GENETICALLY DISSECTING THE RECESSIVE RPG4-MEDIATED WHEAT STEM RUST RESISTANCE LOCUS IN BARLEY A Thesis Submitted to the Graduate Faculty of the North Dakota State University of Agriculture and Applied Science By Deepika Arora In Partial Fulfillment for the Degree of MASTER OF SCIENCE Major Department: Plant Pathology June 2014 Fargo, North Dakota North Dakota State University Graduate School Title GENETICALLY DISSECTING THE RECESSIVE RPG4-MEDIATED WHEAT STEM RUST RESISTANCE LOCUS IN BARLEY By Deepika Arora The Supervisory Committee certifies that this disquisition complies with North Dakota State University’s regulations and meets the accepted standards for the degree of MASTER OF SCIENCE SUPERVISORY COMMITTEE: Robert Brueggeman Chair Maricelis Acevedo Zhaohui Liu Sangita Sinha Approved: 06/27/2014 Jack Rasmussen Date Department Chair ABSTRACT The rpg4-mediated resistance locus (RMRL) in barley provides recessive resistance against wheat stem rust (Puccinia graminis f. sp. tritici) races including the virulent race TTKSK (a.k.a Ug99). Three genes (HvAdfF3, Rpg5, and HvRga1) at the RMRL are required together for resistance. Allele characterization of these three genes identified the dominant rye stem rust resistance gene Rpg5 gene as the polymorphic R-gene conditioning the recessive rpg4-mediated wheat stem rust resistance. The Rpg5 gene contains an atypical R-gene structure encoding a nucleotide-binding site, leucine rich repeat, and serine/threonine protein kinase (STPK) domains. Genetic analysis of crosses between the resistant cultivar Q21861 with a functional Rpg5 allele and different susceptible varieties determined that most susceptible cultivars contain the susceptibility gene HvPP2C.1 in place of Rpg5 STPK domain. Genetic analyses determined that HvPP2C.1 conditions the recessive nature of the reaction, possibly negatively regulating the signaling pathway initiated by the Rpg5 kinase domain. iii ACKNOWLEDGEMENTS My sincere gratitude goes to my major advisor, Dr. Robert S. Brueggeman for his expert guidance and continuous support for my MS study and research. His patience, encouragement and immense knowledge have helped me a lot during my research and while writing this thesis. I can’t thank him enough for giving me an opportunity to work on the project funded by the prestigious National Science Foundation (NSF) grant as part of my thesis. In addition, a special thanks goes to my committee members, Dr. Maricelis Acevedo, Dr. Zhaohui Liu and Dr. Sangita Sinha for their insightful questions, comments and suggestions. I would like to thank all the faculty members of the Department of Plant Pathology for the knowledge that they have passed on to me through their teaching and all the people associated with the plant pathology department for their help and support at many points of time during my stay at NDSU. I will always cherish the time spent here and the friends and colleagues I met here. I am heartily thankful to my parents, my father Mr. Ravinder K. Arora, and my mother Mrs. Raj R. Arora for the unconditional love, care and moral support they have bestowed on me. Without their blessings and motivation for education, it would have never been possible to come this far and be what I am today. iv DEDICATION I dedicate this thesis to my family, my father Mr. Ravinder K. Arora, my mother Mrs. Raj R. Arora, and my loving sibling, Mr. Kapil Arora. Thank you. v TABLE OF CONTENTS ABSTRACT ................................................................................................................................... iii ACKNOWLEDGEMENTS ........................................................................................................... iv DEDICATION ................................................................................................................................ v LIST OF TABLES ....................................................................................................................... viii LIST OF FIGURES ....................................................................................................................... ix 1. LITERATURE REVIEW ......................................................................................................... 1 1.1. Historical significance of stem rust............................................................................... 1 1.2. Major stem rust epidemics in North America ............................................................... 2 1.3. The pathogen- Puccinia graminis ................................................................................. 4 1.4. Stem rust resistance in barley ....................................................................................... 7 1.4.1. Rpg1 resistance, cloning and characterization ........................................................ 9 1.4.2. rpg4-mediated resistance locus or RMRL ............................................................ 11 1.5. Literature cited ............................................................................................................ 14 2. CHAPTER ONE: ALLELE CHARACTERIZATION OF GENES REQUIRED FOR RPG4-MEDIATED WHEAT STEM RUST RESISTANCE IDENTIFIES RPG5 AS THE R-GENE ......................................................................................................................... 20 2.1. Abstract ....................................................................................................................... 21 2.2. Introduction ................................................................................................................. 22 2.3. Materials and methods ................................................................................................ 29 2.3.1. Barley germplasm ................................................................................................. 29 2.3.2. Stem rust isolates and phenotyping....................................................................... 30 2.3.3. Allele sequencing and analysis ............................................................................. 31 2.3.4. RNA extraction ..................................................................................................... 33 2.3.5. Reverse transcription and reverse-transcription PCR ........................................... 35 vi 2.4. Results ......................................................................................................................... 36 2.4.1. Stem rust phenotyping .......................................................................................... 36 2.4.2. Allele sequencing and analysis ............................................................................. 37 2.4.3. Expression analysis ............................................................................................... 40 2.5. Discussion ................................................................................................................... 41 2.6. Acknowledgements ..................................................................................................... 44 2.7. Literature cited ............................................................................................................ 44 3. CHAPTER TWO: A PROTEIN PHOSPHATASE 2C GENE AT THE RPG4- MEDIATED RESISTANCE LOCUS DETERMINES THE RECESSIVE NATURE OF THE ONLY UG99 RESISTANCE IN BARLEY ............................................................ 48 3.1. Abstract ....................................................................................................................... 48 3.2. Introduction ................................................................................................................. 49 3.3. Materials and methods ................................................................................................ 52 3.3.1. HvPP2C.1 genetic analysis ................................................................................... 52 3.3.2. HvPP2C.1 cloning ................................................................................................ 53 3.3.3. HvPP2C.1 functional analysis: VIGS ................................................................... 54 3.4. Results ......................................................................................................................... 55 3.4.1. HvPP2C.1 genetic analysis ................................................................................... 55 3.4.2. HvPP2C.1 functional analysis .............................................................................. 56 3.5. Discussion ................................................................................................................... 60 3.6. Literature cited ............................................................................................................ 62 4. PROLOGUE ........................................................................................................................... 64 4.1. Literature cited ............................................................................................................ 66 vii LIST OF TABLES Table Page 1. Reaction of barley lines to Puccinia graminis f. sp. secalis isolate 92-MN- 90 and P. graminis f. sp. tritici pathotypes QCCJ, HKHJ and MCCF. ............................ 29 2. Primers used for Rpg5, HvRga1, and HvAdf3 allele analysis. .......................................... 32 3. Primers used for expression analysis of the Rpg5, HvRga1, and HvAdf3 alleles. ............................................................................................................................... 34 4. F1 reactions and F2 analyses to determine the fitness of segregation ratios obtained for crosses made to validate the HvPP2C.1 as a dominant susceptibility gene. ............................................................................................................ 57 5. Infection types (IT) of parental lines (Q21861 and Steptoe) and F1s (Q X S) in response to pathotypes of Puccinia graminis f. sp. tritici QCCJ obtained for HvPP2C.1 functional analysis using VIGS. ................................................. 59 viii LIST OF FIGURES Figure Page 1. Physical map of the rpg4/Rpg5-mediated resistance locus and characterization of ............................................................................................................. 24 2. Protein domain structures and amino acid alignment for the Rpg5, HvRga1, and HvAdf3 alleles. ........................................................................................................... 33 3. Expression analysis of the Rpg5, HvRga1, and HvAdf3 alleles. ......................................... 35 4. Predicted gene and protein architecture at RMRL for parental haplotypes and F1 progeny as well as the predicted functional proteins translated from each loci from the parental and F1 progeny and the observed typical reactions of the F1 and F2s progeny after QCCJ inoculation. .......................................... 58 5. Predicted full length gene architecture of HvPP2C.1 gene shown to scale.. ...................... 58 6. pSL38.1 and DDS32 plasmid DNA. ................................................................................... 59 ix 1. LITERATURE REVIEW 1.1. Historical significance of stem rust Stem rust or black rust was historically the most devastating and widespread diseases of small grain crops including wheat, barley, oats and rye and was considered one of the biggest threats to world food security. It is caused by the obligate, biotrophic fungal pathogen Puccinia graminis (P. graminis). Stem rust is believed to have infected grasses for millions of years and it has probably infected grain crops since the beginning of their cultivation in the Fertile Crescent region. The earliest recorded documentation of plant diseases including stem rust is found in the Bible where the epidemics of cereal rusts and smuts were believed to be inflicted upon the Israelites as punishment to their sins (Deut, 28:22). The spores of P. graminis have been found in archeological sites containing wheat storage jars in Israel dating from 1300 BC (Kislev, 1982). Words about stem rust also appeared in the Mishnah, which described it as a kind of spreading plaque of wheat (Taanith, 3:5). Stem rust epidemics were considered a problem in ancient Greek and Rome empires and an ancient practice by the Romans as described by Numa Pompilius (715–672 BC) was to sacrifice red animals such as foxes, dogs, and cows to the rust God in the spring time festival known as the Robigalia hoping the god would be appeased and spare their wheat crops of the devastating red rust (Chester, 1946). Stem rust was observed and recognized as early as 384 B.C. making it one of the earliest studied plant diseases by ancient plant pathologists Aristotle and Theophrastus who associated cereal rust epidemics with warm, wet weather (Chester, 1946). As documented, a string of rainy seasons in the historical times resulted in high stem rust severity and reduced wheat harvests and is speculated to have played a major role in the fall of the Roman Empire. So, through prehistory until our current times stem rust has been threatening food security. 1