GENETIC BASIS FOR HOST RESPONSE TO HOP STUNT VIROID By JEFF MARTIN BULLOCK A dissertation submitted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY WASHINGTON STATE UNIVERSITY Department of Plant Pathology MAY 2016 ©Copyright by JEFF MARTIN BULLOCK, 2016 All Rights Reserved ©Copyright by JEFF MARTIN BULLOCK, 2016 All Rights Reserved To the Faculty of Washington State University: The members of the Committee appointed to examine the dissertation of JEFF MARTIN BULLOCK find it satisfactory and recommend that it be accepted. ____________________________ Kenneth C. Eastwell, Ph.D., Chair ____________________________ Hanu R. Pappu, Ph.D. ____________________________ Brenda K. Schroeder, Ph.D. ____________________________ Paul D. Matthews, Ph.D. ii ACKNOWLEDGEMENTS I have been very fortunate to have Dr. Kenneth C. Eastwell as my advisor and mentor throughout this process. His guidance has been invaluable and his efforts on my behalf have been extraordinary! I was one of his first graduate students working on a master’s degree in 1986, now 30 years later I will be his last graduate student to complete a Ph.D. under his guidance. I cannot think of a better person to have guided my path, he has been a huge influence on my scientific training. I would also like to thank my committee members, Dr. Hanu R. Pappu, Dr. Brenda K. Schroeder, and Dr. Paul D. Matthews for their constant encouragements, technical assistance and support. In addition I am grateful to the Washington Hop Commission and the Hop Research Council for funding this project and to all the members of the Northwest Clean Plant Center, namely: Jan Burgess, Shannon Santoy, Tina Vasile, Syamkumar Sivasankara, Piotr Kowalec, Debbie Woodbury, Dan Villamor, Holly Ferguson, and Eunice Beaver-Kanuya for all their help and reassurances. I would like to especially thank Martin Joseph for his assistance in plant propagation from tissue culture to greenhouse maintenance and Madhu Kappagantu, my fellow graduate student, for her help with plant care and her important insights in bioinformatics. Most importantly I would like to thank my parents Gil and Jean Bullock, and my wife Jan for their ever present love and unwavering support, without them this goal could not have been reached! iii GENETIC BASIS FOR HOST RESPONSE TO HOP STUNT VIROID Abstract by Jeff Martin Bullock, Ph.D. Washington State University May 2016 Chair: Kenneth C. Eastwell, Ph.D. Hop stunt viroid (HSVd) was first detected in hop orchards in Washington State in 2004. A 2012 survey for the presence of HSVd, in three major hop growing regions in Washington State indicated an overall incidence of 17.3 percent. A five year yield study comparing HSVd infected and uninfected hop cultivars ‘Glacier’ and ‘Nugget’ revealed that, of those tested, ‘Nugget’ was the most tolerant hop cultivar to HSVd infection and ‘Glacier’ was the most severely affected. In this study, whole genome and RNA sequence analysis identified potential genes associated with expression of hop stunt disease, and identified DNA-markers that distinguish HSVd-tolerant plants from HSVd-sensitive cultivars. Healthy plants and HSVd-infected ‘Glacier’ and ‘Nugget’ revealed different gene expression profiles that suggested that a pathogenesis- related protein, thaumatin-like protein (TLP), is a candidate marker for HSVd sensitivity. TLP was observed to be down regulated 2.6 fold in ‘Glacier’ infected with HSVd compared to HSVd-free ‘Glacier’; no change in TLP expression was observed in ‘Nugget’ iv between HSVd-infected or HSVd free plants. RNA sequence analyses were confirmed using qRT-PCR for TLP expression levels normalized to Glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Alignments of the TLP coding sequence (CDS) and the region immediately up-stream of the TLP CDS for each cultivar revealed no sequence variations 100 bp upstream, which includes the ATG translation start site, TATA box and the transcriptional start site. However, 23 nucleotide variations exist between ‘Glacier’ and ‘Nugget’ in a 397 bp region up stream of the first CAAT cis-regulatory element (CRE). These variations create eleven unique CRE differences between ‘Glacier’ and ‘Nugget’. Additionally, within the TLP CDS, nineteen single nucleotide polymorphisms (SNPs) were observed in ‘Glacier’ relative to ‘Nugget’. Several of these SNPs result in amino acid substitutions. Four of the SNPs within the ‘Glacier’ TLP CDS result in amino acid substitutions that were not observed in five other cultivars of hop. In total, 42 SNPs were identified that potentially may be used in marker assisted breeding programs to distinguish potential HSVd-tolerant plants from HSVd-sensitive plants. Evaluation of additional hop genotypes will be necessary to confirm this relationship. v TABLE OF CONTENTS Page ACKNOWLEDGEMENTS iii ABSTRACT iv LIST OF FIGURES viii LIST OF TABLES xi LIST OF ABBREVIATIONS xiii CHAPTER 1. GENERAL INTRODUCTION 1 Taxonomy and breeding history of genus Humulus 1 Hop compounds essential to brewing 6 Viroids 17 Operons 28 Gene regulation 30 Small RNAs 36 Pathogenesis related proteins 43 Summary and Project goals 45 vi 2. MATERIALS AND METHODS 49 Cloning 50 Cucumber and hop inoculations 53 RNA extractions and high through-put sequencing 59 RNA sequence expression analysis and gene discovery 61 Sequence variations between hop cultivars 66 Relative HSVd concentration and TLP expression levels 68 Survey for the presence of HSVd 69 RNA extraction from FTA cards and RT-PCR for the presence of HSVd 70 3. RESULTS and DISCUSSION 71 Infectious clone of HSVd 71 High through-put sequencing 79 TLP discovery and expression differences between treatment groups 85 Relative HSVd concentration and TLP expression levels 89 TLP coding sequence and sequence analysis 91 Plant specific TLP isoforms 93 Sequence variations in the TLP CDS and regions upstream 96 Survey for the presence of HSVd in Washington State 103 Discussion 104 References 119 vii LIST OF FIGURES CHAPTER 1 1 Leaf pattern common to Humulus lupulus and Humulus japonicas. 2 2 Female seed cones (strobiles), female inflorescences, and male inflorescences. 4 3 Female hop cone showing bracts, bracteoles, and cutaway view of hop cone exposing lupulin glands. 8 4 Pathway for humulone synthesis using the branched chain acyl-CoA thioester; isovaleryl-CoA, as a precursor in the presence of valerophenone synthase (VPS) to produce the first intermediate, phlorisovalerophenone (PIVP), in the alpha acid pathway. 10 5 The acyl-CoAs, isovaleryl-CoA, isobutyryl-CoA and 2-methylbutyryl-CoA used in alpha and beta acid synthesis are break down products produced by the catabolism of the branched-chain amino acids (BCAA) leucine, valine and isoleucine. 11 6 BCAA biosynthesis from pyruvate in plants. Valine and isoleucine are produced via parallel pathways; both use identical enzymes. 14 7 Multibranched secondary structure showing hairpins, pseudoknots, and loops that form hammerhead ribozymes, which are unique to Avsunviroidae viroids; less complex secondary rod like structure of Pospiviroidae viroids. 21 viii CHAPTER 3 1 Capillary gel electrophoresis of PCR amplification products using plasmid F56 purified from transformed One Shot Cells as a template. 71 2 Sequence of F56 plasmid. 72 3 Sequence of monomer from F56 plasmid aligned to hKFKi a well characterized HSVd isolated from hop. 73 4 Mock inoculated cucumber (Cucumis sativus L. cv. Suyo Long). HSVd (F56) inoculated cucumber (Cucumis sativus L. cv. Suyo Long). 74 5 Capillary gel electrophoresis of RT-PCR amplification products using total RNA extracted from cucumber leaf tissue. 75 6 Sequence alignment of HSVd RT-PCR product produced using total RNA extracted from F56 inoculated cucumber as a template and HSVd isolate hKFKi.76 7 ‘Glacier’ plants 140 days post inoculation. 77 8 ‘Nugget’ plants 140 days post inoculation. 78 9 ‘Nugget’ and ‘Glacier’ plants 140 days post inoculation. 79 10 Leaves mock inoculated and HSVd inoculated. 80 11 RNA extraction resolved on a 1% non-denaturing agarose gel. 81 ix 12 Alignment of 58 bp region of contig LA331881 to Brassica napus thaumatin-like protein and alignment of LA331881 putative TLP region to scaffold LD139544. 85 13 Simple linear regression analysis of qRT-PCR results showing the correlation between normalized TLP levels and HSVd levels relative to an HSVd standard for ‘Glacier’ and ‘Nugget’ HSVd infected plants. 90 14 Identified coding sequence from LD139544 region 29612 to 30283 91 15 Translation product, 223 amino acids from identified open reading frame using LD139544, region 29612 to 30283 and PBLAST analysis identified the amino acid sequence as a TLP specific to the plant subfamily. 92 16 Sequence 200 bp upstream of the TLP ATG translation start site 93 17 Amino acid sequence of different plant specific TLP isoforms 94 18 Nineteen single nucleotide polymorphisms in ‘Glacier’ relative to the hop cultivar ‘Nugget’ 97 19 Thaumatin amino acid variations between hop cultivars ‘Nugget’, ‘Glacier’ ‘Cascade’, ‘Columbus’, ‘Galena’, and ‘Willamette’ 98 20 Positions of single nucleotide substitutions and multi nucleotide insertions and position of TATA box and CA transcriptional start within the 41 bp GC rich region and cis-regulatory elements unique to ‘Glacier’ and ‘Nugget’ 100 x LIST OF TABLES CHAPTER 1 1 Representative sample of viroids, their known host and the associated viroid family Pospiviroidae or Avsunviroidae 19 CHAPTER 2 1 Hop rooting media. 58 CHAPTER 3 1 Relative fold change between BLAST identified sequences in HSVd infected and mock inoculated plants from ‘Nugget’ and ‘Glacier’ treatment groups. 82 2 Relative fold change for TLP between, HSVd infected and mock inoculated (HSVd free) plants from ‘Glacier’ and ‘Nugget’ treatment groups. 86 3 Results from qRT-PCR, normalized to GAPDH showed no change between HSVd infected and mock inoculated ‘Nugget’ plants. 87 4 Results from qRT-PCR, normalized to GAPDH showed a 2.6 fold change (down regulation) for HSVd infected ‘Glacier’ plants compared to mock inoculated plants. 88 5 Concentration levels of HSVd in infected tissue correlated to TLP expression levels, normalized to GAPDH using qRT-PCR.