v .H. ,-~v ‘-3 " -.'-—I..r'.rl'.l«<‘x,uv—~; .31th lllllllllllllllllllllllllllllllllllllHllllllllllllllllllllll 1293 01051 7955 This is to certify that the dissertation entitled GENETIC AND PHYSIOLOGICAL CHARACTERIZATION OF NATURALLY- OCCURRING MULTIPLE POTYVIRUS RESISTANCE IN THE CHINESE CUCUMBER LINE TMG-l presented by Thanda Wai has been accepted towards fulfillment of the requirements for Ph.D. BOTANY AND PLANT PATHOLOGY degree in AND GENETICS 71 W 7/117 Major professor Dr. Rebecca Grumet Date aim a 11 MM MS U is an Affirmative Action/Equal Opportunity Institution 0- 12771 LIBRARY Mlchlgan State Unlvorslty ‘ PLACE N RETURN BOX to romovo this checkout flom your record. TO AVOID FINES rotum on or bdoro dot. duo. DATE DUE DATE DUE DATE DUE 1| II I MSU to An Affirmative Mon/Equal Opportunlty Institution W ulna-9.1 GENETIC AND PHYSIOLOGICAL CHARACTERIZATION OF NATURALLY- OCCURRING MULTIPLE POTYVIRUS RESISTANCE IN THE CHINESE CUCUMBER LINE TMG-l BY Thanda Wai A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology in conjunction with The Genetics Program 1994 ABSTRACT GENETIC AND PHYSIOLOGICAL CHARACTERIZATION OF NATURALLY- OCCURRING MULTIPLE POTYVIRUS RESISTANCE IN THE CHINESE CUCUMBER LINE TMG-l BY Thanda Wai Cucurbit potyviruses cause severe crop losses around the world. The inbred Chinese cucumber line TMG-l is resistant to three related potyviruses: zucchini yellow mosaic virus (ZYMV), watermelon mosaic virus (WMV), and the watermelon strain of papaya ringspot virus (PRSV-W). The goal of this research was to understand the genetics of this multiple potyvirus resistance by determining the inheritance of resistance to each virus and the relationship of the resistances to each other. Linkage associations with known genetic markers were tested. Physiological studies were performed to examine the spatial and temporal distribution of virus in the resistant line in comparison with the susceptible parent. Tissue-specific differences in expression of the resistances were also investigated. TMG-l was crossed with WI-2757, an inbred line that is susceptible to all three viruses. The parents, F1 (WI-2757 X TMG-l), F2, and backcross (F1 X TMG-l and WI-2757 x F1) generations were inoculated with virus and their response _monitored by symptom expression and ELISA. Direct progeny screening, sequential virus inoculations, and tests of clonally propagated F2 individuals were performed. Inheritance of resistance to PRSV-W is due to a single Thanda Wai dominant gene, while resistance to ZYMV is conditioned by a single recessive gene. Two independently asserting recessive resistance factors confer resistance to WMV: the first resistance is due to a single recessive gene, while the second results from the epistatic interaction of a single recessive gene from TMG-l and a single dominant gene from the WI-2757 parent. The two separate resistances to WMV are expressed in different tissues. The resistance that is conferred by the single recessive gene is expressed in the cotyledon and throughout the plant. The resistance that results from the epistatic interaction is expressed only in true leaf tissue. The ZYMV resistance gene mapped to the same locus as the epistatic recessive resistance gene to WMV. Linkage mapping showed that sex expression (the F locus on Linkage Group I) is linked to the cotyledon-expressed WMV resistance, but not ZYMV resistance; Resistance to PRSV is linked to the bitterfree (bi) locus on Linkage Group I. Monitoring of virus spread in each leaf over time indicated a reduced rate of ZYMV and WMV accumulation in TMG-l relative to WI-2757. The apparent resistance to PRSV- W is likely to be tolerance since high levels of virus were detected despite an absence of symptoms. In summary, TMG-l has two to three different resistance mechanisms that employ a total of four to five resistance genes to confer resistance to three related potyviruses. Copyright by THANDA WAI 1994 T0 fill. THOSE IIJHD BELIEUED IN ME... I would like to dedicate this work to all those who have greatly influenced my life. First and foremost, I would like to dedicate this work to the members of my family who have unconditionally supported my academic pursuits from my youth: my mother Mya Thanda Poe, my father U Tha Gyaw Wai, my stepfather James D. Poe, and to my late brother Mra Gyaw Wai and grandmother Daw Mya Sein. I would also like to recognize others who tremendously influenced my educational opportunities: Mr. James Whittier Lewis, headmaster of the Holton—Arms School, who gave me the opportunity to receive a superlative education during my formative years. Terrence Oun Sein, my surrogate brother, who made it possible for me to pursue that education. Dr. Stephen J. Keller, my masters thesis adviser, who not only taught me how to do good science, but also to find and believe in myself. ACKNOWLEDGMENTS I would like to thank the members of my advisory committee (Rebecca Grumet, Ray Hammerschmidt, Richard Allison, Don Ramsdell, Jim Hancock, and more recently Barb Sears) for their support through this program. Off course, a special thanks goes to Rebecca Grumet for the many hours that we spent together testing various genetic models, and especially for the time she spent helping me to complete the writing process. I would also like to thank Dr. Jack Staub, USDA, University of Wisconsin at Madison, who suggested and provided technical support for the work on mapping of potyvirus resistance genes. A special thanks goes to his technicians Linda Crubaugh and Mary Palmer, who worked with me to perform the various disease screens. In addition, I would like to thank his graduate students Larry Knerr and Vladimir Meglic for their help with the isozyme work. I would like to thank all the people who have helped me in the lab and in the greenhouse. Thanks to Sue Hammar for filling in when needed and to the myriad of undergraduates, who have helped with the laborious greenhouse duties. I would like to acknowledge support services provided by Dave Freville, Jim Klug, and their staff in maintaining the greenhouse. I would also like to thank Dr. Alan Jones and Dr. Jim Hancock for providing extra greenhouse space when it vi was needed. A.deep appreciation is extended to Dr. Mike H Thomashow and the people in his lab for their friendship and support (Sarah Gilmour, Nancy Artus, Julia Bell, Dave Horvath, Wei-wen Guo, Chentao Lin, Kathy Wilhem, Deane Lehman, Brett McLarney, Stokes Baker, Lenny Bloksberg, and Kevin O'Connell). I would also like to acknowledge the various funding sources that have supported me through my degree program. Thanks to all those who were involved in obtaining the Patricia Roberts Harris Fellowship that supported the first two and half years of my program. Thanks to Rebecca Grumet for funding me through various sources, thereafter. In particular, I would like to thank Larry Graham for his support and friendship through the last part of my program. I particularly appreciated his help in preparing slides for my defense seminar and in helping me keep my sanity through this particularly stressful time in my life. My utmost gratitude goes to Dr.-Gene Alvin Homandberg, my former boss at Abbott Laboratories in North Chicago, Illinois, for his resolute insistence that I continue my 'education and obtain my Ph.D. I entered this program because of his encouragement and unwavering belief in my success. In general, I would like to thank all those who have directly or indirectly made this experience a valuable one. I would also like to acknowledge support services provided by Jacqueline Guyton, Barb Hoefler, and others not mentioned here. vii TABLE OF CONTENTS Page LIST OF TABI‘ESOOOOOOOOOOOOOOOO0......OOOOOOOOOOOOOOOOOOOI Xi LIST OF FIGUMSOO00....OOOOOOOOOOOOOOCOOOOO0.0.0.0000... Xiii LIST OF ABBREVIATIONSOO0......OOOIOOOOOOOOOOOOOOOO00....O Xiv CHAPTER INTRODUCTION AND LITERATURE REVIEW........... Introduction............................. 1 Some Definitions Related to the Study of Resistance Mechanisms............... 2 Genetics and Mechanisms of Naturally- Occurring Resistance to Plant Viruses.. 5 .1 Resistance Mediated at the Level of Recognition and/or Signal Transduction.................... 7 1.3.2 Resistance Targeted at Inhibition of Viral Replication................... 7 1.3.3 Resistance at the Level of Virus Movement......................... 8 1.3.3.1 Resistance at the Level of Long Distance Movement...................... 9 1.3.3.2 Resistance at the Level of Cell-to- Cell Movement.......................... 11 1.3.4 Viral Determinants That Affect Symptom Expression..................... 13 I-‘ I-' t—II—IH O O O O O. (A to NH 1.4 Possible Use of Non-Host Products to Confer Virus Resistance/Tolerance...... 13 1.5 Future Directions in the Study of Resistance Mechanisms.................. 15 1.6 Multiple Disease Resistance and the Concept of a Linkat.................... 16 1.7 Sources of Resistance in Cucumber to ZYMV, WMV, and PRSV and the Occurrence of Multiple Potyvirus Resistance in TMG-l.................... 19 1.8 Purpose of this Thesis................... 19 REFERENCES............................... 21 viii . CHAPTER 2: CHAPTER 3 : CHAPTER 4 : INHERITANCE OF MULTIPLE POTYVIRUS RESISTANCE IN THE CHINESE CUCUMBER LINE TMG-l ABSTRACT..................................... INTRODUCTION................................. MATERIALS AND METHODS........................ RESULTS...................................... DISCUSSION................................... REFERENCESCOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO .LINKAGE MAPPING OF MULTIPLE POTYVIRUS RESISTANCE IN THE TMG-l CUCUMBER LINE ABSTRACT..................................... INTRODUCTION................................. MATERIALS AND METHODS........................ RESULTS AND DISCUSSION....................... REFERENCES.0......0.0.0.0000...OOOOOOOOOOOOOO TISSUE-SPECIFIC EXPRESSION OF DIFFERENT RESISTANCES TO WATERMELON MOSAIC VIRUS IN CUCUMBER ABSTRACT...0......OOOOOOOOOIOOOOOOOOOOOO0.0... INTRODUCTIONOOCOCOOOOOOOO.COCOOCOOOOOOCOCOOCC MATERIALS AND MTHODS. O O C C O O O C O O O O C O C O O O O O O O . RESIJI’TSCCCO0......OCOOIOOCCOCOCOOOOCC..00.... DISCUSSION.OOOOOOOOOOOOOOOOOOOO0......OOOOOOOC REFERENCES.0......0......OOOOOOCOOOOOOOOOOOOC. ix 28 29 31 37 48 51 54 55 56 62 74 75 76 77 77 84 87 CHAPTER 5: SPATIAL AND TEMPORAL DISTRIBUTION OF ' POTYVIRUS INFECTION IN THE RESISTANT CUCUMBER GENOTYPE TMG-l ABSTRACTOOOOOOOOOOOOOOOOO0.0...0.00.00.00.00. 88 INTRODUCTION. 0 I O O O O O O O O O O O O O O O O O O O I O O O O O O O O O O 89 MATERIAI’S AND MTHODS. O O O O I O O O O O O O O O O O O O O O O O O 90 RESIII’TSOOOOOIOOOOOOOOCOO0.00...0.00.00.00.00. 90 DISCUSSION. 0 O O O O O O O O O O O I O O O O O O O O O O O O O O O O O O O O O 95 REFERENCESOOOOOOOOOOOOOOOCOO...0.00.00.00.00. 96 SUMMARY, CONCLUSIONS, AND FUTURE DIRECTIONS.............. 97 APPENDIX A: A QUICK AND EASY METHOD TO ISOLATE LARGE QUANTITIES OF HIGH QUALITY POTYVIRMJ RNACOCCOOOOOOCOOCCCOCOOOO0.0...I 99 REEENCESCOOCCOOOOOOCOOOOOOOO0......0....-.106 APPENDIX B: TMG-l IS RESISTANT TO APHID TRANSMISSION OF ZYMVANDPRSV-Wooooooooooooooooooooooo.107 APPENDIX C: CUCUMBER SCAB DISEASE SCREEN PROTOCOL.......110 APPENDIX D: CUCUMBER FUSARIUM WILT DISEASE SCREEN PROMOLOOOOOOCOOOOOOOOOOOOOOOOOOOOOOOOOO.114 Table Table Table Table Table Table Table Table Table Table Table Table Table 2.7 3.4 4.1 4.2 LIST OF TABLES Page Segregation of Resistance to PRSV-W........... 38 Segregation of Resistances to WMV............. 39 Segregation of Resistance to ZYMV............. 41 Predicted Genotypes and Phenotypes for Resistance to WMV....................... 43 Relationship Between Resistances tOWMvand ZYMVOOOOOOOOOOOOOOOOOOOOOO0...... 44 Sequential Virus Inoculation Data: Inoculation of ZYMV-Resistant WithWMvO00.00.0000...-OOOOOOOOOOOOO0.0...O 46 Comparison of Symptom and ELISA Data for Young, Fully Expanded Leaves of TMG-l, WI-2757, and Their F1 ProgenYooooooooooooooo 47 Single Trait Goodness of Fit Tests............ 63 F2 Phenotypic Classes and Their Frequencies in the Cross WI-2757 x TMG-IOIOOOOOOOOCOOCOC 64 Test for Linkage Association Between Resistance to PRSV and Bitterfree Cotyledon in the Backcross WI—2757 x F1................................ 71 Tests for Linkage Associations Between Resistance to PRSV and Genes on Linkage Group I in an F2 Population................. 72 Response of Progeny of TMG—l and WI-2757 to Inoculation with WMV at the COtYJ-edon StageOOOOOOOOOOOOO0.0......0...... 79 Intermediate Symptom Expression by a Subset of the Backcross (F1 x TMG-l) Individuals Susceptible to WMV After Cotyledon Inoculation................................. 80 xi . Table 4.3 Table 4.4 Table B.1 Table 3.2 Segregation of Resistances to WMV Following Inoculation of Either the (A) Cotyledons or (B) True Leaves.......................... 81 Test for Linkage of the Cotyledon-Expressed WMV Resistance to the F'locus in the F1XTMG"1BaCRCIOSSoooooooooooooooooooooooo 83 Resistance of TMG-l to aphid- transmitted ZYMV00000000000000000000000000000108 Resistance of TMG-l to aphid- transmitted PRSV-W0000000000 OOOOOOOOOOO 000000109 xii Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure 3.1A 3.1B 3.1C 3.1D 3.1E U" UIUIU'I o co U" 9““) LIST OF FIGURES Page Visualization of Cucurbitacins by TLC....... 58 Starch Gel Electrophoresis of ISOZY'meS (MI-1orPGTT-l)ooooooooooooooooo 59 Illustration of the Morphological Markers Numerous Spine and Tuberculate.... 59 The Effect of Cladosporium cucumerinum (scab) on the Susceptible TMG-l........... 61 The Effect of Fusarium oxysporum f. sp. cucumerinum in an F2 Population........... 61 Cucumber Linkage Map For Groups I and IV ... 67 Comparison of Symptoms Produced in Response to ZYMV, WMV, and PRS-W in zuCChini squa8h000000000000000000000000 91 Comparison of Virus Spread Throughout Various Stages of Infection for ZYMVI WMV, and PRSV-W000000000000000000000 93 Comparison of PRSV-W and ZYMV Accumulation in TMG-l as a Percentage of the Level in the Susceptible WI-2757................ 94 Visualization of the Purity of ZYMV RNA Isolated Under Different Conditions.......104 xiii Viruses ZYMV WMV PRSV-W LIST OF ABBREVIATIONS zucchini yellow mosaic virus watermelon mosaic virus (also known as watermelon mosaic virus-2) papaya ringspot virus - watermelon strain (also known as watermelon mosaic virus-1) Cucumber Cultivars TMG- 1 WI-2757 Taichung Mau Gua - resistant to ZYMV, WMV, and PRSV Wisconsin 2757 - susceptible to ZYMV, WMV , and PRSV Genetic Loci and Phenotypes bi F Tu ns Foc Ccu Mpi-l Pgm-l zymv wmv—Z wmv—3 wmv-4 Prsv-Z bitterfree cotyledon (WI—2757) sex expression (WI-2757 - Female flowers) tuberculate or warty fruit/flowers (TMG-l) numerous spine on fruit/flowers (WI-2757) fusarium resistance (WI-2757) scab resistance (WI-2757) mannosephosphate isomerase (codominant) phosphoglucomutase (codominant) resistance to ZYMV resistance to WMV identified in this study, acts independently and is expressed in the cotyledon and throughout the plant (mo-1) resistance to WMV identified in this study, acts epistatically with a dominant gene from WI-2757 to confer resistance in true leaves resistance to WMV identified in this study (WI-2757) that acts in concert with a recessive resistance gene in TMG-l resistance to PRSV-W identified in this study xiv Others ELISA Enzyme-linked immunosorbent assay EM Electron microscopy TLC Thin layer chromatography XV CHAPTER I INTRODUCTION AND LITERATURE REVIEW 1.1 Introduction In order to further the understanding of host-pathogen interactions and to elucidate mechanisms of plant virus resistance by the host plant, it is useful to study resistance phenotypes conditioned by monogenic or oligogenic resistances. To this end, this study addresses questions concerning naturally-occurring resistances to potyviruses that infect cucurbit crops. The inbred, Chinese cucumber line Taichung Mau Gua (TMG-l) is resistant to three, related cucurbit potyviruses: zucchini yellow mosaic virus (ZYMV); watermelon mosaic virus (WMV), formerly known as watermelon mosaic virus 2; and the watermelon strain of papaya ringspot virus (PRSV-W), formerly known as watermelon mosaic virus 1 (Provvidenti, 1985). Resistance to ZYMV was found to be due to a single, recessive gene (Provvidenti, 1987a). The inheritance of resistance to WMV and to PRSV had not yet been characterized. The goals of this research were to study the genetics of inheritance of resistance to these three related viruses, to determine the relationship of the resistances to each other, to examine the genome organization of multiple disease resistances, and to gain some insight into mechanisms of resistance to potyviruses. 2 1.2 Some Definitions Related to the Study of Resistance Mechanisms In order to discuss phenomena related to plant virus resistance mechanisms, it is first necessary to define terminology used in this study. Vanderplank (1963) utilized the terms "vertical" and "horizontal" resistance to describe specificity involved in the resistance reaction. Vertical resistance is generally conferred by one to a few genes and exhibits a race-specific response that controls the disease phenotype in a significant manner. In contrast, many genes are believed to condition horizontal, or non- specific resistance. These terms are discussed in more detail below. Cooper and Jones (1983) proposed terms as discussed by the Virology Group Committee of the Federation of British Plant Pathologists (FBPP). A species that cannot be infected by any biotype of the pathogen is considered immune (i.e. a nonhost). In the past, many investigators (plant breeders, in particular) called a highly resistant cultivar immune. The term "infectible" has been proposed as the opposite of immune. Infectible plants may be "susceptible" or "resistant." A resistant plant is one in which virus infection and/or replication and/or invasion is restricted. This term applies to the cultivar type resistance studied in this thesis. A susceptible plant is one in which virus readily infects and/or replicates and/or invades. A susceptible plant can then be subdivided into ”sensitive" or "tolerant" plants, depending on the range of the response exhibited by the host plant. A sensitive plant reacts severely to the pathogen, while a tolerant plant shows few or no symptoms. From the point of view of a plant breeder, a tolerant plant is considered resistant because the pathogen has little effect on quality and yield of the product. However, a plant virologist views tolerance as a form of susceptibility since the host plant suffers from a full-scale viral infection. Tolerance is a difficult term to define since the ability of the plant to suppress disease development may be due to a low level of resistance, i.e. a slight reduction in multiplication and long distance spread. However, since these terms are not universally used, definitions proposed by Walkey (1985) will be used in this study. The terms defined here apply to genetically controlled vertical, cultivar resistance. Other types of resistance, such as induced (or acquired) resistance and cross-protection are not included in this discussion. A plant is considered to be susceptible if virus can easily infect and multiply within it. Susceptibility may be high or low. A resistant plant is one that suppresses or retards . multiplication of a virus or development of pathogenic symptoms. Resistance may be classified as high (extreme), moderate, or low. Tolerance describes the response by the host plant, in which almost normal levels of virus movement and concentration cause mild or virtually no symptoms at 4 i all. These terms should be clarified in the future as more research on mechanisms of virus spread and symptom expression are elucidated. Dawson and Hilf (1992) view virus-host interactions by the extent of virus multiplication and spread and the degree of symptom expression in the host, whether natural or experimental. They observed seven types of virus-host interactions, ranging from "total susceptibility" to "immunity." Plants that suffer "total susceptibility" exhibit marked reductions in growth and yield, and those that do not show obvious symptoms are considered "tolerant," as proposed by Cooper and Jones (1983). They noted varying degrees of susceptibility, in which there is decreased virus replication to reduced or limited virus spread in the host plant. Under laboratory conditions, it is sometimes possible to detect viral replication in protoplasts of plants that are believed to have species resistance (Cheo, 1970; Sulzinski and Zaitlin, 1982; Paje-Manalo and Lommel, 1989; Stenger et al., 1992). In this type of host-pathogen interaction, the resistant plant is called a "subliminal host," i.e. one that is able to support virus replication at the cellular level, but does not permit virus spread or symptom expression (Cheo, 1970). Cooper and Jones (1983) reserve the term true "immunity" for absolute resistance at the species level, i.e. plants that do not support virus replication even in protoplasts. Holmes (1955) proposed that non-host immunity 5 A is conferred by the additive effect of 20 to 40 individual resistances, a concept later called horizontal resistance by Vanderplank (1963). Bald and Tinsley (1967) suggested that non-host resistance is due to the lack of "susceptibility factors" needed by the virus to undergo pathogenesis. It is now generally believed that susceptibility is due to host plant factors that aid in virus multiplication and spread (Fraser, 1990a). The types of resistance discussed here are vertical (Vanderplank, 1963), i.e. resistance phenotypes that result from the action of major gene effects. 1.3 Genetics and Mechanisms of Naturally-Occurring Resistance to Plant Viruses The topics of host-range determinants of plant viruses and possible correlations between the genetics of resistance and their underlying mechanisms have been reviewed by several investigators (Dawson and Hilf, 1992; Ponz and Bruening, 1986; Fraser, 1987, 1990a, 1990b). According to Fraser (1987; 1990), the major processes involved in virus- host interactions include recognition between virus- and host-encoded factors, signal transduction, and the response. Recognition is probably the main factor involved in nonhost immunity and in cultivar resistance. A defect or lack of a recognition molecule (i.e., reduction or absence of susceptibility) necessary for plant virus multiplication could result in resistance. This type of negative mechanism is most likely to be recessive for a go/no go recognition 6 - event and may be considered a passive resistance. In contrast, a positive resistance mechanism proposes that the product of a host resistance gene interacts directly with a virus-encoded function as either a direct inhibitor, or elicits a signal transduction pathway. This type of resistance mechanism is likely to be dominant if recognition is a go/no go event. Both types of mechanisms can sometimes exhibit gene-dosage dependence. Fraser (1990b) also attempted to correlate dominance and recessiveness of resistance genes with the resistance phenotype expressed. Dominant resistance genes are frequently associated with resistance mechanisms that limit the spread of virus, such as the formation of (necrotic) local lesions. Incompletely dominant or gene-dosage dependent resistances often confer moderate resistance, resulting in partial localization of the virus (such as localization to the inoculated leaf), or a decrease in the overall multiplication of the virus. Some recessive- resistance genes appear to confer extreme resistance, such that there are no detectable levels of virus. In summary, plant virus resistance mechanisms exhibit a continuum of processes: (1) mechanisms that involve localization of virus infection tend to be inducible and conferred by dominant genes; (2) those that confer a very high level of resistance tend to be recessive and constitutively expressed; and (3) those that are expressed systemically and affect virus multiplication or long-distance spread tend to .7 I be incompletely dominant (i.e., exhibit a gene-dosage effect). These generalizations probably have many exceptions and are meant to provide a general theoretical framework in the study of the genetics of plant virus resistance mechanisms. 1.3.1 Resistance Mediated at the Level of Recognition and/or Signal Transduction The mode of action of a host plant resistance gene product is known only for a few systems to date. The product of the tobacco N gene elicits a range of reactions that result in a localization type of resistance known as a hypersensitive response. This extremely complex set of reactions reviewed by various authors are not covered here (Dawson and Hilf, 1992; Dumas et al., 1991). 1.3.2 Resistance Targeted at Inhibition of Viral Replication “ A host-encoded gene product that directly inhibits plant viral replication is found in the Arlington cowpea (Bruening et al., 1987). A single, dominant gene in Vigna unguiculata cv. Arlington codes for a protease inhibitor that specifically inhibits processing of polyproteins of cowpea mosaic virus (CPMV). In an extremely thorough study of the mechanism of resistance, the investigators found cosegregation of the protease inhibitor and resistance to 8 ' CPMV-SB. Enzyme inhibition studies were performed in protoplasts of F2 populations. Similarly, the Tm-l gene on tomato was found to confer resistance to TMV in protoplasts (Motoyoshi and Oshima, 1975, 1977). The Tm-l gene inhibits viral replication, but allows TMV to move through the plant (Fraser and Loughlin, 1980). Recent evidence suggests that it may act at the level of replicase inhibition (Watanabe et al., 1988). These two resistances are examples of the positive, active resistance model that acts in a dominant manner, as proposed by Fraser (1990). Lobenstein and Gera (1990) have identified a 23 kDa protein (called IVR or inhibitor of viral replication) from protoplasts made from tobacco plants that contain the N gene. This protein inhibits replication of several plant viruses and is somehow believed to take part in the localization reaction. Whether this protein is the N gene product, or if is related to the N-gene mediated hypersensitive reaction remains to be seen. 1.3.3 Resistance at the Level of Virus Movement Since invasion of a host plant by viruses requires both cell-to-cell and long distance transport through the vascular system, recent interest has focused on these processes. While there is a great deal of information on the structure of plasmodesmata (review by Robards and Lucas, 1990), this discussion will concentrate on movement of virus 9 A in infected plants (reviews by Hull, 1989; Maule, 1991; Godefroy-Colburn et al., 1991; Been et al., 1992). After entry of a plant virus into a cell that supports replication, the virus must move cell to cell through membrane-lined plasmodesmata, which are interconnections that provide continuous symplastic connections throughout the tissue until it reaches the vascular system. Viruses have been observed to move long distance through the phloem as virions (Esau and Cronshaw, 1967). Once the virus is in the phloem, it is believed to be carried along the vascular stream with photosynthates to a sink (Agrios, 1988; Leisner et al. 1992). A recent report (Leisner et al., 1992) correlated the movement of cauliflower mosaic virus in infected turnip plants with the flow of photoassimilates both long distance and within individual leaves. The concentrations of both virus and photoassimilates paralleled source (older leaves) - sink (younger leaves) relationships. They also concomitantly decreased basipetally, from the leaf tip to the base. 1.3.3.1 Resistance at the Level of Long Distance Movement The form in which plant viruses achieve long distance movement is not known. Saito et al. (1990) showed that mutant TMV defective in coat protein assembly is greatly compromised in its ability to spread long distance. Dorokhov et al. (1984) suggested that plant viruses move long distance as informosome-like ribonucleoprotein 10 ' complexes. Cytological evidence presented by deZoeten and Gaard (1983) showed that pea enation mosaic virus replication complexes may be transported in vesicles. The authors suggested that this type of transport may be common to most or all plant viruses that undergo a dsRNA intermediate and utilize a membrane as a site for replication. Cytological evidence suggests that resistance may occur at the level of long distance spread in at least two studies. The tobacco masked strain of tobacco mosaic virus first described by Holmes (1934) is either symptomless or produces mild chlorosis. There is a delay in the accumulation of virus in the upper leaves (Ding et al., 1993). This strain was shown to produce less movement protein than a more virulent strain (Watanabe et al., 1987). While the attenuated phenotype had been correlated with reduced replication (Holt et al., 1990), recent cytological evidence suggests that the masked strain has difficulty in accumulating in the vascular cells of the inoculated leaf (Ding et al., 1993). Resistance to cucumber mosaic virus in resistant pepper lines also appears to be related to a reduction in the efficiency with which virus can invade phloem tissue (Dufour et al., 1989). In resistant varieties, virus spread was limited to cortical cells and a few phloem bundles. It is possible that symptom expression is attenuated if not enough ll virus is present in young leaves before expansion takes place. A resistance identified in Arabidopsis thaliana ecotype Dijon to infection by turnip crinkle virus also seems to affect long distance virus spread (Simon et al., 1992). There appeared to be little difference in viral RNA accumulation in protoplasts of both the susceptible and resistant ecotypes. In addition, when inoculated, and corresponding opposite leaves of the resistant Dijon ecotype showed accumulation of viral RNA, but further virus spread was not observed.' Resistance in the Dijon ecotype appears to be mediated through long distance spread of the virus. 1.3.3.2 Resistance at the Level of Cell-to-Cell Movement The 30 kDa protein encoded by TMV is believed to be the movement protein (Deom et al., 1987). When this protein was expressed in transgenic plants, molecular exclusion size of the plasmodesmata was increased from 800 to 9,400 daltons (Wolf et al., 1989; Wolf et al., 1991). The role of movement protein in the transport of TMV particles through plasmodesmata has been studied in detail (Citovsky, 1993; Citovsky and Zambryski, 1993). The TMV movement protein has several domains: two that bind single-stranded nucleic acids, a folding domain, a phosphorylation domain, and another domain that allows it to bind plasmodesmata. Passage of movement protein-nucleic acid complexes through plasmodesmata has been observed under EM (Citovsky et al., 12 I 1992). These complexes may be analogous to the informosome- like virus-specific ribonucleoprotein (vRNP) particles proposed by Dorokhov et al. (1984) to be involved in both long distance and cell-to-cell movement of plant viruses. Movement protein is believed to be inactivated by a plant cell wall-associated protein kinase (Citovsky and Zambryski, 1993). Citovsky et al. (1993) have discovered a developmentally regulated plant cell wall-associated protein kinase that phosphorylates TMV movement protein. The protein kinase activity is found primarily in leaves and is expressed as a function of leaf maturation, i.e. enzyme activity closely follows basipetal leaf development. These discoveries suggest possible mechanisms of reSistance to plant virus infection at the level of cell-to-cell spread. Mutations in the movement protein have been associated with virus accumulation and symptom severity. Since the Tm- 2 gene and its allelic variant Tm-Zz do not inhibit virus replication in protoplasts, they are believed to cause resistance at the level of virus spread (Motoyoshi and Oshima, 1975, 1977). The Tm-Z allele has been shown to prevent movement of TMV from infected cells (Nishiguchi and Motoyoshi, 1987). A mutation that allows TMV to overcome the Tm-z resistance was found in the 30 kDa cell-to-cell movement protein (Meshi et al., 1988). The allelic Tmzz gene is also believed to confer resistance at the level of cell-to-cell spread (Niguchi and Motoyoshi, 1987). Tsai and Dreher (1993) reported that a single amino acid substitution 13 A in the turnip yellow mosaic virus movement protein resulted in a fourfold increase in accumulation of viral proteins and a concommitant increase in the severity of symptoms. In summary, it appears that the rates of virus movement, both cell-to-cell and long distance, affect virus accumulation and titer, which in turn can greatly affect symptom expression. 1.3.4 Viral Determinants That Affect Symptom Expression Genetic engineering of plant virus genomes has been used to study the effects of various changes on symptom expression. Point mutations in Gene VI (unknown function) of cauliflower mosaic virus have revealed virus determinants that affect symptom expression in the host plant (Daubert and Routh, 1990). The role of the coat protein in viral pathogenesis has been examined through site-directed modifications of the TMV coat protein (Saito et al., 1987; Culver et al., 1991; Lindbeck et ali, 1992). Similarly, further dissection of viral determinants that affect symptom expression may lead to an understanding of mechanisms by which a host plant expresses resistance to viral infections. 1.4 Possible Use of Non-Host Products to Confer Virus Resistance/Tolerance Recently, non-host encoded gene products have been found to have inhibitory effects on some plant viruses. It has been reported that high levels of human cystatin C, an 14 I inhibitor of cysteine proteases, interfere with autoprocessing of the plum pox potyvirus HCPro and NIa proteases (Garcia et al., 1993). In addition, one of the pokeweed antiviral proteins (PAP) that imparts resistance to tobacco plants from infection by tobacco mosaic virus (TMV) was identified as a ribosome inactivating factor (RIF) using in vitro assays (Chen et al., 1993). The mechanism involves removal of a specific adenine base in a conserved loop of the large rRNA. This depurination process prevents ribosomes from binding elongation factors and blocks translation as a result. The protein apparently does not block translation in the host pokeweed plant. The in vivo function of this protein and its role in the inhibition of viral replication are not yet understood well. However, such non-host specific antiviral proteins may be useful in the future as genetically engineered resistances. Little is known about what actually causes symptom expression and modulation in plants; Efforts to utilize genetically engineered plant virus resistance have shed some light on the processes involved in pathogenesis of plant viral infection. Current strategies to confer pathogen- derived plant virus resistance have been reviewed by a number of investigators (Beachy, 1988; Baulcombe, 1989; Grumet, 1990; Scholthof et al., 1993). These strategies include cloning and expression of (1) portions of the viral genome, such as the coat protein or replicase in both the sense and antisense forms; (2) satellite RNAs that have been 15 ' observed to decrease symptom expression in nature; and (3) other novel approaches such as the use of a capsid-nuclease fusion protein (Natsoulis and Boeke, 1991). Possible mechanisms of coat-protein mediated cross protection have been reviewed (Nelson et al., 1990; Nejidat et al., 1990; Grumet, 1994). Proposed mechanisms of coat-protein mediated cross protection range from preventing uncoating of the incoming virus, competition for replication factors, to interference with cell-to-cell or long distance spread. It is likely that different mechanisms act to confer resistance/tolerance in different virus groups, although similar genetic engineering approaches have been attempted. Carr and Zaitlin (1991) demonstrated that expression of a non-structural sequence (54 kDa protein of TMV) results in markedly decreased levels of virus-specific RNAs in tobacco protoplasts. It is possible that some of the naturally- occurring resistances utilize mechanisms similar to those currently employed by genetic engineering. 1.5 Future Directions in the Study of Resistance Mechanisms There are several possible resistance mechanisms not yet explored at this time. The role of the cytoskeleton in plant virus replication and movement is not known. The fluidity of the membrane systems may influence the ability of the replication complex to function properly. Inability of a virus replication complex to anchor in the host cell membrane may be involved in some resistances that are 16 lexpressed only at high temperatures. Certain integral components of membranes that may be involved in recognition of the virus complex may also affect virus replication and movement. Some mechanisms of tolerance may be due to the ability of the host plant to divert nutrient utilization into alternate pathways. The ability to chelate, transport, or utilize micronutrients that are essential cofactors such as iron or sulfur may be altered to allow the plant to function normally. Much work lies ahead to understand plant virus-host interactions and mechanisms that allow the host plant to resist or tolerate the invading pathogen. 1.6 Multiple Disease Resistance and the Concept of a Linkat Several examples of multiple potyvirus resistance exist in the literature. The most well characterized is the I gene in Phaseolus vulgaris cv. Black Turtle Soup 1. The dominant I allele confers resistance to five potyviruses: been common mosaic virus, blackeye cowpea mosaic virus, cowpea aphid-borne mosaic virus, soybean mosaic virus, and watermelon mosaic virus (Kyle and Dickson, 1988). Another example is in Pisum sativum, in which multiple potyvirus resistance is conferred by two tightly linked loci. Five genes that occur as a cluster on chromosome 2 confer resistance to six potyviruses. The temperature-independent genes (bcm, cyv-l, pmv, and sbm-Z) confer resistance to the NL-S pathotype of been common mosaic virus (BCMV NL-8), st: no: Gr: 195 the Ca; lin Con tha tha- Thi: Con: hist thrc the} loca Stru 17 . clover yellow vein virus (CYVV), pea mosaic virus (PMV), and the lentil strain of pea seed-borne mosaic virus (PSbMV-L1), respectively (Provvidenti, 1987b; Provvidenti, 1990; Provvidenti and Alconero, 1988a). A fifth temperature sensitive gene, mo, confers resistance to bean yellow mosaic virus (BYMV) and WMV (Marx and Provvidenti, 1979). A second cluster of five tightly linked genes on chromosome 6 (sbm-l, sbm-3, sbm-4, wlv, and cyv-Z) confer resistance to three strains of PSbMV (standard, lentil, and P4), white lupine mosaic virus (WLMV) and CYVV, respectively (Hagedorn and Gritton, 1973; Provvidenti, 1987b; Provvidenti and Alconero, 1988b). In addition, genes that confer resistance to more than one virus have also been observed in Solanum and Capsicum (Cook, 1960; Cockerham, 1970). A possible explanation for the existence of tightly linked loci that confer resistance to several viruses is the concept of a linkat, a cluster of duplicated linked genes that appear to be semi-stable functional units. Demarly (1979) proposed that gene duplications may have occurred that allow for multiple resistance genes at the same locus. This hypothesis is based on trends that he noted in conserved genes, such as those that encode hemoglobins, histones, and ribosomal RNAs. Gene duplications that arise through evolution are maintained in linked clusters because they have coadapted functions. "Antimutator" genes that locally control linkats are proposed to maintain the structure and function of these units. Decreased in 91 CO 18 irecombination rates are associated with genes within a linkat. Such an occurrence may explain the clustered multiple virus resistance genes on chromosomes two and six in Pisum sativum (Kyle and Provvidenti, 1993). Examination of the molecular genetic structure of disease resistance loci is in progress in tomatoes (Martin et al., 1992; 1993a,b). Yeast artificial chromosome libraries have been made in order to clone the Tm-Za and Pto loci that confer resistance to TMV and Pseudomonas syringae pv. tomato. Map-based positional cloning was used to isolate the Pto locus. A tightly linked phenotype with Pto is resistant to damage by fenthion, an organophosphate insecticide that causes necrotic lesions on plants. Recent evidence has shown that this locus is composed of at least six to seven duplicated regions (Martin, 1993b). Resistance to fenthion damage segregated from resistance to P.s. pv. tomato after screening more than 1300 F2 individuals. It appears that the duplicated genes have diverged. Molecular characterization of this locus in currently under study. The results of this work will aid in our understanding of the genetic organization and biochemical reactions involved in plant disease resistance responses. While this study elucidates the mechanism of a bacterial resistance gene, the concept of a linkat seems to apply. 19 '1.7 Sources of Resistance in Cucumber to ZYMV, WMV, and PRSV and the Occurrence of Multiple Potyvirus Resistance in THC-1 Sources of potyvirus resistance in cucurbits have been reviewed by Provvidenti and Hampton (1992) and Provvidenti (1993; 1989). There are a limited number of resistances to potyviruses in cucumbers. In the Japanese cultivar Kyoto 3 feet long, a single dominant gene confers resistance to WMV (Cohen et al., 1971). The cultivar Surinam Local carries a single recessive resistance gene to PRSV-W (Wang et al., 1984). This resistance appears to limit long distance spread of the virus, as high titers of virus were detected by ELISA in the lower leaves but not in the upper ones. >While Provvidenti (1985) determined that a number of Chinese cultivars were resistant to ZYMV, a single plant selection was made from the cultivar TMG-l that was resistant to three cucurbit potyviruses: ZYMV, WMV, and PRSV. Resistance to ZYMV is due to a single recessive gene in TMG-l (Provvidenti, 1987). However, the genetic basis of the multiple potyvirus resistance was not characterized. 1.8 Purpose of this Thesis In this study, inheritance of multiple potyvirus resistance in the inbred, Chinese cucumber line TMG-l was performed in order to determine (1) the genetics of resistance to each virus; (2) the mode of inheritance, whether the resistance is dominant or recessive; and (3) the 20 relationship of the resistances to each other, i.e. does one gene confer resistance to all three viruses? The mechanisms of resistance were studied by examining the spatial and temporal distributions of each virus in the resistant and susceptible lines. The relationship of the resistances to each other was further examined by attempting to establish linkage markers to each resistance. A physiological study was also undertaken to determine the underlying cause of different segregation ratios obtained when different tissues were inoculated with WMV. A tissue-specific mechanism was found and is further described. BpOY BREA‘CC CIIVAH (house‘- ( (Ir‘ul (ECIIVI 21 ‘ REFERENCES Agrios, G.N. 1988. Plant diseases caused by viruses. Pages 622-722 in: Plant Pathology. 3rd ed. Academic Press, New York, N.Y;. Bald, J.G., Tinsley, T.W. 1967. A quasi-genetic model for plant virus host ranges. II. Differentiation between host ranges. Virology 32:321-327. Baulcombe, D. 1989. Strategies for virus resistance in plants. Trends in Genetics 5:56-60. Beachy, R.N. 1988. Virus cross-protection in transgenic plants. Pages 313-328 in: Temporal and spatial regulation of plant genes. D.P.S. Verma and R.B. Goldberg, eds. New York: Springer-Verlag, New York, N.Y. Bruening, G., Ponz, F., Glascock, C., Russell, M.L., Rowhani, A., Chay, C. Resistance of cowpeas to cowpea mosaic virus and to tobacco ringspot virus. 1987. Pages 23-37 in: Plant resistance to viruses, Ciba Foundation Symposium 133. John Wiley & Sons, New York, N.Y. Carr, J.P., Zaitlin, M. 1991. 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CHAPTER 2 INHERITANCE OP MULTIPLE POTYVIRUS RESISTANCE IN THE CHINESE CUCUMBER LINE THO-1 ABSTRACT The inbred Chinese cucumber line TMG-l is resistant to three potyviruses: zucchini yellow mosaic virus (ZYMV), watermelon mosaic virus (WMV), and the watermelon strain of papaya ringspot virus (PRSV-W). The genetics of resistance to these viruses and the relationship of the resistances to each other were determined. TMG-l, WI-2757 (a susceptible inbred line), and their F1, F2, and backcross progeny were screened for resistance to all three viruses by monitoring symptom expression and virus level using enzyme-linked immunosorbent assay (ELISA). Segregation data indicated that resistance to PRSV-W was due to a single dominant gene. Two independently asserting recessive resistance factors conferred resistance to WMV: the first resistance was due to a single recessive gene and the second resistance resulted from an epistatic interaction between a recessive gene from TMG-l and a dominant gene from WI-2757. The gene conferring resistance to ZYMV was the same as, or was very tightly linked to, the recessive gene in the epistatic resistance to WMV. ELISA data suggested that the mechanism of resistance to PRSV-W was different from that of ZYMV and WMV. 28 V: 15 ge Se do: t8] 1110: box Vir Dic 29 INTRODUCTION Potyviruses cause great economic loss to a wide variety of crops. At least three potyviruses, zucchini yellow mosaic virus (ZYMV) (Lisa and Lecoq, 1984), watermelon mosaic virus (WMV) (Purcifull et al., 1984), and the watermelon strain of papaya ringspot virus (PRSV-W) (Purcifull and Gonsalves, 1984a), cause severe losses in cucurbit crops (Nameth et al., 1985; Nameth et al., 1986; Davis, 1987; Sammons et al., 1989; Perring et al., 1992). Provvidenti (1985) identified resistance to all three viruses in a single plant introduction from the Chinese cucumber cultivar Taichung Mau Gua (TMG-l). Cultivars that are resistant to all three cucurbit potyviruses would be very valuable, especially because of the frequent occurrence of mixed infections (Nameth et al., 1985; Davis and Mizuki, 1987). H Multiple potyvirus resistance could be conditioned by a gene at a single locus, by a cluster of linked genes or by several independent genes. In Phaseolus vulgaris, the dominant I allele confers a systemic necrotic resistance at temperatures above 30 C to five potyviruses: bean common mosaic virus, blackeye cowpea mosaic virus, cowpea aphid- borne mosaic virus, soybean mosaic virus, watermelon mosaic virus, and possibly passionfruit woodiness virus (Kyle and Dickson, 1988). To date, it has not been possible to break 30 I the linkage among the resistances; Pisum sativum has two tightly clustered arrays of multiple potyvirus resistance genes on different chromosomes, as well as the mo locus that confers resistance to both been yellow mosaic virus and WMV (Marx and Provvidenti, 1979; Provvidenti, 1987b ; Provvidenti, 1990; Provvidenti and Alconero, 1988a, 1988b). Other examples of an allele conferring resistance to more than one virus exist in Capsicum annuum (Cook, 1960) and in Solanum (Cockerham, 1970). In cucumber, several monogenic resistances have been characterized. Resistance to PRSV is controlled by a single recessive gene in the cultivar Surinam Local (Wang et al., 1984), resistance to WMV in Kyoto 3 feet long is due to a single dominant gene (Cohen et al., 1971). Resistance to ZYMV is due to a single recessive gene (Provvidenti, 1987a). In this work, we sought to determine the genetics of the multiple potyvirus resistance in TMG-l. TMG-l was crossed with WI-2757, an inbred line that is susceptible to all three viruses. F1, F2, and backcross progeny were examined for response to inoculation by each virus. Our data indicate that multiple potyvirus resistance in TMG-l is conferred by four (possibly five) separate genes. 31 ' MATERIALS m xnrnons Maintenance of Virus Inocula ZYMV (CT strain, originally supplied in 1987 by R. Davis, Rutgers State University, New Brunswick, NJ), WMV (ATCC PV379), and PRSV-W (ATCC PV380) were propagated in zucchini squash plants (cucurbita pepo cv. Blackjack) (Petoseed Co., Inc.; Saticoy, CA). Cotyledons of one-week old seedlings were lightly dusted with 320-grit carborundum (Fisher Scientific) and mechanically inoculated using sponge plugs. Virus-infected tissue (lyophilized, frozen, or fresh) was macerated in ice cold 20 mM sodium phosphate buffer, pH 7.0, in a pre-chilled mortar and pestle. All non-biological materials were sterilized prior to usage. Virus-infected leaves were harvested to use as inocula sources at the time when symptoms were expressed optimally. Several tests were performed routinely to verify the identity and purity of each virus. ”PRSV-W did not cross- react with antibody to ZYMV or WMV, and so was verified to be free of contamination by ELISA (antibody purchased from Agdia) (Clark and Adams, 1977). Since ZYMV-CT and WMV both react with ZYMV antibody, a differential host, Phaseolus vulgaris cv. Black Turtle 2, was used (Provvidenti and Gonsalves, 1984b): WMV elicits strong, systemic, mosaic symptoms, while ZYMV only produces red, necrotic, local lesions on the inoculated leaves. 32 I Cucumber Genotypes The inbred cucumber (Cucumis sativus L.) lines TMG-I, resistant to ZYMV, WMV, and PRSV-W, (Provvidenti, 1985) and WI-2757, susceptible to all three viruses, (Peterson et al., 1982) were provided by Dr. Jack Staub (USDA, University of Wisconsin at Madison). The F1 progeny (WI-2757 X TMG-l) were either self- or sib-pollinated to produce the F2 generation. Backcrosses were made with both parents: WI-2757 X F1 and F1 X TMG-l. In each cross, the source of resistance studied came from the male parent. The inbred line Straight 8 (Stokes Seeds, Inc., Buffalo, NY) was used as an additional control genotype that is susceptible to all three viruses. Propagation of Rooted Cuttings Rooted cuttings of TMG-l, WI-2757, and their F1 and F2 progeny were made by cutting plants with an ethanol- sterilized razor blade two nodes below the growing tip. After removing the leaf at the lowest node, the cutting was dipped in a fungicide (Captan, Zeneca Agricultural Products, Wilmington, DE) and placed in an 1 1/4 X 1 X 1 1/2 inch rooting cube (Smithers-Oasis; Kent, Ohio). Trays were filled with 2 cm of water from the bottom and the cuttings covered with plastic wrap to maintain high humidity for 5 days. The plastic was peeled back slowly on a daily basis until rootlets emerged through the rooting cubes (approximately 2 weeks). Plantlets were transplanted to wet 33 ' Baccto Professional Planting Mix (The Michigan Peat Company, Houston, TX) and allowed to grow for 2 to 3 weeks prior to inoculation with virus. Experimental Designs Plants were mechanically inoculated with virus-infected sap at either the cotyledonary stage (ZYMV and PRSV experiments) and/orthe true leaf (WMV and ZYMV) stage. Rows of susceptible Straight 8 plants were evenly spaced throughout each experiment in order to detect any possible variation in inoculation technique and symptom expression. For the F2 experiments, ten rows (10 plants/row) of F2 individuals were interspersed with five internal control rows. Complete blocks that consisted of inoculated and mock-inoculated TMG-1, WI-2757, and F1 plants were arranged in either a Latin square or repeating pattern. Backcross experiments (20 to 120 individuals) also contained evenly spaced control rows. Secondary Inoculation of Resistant Plants and F2 Cutting Experiments To differentiate between possible genetic models for the number of genes conferring resistance to ZYMV and to WMV, experiments were performed in two ways. (1) Clonally propagated sets of genetically identical F2 individuals were prepared as described above and inoculated with either ZYMV or WMV. Rooted cuttings of TMG-l, WI-2757, and their F1 34 progeny were included as controls. Sequential inoculations were performed on F2 and BC (F1 X TMG-l) progeny individuals were first inoculated with ZYMV. Those with symptoms were discarded; those without were assayed by ELISA to verify that they were free of virus. In some experiments, they were inoculated with ZYMV a second time to ensure that there were no escapes. Half fully expanded leaves of the virus- free individuals then were inoculated with WMV. Additional inoculated and mock-inoculated centrol rows were added to experiments that were infected with a second virus. Data Analyses Data from individual and combined experiments were analyzed by chi square analysis. The number of times each experiment was performed is included in each table; experiments termed as independent were performed at different times in the greenhouse. Genetic models proposed are the simplest ones that explained the collective data sets. Scoring of Symptoms Plants were scored when symptom development was optimal. Symptoms of ZYMV and WMV generally developed within 7 to 10 days after inoculation; symptoms of PRSV-W took 3 to 6 wk. to appear. Susceptibility of an individual plant to virus infection was scored visually and/or by ELISA. Symptoms of cucurbit potyviruses include the 35 A presence of mosaic, severe leaf distortion, or rugosity. The rating system used a point scale from 0 to 5, where: 0 - healthy with no evidence of virus; 1 = light mosaic on at least one leaf; 2 = moderate mosaic on one or more leaves; 3 = strong mosaic on one or more leaves; 4 = strong mosaic on several leaves, has spread to terminal leaves, and often accompanied by severe stunting; and 5 = death of the plant due to virus. Many experiments were scored by two people independently. There was agreement to within one point regarding the symptom ratings given to each plant. When assigning a simple classification of resistant or susceptible, any score of 1 or greater (any symptom expression) was classified as susceptible. Detection of Systemic Virus Spread by ELISA Virus levels were determined via ELISA. One or two leaves at the half to first fully expanded stage were harvested from each plant and stored at either 4 C or -80 C. Assays were either performed using standard sandwich methods as described by Clark and Adams (1977) or using a modified version of the leaf disk procedure of Romaine et al. (1981). The two methods were verified to give comparable results. At least four or more healthy controls were included on each plate. Healthy and mock-inoculated controls of all the genotypes (i.e. TMG-l, WI-2757, their F1 progeny, Straight 8, and Blackjack zucchini squash) gave comparable readings. Buffers were prepared according to Clark et al. (1986). 36 A Anti-PRSV-W and some anti-WMV antibody were purchased from Agdia (Elkhardt, IN). ZYMV and WMV were detected with anti- ZYMV (CT strain) polyclonal rabbit IgG antibody (Hammer and Grumet, unpublished). For the sandwich assays, the anti- ZYMV antibody was conjugated with alkaline phosphatase. Absorbance at 405 nm was monitored using an EIA Reader Model EL-307 (Bio-Tek Instruments, Inc., Laboratory Division, Winooski, VT) after reacting with p-nitrophenyl phosphate (Sigma 104 phosphatase substrate). PRSV-W was detected with horseradish peroxidase-labelled conjugate and the substrate o-phenylenediamine dihydrochloride (OPD) supplied by the I Agdia kit. Absorbance was monitored at 490 nm. When performing the leaf disk assays, leaf tissue was sampled with a 6 mm paper hole puncher. Disks were directly placed into ELISA wells along with 200 ul/well coating buffer. Unused wells were filled with buffer. To affix the antigen to the well, samples were either incubated directly or frozen and thawed prior to incubation. Either method worked equally well. Samples were then reacted with 100 ul/well of 1 ug/ml anti-virus-specific antibody in virus buffer at pH 7.4 (Hammer and Grumet unpublished for ZYMV and WMV or Agdia for PRSV-W). Goat anti-rabbit IgG (Sigma Immuno Chemicals A-8025 alkaline phosphatase conjugate) was used to indirectly detect virus-specific antibody at 405 nm as described above. RES mos TM: the gre inc seq prc ’4 .1 . . su] pa: CO? Op an gr (r re Pr re re ir Inc re ti. 37 ’ assures When inoculated with PRSV-W, the WI-2757 parent showed mosaic and vein banding symptoms, while the resistant parent TMG-I grew vigorously and remained symptom-free throughout the life of the plant. The F1 progeny of WI-2757 X TMG-l grew vigorously and also did not exhibit symptoms when inoculated with PRSV-W (Table 2.1). The F2 progeny segregated in a 3:1 (R:S) ratio, the WI-2757 x F1 backcross progeny segregated in a 1:1 ratio (R:S) ratio, and the F1 X TMG-l backcross progeny were all resistant. These data support a model in which a single dominant gene in the TMG-l parent confers resistance to PRSV-W. Environmental conditions can affect the expression of resistance to PRSV- W. Full expression of resistance to PRSV-W depended on optimal growing conditions for the plants, i.e. high light and warm temperatures. Under cool temperatures and slow- growing conditions, the F1 progeny showed very mild symptoms (ratings of 1 vs. 3-4 for WI-2757), while the TMG-l parent remained asymptomatic. In contrast to PRSV-W, when inoculated with WMV, the F1 progeny developed symptoms as would be expected for a recessive trait (Table 2.2). F2 and backcross segregation ratios, however, suggested that more than one gene was involved in the resistance to WMV. Three possible genetic models are presented in Table 2.2. Segregation ratios of resistance in the F2 population suggest that either of the two independently assorting recessive genes confer 38 .moo.o u emxmwx Nx .255 u memmx .ooo.o n memmx mounucu omnowomua 93 mpg acme—Humane comm .mucofiuomxo ucoocomoocfl .30“ Son“ ooaoom moon 0 .moo.o u maxmwx new .aeo.o u Noxmmx .moo.o u Hexmmx moans“ omuoeooue on» mpflw ucguomxo zoom .mucmefiummxo ucoocoaoocw mouse Eon.»— ooHoom sumo o .Hooos memo pcmcfleoo .oamcflm o co comma woeuou countdown m m: mH.o H u H emH omH 0 He x smsmqu o u H o .ms H-029 x Hm ma Hoo.o H u m mm new n mm o «a Hm mm o smswuHs o mm Huoza a House oeuom mx oouoomxm oaoflumoomsm pcmumflmom oguocow Imucloam mo Hooscz 3|>mmm ou mocmpmwmom Ho coaummmwmom H.N magma I39 .mHo.o a nanomx one .ooo.o u «x .mmoo.o u ux .Hooos ones In on» How no.3!» vofigoum on» even vane—Hugo noon .nvaefiwuomxe undocumoofi. Gena» Bonn 00.309 oven 0 H93 N93 H93 .mmo.c u maxomx.uqa .m~o.o : ~x.«n.H a In on». 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H.o H.o H.o NH c He x smsmqu ma ado man 3%.” En 1&6 as." mm co." 0 H.628 x H.» on on; mmnmn an .36 m3. «eNKv min hmH own a N...— ON 0 ah on o 35le o no H.193. «x m3" Nx mam «x m.“ Hence. once m Hoooe econ N HOUOE coon H m m 0950080 . 3.83% 3 >23 0» neosveaeom no coaueueuuem ~.~ 59E I85 Ho. inc pr: ep: (30] 9P 51. If}! Ta We 9e re Po SE of We Dc re 9e 4O 1 resistance to WMV in the progeny of TMG-l and WI-2757. However, the F1 X TMG-I backcross screens gave ratios of 5:3 (R:S), instead of the 3:1 (R:S) ratio expected for two independent recessive genes. This result implies the presence of an additional genetic factor. The model that best explains all of these data proposes the presence of an epistatic dominant gene from WI-2757. The first resistance consists of a single recessive gene (proposed genotype: wmv-Zwmv-Z), and the second resistance the result of an epistatic interaction between a single recessive and a single dominant gene (proposed genotype: wmv-3wmv-3, WMV-4,__) . Segregation of resistance to ZYMV is summarized in Table 2.3. Consistent with the results of Provvidenti (1987), resistance was conferred by a single recessive gene. We next sought to determine whether the single, recessive gene that confers resistance to ZYMV is also one of the recessive genes that confers resistance to WMV. This possibility was studied using two approaches. In the first set of experiments, the individual members of clonal pairs of vegetatively propagated, genetically identical F2 plants were inoculated with either WMV or ZYMV. Four possible models are presented: (1) the ZYMV resistance gene is the same recessive gene that independently confers resistance to WMV (zymv = wmv-Z); (2) the ZYMV resistance gene is the second recessive gene that is involved in 41 .35 u eoxmmx new $85 u Nxamoa n Nexm~x.Hm.o u "833 636368 .23 one“ pauseuomxm comm .mucoewuomxo ucoocommoce meow scum ooHoom moon 0 make Home x m .ooo.o u mmxmmxocm .36 u make EH8 x a a a «for; u N .36 u m mm~x§~.o u N NGNXJHS u H nomx "mounumu ocuOHomHm one made ucoaflummxo comm .mucoEHHomxo ucoocommocw New ECHH ooaoom upon a .Hoooe ocom o>HmmoooH .oamcem o co comma mOfluoH oopommxm m H u o ea c Hm x smemqu m: em.o H u H mHH on o Huoze x Hm me Go.o m u H can emH n we me c He mm o smemqu o mm H-029 Hmumv oeumm «x o oopoomxm m m oompocoo mUCMHm HO HCQEHHZ >2HN Op mocmumwmom no coepomoumom m.~ mummy 42 . resistance to WMV (zymv = wmv-3); (3) the ZYMV resistance is conferred by the second resistance, in which a single recessive resistance gene from TMG-l acts in concert with a single dominant gene from WI-2757 (zymv = wmv-3 + wmv-4); and (4) four independently assorting genes confer resistance to WMV and ZYMV (three genes that confer resistance to WMV, and a fourth one that confers resistance to ZYMV). The expected phenotypic and genotypic ratios for these models are presented in Table 2.4. Models 1 and 3 predict that there would be no individuals that are resistant to ZYMV, but susceptible to WMV. These models are not likely since this class was observed (Table 2.5). Note that Model 3 also can be ruled out because the ZYMV segregation data are not consistent with that of a recessive and a dominant gene acting epistatically. The F1 X TMG-l backcross generation segregated 1:1 (R:S) for ZYMV (Table 2.3), and not 1:3 (R:S). Model 4, proposing that the ZYMV resistance is' completely independent of the WMV resistances also was not_ supported by the observed segregation ratios from the clonal pairs experiments (Table 2.5). The model that best fits the observed data predicts that zymv is actually wmv-3. However, it is not possible to distinguish between this model and the possibility that zymv is tightly linked with wmv-3. The second test of the relationship between the resistance to ZYMV and WMV was performed by sequential 43 .25 on oHoHumoomsm use as ou ucoumfimon mmoHo anemone 3905.5 .4. m ”H “om HuH ”om HuH ”om mHum "mm muH «mm muH «um m2... mmumm oeuou mum Hence m m H H m 333 m3m3 ~3N3 m m H m m lea mam: News m m H m m 333 IlmB NBNB m m H m m IE I? News m 9. m H m _. m «335 M395 '9» m m H m m lee name I? m m H a m e335 '9» INS m m H R .0. leg Inc I? T532 + muses mike: N155 um mm omauocozm $93950 u EN u Baku u .25? mounucm m H Homo: woeumm oahuocmnm mommuococm >zwu oopoflooum mocmumflmom >23 0090mmxm m d .mcoo c9550 m an ooHHoHEoo me >23 one an on 93:33me Hun moahuococm >z~u oopoHooum .m .Hoooz mcoOIoousa on» ecu mOHumm omxuococm oocmumHmom >23 oouoomxm no HGT—Eon .HH $23 on oocmumflmom How momauococm one momauocow oouoHHooum Him 033. 44 .omcHEHmpmo ma poccmo ax .mmMHo m3\mN map :H mHmsuH>HocH mo mucmmwum may ou mac umuownmn mum meuoE wmmne .cmcHEumuwu uocu U: n .mucwEHummxw ucmncmmmccH mmnnu Eon“ umHoom mama m .Ho.o W m .«* «Hmooa UHumcmm cmmomoum map uuommsm no: on mumc U¢>Hmmno map was» mmHMOHocH msHm> Nx HGMOHuHcmHm «« .>2HN no >22 HmzuHm nu 3VumumHsoo=H mm3 mpcme mm HMOHuchH mHHMOHumcmm .omummwacum >Hw>Humpmmm> no HHmm m mo HmnEmE 20mm ** mm.NH a v: m: H¢.¢ n v: “ Nx HNHV mN Hmm. NH Homv mH HHHV mH mN m m ANNV am Hey o Hwy m Hov o «H m m HNmV mN Hmmv NH Hmm V NH Hva a NH m m HHNV NHH HNoHV mm HooHv mm HmoHv mm .mm m m mmcmo vu>§3 + mu>53 mubEE NI>E§ ucmucmmmccH v u bsxu u >Exu u.>Exu m cm>ummno >23 >2>N muduwm wo umaamz H m N H mmauoamam Hmnoz Hmuwnsszv moHumm cmuoHomum EN UGM >23 Cu. m¢OGMHmHm0~m ngflwm QHSMCOHHmem Mom M.HEB 45 ' inoculations. F2 and F1 X TMG-l backcross progeny were first inoculated with ZYMV; resistant individuals were then tested for susceptibility to WMV. The results from these experiments (Table 2.6) closely paralleled the results from the clonal pairs experiments. Again, there was a class of individuals that were resistant to ZYMV but susceptible to WMV. These findings make it unlikely that zymv is wmv-z (Model 1), or that it is equivalent to the epistatic interaction of wmv-3 and Wmv-4 (Model 3). Again, a significant chi square value was obtained for the model that the ZYMV resistance is completely independent of the WMV resistances (Model 4). Finally, the hypothesis that zymv is the same or very tightly linked to wmv-B (Model 2) is supported by these observations. Both experimental approaches led to the same hypothesis. The relationship between symptom level and virus level was also examined for all three viruses (Table 2.7). TMG-l showed no symptoms (rating of 0), while WI-2757 exhibited full symptoms (rating of 4). The F1 progeny showed symptoms in response to ZYMV and WMV but not to PRSV-W. These results reflect the data presented thus far, that the resistances to ZYMV and WMV are recessive, while resistance to PRSV-W is dominant. However, when virus titers were examined, a different scenario was observed. As might be expected, high virus titers were detected in the susceptible WI-2757 for all three viruses and for ZYMV and WMV in the symptomatic F1 progeny. 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On the other hand, high levels of PRSV-W were found in the TMG-l and F1 genotypes, despite the absence of symptoms. DISCUSSION We have examined the inheritance of multiple potyvirus resistance in the TMG-l cucumber line. Resistance to PRSV-W was due to a single dominant gene; however, there were two separate resistances to WMV that were controlled by a total of three separate genes. The first resistance was due to a single recessive gene. The second resistance was due to the epistatic interaction of a single recessive and a single dominant gene. Resistance to ZYMV was due to a single recessive gene as was published by Provvidenti (1987a). Therefore, the TMG-l cucumber line has four to five different resistance genes to confer resistance to three related potyviruses (PRSV-W, WMV, and ZYMV). The gene for resistance to PRSV-W in TMG-l is not likely to be the same as one of the recessive genes conferring resistance to ZYMV or WMV. The mechanisms of resistance also appear to be different. Little or no virus was detected in the upper leaves of TMG-l inoculated with ZYMV or WMV. In contrast, the resistance to PRSV in TMG-l may be more appropriately described as tolerance since in the asymptomatic parent and F1 progeny, the upper leaves contained virus levels that were comparable to those found in the susceptible WI-2757 parent. 49 In contrast to the relationship between the resistance to PRSV and ZYMV or WMV, resistance to ZYMV appears to be the same as, or tightly linked with the recessive resistance gene (wmv-3) that acts in concert with a dominant gene to confer resistance to WMV. The additional factor encoded by WMv-4 may be necessary to confer specificity to WMV vs. ZYMV. It is also possible that zymv and wmv-3 are actually separate, but tightly linked genes. It will be difficult to distinguish between these possibilities unless the resistances segregate from each other, or if the actual genes are cloned and their regulation studied at the molecular level. Several multiple potyvirus resistance systems have been reported in the literature. The finding that TMG-l has two independently assorting resistances to WMV is not unprecedented. Kyle and Provvidenti (1987) reported two separate resistances to WMV in Phaseolus vulgaris. Similarly, genes located on separate linkage groups in Pisum sativum have been found to confer resistance to clover yellow vein virus and pea seed-borne mosaic virus (Provvidenti, 1987b, 1990; Provvidenti and Alconero, 1988a). The possibility that a gene at an apparently non-segregating locus can confer resistance to more than one virus has been reported also in several species including Phaseolus vulgaris L. (Kyle and Dickson, 1988), Pisum sativum (Marx and Provvidenti, 1979), Solanflm (Cockerham, 1970), and capsicum annuum (Cook, 1960). It is interesting to note 50 I that multiple virus resistance conferred by a single locus has, thus far, been reported only for potyviruses (Kyle and Provvidenti, 1993). Whether the ability to confer multiple virus resistance to potyviruses is a mechanism peculiar to this particular virus group, or whether it is the genetic mechanism evolved by these particular plant species to fight off parasitic infections remains to be seen. The resistances to PRSV-W and WMV derived from TMG-l can be contrasted with other resistances reported in the literature. The resistance to PRSV from TMG-l was dominant, whereas the resistance described in the cultivar Surinam Local was recessive (Wang et al., 1984). The resistance in Surinam Local appears to act at the level of movement, since virus was detected by symptom and ELISA on the first few leaves but not in upper leaves. In contrast, TMG-l shows no symptoms of PRSV-W, but has high virus titers in the upper leaves. It appears that both the genetics and mechanism of resistance differ between TMG-l and Surinam Local. Resistance to WMV in TMG-l also appears to be different from that reported in the literature. A dominant resistance gene was reported in Kyoto 3 feet long (Cohen et al., 1971), whereas both resistances in TMG-l are recessive. Since the inheritance appears to be different for the resistances to WMV in Kyoto 3 feet long and TMG-l, they may not be allelic. Tests for allelism have been initiated to determine whether the resistances to WMV and PRSV-W in TMG-1 are at the same loci as those in Kyoto 3 feet long and Surinam Local. 51 REFERENCES Clark, M.F., Adams, A.N. ~1977. Characteristics of the microplate method of enzyme-linked immunosorbent assay for the detection of plant viruses. Journal of General Virology 34:475-483. Clark, M.E., Lister, R.M., Bar-Joseph, M. 1986. ELISA techniques. Methods in Enzymology 118:742-780. Cockerham, G. 1970. Genetical studies on resistance to potato viruses X and Y. Heredity 25:309-348. Cohen, S., Gertman, E., Kedar, N. 1971. Inheritance of resistance to melon mosaic virus in cucumbers. Phytopathology 61:253-255. Cook, A.A. 1960. Genetics of resistance in Capsicum annuum to two virus diseases. Phytopathology 50:364-367. Davis, R.F., Mizuki, M.K. 1987. Detection of cucurbit viruses in New Jersey. Plant Disease 71:40-44. Kyle, M.M., Dickson, M.H. 1988. Linkage of hypersensitivity to five potyviruses with the B locus for seed color in Phaseolus vulgaris L. Journal of Heredity 79:308-311. Kyle, M.M., Provvidenti, R. 1987. Inheritance of resistance to potato y viruses in Phaseolus vulgaris L. 1. Two independent genes for resistance to watermelon mosaic virus-2. Theoretical Applied Genetics 74:595-600. Kyle, M.M., Provvidenti, R. 1993. Genetics of broad spectrum resistance in bean and pea. Pages 153-166 in: Resistance to viral diseases of vegetables: genetics and breeding. M.M. Kyle, ed. Timber Press, Portland, OR. Lisa, V., Lecoq, H. 1984. Zucchini yellow mosaic virus. Descriptions of Plant Viruses No. 282. Kew, Surrey, England: Commonw. Mycol. Inst./Assn. Appl. Biol. Marx, G.A., Provvidenti, R. 1979. Linkage relationships of mo. Pisum Newletter 11:28-29. Nameth, S.T., Dodds, J.A., Paulus, A.0., Laemmlen, F.F. 1986. Cucurbit viruses of California. Plant Disease 70:84 11. Nameth, S.T., Laemmlen, F.F., Dodds, J.A. 1985. Viruses cause heavy melon losses in desert valleys. California Agriculture July-August:28-29. 52 ' Peterson, C.E., Williams, P.H., Palmer M., Louward, P. 1982. Wisconsin 2757 cucumber. HortScience 17:268. Perring, T.M., Farrar, C.A., Mayberry, K., Blua, M.J. 1992. Research reveals pattern of cucurbit virus spread. California Agriculture 46:35-40. Provvidenti, R. 1985. Sources of resistance to viruses in two accessions of Cucumis sativus. Cucurbit Genetics Cooperative Report 8:12. Provvidenti, R. 1987a. Inheritance of resistance to a strain of zucchini yellow mosaic virus in cucumber. HortScience 22:102-103. Provvidenti, R. 1987b. Inheritance of resistance to clover yellow vein virus in Pisum sativum. Journal of Heredity 78:126-128. Provvidenti, R. 1990. Inheritance of resistance to pea mosaic virus in Pisum sativum. Journal of Heredity 81:143- 145. Provvidenti, R., Alconero, R. 1988a. Inheritance of resistance to a lentil strain of pea seed-borne mosaic virus in Pisum sativum. Journal of Heredity. 79:45-47. Provvidenti, R., Alconero, R. 1988b. Inheritance of resistance to a third pathotype of pea seed-borne mosaic virus in Pisum sativum. Journal of Heredity 79:76-77. Purcifull, D., Gonsalves, D. 1984a. Papaya ringspot virus. Descriptions of Plant Viruses No. 292. Kew, Surrey, England: Commonw. Mycol. Inst./Assn. Appl. Biol. Purcifull, R., Gonsalves, D. 1984b. Occurrence of zucchini yellow mosaic virus in cucurbits from Connecticut, New York, Florida, and California. Plant Disease 68:443-446. Purcifull, D., Hiebert, H., Edwardson, J. 1984. Watermelon mosaic virus 2. Descriptions of Plant Viruses No. 293. Kew, Surrey, England: Commonw. Mycol. Inst./Assn. Appl. Biol. Romaine, C.P., Newhart, S.R., Anzola, D. 1981. Enzyme- linked immunosorbent assay for plant viruses in intact leaf tissue disks. Phytopathology 71:308-312. Sammons, B., Barnett, 0.W., Davis, R.F., Mizuki, M.K. 1989. A survey of viruses infecting yellow summer squash in South Carolina. Plant Disease 73:401-404. 53 . Wang, Y.J., Provvidenti, R., Robinson, R.W. 1984. Inheritance of resistance to watermelon mosaic virus 1 in cucumber. HortScience 19:587-58 CHAPTER 3 LINKAGE MAPPING OP MULTIPLE POTYVIRUS RESISTANCES IN THE THC-1 CUCUMBER LINE ABSTRACT The cucumber genotype TMG-1 is resistant to three related potyviruses, zucchini yellow mosaic virus (ZYMV), watermelon mosaic virus (WMV), and the watermelon strain of papaya ringspot virus (PRSV-W). Resistance to ZYMV is due to a single recessive gene (zymv), WMV to two recessive resistance factors (wva and wmv3 Wmv4), and PRSV-W to a single dominant gene (Prsv-Z). Possible linkage relationships between these resistances and various physiological, morphological, electromorphic, and phytopathological markers were studied. TMG-1, WI-2757, (an inbred line susceptible to all three viruses), and their F2 and backcross progeny were screened for various single gene characters that differ between the two parents. Linkages were detected between resistance to WMV and PRSV-W and traits on Linkage Group I: resistance to WMV was associated with sex expression (F) and resistance to PRSV-W was associated with bitterfree cotyledon (bi). The proposed sequence on this linkage group is Prsv-Z, bi, F, and wmv-z. Resistance to WMV was closely associated with ZYMV, but resistance to ZYMV was not linked to bitterfree, suggesting that zymv and the second gene conferring resistance to WMV are not located on Linkage Group I. 54 55 ' turnonucrxou The inbred, Chinese cucumber line TMG-l is resistant to three related cucurbit potyviruses: zucchini yellow mosaic virus (ZYMV), watermelon mosaic virus (WMV), and papaya ringspot virus (PRSV) (Provvidenti, 1985). The genetics of resistance to each virus has been characterized. Resistance to ZYMV is due to a single recessive gene (Provvidenti, 1987). A single, dominant gene confers resistance to PRSV (Wai and Grumet, 1994). The inheritance of resistance to WMV is more complex (Wai and Grumet, 1994). The progeny of TMG-1 and WI-2757, a susceptible line, possess two independently assorting recessive resistances. The first resistance is conditioned by a single recessive gene (wmvz), while the second resistance results from the epistatic interaction of a single recessive gene from TMG-l (wmv3) and a single dominant gene from WI-2757 (wmv4). To further understand the relationship of resistances to each other and to try to assign the virus resistance genes to linkage groups within cucumber, linkage relationships with markers were studied. The current morphological linkage map contains fewer than 40 map positions on six linkage groups (Pierce and Wehner, 1990). More recently, Vakalounakis (1992) placed the heart leaf shape marker onto Linkage Group IV and Knerr and Staub (1992) assigned 12 isozymes into four different linkage groups. The isozyme linkage groups have not yet been 56 ' integrated onto the morphological map assembled by Pierce and Wehner (1990). The potyvirus resistance genes were tested for associations with both morphological and isozyme markers. Single gene markers that differ between the resistant TMG-l and susceptible WI-2757 parents were used to establish linkage relationships with resistance to ZYMV, WMV, or PRSV. Our results indicate that resistance to WMV is linked to the F locus for sex expression, and resistance to PRSV is linked with bitter cotyledon, the bi locus. MATERIALS AND METHODS Linkage tests between all the characters described below and resistance to ZYMV or WMV were performed on clonally propagated genetically identical F2 cuttings as described by Wai and Grumet (1994). Linkage associations between resistance to PRSV-W and bitterfree cotyledon and the sex expression were performed in an F2 population and the linkage distance between the resistance to PRSV-W and bitterfree cotyledon were estimated from WI-2757 X F1 backcross populations. Presence of Cucurbitacins (Bitter Principle). iThe presence of cucurbitacins was assayed by tasting a small piece of the cotyledon. WI-2757 is sweet (bibi), i.e. the absence of cucurbitacin, and TMG-1 and the F1 (WI-2757 X TMG-1) progeny are bitter (Bi_). A few representative samples were also 57 ‘ examined for cucurbitacins by thin layer chromatography (TLC) (Figure 3.1A). , Blectromorphic variants. Two isozymes that differ between the two parents are mannosephosphate isomerase (MPI-1) and phosphoglucomutase (PGM-l) (Figure 3.18). Samples were prepared and separated by standard starch gel electrophoresis using the protocol of Knerr et al. (1989). Sex Expression. WI-2757 is a gynoecious cucumber line (FF) that only produces female flowers under normal conditions. Male flowers may be induced by exogenous treatments such as silver nitrate. TMG-1 is andromonoecious (ff), that is it produces many male flowers and some female flowers under normal conditions. Spine Number and Tuberculate Fruit. The fruit characters spine number and tuberculate were scored visually. WI-2757 is homozygous recessive for numerous spine (ns) and smooth fruit/flower (tu); TMG-l is homozygous dominant for few spine (Ns) and warty fruit/flower (Tn) (Figure 3.1C). Resistance to Scab. Conidia of Cladosporium cucumerinum were prepared as described by Abul-Hayja (1975) and Vakalounakis (1993). The growing point and young leaves of rooted plants were sprayed to runoff with 4 X 105 conidia/ml in distilled water using an artist's airbrush at 10 p.s.i. Plants were incubated in a biotron for 48 h in the dark at 20 C, 100% RH, followed by 4 days at 20 C, 12 h photoperiod, 40 to 50% RH. 58 w1-27s7 TMG-l F1 Figure 3.1A. Visualization of cucurbitacins by TLC. The presence (indicated by the arrow) or absence of purported cucurbitacins in the parental and F1 genotypes was visualized by TLC (Hammerschmidt, unpublished). Cotyledon tissue was homogenized in 2 ml/g fresh weight in chloroform: methanol (1:1, v/v), and then dried. In each set of three lanes, approximately 1/6, 1/3, and 1/2 of an expanded cotyledon were homogenized. Extracts were solubilized in_ methanol, spotted on a silica gel plate, and developed in a solvent containing 95% chloroform and 5% methanol. The plate was dried, sprayed with a solution containing 3% vanillin solubilized in ethanol and 0.5% (v/v) sulfuric acid, and then baked at 95 to 100 C for 10 minutes. The presence of a band (Rf of 0.49) in TMG-l and F1 and its absence in WI-2757 correlate with the bitter and sweet phenotypes. Figure 3.1B Starch gel electrophoresis of isozymes (MPI-1 or PGM-l). Electromorphs are labelled 1 and 2 for fast (lower) and slow (upper) bands. WI-2757 TMG-l Figure 3.1C Illustration of the morphological markers numerous spine and tuberculate. Female flowers/fruits of WI-2757 have numerous spines and are smooth, whereas those of TMG-1 have few spine and are warty. 6O Symptoms of susceptible plants ranged from tip maceration to death. WI-2757 carries a single dominant gene to scab (Ccu), whereas TMG-l is susceptible. A comparison of symptom observed in the susceptible TMG-1 is shown in comparison with the resistant WI-2757 (Figure 3.1D). Resistance to Fusarium Wilt. Macroconidia of FUsarium oxysporum f. sp. cucumerinum were prepared as described by Vakalounakis (1993) and in Appendix D. Rooted plants in rooting cubes were placed into a solution containing 105 macroconidia/ml. The inoculum was immediately soaked into the rooting cubes (approximately 12 ml/cube). The plants in cubes then were placed into metal pans filled with sand. The plants were watered daily and maintained in water baths at 28 C for one week until symptoms appeared. Symptoms of susceptible plants ranged from severe wilt to death (Figure 3.1E). WI-2757 is resistant to fusarium (Foc); TMG-l is. susceptible. Resistance to ZYMV and WMV. Half-expanded true leaves were mechanically inoculated with ZYMV and WMV as described by Wai and Grumet (1994). TMG-l is resistant (zymvzymv,wmv2wmv2,wmv3wmv3,wmv4wmv4) and WI-2757 susceptible. The inheritance of resistance is described by Wai and Grumet (1994). 61 l \ Afll'lll .I .—— ‘ H ,1- Figure 3.1D The effect of Cladosporium cucumerinum (scab) on the susceptible TMG-l (arrow points to leaf maceration caused by the fungus). Note that WI-2757 is healthy. .‘ I: ‘ VHF ‘_ .m . Figure 3.1E The effect of Fusarium oxysporum f. sp. cucumerinum in an F2 population. Susceptible plants are severely wilted or dead, whereas resistant plants remain standing and are green. 62 ' Analysis of Data Linkage relationships between single gene characters were analyzed using the LINKAGE 1 (version 3.5) program. Chi square analyses for associations with WMV resistance were calculated using linear models. RESULTS AND DISCUSSION The selected markers were easily scored, single gene, homozygous traits of inbred lines that differed between the parents and fit the expected segregation ratios. The following morphological, physiological, disease resistance, and isozyme characters were utilized to map the potyvirus resistance genes:. bitterfree cotyledon; sex expression; spine number; tuberculate fruit; isozymes of mannosephosphate isomerase and phosphoglucomutase; and resistance to scab, Fusarium wilt, ZYMV, WMV, and PRSV. Single trait goodness-of-fit tests show that each character used in the analyses fit expected ratios (Table 3.1). There was close agreement between linkage values observed in our study and those already cited in the literature for genes in Linkage Group I and Group IV (Table 3.2, Figure 3.2). The loci for bitterfree and female (sex expression) on Linkage Group I were linked with an estimated map distance of 0.34 vs. 0.37 cM (Fanourakis and Simon, 1987). A linkage association found between the genes encoding the morphological markers for numerous spine and 63 Table 3.1 Single Trait Goodness of Fit Tests Locus Expected Observed Chi Square P Value Ratio Ratio and Phenotype Value Scab 3:1 72:26 R:Sa 0.12 0.73 Fusm 3:1 61:25 S:R 0.76 0.38 Male 3:1 76:30 F:f 0.62 0.43 ZYMV 3:1 56:20 S:R 0.07 0.79 WMV 39:25 50:30 S:R 0.08 0.33 Bitr 3:1 80:30 Bi:bi 0.30 0.58 MPIl 1:2:1 28:54:28 LL:LU:UUb 0.04 0.98 PGMl 1:2:1 30:55:25 LL:LU:UU 0.45 0.80 SpNo 3:1 30:14 Ns:ns 1.09 0.30 Wrty 3:1 32:12 Tu:tu 0.12 0.73 The following are the genetic designations for the abbreviations above: Scab = resistance to scab (Ccu) Fusm = resistance to fusarium (FOG) Male = sex expression (F’is dominant and is strongly female) ZYMV = resistance to ZYMV (zymv) ‘ WMV = resistance to WMV (wmv-Z or wmv-mev—3,Wmv-4_) Bitr = bitterfree cotyledon (bi) MPIl = mannosephosphate isomerase (Mpi-I) PGMl = phosphoglucomutase (Pgm-l) SpNo = spine number or numerous spine (ns) Wrty = warty or tuberculate fruit or flowers (Tu) a R and S indicate resistant and susceptible classes, respectively. b L and 0 indicate lower and upper bands, respectively. 64 Noo.o e~.m m e 6 mm sun: 6:8 026m mH.o me.H N OH NH om mums can uuHm m~.o mH.H m m m «N ozmm can uuHm mm.o 5H.o H e s 5H mugs can >=as no.o mH.n 0 0H m «H ozmm new >=HN mm.o mm.o w mH mH He uuHm can >=ws mm.o mm.o m m 5 mm >003 6:0.smsm oH.o ~e.~ H HH a mH ozmm can swam Hm.o e~.o 6 mH mH me upHm can swam OH.o mm.~ m m NH mm >=wu cam swam mm.o m~.o m SH RH me mHms cam swam m~.o 6H.H H HH 5 «m anus new amom mH.o om.H H MH e H mm ozdm can amom ee.o mo.o m om mH mm uuHm cam amom e~.o -.H m pH «H em >zwu can amom Ho.o «H.m NH mH «H em 0H0: new amom m¢.o e¢.o m 6H mH ms amnm can anon mm.o Hm.o v m m «N suns cam 0H0: mm.o mm.o m HH m Hm ozdm 6:8 mHmz vo.o «H.¢ e mm mm Hm when 6:0 0H6: me.o mo.o m sH mH He >=wu 6:0 0H0: 051.5 m 0=Hm> 35.5 Tbs: 333 IE: AHumumumv Nx «mmflocmsvmum mumuomumso HIQZB x hthIHB mmOHU on» cH mwflocmsvmum mamas can mommmHo vamauocmzm mm ~.m manna 65 N ONO .03? m 34 $.ng O .5 as H NH 35: N N 3.3 H HHuNuHuNuO«N.H.NuH. 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AmmumNummuNHHv Nx mmflocwsvmnm mumpomumzu mm.H NM.H m ca 5 NH NN 0H HHm2 Ocm >23 H¢.o o.N m ¢H . 0H NH mm oH H20m new >23 039. m 032, 25;: 23;: 33.5 25.3 23.8 13...: mmflocmsvmnm mumuomnmnu AmNuomumNummumhummv Nx HomscHucooHHM.m mHnme 67 coaumcflnaoown mm copuommnv mommxcaq .>H new H masonw momxcflq How HommHv Hounds cam moumflm an cosmflansm 965 on» on w>wumamu Ammmu moan UHOD GOV acsum was» CH om>ummbo Hmmflocmsvmuu .>H can H mmsouw How one momxafla Honasoso N.m musmflm 1' I‘ 86 ON6 3 NN. :8 OO. 8 :3 ON. 3 NH. :5 ON. mm 3. m: cuEQ a Q_um a mm. as «thee. H.oH.o v m .momxcfla mmooH mHnHmmomv an am. not hm. _bmflm >H msouw mwmxcwq \ em. \ Nm. \ \ HN. \ mm. \ I H34 6H6 .8 NOE Tm .8 mum .6 ...H 06 .3 H6 NO NO 7.63 «.25 OIIO . ..H w...” . 3 ON . «.593 H gnome womxcflq 68 mwoowmmmo whomwmcxwou an cmmsmo 90mm mama umouwu on oozmvmflmwu mmocomucpcm Ho H moon on mocmumHmmH EswhmchMH EscoHHuOumHHoo an cmmsmo mmocomuzpcm ou mocmumflmmu mwmcmnsu whomwocohmmopsmmm 3n oomnmo_3wcHHE hczoc on wocmpmflmmu mumonH HHMEm unuocma Hmoocuwucfl commmuomo .m.H .pommeoo .3moHHE mumnzom “Hhuoooman I zwcHHE humczom uHsHH Ho :me Rocco» HoHoo HHSHH musuMEEH EHOHHG: :me uHSHH HHso anymoocmnpumm uHsHH hpum3 Ho mumHsoumnsu mchm HHmEm mcwmm msoumesc >H msouw ucmomwnsm hauanHm mmcHEMH “msounmam mum mmHOHumm 0cm Emum .wumunmam mcHHmonH cmHMHmo mHHHmum mes ochm xUMHb msoaowcmo cowmmmumxm xmm mHmEmH Ho wmummc cmHn .mHmemm pans: muwcfleumuwo coomepoo mmuuumpufln HmoH oon>Ho moocuwucfl pmHHH on» cam Hhuooomhn may Ho mawcmunonm I cuzoum omameo mmeonowup o: .m.H .uH5HH Ho mm>mmH msounmao Hmoon EmcHusm CH cmHHHucmoH zu>mmm o» mocmumflmmu o>HmmmomH H msouw mmwxcflq 3O up .NImE vim mum xm .m on we H>p Hp Hm HI>mHQ momxcflq «OonHoH mm mum csonm HooH oHumcmm one Homscflpcoov ~.m mHsOHm 69 tuberculate fruit on Linkage Group IV was consistent with the literature (Fanourakis and Simon, 1987). Genes that assorted independently gave very low chi square values. Therefore, the newly observed linkages are likely to be real. A very strong association was detected between resistance to ZYMV and WMV. This finding supports the proposed genetic model that resistance to WMV is due to two genetic factors (wmvzwmvz, wmv3wmv3Wmv4_), one of which (wmv3wmv3Wmv4_)is the same or is very tightly linked with the resistance to ZYMV (Wai and Grumet, 1994). Resistance to WMV is also linked to the F locus on Linkage Group I. Since resistance is not linked to bitterfree, it is predicted to lie approximately 34 cM to the other side of F, near delayed flowering and glabrate. Resistance to ZYMV does not appear to be linked to the F locus, suggesting that it is the independent WMV gene wmv-z, that is linked to F. In addition, there is a possible loose linkage between resistance to Fusarium wilt and spine number, between resistance to ZYMV and resistance to fusarium, and ZYMV and spine number, possibly putting zymv and wmv-3 inLinkage Group IV. In our experiments, the resistance to Fusarium wilt from WI-2757 appeared to be recessive. The F1 progeny died from the disease, while approximately 25% of the F2 survived the treatment (Table 3.1). In earlier studies, the resistance was reported to be due to a dominant allele 7O (Netzer et al., 1977; Vakalounakis, 1993). Vakalounakis (1993) has found absolute linkage between resistance to scab and resistance to race 1 of fusarium. A possible explanation to account for this difference is a difference in the disease screen protocol. Our tests were performed with cuttings in rooting cubes and so the plants absorb more inoculum than the standard assay in which roots of young seedlings are dipped into the Fusarium inoculum for only a limited period of time. It is also possible that the gene observed in this study is actually a different resistance gene from the ones that have been described by Netzer (1977) and Vakalounakis (1993). In our study, the genes conferring resistance to scab and fusarium not only are dominant and recessive, respectively, but also assort independently. In addition, environmental and epistatic interactions may vary in different genetic backgrounds and influence the results observed. A separate set of experiments was performed to map resistance to PRSV. The recessive gene that confers resistance to PRSV in the cultivar Surinam Local was reported to be linked with bi (Wang et al., 1984). Therefore, we tested for possible cosegregation between our resistance to PRSV with loci on Linkage Group I, bi and F (Tables 3.3 and 3.4). Our results showed that resistance to PRSV is linked to bi at a distance of approximately 28 cM in the WI-2757 X F1 backcross (Table 3.3). We further tested for linkage with the F and bi loci in an F2 population, 71 Table 3.3 Test for Linkage Association Between Resistance to PRSV and Bitterfree Cotyledon in the backcross WI-2757 x F1. Phenotype Observeda Expected Ratio R Bb (TMG-l) 76 1 R b 31 1 S B 31 1 s b (WI-2757) 82 1 x2 42.2** Single B:b 107:111' 1:1 Trait x? 0.04 ns R:S 107:111 1:1 x2 0.04 ns ** Deviation from the predicted values for independently assorting genes is significant: P < 0.01. a Each experiment fits the predicted single-trait ratio for resistant to susceptible and for bitter to sweet. Data pooled from two independent experiments: XZR:Sexpl = 0.008, 2 = 2 o = 2 0 = x B:bexpl 0.008, X R.Sexpz 0.25, and X B.bexp2 0.09. b R = resistant, S = susceptible; B = bitter, b = bitterfree (sweet). The estimated map distance for the combined experiments is 28 on between resistance to PRSV and bitterfree cotyledon (bi). 72 .mHms u z .mHMEwH n m “Hummzmv mmuuumuqu u n .Houqu u m HmHanmmomsm u m .ucmumHmmH u m .Ho.o v m «ucmofluflcmflm OH mmcmo mafiuHOmmm >HucmoswmmocH How mmsHm> omuoflomum on» EOHH coaumH>wo OO ma moo.o u Nx m: moo.o n Nx Hnm qumm mam Hum mmumm mum O: OO.O .. Nx O: OH.O u Nx Hum Nmuom Sum Hum ONONO nnm .wummu pHH Ho mmwcooom\uHme wHoch HMO .mumwu uHH Hoummbo wmauosmnm owuommxm mom>ummno mmapocmnm .m osmiwnbmum cwmsumb COHumommowmoo HHe .HQ cam Musmum cmmzumb QOHumomHowmoo HH. .h cam NI>mHm :wm3uwn mmmxcflH HoH name .m .Hn cam N|>mhm cmm3pmn momxcHH HoH page .< .:0HumH=mom «m cm :HHHEV scammmumxm Rom cam AHA. coomHauoo meHHmuqua H msouw momxcwq so mwcmu cam .mnbmhmv >mmm ou mocmumHmmm :wmsuwm macapmHUOOmm momxcHA HoH mumma ¢.m anma 73 which segregates for all three characters (Table 3.4). In correspondence with the backcross data, we found linkage between bi and resistance to PRSV at an estimated distance of 22.4 cu. Because resistance to PRSV was found to be independent of the F locus (Table 3.4), we have tentatively placed it to the left of bi (Figure 3.2). Since both the dominant resistance to PRSV from TMG-l and the recessive resistance from Surinam Local were placed on Linkage Group I to the left of bi, it will be especially interesting to perform tests of allelism between these two resistances. In summary, these studies have placed two additional genes wmv-z and Prsv-z onto Linkage Group I. The proposed gene sequence is Prsv-2 - bi - F— wmv-z. In order to further map useful genes, such as those that confer resistance to plant viruses, it will be necessary to develop a more saturated genetic 74 REFERENCES Abul-Hayja, Z.M. 1975. Multiple disease screening and genetics of resistance in cucumber. Ph.D. Thesis, University of Wisconsin-Madison. 149 pp. Fanourakis, N.E., Simon, P.W. 1987. Analysis of genetic linkage in the cucumber. The Journal of Heredity 78:238- 242. Knerr, L.D., Staub, J.E., Holder, D.J., May, B.P. 1989. Genetic diversity in Cucumis sativus L. assessed by variation at 18 allozyme coding loci. Theoretical and Applied Genetics 78:119-128. Netzer, D., Niegro, S., and Galun, F. 1977. A dominant gene conferring resistance to fusarium wilt in cucumber. Phytopathology 67:525-527. Pierce, L.K., Wehner, T.C. 1990. Review of genes and linkage groups in cucumber. HortScience 25:605-615. Provvidenti, R. 1985. Sources of resistance to viruses in two accessions of Cucumis sativus. Cucurbit Genetics Cooperative Report 8:12. Provvidenti, R. 1987. Inheritance of resistance to a strain of zucchini yellow mosaic virus in cucumber. HortScience 22:102-103. Vakalounakis, D.J. 1992. Heart leaf, a recessive leaf shape marker in cucumber: linkage with disease resistance and other traits. The Journal of Heredity 83:217-221. Vakalounakis, D.J. 1993. Inheritance and genetic linkage of fusarium wilt (Fusarium oxysporum f. sp. cucumerinum race 1) and scab (Cladosporium cucumerinum) resistance genes in cucumber (Cucumis sativus). Annals of Applied Biology. 123:359-365. Wai, T., Grumet, R. 1994. Inheritance of multiple potyvirus resistance in the Chinese cucumber line TMG-l, manuscript in preparation. CHAPTER 4 TISSUE-SPECIFIC EXPRESSION OF DIFFERENT RESISTANCES TO NRTERNELON MOSAIC VIRUS IN CUCUMBER ABSTRACT The Chinese cucumber line TMG-l is resistant to the potyvirus watermelon mosaic virus. The resistance can be conferred by either of two independently segregating factors. To further characterize these resistances, the parents, F1, F2, and backcross progeny of a cross between TMG-l and a susceptible line WI-2757 were monitored for symptom expression and virus accumulation by ELISA. Segregation ratios differed depending on whether cotyledons or true leaves were inoculated. Cotyledon-inoculated backcross progeny segregated 1:1 as would be expected for a single recessive gene, whereas segregation ratios for true- leaf-inoculated progeny were 5 resistant: 3 susceptible. Similar differences were observed between cotyledon and true-leaf-inoculated F2 progeny. These results suggest that the two resistances are expressed differently: one at the cotyledon stage and throughout the plant, the second only at the true leaf stage. The cotyledon-expressed gene was found to be linked to sex expression (F) in Linkage Group I. 75 76 INTRODUCTION The cucumber cultivar TMG-l is resistant to several potyviruses including watermelon mosaic virus (WMV) and zucchini yellow mosaic virus (ZYMV) (Provvidenti, 1987; Wai and Grumet, 1994). When crossed with the susceptible genotype WI-2757, two independently assorting resistances to WMV were observed (Wai and Grumet, 1994). The first resistance is due to a single recessive gene (wmv-Z), whereas the second resistance is the result of an epistatic interaction between a single recessive gene from TMG-l (wmv- 3) and a single dominant gene from WI-27S7 (WMv-4). The second recessive gene, wmv-3, appears to be the same as, or tightly linked with, the recessive gene (zymv) conferring resistance to ZYMV. Linkage analyses with these resistances indicated that the resistance to WMV, but not ZYMV, is associated with the F locus for sex expression located on cucumber Linkage Group I (Wai et al., 1994). During the course of inheritance and linkage studies, a difference was noted in the response to WMV inoculation depending on whether true leaves or cotyledons were inoculated. This study was initiated to differentiate further between the two resistances to WMV. We found that the two resistances showed different tissue-specific expression; wmv-Z was expressed in cotyledons and true leaves, the epistatic wmv3,Wmv4 resistance was expressed only in true leaves. 77 NATERIALS AND METHODS Cucumber Genotypes The inbred cucumber (Cucumis sativus L.) lines TMG-l, resistant, (Provvidenti, 1985) and WI-2757, susceptible, (Peterson et al. 1982) were provided by Dr. Jack Staub (USDA, University of Wisconsin at Madison). The F1 progeny (WI-2757 X TMG-l) were either self- or sib-pollinated to produce the F2 generation. Backcrosses were made with both parents: WI-2757 X F1 and F1 X TMG-l. In each cross, the source of resistance came from the male parent. The inbred line Straight 8 (Stokes Seeds, Inc., Buffalo, NY) was used as an additional control genotype that is suceptible to all three viruses. Experimental designs and methods for virus maintenance and verification, inoculation, and leaf disk ELISA are as described by Wai and Grumet (1994). To study the different resistances that are expressed in different tissues, cotyledons or true leaves were mechanically inoculated. Linkage relationship between the resistances and sex expression (F locus) were determined as described in Wai et a1. (1994). RESULTS Inoculation of true leaves of F2 or backcross progeny of WI-2757 X TMG-l with WMV indicated the presence of two independently assorting resistance factors, one controlled by a single recessive gene (wmvzwmvz), the second by the 78 epistatic interaction of a recessive and dominant gene (wmv3wmv3,Wmv4_) (Wai and Grumet, 1994). However, other experiments, in which cotyledons rather than true leaves were inoculated, gave different results. When cotyledons of the F1, F2 and backcross progeny were inoculated, segregation ratios indicated that resistance to WMV was due to a single recessive gene (Table 4.1). Possible explanations for these differing results include: different environmental conditions when the experiments were performed, different ages of the plants, or tissue-specific expression (cotyledon vs. true leaf). Upon closer inspection of the susceptible individuals in cotyledon-inoculated experiments, two levels of symptom expression were detected (Table 4.2). In experiments using F1 X TMG-1 backcross progeny, the individuals again segregated as a single gene trait, 1:1 resistant: susceptible. In one experiment, 1/4 - 1/2 of the susceptible class showed a mild flecking type of mosaic pattern, while the remainder exhibited a more severe mosaic pattern. An approximately 3:1 (susceptible:partially resistant) segregation within the susceptible class would be predicted if the second gene could confer only partial resistance once an infection became established in the cotyledons. In the second experiment, again two levels of symptom expression were observed in an approximately 3:1 ratio. About one quarter of the susceptible class showed symptom spread approximately one tenth of the way down the 79 .om.o n mmxmwx .ooo.o u meowx .mO.o u memwx «OOHDMH UODOHcmHm on» mpHH pcwEHHmme zoom .ODCOEHHmmxm ucoocmmwccfl m>HHOEOHH cmHoom mumo b .chos mama m>HmmmomH mHmch m co comma mOHumH pmpoomxm m H n O NN O HO x NONNuHs Os HH0.0 H u H HON OOO bHuoze x HO O: ON.O O u H ONN HO NO NN O HO OO O NONNqu O OOH Huwze O Hmnme OHHOO Nx cwuommxm mHnHumwomsw ucmumflmmm 095980 MpcmHm Ho Honssz .mOOuO cocmHHuoo may um >23 nuH3_OoHuOHsoo:H 0» NONNuHs OOO H-629 Oo OcmOoHO Ho mOOoaOmm H.O mHmOa 80 leaf, while the remainder ofthe susceptible class exhibited a more extensive mosaic on the leaves. These results gave further evidence for two separable resistances, and led to the hypothesis that the second resistance was not expressed until the true leaf tissue formed. Table 4.2 Intermediate Symptom Expression by a Subset of the Backcross (F1 X TMG-l) Individuals Susceptible to WMV After Cotyledon Inoculation. Resistant (without symptoms) 90 Susceptible 84 mild symptoms 20 strong symptoms 64 Data were combined from two experiments. To differentiate among possible explanations for the different ratios observed in cotyledon and true leaf experiments, sets of experiments were performed where cotyledon only, cotyledon and true leaf, or true leaf only, inoculations were made simultaneously on groups of plants of the same age (two true leaf stage). Again, different segregation ratios were observed depending on whether true leaves or cotyledons were inoculated (Table 4.3). Inoculation of cotyledons alone indicated a single, recessive gene; inoculation of true leaves indicated two resistances. Inoculation of both true leaves and cotyledons 81 000 000 000.000 000000000 000 000.0 000500080 0000 on» .80 333 00.6893 on» 3.3 08500000 noun .OHo.o I . 0009500000 0000000000.... 030 8000 00H000 0000 0 .oH.o I . 00000000000 00000000000. 030 8000 00H000 0000 a «0.00 H000 x Nx us. HOO.O .. 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V H0000 0000|n ccnh.mnH MOH «cmn.mH MHOM NX mum Alvkmknhv H0002 0000|N 0H0000H0u c0uv.on mOH ¢v~.n MH.n «x mum Alflamtnav H0000 0000I~ 0H0000H0u «cm.NH H.H ¢Hv.¢ MOH Nx mum HNRN... V H0005 0000IH 0d vN0.0 HOH ad on.o nun «x 9m 3ng 0 H000.0 0000IH ON NOH o H-020. 0 H0 OO Om N0 0 0 00000000 0H00 0000.3 H00H 0000 00 00H00H0000H 0 OO NO n 70.0 0 H... NN ON N0 0 0 00000000 00H00H0000H 0000Hh000 4 .0203 0000. 3. 0o 00000H0000 H5 000 0000.3 00 00H00H0000H 00H30HH00 2.5 00 0000003000 00 0930000000. n.v 5000. 82 gave the same segregation ratios as when cotyledons alone were inoculated: 21:28 (R:S) (X2 = 0.73) resistant in the F1 X TMG backcross progeny. Inoculation of the second true leaf or the eighth true leaf both gave similar results (data not shown). These results suggest that the observed difference is due to the tissue that is being inoculated, and not differences in plant age at the time of inoculation or environmental conditions. These results further indicate that the resistance expressed in the cotyledon is due to the single recessive gene that acts independently (wmvz) rather than the epistatic interaction between the recessive and dominant genes (wmv3, Wmv4) (Table 4.2). Previous studies had identified a linkage relationship between WMV resistance and the F locus for sex expression (Wai et al., 1994). It was now possible to determine which of the two WMV resistances, cotyledon-expressed (wmv-Z), or true leaf only (wmv3, Wmv4), was linked to the F locus. The two single traits, resistant vs. susceptible, and male vs. female, gave the predicted 1:1 ratios in the backcross generation (Table 4.4). Analysis of cosegregation indicated a linkage association between the cotyledon-expressed gene and the F locus; thus wmv2 is linked to the F locus. These results are consistent with previous obervations (Wai et al., 1994) that: (1) the resistance to ZYMV is due to the same gene, or is tightly linked to the gene that acts in concert with the dominant gene to confer resistance to WMV (wmv3), and (2) the resistance to ZYMV is not linked to the 83 Table 4.4 Test for Linkage of the Cotyledon-Expressed WMV Resistance to the E'locus in the F1 x TMG-l Backcross. Cotyledon Inoculation Phenotype Observeda Expected Ratio R Mb (TMG-l) 134 1 R F '66 1 s M 82 ‘ 1 s F (WI-2757) 130 1 x2 34.0** Single MzF 216:196 1:1 Trait x? 0.88 ns R:S 200:212 “ 1:1 x? 0.29 ns ** Deviation from the predicted values for independently assorting genes is significant: P < 0.01. a Data from each experiment fit the predicted single- trait ratios for resistant to susceptible and for the number of male to female flowers. Data pooled from two independent experiments: sz:S = 0.000, XZM:Fe expl = 0.61, xzn:s = 0.02. xpl = 0.52, and sz:F exp2 exp2 b R = resistant, S = susceptible; M = male, F = female. 84 F locus. Physiological separation of the two WMV resistances also made it possible to map the wmv2 gene more accurately relative to the F locus; the estimated distance is 35 cu. DISCUBBION A novel tissue-specific plant virus resistance has been identified. In the progeny of TMG-l and WI-2757, two independently assorting resistance factors to WMV were identified (Wai et al., 1994). The first resistance is due to the action of a single recessive gene, while the second resistance is conditioned by the epistatic interaction of a recessive gene from TMG-l and a dominant gene from WI-2757. This work demonstrates that there is differential developmental control of the two resistances. The resistance conferred by the single recessive gene is expressed in the cotyledon and throughout the plant. In contrast, the epistatic resistance is expressed only in true leaf tissue. These experiments cannot distinguish whether it is the recessive factor from TMG-l, the dominant factor from WI-2757, or both that are not expressed until the true leaf stage. We also cannot determine whether the requirement for the epistatic factor from WI-2757 is unique to that genotype or would occur in other crosses with other genotypes susceptible to WMV. 85 Interestingly, although the resistance requiring recessive gene wmv3 is not expressed until the true leaf stage, genetic analysis indicates that this gene is the same as, or tightly linked to, the recessive gene zymv for resistance to ZYMV (Wai and Grumet, 1994). The resistance due to zymv is expressed at the cotyledon stage (Provvidenti, 1987; Wai and Grumet, 1994). Possible explanations for this difference are that an additional factor is necessary for the zymv gene product to be able to confer resistance to the related potyvirus WMV or that the two genes are separate, but closely linked. We have not found other reports of tissuefspecific expression of virus resistance. However, standard virus tests usually employ one type of inoculation. In our studies, the first differences appeared when we used vegetatively propagated clonal sets of F2 individuals to perform direct comparisons of WMV and ZYMV resistance, rather than standard seedling screens (Wai and Grumet, 1994). Developmentally expressed resistances have been described in other systems, for example, in bacteria and fungi, as in the case of resistance to damping off, described to be expressed at the true leaf stage and not in cotyledons. In addition, virus resistance that is only expressed in true leaf tissue is useful, since aphids normally feed on young leaves and not on cotyledons. Recently, a developmentally regulated plant cell wall- associated protein kinase has been described (Citovsky et 86 al., 1993). This protein kinase was shown to phosphorylate the movement protein of TMV in vitro. It is expressed primarily in leaves and increased activity is found as a function of leaf maturation, with the greatest activity found at leaf tips and the least at the base. The protein kinase activity is also correlated with the development of secondary plasmodesmata in mature leaves. Phosphorylation of movement protein is believed to inactivate it (Citovsky and Zambryski, 1993). It is conceivable that a host plant resistance that is expressed only in true leaves may block cell—to-cell movement of the virus. The observations made in this study may be explained by such a model. A subpopulation of plants that exhibit symptoms only at the very base of the leaf correlates well with such a hypothesis. In addition, recent characterization of calcium-regulated protein kinases in zucchini has shown that cotyledons contain the fewest protein kinases (Verhey et al., 1993). This finding also supports a model in which a developmentally regulated protein kinase activity may be involved in a resistance that is only expressed in true leaves. Although we cannot rule out other possible mechanisms, such as reduced rates of replication or inhibition of long distance movement, it would be very interesting to study further whether this tissue-specific resistance acts at the level of cell-to-cell movement. 87 REFERENCES Citovsky, V., McLean, B.G, Zupan, J.R., Zambryski, P. 1993. Phosphorylation of tobacco mosaic virus cell-to-cell movement protein by a developmentally regulated plant cell wall-associated protein kinase. Genes and Development 7:904-910. Citovsky, V., Zambryski, P. 1993. Transport of nucleic acids through membrane channels: snaking through small holes. Annual Review Microbiology 47:167-197. Peterson, C.E., Williams, P.R., Palmer M., Louward, P. 1982. Wisconsin 2757 cucumber. HortScience 17:268. Provvidenti, R. 1987. Inheritance of resistance to a strain of zucchini yellow mosaic virus in cucumber. HortScience 22:102-103. Verhey, S.D., Gaiser, J.G. Lomax, T. 1993. Protein kinases in zucchini. Plant Physiology 103:413-419. Wai, T., Grumet, R. 1994. Inheritance of multiple potyvirus resistance in the Chinese cucumber line TMG-l, manuscript in preparation. Wai,T., Staub, J.E., Grumet, R. 1994. Linkage characterization of multiple potyvirus resistance in the Chinese cucumber line TMG-l, manuscript in preparation. CHAPTER 5 SPATIAL AND TEMPORAL DISTRIBUTION OP POTYVIRUS INFECTION IN THE RESISTANT CUCUMBER GENOTYPE THO-1 ABSTRACT The inbred Chinese cucumber line TMG-l is resistant to three related potyviruses: zucchini yellow mosaic virus (ZYMV), watermelon mosaic virus (WMV), and the watermelon strain of papaya ringspot virus (PRSV-W). The genetics of resistance to three viruses is different: ZYMV is due to a single recessive gene, WMV to two recessive resistance factors, and PRSV-W to a single dominant gene. We sought to determine if the resistances also differ in their effect on systemic spread. The kinetics of virus accumulation were studied in the resistant TMG-l genotype and compared with the pattern in WI-2757, an inbred line that is susceptible to all three viruses. While the spread of PRSV-W is retarded initially, eventually levels of virus detected by ELISA are comparable to those in WI-2757. ZYMV and WMV, however, spread more slowly and do not attain as high a titer in TMG-l as in WI-2757. More than one resistance mechanism appears to be acting in the resistant TMG-l cucumber. 88 89 INTRODUCTION The inbred Chinese cucumber line TMG-l is resistant to three related potyviruses: zucchini yellow mosaic virus (ZYMV), watermelon mosaic virus (WMV), and the watermelon strain of papaya ringspot virus (PRSV-W). The genetics of resistance to the three viruses is different; resistance to the ZYMV is due to a single recessive gene (Provvidenti, 1987), resistance to WMV is due to two recessive resistances, and resistance to PRSV-W is due to a single dominant gene (Wai and Grumet, 1994). There is also a difference in the time that each virus takes to show full symptom expression in susceptible genotypes: approximately seven to ten days for ZYMV, ten to fourteen days for WMV, and three to six weeks for PRSV-W. Since different genetic mechanisms (e.g. dominant vs. recessive) have been associated with different resistance phenotypes (Fraser, 1990), the resistances in TMG-l were examined for differences in mode of action. Initial studies suggested that the phenotype of the dominant PRSV-W resistance trait differs from that of the recessive ZYMV and WMV resistances (Table 2.7, Chapter 2). There were high PRSV-W titers in young, fully expanded leaves of the symptomless TMG-l and F1 genotypes. In contrast, little or no virus was detected in comparable leaves when TMG-l was inoculated with ZYMV or WMV. In this study, the kinetics of accumulation of each virus was monitored in the resistant 90 THC-1 genotype and compared with the pattern in WI-2757, an inbred line that is susceptible to all three viruses. NATERIALS AND NETHODS Maintenance of virus inocula, method of inoculation, and detection of virus by the leaf disk method were performed as described by Wai and Grumet (1994). Plants were inoculated at the cotyledonary stage. Virus spread to each leaf position was assayed by indirect ELISA, using the leaf disk method. The experiments for all three viruses were performed simultaneously under identical environmental conditions. RESULTS Figure 5.1 shows a comparison of symptoms produced in response to each virus in zucchini squash. Each virus induces distinct symptoms. ZYMV characteristically produces a striping type of interveinal chlorosis, while WMV produces a fine mosaic or mottling. Infection with PRSV-W produces well-defined rugosity. Table 2.7 (Chapter 2) presents data that suggest that the mechanisms of resistance may be different for each virus. TMG-l plants showed no symptoms in response to each virus. Little or no ZYMV or WMV was detected in the young, fully expanded leaves of TMG-l. 91 .muflmomsu confluwolaag m moosooum >93 :33 coauowucH .mcflpuoe whoa moosooum >23 03:3 .mHmouoHno Hmcfloampcfl no 093 mcflmfluum m monsooum haamoaumfluwpomumao EN .mEOpQEm pocflumflo moosocfl mafia, comm vac» opoz .nmmsvm flcflcoonfi ca >mmm new 523 .EN 0» mmcommmn :H couscoum mEoumEm no conflummfioo H.m wusmflm 92 However, equally high levels of PRSV-W were detected in young, fully expanded leaves of TMG-l as were observed in the symptomatic WI-2757 leaves. Figures 5.2, 5.3, and 5.4 display a comparison of virus spread throughout various stages of infection. Each figure illustrates virus titer at each leaf position in both the resistant TMG-l and in the susceptible WI-2757. Panel A of each figure shows virus accumulation at week 1, Panel B at week 3, and Panel C at week 6. All three experiments were performed at the same time under identical environmental conditions. Each point represents the average of five replicate plants. Even during the first week of infection, WI-2757 supports markedly higher levels of virus than the resistant TMG-l for all three viruses. By week 3, ZYMV and PRSV-W appear to spread to the second leaf in TMG-l; however, virus levels drop off quickly in subsequent leaves. The levels of WMV appear to remain at a constant low level in TMG-l. By week 6, PRSV-W has spread to a higher virus titer in the upper nodes of the resistant plant than either ZYMV or WMV. Figure 5.5 shows a comparison of PRSV-W and ZYMV accumulation in TMG-l as a percentage of the level in the susceptible WI-2757. Levels of ZYMV sharply drop off after the third leaf, whereas high titers of PRSV-W were detected through leaf 9. The first fully expanded leaf (approximately the third leaf from the apex) was usually 93 .mucmfiflnmmxo mmonu ca oom.o maoumaflxoummm um ommuobm maw>ma ocsoumxomn hauamom .mpcmam oumoaamon m>flu no momuo>m on» mucmmoumon ucfiom comm .mcowuflocoo Hmucmeconw>cw Hmowucmofl nouns mean meow map um omEHOHHmm mums mucoafiuomxo omucu Had. .m x003 um 0 House com .m 3003 um m Hmong .H #003 um coapmasssoom msuw> mzonm musmflu comm mo 4 Hocmm .nmhmuH3_oHnHumoomsm cH new H1029 pcmumamwu on» anon ca coauflmom “mod comm um nova» msnfl> moumuumsaafl musmau comm .coauomucfl no mommum mSOHHm> psonmsousw ommumm msufl> no GOmHHMQEoo .A3|>mmmv ¢.m can .A>zzv m.m .A>:MNV ~.m mousuflm 8.. .23 .. . . zoE zoEmE Em: 2950.. 5a.. «u. n a. n .v d d. . w m— 0 m . w _ OE \t . . 1 \‘Ill 01 II‘IIIIOII’I inc \ J I m 1.3 :3 m. S m I S m s v V .r Y Li A 1 , A v m. . w m 1 n — U gvnu— m 11“.— . U o e a . nln.° . nth.° run-o .. l. m e3 :3 . n.— m ._r r. - . Hwy—1.4%. 1.502... .. .62... ... I: I(\ and / ..nd ..nd 82.5 /. .. ERIE < .- < < 5:23 . . m and g >23 m m H .m .2 >zwn ~.m musmam 1:; zn>mmm e.m mnsmflm -3. 94 PRSV-W 0.5- % ELISA VALUE LEAF POSITION Figure 5.5 Comparison of PRSV-W and ZYMV accumulation in TMG-l as a percentage of the level in the susceptible WI- 2757. In this comparison, the healthy background levels have been subtracted. 95 sampled. Virus titers in the upper leaves are markedly different for ZYMV and PRSV. DISCUSSION Comparison of virus titers in young, fully expanded leaves of TMG-l (sampled at the time of full symptom expression in WI-2757) suggested that the mode of resistance to PRSV-W may differ from that of WMV or ZYMV. A more detailed analysis over time suggests that a similar response might exist for all three viruses, but with differences in the kinetics of the resistance phenotype. Although PRSV-W accumulation is delayed in TMG-l relative to the susceptible WI-2757, it appears to move more quickly than either ZYMV or WMV. Moyer et al. (1985) described similar findings in a Cucumis melo L. line 91213 that exhibits resistance to WMV. A reduction in accumulation of virus concentration was detected in leaves of the resistant line. The authors called this phenomenon a "suppressive virus resistance." Further characterization of this resistant line by differential temperature treatment of upper and lower leaves suggested that resistance is correlated with reduced movement of virus within leaves, possibly at the level of cell-to-cell movement (Gray et al., 1988). During the first week of infection, the susceptible WI- 2757 supports higher levels of all three viruses. This trend persists over time; accumulation of all three viruses 96 is delayed in TMG-l, indicating that none of the resistance genes confers resistance at the level of complete inhibition of viral replication. A possible reason for high virus titers in the lower leaves in the absence of symptom expression, is that sufficient virus titers do not accumulate in the leaf at the critical time for symptom development. Further experiments would be required to distinguish between resistance mechanisms acting at the level of viral spread versus reduced virus multiplication. REFERENCES Fraser, R.S.S. 1990. The genetics of resistance to plant viruses. Annual Review of Phytopathology 28:179-200. Gray, S.M., Moyer, J.W., Kennedy, G.G. 1988. Resistance in Oncumis melo to watermelon mosaic 2 correlated with reduced virus movement within leaves. Phytopathology 78:1043-1047. Moyer, J.W., Kennedy, G.G., Romanow, L.R. 1985. Resistance to watermelon mosaic virus II multiplication in Cucumis melo. Phytopathology 75:201-205. Provvidenti, R. 1987. Inheritance of resistance to a strain of zucchini yellow mosaic virus in cucumber. HortScience 22:102-103. Wai, T., Grumet, R. 1994. Inheritance of multiple potyvirus resistance in the Chinese cucumber line TMG-l, manuscript in preparation. SUMMARY, CONCLUSIONS, AND FUTURE DIRECTIONS TMG-l is resistant to ZYMV, PRSV-W, and WMV. Resistance to ZYMV was due to a single recessive gene, resistance to PRSV-W was conferred by a single dominant gene, and resistance to WMV was due to two independently assorting recessive resistance factors, either one of which may impart resistance to WMV. The first resistance to WMV was due to a single recessive gene and the second was the result of an epistatic interaction between a single recessive gene from TMG-l and a single dominant gene from WI-2757. The recessive resistance gene to ZYMV (zymv) appeared to be at the same locus (either the same, or tightly linked) as the recessive resistance gene that is part of the epistatic interaction. Linkage mapping placed resistance to PRSV-W (linked with bitterfree cotyledon, but not sex expression) and WMV (linked with sex expression, but not bitterfree cotyledon) on Linkage Group I, but at different locations. The two resistance factors to WMV were found to be expressed in different tissues. The single recessive gene was found to be expressed in cotyledon tissue and throughout the entire plant, whereas the resistance that resulted from an epistatic interaction between a single recessive and a single dominant was expressed only in true leaf tissue. The single recessive gene to WMV that is expressed in cotyledon tissue was found to be linked to sex expression. Different mechanisms of resistance were also found to be active in TMG-l. ELISA results showed that 97 98 resistance to ZYMV and WMV were most likely due to reduced multiplication, whereas resistance to PRSV-W was probably tolerance. A time course study showed that little or no ZYMV or WMV were found in the upper leaves of TMG-l, while high titers of PRSV-W were present in comparable positions. Several lines of experiments are possible to continue this work. The first would be to perform tests for allelism between the resistances to PRSV-W and WMV in TMG-l with those described in the literature. The cultivar Surinam Local carries a single recessive gene to PRSV-W and Kyoto 3 feet long has a single dominant gene to WMV. Secondly, further mapping of the resistance genes by RFLP's and RAPD's might uncover closer markers. It may be possible to map the cotyledon-expressed WMV resistance closer to delayed flowering under the appropriate environmental conditions. It may be possible to study mechanisms of resistance by the tissue print method. This method maybe useful in differentiating between resistance that is expressed cell- to-cell vs. long distance spread. The hypothesis that the true-leaf-expressed WMV resistance may act at the level of cell-to-cell movement could be studied by the tissue print method also. Separation of resistances to ZYMV and WMV by creating F3 and F4 lines would allow for further dissection of the different mechanisms of resistance. Thus, many interesting questions remain to be investigated in this system. APPENDICES APPENDIX A APPENDIX A A QUICK AND EASY METHOD TO ISOLATE LARGE QUANTITIES OF HIGH QUALITY POTYVIRAL RNA One approach to study resistance mechanisms is to determine whether the resistance is expressed at the cellular or at the organismal level. A method to distinguish between these two possibilities is to determine whether viral replication occurs at the same rate and to the same extent in the resistant cultivar in comparison with the susceptible one. This approach was used to elucidate the mechanism of resistance in the Arlington cowpea (Bruening et al., 1987). The investigators found that the resistance mechanism was due to a protease inhibitor. Potyvirus replication in a protoplast system has been studied by introducing naked viral RNA into the cell via electroporation (Luciano et al., 1007). Luciano et al. used 20 to 80 pg of viral RNA per sample and then tested for increased levels of viral RNAs to determine whether virus replication had taken place. I tried to study virus replication in a cucumber protoplast system using a similar approach. The standard method of isolating viral RNA involves isolating whole virions in a caesium sulphate gradient by ultracentrifugation, and then isolating RNA in a sucrose density gradient by ultracentrifugation, as was done for 99 100 ZYMV by Grumet and Fang (1990). A more efficient method of isolation was needed to obtain the large quantities of viral RNA required to study of virus replication in vitro. When virus was isolated in a caesium sulfate gradient' made in phosphate buffer (pH 7.5) and RNA extracted by phenol chloroform extraction, the virus preparations were free from other proteins, but were contaminated with nucleic acids. Different treatments, for example digestion of virions with DNAase I, were tried to remove the host DNA. A report that indicated the use of HEPES buffer could remove contaminating nucleic acids was tested. It was also necessary to develop a method to destabilize the virus coat protein to extract the viral RNA. The standard dissociation buffer involved the use of 808, which precipitates with the RNA in the ethanol precipitation step. The presence of SDS in RNA preparations was detrimental to cucumber protoplasts. After many trials of varying buffers at different pH, an extremely simple method of purifying virions and RNA without any ultracentrifugation steps was developed. The procedure developed is as follows: isolate virions as decribed by Grumet and Fang (1990), but substitute the basic buffer component with one that contains 10 mM EDTA with 20 mM HEPES at pH 7.0 or 6.5 or 20 mM MES at pH 6.5. The procedure requires that the virus isolation is performed only up to the second PEG precipitation step. All of the cellular debris precipitates out in the pellet, and the remaining supernatant contains pure virions without any associated 101 contaminating nucleic acid. Virus isolated in grinding buffer that consisted of 10 mM EDTA and 20 mM HEPES at pH 7.5 had contaminating nucleic acid on the surface of the virions (Figure A.1, lane 2). It is possible that changing the pH of the phosphate buffer from pH 7.5 to pH 7.0 would also eliminate the contaminants. This possibility was not pursued since the current procedure works well. The procedure recovers almost 90% of the full-length viral RNA from the starting material. An additional check gel showed that approximately one tenth (by visual quantitation) of the total full-length viral RNA is lost in the second pellet and none in the first pellet. An average yield from this procedure would be as follows: approximately 100 g of fresh weight leaf material would yield 2-10 mg of virus (with an absorbance 260/280 ratio of 1.3 to 1.6), from which approximately 2-10 pg of full-length RNA may be isolated by the standard phenolzchloroform extraction method. Yield may be increased by re-extraction of the proteinaceous material at the interface with phenolzchloroform. The main advantage of this procedure is that very large quantities (1 to 2 kg) of leaf material may be processed at one time. In addition, both of the time-consuming and expensive caesium sulfate and sucrose ultracentrifugation gradients have been eliminated. 102 Several different methods of dissociating virus coat protein work equally well. Stocks may be made 2x, 5X, or 10X of the final concentration. Final concentrations are given below. (1) Standard RNA prep buffer 100 mM ammonium carbonate, pH 9.0 1 mH EDTA 1% SDS 0.125 ug/ml proteinase K (2) Tris, EDTA, SDS, Proteinase K 100 mM Tris, pH 9.0 1 mM EDTA 1% 808 0.125 ug/ml proteinase K (3) Ammonium carbonate, EDTA, Sarkosyl, proteinase K 100 mM ammonium carbonate, pH 9.0 1 mM EDTA 1% to 1.5% Sarkosyl 0.125 ug/ml Proteinase K (4) Tris, EDTA, Sarkosyl, Proteinase K 100 mM Tris, pH 9.0 1 mM EDTA 1% to 1.5% Sarkosyl 0.125 ug/ml Proteinase K (5) Tris, EDTA, Proteinase K (*NO DETERGENT) 100 mM Tris, pH 9.0 1 mM EDTA 1 ug/ml Proteinase K 103 All materials used should be RNAase free. Solutions and materials should be autoclaved twice or baked, if possible. (1) Add at least 50 ug of virus to the tube in at least 100 pl of total volume with buffer. (2) Add bentonite to at least a final concentration of 1 ug/ml. (3) Use dissociation buffer of choice. (4) Add Proteinase K. (5) Leave on bench top for 20 minutes. (6) Extract three times with equal volume of phenolzchloroform:isoamyl (50:48:2), pH 6.5 to 8.0. (7) Add NaOAc to a final concentration of 0.3 M if using the Tris dissociation buffer. It is not necessary to add if ammonium carbonate is used. (8) Add 2.5 to 3 volumes of 95% to 100% ethanol. Spin down precipitate RNA._ Verify RNA by standard gel electrophoresis. 104 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Figure A.1 Visualization of the purity of ZYMV RNA isolated under different conditions. The different pH indicated are the pH of the grinding buffer used to isolate virions. Samples were separated on a 0.6% agarose gel, pre-run with ethidium bromide. The gel was run for 12 minutes at 150 volts and 65 mAmps. Lanes 1 and 14 are Lambda Hind III markers; lanes 2 - 5 are isolated whole virions: 2 is HEPES + EDTA at pH 7.5 3 is HEPES + EDTA at pH 7.0 4 is HEPES + EDTA at pH 6.5 5 is MES + EDTA at pH 6.5 lanes 6 - 9 are apparent full-length viral RNAs isolated by the standard phenol / chloroform method 6 is HEPES + EDTA at pH 7.5 7 is HEPES + EDTA at pH 7.0 8 is HEPES + EDTA at pH 6.5 9 is MES + EDTA at pH 6.5 lanes 10 and 11 were treated with RNase at 37 C, 15 min. 10 is HEPES + EDTA at pH 7.0 11 is MES + EDTA at pH 6.5 lanes 12 and 13 were treated with DNase at 37 C, 15 min. 12 is HEPES + EDTA at pH 7.0 13 is MES + EDTA at pH 6.5. p: (11 cc e] pr Ina 105 Lanes 2 and 6 show that isolation of virus in the HEPES + EDTA buffer at pH 7.5 brings along contaminating nucleic acids. 0n the other hand, virus that was isolated in buffers at pH 7.0 or pH 6.5 were free of these contaminants (lanes 3, 4, 5, 7, 8, and 9). MES was chosen as an alternate buffer because the buffering capacity of HEPES is not optimal at pH 6.5. Lanes 10 and 11 confirm that the nucleic acids that were isolated are sensitive to RNase treatment, and, therefore, are probably viral RNAs. Digestion with DNase (lanes 12 and 13) does not eliminate the nucleic acids. The degradation observed is probably due to contaminating RNases that are frequently found with DNases. In summary, the virus and viral RNA extraction procedures presented here allow for isolation of good quality viral RNAs that are free from nucleic acid contamination. Both ultracentrifugation steps have been eliminated, so that large quantities of leaf material can be processed at greatly reduced expenditure of time and materials. 106 REFERENCES Bruening, G., Ponz, F., Glascock, C., Russell, M.L., Rowhani, A., Chay, C. Resistance of cowpeas to cowpea mosaic virus and to tobacco ringspot virus. 1987. Pages 23-37 in: Plant resistance to viruses, Ciba Foundation Symposium 133. John Wiley & Sons, New York, N.Y. Grumet, R., Fang, G. 1990. cDNA cloning and sequence analysis of the 3'-terminal region of zucchini yellow mosaic virus RNA. Journal of General Virology 71:1619-1622. Luciano, C.S., Rhoads, R.E., Shaw, J.G. 1987. Synthesis of potyviral RNA and proteins in tobacco mesophyll protoplasts inoculated by electroporation. Plant Science 51:295-303. APPENDIX B Appendix B THC-1 IS RESISTANT TO APHID TRANSMISSION OF ZYMV AND PRSV-W In Chapter 1, resistances to ZYMV and PRSV-W were characterized via rub inoculation of cotyledons and/or true leaves. However, the question arises whether TMG-l also confers resistance to aphid transmission of these viruses, since transmission of these viruses in nature occurs by aphids. Aphids (Myzus persicae Sulz.) were maintained on tobacco plants (Nicotiana tabacum L. cv. Burley) in a growth chamber. Plants for aphid transmission experiments were grown to the one to two-leaf stage. Aphids were harvested by tickling their underside using an artist's paintbrush. The insects were collected in Petri plates and sealed with parafilm until feeding time (1-2 hours). Aphids were then placed on detached symptomatic zucchini leaves and allowed to feed for at least 1 minute. Those that crawled off the leaves were killed. Ten aphids were placed on each plant and allowed to feed for 2 to 3 hours. Visible aphids were removed using the artist's paintbrush and squashed by hand. Plants were then sprayed to drip with 5 ml/gallon 50% Malathion Emulsifiable Concentrate (Meijer, Inc., Grand. Rapids, MI) in a chemical hood, allowed to dry overnight and sprayed again the next day. Plants were placed in a random 107 108 arrangement and grown in a growth chamber set at a constant temperature of 26 C and 16 hours of light. In each experiment, 10 plants each of the resistant THG-l, susceptible WI-2757, and their F1 progeny were planted, 2 plants/6 inch plastic pot. Two plants of each genotype were mechanically inoculated as controls, the remaining eight plants were inoculated by aphids. Plants were monitored for symptom expression daily. Full symptoms took 2 to 3 weeks to appear for both ZYMV (Pickens and CT2, aphid transmissible strains as described by Bada and Grumet, unpublished) and PRSV-W. Table B.1 Resistance of TMG-l to aphid-transmitted ZYMV Exp’t 1 - ZYMV-Pickens Exp't 2 - ZYMV-CTZ N9. Plants Symptomatic ~ No. Plants S tomat c No. Inoculated No. Inoculated Genotype Rub Inoc. Aphid Rub Inoc. Aphid TMG-l 0/2 0/8 0/2 0/8. w1-2757 2/2 8/8 2/2 5/6 F1 2/2 8/8 2/2 8/8 109 Table 8.2 Resistance of TMG-l to aphid-transmitted PRSV-W Exp’t 1 - PRSV-W Exp’t 2 - PRSV-W No. Plants Symptomatic go. Plants Symptomatic No. Inoculated No. Inoculated Genotype Rub Inoc. Aphid Rub Inoc. Aphid TMG-l 0/2 0/8 0/2 0/8 WI-2757 ?/2 8/8 2/2 8/8 F1 0/2a 0/8a 0/2b 0/8b ? Symptoms were not clear. a F1 plants were stunted in growth, in comparison with the normal hybrid vigor they exhibit. b Mild symptoms were observed on lower leaves of F1 plants. These experiments demonstrate that TMG-l is resistant to ZYMV and PRSV-W when transmitted by aphids. The F1. progeny showed symptoms when inoculated with ZYMV, as was observed for mechanically inoculated F1 plants. This result supports the previous finding that resistance to ZYMV in TMG-l is recessive (Chapter 2). In contrast, the F1 progeny showed little or no symptom in response to aphid-inoculation with PRSV-W. This finding is consistent with the observation that resistance to PRSV-W in TMG-l is dominant (Chapter 2). Therefore, the resistances to ZYMV and PRSV-W in TMG-1 are effective in protecting the plant in a natural infection situation. APPENDIX C Appendix C CUCUMBER SCAB DISEASE SCREEN PROTOCOL Disease: Scab, Cladosporium rot, spot rot, spotting of cucumber, leaf blight, fruit, gummosis. Pathogen: Cladosporium cucumerinum Ellis and Arth. Culture Description: On potato dextrose agar (PDA) hyaline when young, but greenish to black with age. Microscopic Description: Mycelium: septate and pigmented. Spores: conidia mostly one-celled, some septate; colored; oblong (4.1-5 X 15.2-18.8 microns); borne on short branched dark conidiophores. 8011300 8 M.J. Havey, Department of Horticulture, University of Wisconsin, 1575 Linden Drive, Madison, WI 53706. Relative Stability: No races reported. No loss of pathogenicity after 10 years of periodic transfer on PDA. ' Variants: Sectoring occurs to give non-sporulating culture. storage and Retrieval: ~ Store at 4 C on PDA for 3 months or on PDA under sterile minieral oil for 1 yr. For retrieval aseptically remove a piece of mycelium, place on PDA, and incubate at 20 C. Inoculum Increase: Add a few mls of sterile distilled water to a PDA slant culture. Scrape with a sterile bacteriological loop to dislodge spores. Aseptically remove a loopful of spores and streak over entire surface of PDA slant Incubate at 20 C. Inoculum Preparation: Add a few mls of distilled water to a 3-5 day old scab culture grown on a PDA slant. Scrape with sterile bacteriological loop to dislodge spores. Filter through a single layer of cheesecloth to remove mycelial fragments. 110 111 Quantification: Method 1: Count spores with a hemacytometer. Adjust spore concentration to 4 X 105 spores/ml with distilled water. Method 2: Count spores. Adjust concentration to 2 X 105 spores/ml. Method 3: Dilute with distilled water until water until spore suspension is light green in color when held to the light. Check percent germination of spore suspension on water agar after 24 hr. Inoculun Distribution and Delivery: Method 1: Spray hypocotyl of plant in cotyledon stage with inoculum (4 X 105 spores/ml) using an airbrush. Method 2: Using a Pasteur pipette, place a 0.01-0.03 ml droplet of inoculum (2 X 10 spores/ml) on cotyledon when cotyledons are just expanded. Inoculating cotyledons beyond this stage may give a resistant reaction on susceptible plants. Method 3: Spray inoculum (light green in color) on growing point and young leaves of plants in cotyledon stage up to the fifth leaf stage. Some growing points of resistant plants may be damaged using this method. All seedlings should be marked, by punching the tip of the cotyledon with a Pasteur pipette, at inoculation so late germinating, uninoculated seedlings can be distinguished from resistant plants. Host: Cucumis sativus L., cucumber. Source of resistance: Wisconsin SMR18. Differentials - Controls: Susceptible check - Straight 8. Resistant check - Wisconsin SMR18. 112 ' Growth of Host: Cucumber seeds are sown in steam sterilized coarse grade vermiculite in wooden flats (52 X 36 X 7 cm). Each flat contains 10 rows, 25 seeds/row. Resistant and susceptible checks are sown in row 6. The flats are placed on a heated germination bench. Vermiculite temperatures of 32 C ensure rapid and uniform germination. Newspaper on top of the flat prevents cooling by evaporation. Newpaper is removed when seeds germinate. If inoculating plants in the true leaf stage, transplant 2 wk old seedlings to steam sterilized soil in 4 inch plastic pots. Soil composed of sand:peat: field soil:field compost (1:1:1:1). Fertilize plants in 4 inch pots once/wk. Tissue Age: Methods 1 & 2: Inoculate plants when cotyledons are just expanded. Method 3: Spray inoculum on plants in cotyledon stage up to the fifth leaf stage. Postinoculation Environment: Incubate plants at 20 C in the dark for 48 hr at 100% relative humidity. If leaves become watersoaked, leaf tissue on older plants may collapse and resistant plants appear susceptible. After incubation, grow plants at 20 C. Warmer temperatures can cause a resistant reaction on susceptible plants. Disease Response: Plants are rated as susceptible or resistant 5-7 days after inoculation. Method 1: The hypocotyl of susceptible plants is girdled or has sunken necrotic lesions. Resistant plants show no reaction or, rarely, a faint watersoaked lesion. Method 2: A sunken lesion develops on the cotyledon of susceptible plants. Resistant plants show no reaction or a glossy spot where inoculum was applied. Method 3: The growing point of susceptible plants is killed and young leaves have necrotic lesions. Resistant plants remain healthy, though some damage to the growing point may occur. Disease Rating Scale: Hypocotyl tissue - no symptoms blisters on hypocotyl restricted tan lesion on hypocotyl small tan lesion(s) on hypocotyl large sunken lesion(s) on hypocotyl dead \o~Jmc»hIo II II II II II I 113 Cotyledon tissue 0 = no symptoms 1 = glossy spot 3 = glossy spot with necrotic flecks 5 = necrotic flecks surrounded by chlorosis 7 = small necrotic lesions surrounded by chlorosis 9 = large sunken necrotic lesion 10 = dead due to inoculum runoff onto growing point or hypocotyl Growing point no symptoms growing point damaged, cotyledons no symptoms growing point dead, cotyledons no symptoms growing point dead or damaged, cotyledons lesions growing point dead or damaged, one cotyledon dead dead DQUIUHO II II II II II Multiple Inoculation: Methods 1 and 2: Simultaneous inoculation with anthracnose, downy mildew, angular leaf spot, or bacterial wilt. Subsequent inoculation with cucumber mosaic virus (CMV) and powdery mildew. With Method 2, scab resistant seedlings inoculated with C. cucumerinum and Colletotrichum orbiculare (anthracnose) had fewer lesions and less disease severity than seedlings inoculated with C. orbiculare alone. Method 3: Previous inoculation with anthracnose, downy mildew, angular leaf spot, bacterial spot, bacterial wilt, CMV, and powdery mildew. Saving Host: Method 2: Both resistant and susceptible seedlings can be transplanted to steam sterilized soil. Methods 1 and 3: Only resistant plants survive. Some of the information in this handout was obtained from: Abul-Hayja, Z.M. 1975. Multiple disease screening and genetics of resistance in cucumber. Ph. D. thesis. University of Wisconsin-Madison. 149 pp. Mary J. Palmer Department of Horticulture University of Wisconsin 1575 Linden Drive Madison, WI 53706 5-07-90 APPENDIX D APPENDIX D CUCUMBER FUSARIUM WILT DISEASE SCREEN PROTOCOL Disease: Fusarium wilt of cucumber, cucumber wilt, foot-rot of cucumber. Pathogen: FUsarium oxysporum (Schlect.) Synd. and Hans. F. sp. cucumerinum Owen. Culture Description: On potato dextrose agar (PDA) white mycelium, purple pigment usually develops with age. Microscopic Description: Mycelium: septate. Spores: macroconidia-crescent shaped; multiseptate; microconidia-single celled; oval chlamydospores - resting structure; thick cell wall. Source: . American Type Culture Collection; 12301 Parklawn Drive, Rockville, MD 20852. Races 1 through 3 available. M. J. Havey, Department of Horticulture, University of Wisconsin, 1575 Linden Drive, Madison, WI 53706. Race 2 available. Relative Stability: Three races reported. Probably loses pathogenicity after periodic transfer on PDA. Variants: No information. Storage and Retrieval: Store in sterile soil at 4 C for several years. Store on PDA or PDA under sterile mineral oil for 3 months. For retrieval, aseptically remove soil or mycelium, place on PDA, and incubate at 24 C. Inoculum Increase: Place a piece of mycelium in 50 ml of potato dextrose broth (PDB) in a 125 ml Erlenmeyer flask. Shake on a rotary shaker 26-30 C for 3-6 days. Inoculum Preparation: Comminute fungus and PDB in Waring blender at low speed for 30 sec. 2 times. Centrifuge at 3,000 rpm for 10 min. to wash the spores. Discard supernatant. Resuspend pellet in distilled water. 114 115 Quantification: Count spores with a hemacytometer. Check percent germination of spore suspension on water agar after 24 hr. Inoculun Distribution and Delivery: Fill metal pan (50 X 29 X 11 cm) with silica sand. Weigh sand. Need 105 spores per gram sand. Calculate ml of concentrated inoculum needed per pan. Mix inoculum with deionized water to 1,500 ml volume Add to sand. Mix thoroughly in a large mising pan. Return sand to metal pan. Make 8 X 1.5 cm furrows. Plant 20 seeds per row. Row 5 contains resistant and susceptible checks. Host: Cucumis sativus L., cucumber. Source of Resistance: Wisconsin SMR18. Differentials - Controls: Susceptible check Straight 8. Resistant check Wisconsin SMR18. Differentials for races: Race 1 MSU 8519 S MSU 441034 (Chipper) S PI 390265 R mcnxiw ”:02!“ R = resistant, S = susceptible. Include cucumber cultivar Ashley to check pathogenicity of race 3. Reference for races: Armstrong, G.M., J.K. Armstrong, and D. Netzer. 1978. Pathogenic races of the cucumber — wilt Fusarium. Plant Disease Reporter 62:824-828. Growth of Host: See inoculum distribution and delivery section. Water with tap water or fertilizer daily. Photoperiod 12 hr. light, 12 hr. dark. Tissue Age: Seeds are sown in infested sand. See inoculum distribution and delivery section. Postinoculation Environment: Place pans in Wisconsin temperature tanks. Sand temperature 28 C. No light first 2 days to prevent drying. Disease Response: Plants are rated on a scale from 0 to 9 three weeks after inoculation. 116 Disease Rating Scale: no symptoms hypocotyl browning, no wilt, no stunting cotyledon lesion, no wilt, no stunting slight wilt, stunted severe wilt, stunted dead \DQUIUI-‘O Plants rated 0 are classified as resistant, 1 or 3 as intermediate, 5, 7, or 9 as susceptible. Multiple Inoculation: No information. Experiments planned. Saving Host: Using this method, resistant and possibly intermediate plants can be transplanted to steam sterilized soil. Mary J. Palmer Department of Horticulture University of Wisconsin 1575 Linden Drive Madison, WI 53706 5-07-90 "711111111111171,1410“