. omnivora chromosomes 83 3.4 Predicted protein profile of P. omnivora 86 3.4.1 Analysis of P. omnivora predicted proteins 88 3.4.1.1 Comparison of P. omnivora predicted proteins with that of other fungi 88 3.4.1.2 Metabolism in P. omnivora 90 3.4.1.2.1 Glycolysis, the pentose phosphate pathway and gluconeogenesis. 90 3.4.1.2.2 Tricarboxylic Acid (TCA) cycle and the glyoxylate shunt 94 3.4.1.2.3 Fructose, mannose and Galactose metabolism 95 3.4.1.2.4 Oxidative phosphorylation 96 3.4.1.2.5 Lipid metabolism 97 3.4.1.2.6 Amino acid biosynthesis 99 3.4.1.2.7 Nucleotide biosynthesis 106 ix 3.4.1.2.8 Storage polysaccharides 108 3.4.1.3 Analysis of predicted transporters in the P. omnivora genome 109 3.4.1.4 Putative pathogenesis genes of P. omnivora 111 Chapter4 Conclusions 115 References 121 Appendix Table 1. Putative identification and classification of EST’s from the vegetative mycelia based on blast homology searches in KEGG, COG and COGEME databases. 138 Table 2. Putative identification and classification of EST’s from sclerotia based on blast homology searches in KEGG, COG and COGEME databases. 158 Table 3.Putative identification and classification of EST’s from spore mats based on blast homology searches in KEGG, COG and COGEME databases. 169 Table 4. Putative identification and classification of EST’s from the carbon, nitrogen starved mycelia based on blast homology searches in KEGG, COG and COGEME databases. 180 Table 5.Putative identification and classification of EST’s from the mycelia exposed to host root exudate based on blast homology searches in KEGG, COG and COGEME databases 185 Table 6. Putative identification and classification of EST’s from the mycelia exposed to non-host root exudate based on blast homology searches in KEGG, COG x and COGEME databases 193 x i List of Tables Table 1. Plant diseases caused by filamentous fungi. 3 Table 2. List of selected completed and draft fungal genomes 8 Table 3. Assembly statistics of P. omnivora cDNA libraries by Newbler 49 Table 4. Description of top 20 most highly expressed transcripts in vegetative mycelia with homology in GenBank 56 Table 5. Description of top 20 most highly expressed transcripts in sclerotia with homology in GenBank 59 Table 6. Description of top 20 most highly expressed transcripts in spore mats with homology in GenBank. 63 Table 7. Description of top 20 most highly expressed transcripts in carbon/nitrogen starved mycelia with homology in GenBank 67 Table 8. Description of top 20 most highly expressed transcripts in mycelia exposed to host root exudate with homology in GenBank 70 Table 9. Description of top 20 most highly expressed transcripts in mycelia exposed to non-host root exudate with homology in GenBank 73 Table 10. P. omnivora whole genome shotgun sequencing and assembly statistics 83 Table 11. Preliminary sequencing results of P. omnivora amplified chromosomes 86 xi i List of Figures Figure 1. Life cycle of P. ominvora as it passes through the vegative, sclerotial and conidial stages. 5 Figure 2. Reported distribution of Phymatotrichopsis omnivora in North America. Adapted from map by Streets and Bloss (1973). 6 Figure 3: The structure of DNA (Watson and Crick, 1953) 13 Figure 4.