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DATE DUE DATE DUE DATE DUE |___J| MSU Is An Affirmative Action/Equal Opportunity Institution email! 37 , GENETIC ENGINEERING OP POTYVIRUS RESISTANCE USING CONSTRUCTS DERIVED FROM THE ZUCCHINI YELLOW ' MOSAIC VIRUS COAT PROTEIN GENE BY GUOWEI FANG A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Plant Breeding and Genetics Program and Department of Horticulture 1992 ABSTRACT GENETIC ENGINEERING OF POTYVIRUS RESISTANCE USING CONSTRUCTS DERIVED FROM THE ZUCCHINI YELLOW MOSAIC VIRUS COAT PROTEIN GENE BY GUOWEI FANG Zucchini yellow mosaic virus (ZYMV) is an extremely aggressive potyvirus, it causes major losses in cucurbit crops (cucumbers, squashes and melons), and is capable of out- competing related viruses in establishing infection. This_ dissertation describes the genetic engineering of resistance to ZYMV and other potyviruses using constructs derived from the ZYMV coat protein gene. The ZYMV coat protein gene was cloned, sequenced, engineered into a form for expression in plants, introduced into muskmelon and tobacco plants, and shown to confer increased resistance to potyvirus infection. The cDNA sequence of a portion of the putative RNA polymerase gene, the complete coat protein gene and the 3’ untranslated region were determined. The predicted amino acid sequence indicated a ZYMV-unique N-terminal region and potyvirus-characteristic central and C-terminal region. The ZYMV coat protein has 279 amino acids and a calculated Mr. of 31 kd. In order to produce transgenic plants that express_viral capsid protein, an efficient Agrobacterium tumefaciens mediated transformation and regeneration system was developed GUOWEI FANG for melon (Cucumis melo L.). The factors affecting transformation efficiency were studied. Three versions of the ZYMV coat protein gene were engineered for expression in plants: the full length coat protein sequence (CP), the conserved core portion of the gene (Core) and an antisense version (AS). These constructs were introduced into muskmelon and tobacco plants, gene expression was verified by PCR, northern and western analysis. Transgenic lg muskmelon plants expressing CP, Core or AS and inoculated with ZYMV showed a lack or delay of systemic symptoms, and. little or no virus accumulation as determined by ELISA. Similar resistance was displayed in R1 transgenic melon plants expressing CP or Core. Furthermore, transgenic R0 and R1 tobacco plants expressing the CP, Core or A8 constructs of ZYMV, a non-pathogen of tobacco, showed a delay in symptom development and reduced virus titer when inoculated with the heterologous potyviruses, PVY and TEV. The transgenic tobacco plants were not protected against the non-potyvirus, TMV. ACKNOWLEDGEMENTS I would like to express sincere appreciation to the members of my guidance committee, Drs. Michael Thomashow, Donald Ramsdell, James F. Hancock, Amy F. Iezzoni, and Rebecca Grumet, for their helpful advice and counsel during the course of my studies here at Michigan State University, and for their efforts in correcting and editing this dissertation. I am especially indebted to Dr. Rebecca Grumet, my major advisor, for her constant academic guidance and technical assistance in the entire program; it would be extremely difficult for me to complete this work without her encouragement and support. I also wish to thank Ms. Sue Hammer, whose assistance and expert technical support is greatly appreciated. Special depth of gratitude and love to my wife Bong Wang for her continuous support, understanding and patience; to my parents and Paul, my lovely son, for their help and sacrifices . throughout the course of my studies. iv TABLE OF CONTENTS LIST OF TABLES.......................................... vii LIST OF FIGURES........................................ viii LIST OF ABBREVIATIONS..................................... x LITERATURE REVIEW......................................... 1 LITERATURECITED....................................... 27 CHAPTER ONE: AGROEACTERIUM TUMERACIENS MEDIATED TRANSFORMATION AND REGENERATION OF MUSKMELON PLANTS...... 39 INTRODUCTION .......................................... 40 MATERIALS AND METHODS... ......... ...................... 41 RESULTS AND DISCUSSION................................. 44 LITERATURE CITED....................................... 55 CHAPTER Two: cDNA SEQUENCE ANALYSIS OF THE 3' TERMINAL REGION OF ZUCCHINI YELLOW MOSAIC VIRUS .................. 57- INTRODUCTION........................................... 53 MATERIALS AND METHODS.................................. so RESULTS AND DISCUSSION................................. 62 LITERATURE CITED ...................................... 70 CHAPTER THREE: GENETIC ENGINEERING OF THE zyuv COAT PROTEIN GENE FOR EXPRESSION IN PLANTS ................... 75 INTRODUCTION .......................................... 76 MATERIALS AND METHODS.................................. 81 RESULTS................................................ 85 DISCUSSION 0000...... ..... . ..... . 000000000000 .00....00. 99 LITERATURE CITED 0.00.00.00.000.00......00...0.00.0... 101' CHAPTER FOUR: PLANTS EXPRESSING CONSTRUCTS OF THE ZYMV COAT PROTEIN GENE ARE PROTECTED AGAINST ZYMV AND HETEROLOGOUS POTYVIRUSES ............................... INTRODUCTION ........ ........... . ....... .............. MATERIALS AND METHODS ................................ RESULTS .................................;............ DISCUSSION............................................ LITERATURE CITED ..................................... APPENDIX 000000000000000 . ........... 00.000.000.0000000000 vi 106 107 109 113 127 133 137 LIST OF TABLES Table Page 1. Effect of A. tumefaciens inoculation time on callus and shoot production by melon explants cultured on MR medium with 50 mg/l kanamycin.......... 46 Effect of co-cultivation period on callus and shoot production by melon explants cultured on MR medium with 75 mg/l kanamycin...................... 46 Summary of dot blot data of progeny of transgenic regenerants................................ 52 Percentage amino acid sequence homology between the ZYMV coat protein and other potyvirus coat proteins... 68 Segregation ratios in R,tmansgenic tobacco plants ... 97 PVY symptom development and virus titer in R0 transgenic tObacco p1ants00000.00.00.00.00000000.0.00.0...000... 121 Relative virus levels of R, transgenic tobacco plants inoculated with PVY or TEV ................... 123 vii LI ST OF FIGURES Figure Page 1. Potyvirus genomic map and putative functionsofgeneproducts.............................. 22 2. Cotyledon explants of melon cultured onmediumwith75mg/lkanamycin........................ 47 3. Southern blot hybridization analysis of genomic DNA from putative transgenic melonp1ants......0.0.0.................0..0.00.00.00.00 so 4. Sample leaf callus assay for expression OfNPTIIgeneO0.00.0.0.00000000000000000000.00.0000... 53 5. The cDNA sequence and predicted amino acid sequence of the 3’ terminal 1546 nucleotideonYMV..................... ..... . .......... 63 6-A. Comparison of the C-terminal region ofpotyviralreplicasegenes ...... 65 6-8. Comparison of potyviral coat protein sequences. ........ 0 ....... ... ...... 00.000.000.000000000 67 7. Genetic engineering of the ZYMV CP gene forexpreSSioninplants...0.0.0.0000000000000......86' 87 8. In vitro transcription and translation of theZYMVCPandcoregenes......0........ ....... 0...... 89 9. Three versions of the ZYMV’CP gene ..... .............. .. 90 10. PCR amplified ZYMV CP DNA fragments from transgenic plants......................... ..... . ....... 93 11. Accumulation of transcripts of ZYMV CP gene constructs in transgenic plants. . . . . . ........ . . . . . 94 12. Detection of ZYMV CP and Core protein intransgenicmelonplants............................. 95 13. ZYMV symptom development in R0 transgenic melon plantSOO0............OOOOOOOOOOOOOOOOO00.0.00... 114 14. Comparison of ZYMV disease symptoms in non-transformed control and CP+ transgenic melonplants...00.0.0.0...OOOOOOOOOOOOOOOO0.000.00.... 115 viii 15. 16. 17. 18. 19. ZYMV accumulation in R0 transgenic melonplantSOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO. 11 ZYMV symptom development in transgenic R, melonplants...............00........0......000......119 PVY symptom development in transgenic R, tObaCCOIines....0......0......0.0...0....0.......... 122 TEV symptom development in transgenic R, tObaCCOIineSOOOOOO ........ .0...00.0.0000000000000000125 TMV symptom development in transgenic R, tObaCCOIineS..........000...0.0.0.000000000000...... 126 ix LIST OF ABBREVIATIONS ABA - abscisic acid; BAP - 6-benzylaminopurine; IAA - indole 3-acetic acid; 2,4-D - 2,4-dichlorophenoxyacetic acid; NPT II - neomycin phosphotransferase II. CP - coat protein Potyviruses Zucchini yellows mosaic virus Watermelon mosaic virus 2 . Watermelon strain of papaya ringspot virus Tobacco etch virus Tobacco vein mottling virus Potato virus Y Plum pox virus Johnson grass mosaic virus (formerly Johnson grass JGMV strain of sugar cane mosaic virus) Other viruses Tobacco mosaic virus Alfalfa mosaic virus Potato virus X Brome mosaic virus Cowpea mosaic virus Tobacco rattle virus ZYMV WMV-Z PRV-W TEV PVY PPV JGMV A1Mv pvx BMV Cva TRV LITERATURE REVIEW INTRODUCTION The development. of 'molecular biology and. plant transformation technologies has made it possible to develop genetically engineered virus resistance in plants. A highly successful strategy has been to use viral coat protein (CP) genes as a source of resistance genes (Beachy et al., 1990; Grumet 1990). My project was to genetically engineer resistance to a group Of potyviruses that infect cucurbit plants using viral coat protein genes. The cucurbit family includes some very important vegetable and fruit crops (e.g. melons, cucumbers, squashes) , that are subject to serious yield losses due to infection by three members of the potyvirus group, zucchini yellow mosaic virus (ZYMV), the watermelon strain of papaya ringspot virus (PRV-W) and watermelon mosaic virus 2 (WMV-Z). WMV-z and PRV-W have been observed in cucurbits since the 1950's and continue to cause serious outbreaks (Davis and Mizuki, 1987; Nameth et a1. 1986) . ZYMV, however, is a relatively new and extremely aggressive member of the potyvirus group. TO engineer plant virus resistance, two factors are necessary: (1) an efficient transformation and regeneration system for the species of interest and (2) knowledge of the genomic organization of the virus to facilitate cloning and engineering of the CP gene into a form that can be expressed in the target plants. My literature review is therefore focused on ’plant transformation 2 7 3 technologies, CP-mediated virus protection and potyvirus biology. A. PLANT TRANSFORMATION The stable introduction of foreign genes into plants represents one of the most significant developments in a continuum of advances in agricultural technology that includes modern plant breeding, hybrid seed production, farm mechanization, and the use of agrichemicals to provide nutrients and control pests (Gasser and Fraley, 1989). The first transgenic plants expressing engineered foreign genes were tobacco plants produced by use of Agrobacterium tumefaciens vectors (Horsch et al. 1984). Transformation was confirmed by the presence of foreign DNA sequences in both primary transformants and their progeny, and by an antibiotic resistance phenotype conferred by a Chimeric neomycin phosphotransferase (NPT) gene. The subsequent development of transformation methods has contributed significantly to the facile and routine Agrobacterium transformation methods that are used today for many dicotyledonous plant species. In addition to the use of Agrobacteria as transformation vectors, a variety of free DNA delivery methods, including microinjection, electroporation, and particle gun technology are being developed for the transformation of monocotyledonous plants such as corn, wheat and rice. In this section, several plant transformation methods are described. 4 1- Wm Insertion of foreign DNA into A. tumefaciens derived plasmid vectors is currently the most frequently used strategy for generating transgenic plants in a wide variety of species (Potrykus, 1991) . Agrobacterium tumefaciens is the etiological agent of crown gall disease and produces tumorous crown galls in infected plants (Chilton.et al., 1977). The utility of this bacterium as a gene transfer system was first recognized when it ‘was demonstrated that. the crown. galls were actually produced as a result of the transfer and integration of genes from the bacterial Ti (tumor inducing) - plasmid into the genome of the plant cells (Chilton et al. 1977) . Incorporation of the transferred DNA (T-DNA) into the nuclear genome of the plant cell and its resultant expression causes the pathogenic response characteristic of the transformed cell. Since then, a variety of studies have led to the development of Ti based vectors for use in efficient and widely used strategies to generate transgenic plants. One of the most important findings facilitating the practical. utilization of the Agrobacterium-Ti system was that disarmed T-DNA, that is T-DNA lacking functional oncogenes, can be transferred and integrated into the plant genome, allowing for the regeneration of transgenic plants (Barton et a1. 1983). In fact, any DNA fragment (size limitation not known) can be transferred from a Ti plasmid as long as it is between the 25 bp direct repeats, called borders, that flank the wild type Ti—plasmid T-DNA (Slightom 5 et al. 1986, Binns and Thomashow 1988). The T-DNA insertion appears to be stable. It has been characterized genetically for a number of generations and behaves as a normal Mendelian, trait (Wallroth et al. 1986). The virulence (vir) genes of the Ti plasmid are also required for T-DNA transfer, which can function in trans to process and transfer T-DNA present on a separate plasmid (Stachel and Nester 1986;, Hoekema et al. 1983). This led to the development of small, easy to handle, binary vectors that can replicate in both E. coli and A. tumefaciens. These ibinary ‘vectors carry' plant. selectable marker(s) (eg. NPT gene), plant scorable marker(s), and various cloning sites, all of which are flanked by the border repeats (Binns 1990). The major advantage of binary vectors is their lack of dependence on a specific Ti plasmid. The vector, may be introduced into virtually any Agrobacterium host containing any Ti plasmid as long as the vir helper functions are provided. There are two basic approaches which have been used to obtain transgenic plants: co-cultivation of regenerating protoplasts (Horsch et al. 1984), and the leaf disc procedure (Horsch et al. 1985). Co-cultivation of regenerating protoplasts was the first procedure used successfully to generate a transgenic plant. The procedure involves Agrobacterium co-cultivation of protoplasts, followed by selection and regeneration to plants. Because it requires a good regeneration protocol for protoplasts, this procedure is 6 .not useful for many important plant species. In the leaf diSC‘ procedure, surface-sterilized leaf pieces, or other axenic explant, are co-cultured on regeneration medium for 2-3 days 'with.Agrobacterium. The best choice of explant is usually one that regenerates well in tissue culture for the species of interest, such as stem or petiole segments, shoot, leaf or cotyledon pieces, tuber disks, root pieces or seed (Hooykaas 1989). During co-cultivation, bacteria bind to plant cells around.the wounded explant, and T-DNA transfer occurs. A.nurse culture Of tobacco cells is sometimes used to increase transformation frequency. Following co-cultivation, the explant are transferred to regeneration/selection medium. This . medium contains antibiotics to kill the Agrobacterium and to select for transformed plant cells. After shoots regenerate, they are excised, rooted on an appropriate medium in presence of the selective agent, and transferred to soil. The leaf disc-based procedures are a great improvement overprotoplasts co-cultivation, because it is not necessary to produce and ' regenerate protoplast. I Although Agrobacterium species are capable of transferring new genes to a wide variety of plant species, their practical use is mainly limited to natural hosts, mostly dicotyledons, even though transgenic plants have been produced in asparagus. with Agrobacterium vectors (Bytebier et al. 1987). Important cereal grains such as rice, corn, and wheat have not been successfully transformed by Agrobacterium. It has. been. 7 suggested that monocots are difficult to transform because they do not have the proper wound response (Potrykus 1991). Extensive efforts have consequently been directed toward the development of systems for theidelivery Of free DNA into these species. . 2-W The systems giving demonstrable transformation of plant cells relied on physical means similar to those used in the transformation of cultured animal cells. Transformation has been achieved in plantprotoplasts through facilitation of DNA . uptake by calcium phosphate precipitation, polyethylene glycol (PEG) treatment, electroporation, or combinations of these treatments (Potrykus et al. 1985; Fromm et al. 1986; Uchimiya et.al. 1986). Integrative transformationican.be very efficient and lead to stable inheritance of predominantly single gene loci of the foreign genes (Negrutiu et al. 1987), but it often leads to multiple integrations (Potrykus, 1991). Transgenic monocots such as rice (Shimamoto et al. 1989), maize (Rhodes et al. 1989), and other cereals (Lorz et al, 1985)have been produced by direct gene transfer in several laboratories. Virtually everyprotoplasts system has proven transformable,h though with different efficiencies (Potrykus 1991). It would be the ideal experimental system for gene transfer if there were not problems with plant regeneration from protoplasts. Although there has been exciting progress recently in plant regeneration from protoplasts (Roest and Gilissen 1989), and 8 although it can be foreseen that this progress will continue, this process will probably always be a delicate one (Potrykus 1991). 3. W The biolistic process is another direct gene transfer method, the process can be defined as the introduction of substances into intact cells and tissues through the use of high-velocity':microprojectiles (Sanford. 1988,1990). It. is primarily a mechanism for breaching cell walls and cell membranes, which are the principal barriers to DNA delivery. By the use of a finely tunable discharge apparatus~ (eg.particle gun), DNA coated metal particles such as tungsten or gold are used to transform plant tissue (Christou et al. 1988, 1990). One of the most important features of the biolistic process is that it is of broad utility, and may prove to be something of a "universal" gene delivery mechanism. It has been demonstrated in a very diverse range of plants as well as microbes and animals (for review, see Sanford 1990). There is no apparent difference in the efficiency of biolistic transformation of monocots vs dicots. It is also effective regardless of tissue and cell type, and is a rapid and simple procedure. In addition, the biolistic process appears to be uniqUely suitable for organelle transformation. Transgenic plants have been recovered from species that have been difficult to be transformed by other methods, such as maize 9 and soybean (Fromn et al. 1990, NcCabe et al. 1988). Some expected that this technique would solve all gene transfer problems, however, limitations do remain. When bombarding totipotent tissues, the process Often gives rise to chimeric plants, which need to be sorted out and stabilized; and the. technique is less efficient than other transformation methods in yielding stable integrative events. 4- W Microinjection uses microcapillaries and microscopic devices to deliver DNA into defined cells in such a way that the injected cell survives and can proliferate. This technique has produced transgenic clones from protoplasts (Miki et al. 1987) and from microspore-derived pro-embryos (Neuhaus and Spangenberg 1990). Like biolistics, microinjection delivers DNA into walled plant cells, and is host range independent. The differences from other direct DNA delivery methods are:, DNA delivery is precise and predictable, is under visual control, and the quantity of DNA delivered can be optimized. Subsequent individual culture of the microinjected cells in microculture systems offers the possibility of avoiding selection and thus the requirement for selectable marker genes. The limitations of the method are: only one cell receives DNA per injection, handling requires more skill and instrumentation, and it is time consuming. All of the above four approaches have led to the jproduction of transgenic plants (Agrobacterium-mediated gene 10 transfer, direct gene transfer to protoplasts, biolistics and microinj ection) ; of them Agrobacterium mediated gene transfer, direct gene transfer toprotoplasts and the use of biolistics are routine and efficient methods. Other approaches that have potential for integrative transformation are: agroinfection (Grimsley 1990) , viral vectors (Ahlquist and Pacha 1990) , macroinjection (DelaPena et al. 1987), electrophoresis (Alwen et al. 1990), liposome fusion (Caboche 1990), and the use of microlasers (Weber et al. 1990). Most of these require further Optimization and improvement, and stability of the integrative transformation events needs to be proven. Muskmelon (Cucumis melo L.) is a high value and important ' crop throughout the world. It is subject to severe losses by several viruses (Nameth et al.1985) , and so the genetic engineering of virus resistance is a primary goal for this crop (e.g. Grumet and Fang, 1990; Gonsalves et al.1991). As described above, Agrobacterium tumefaciens has been the most commonly used vector for the transfer of foreign genes into dicotyledonous plants. Since muskmelon is a suitable host for A. tumefaciens, and efficient regeneration from tissue culture has been achieved in several laboratories (eg. Niedz et al. 1989; Kathal et al.1988), one project of my research was to develop a crop specific protocol for Agrobacterium mediated- transformation in muskmelon. 11 B. COAT PROTEIN MEDIATED PROTECTION AGAINST VIRUS INFECTION Dramatic progress in the development of plant transformation and regeneration systems as a mechanism for the expression of novel genes, has allowed for the genetic engineering of plants for crop improvement. One of the most promising applications has been the development of genetically engineered resistance to plant viruses. Virus resistance was first shown for tobacco mosaic virus (TMV) in tobacco. plants (Powell et al. 1986); it was found that transgenic tobacco plants expressing the coat protein gene from TMV showed a significant delay in symptom development after inoculation with TMV. Coat protein-mediated protection has now been demonstrated for a number of different viruses from at least eight different virus groups, such as the tobamo-, potex-, cucumo-, tobra-, carla-, poty—, and alfalfa mosaic virus groups (for review see Beachy et al. 1990; Grumet 1990) , and more recently the luteovirus group (Kawchuk et al. 1990, 1991) . The list is growing rapidly. Virus inoculated leaves of the transgenic plants show fewer chlorotic or necrotic lesions relative to control plants, and systemic spread of infection is either prevented, delayed or reduced (Cuozzo et al. 1988; Hemenway et al. 1988; Hoekema et al. 1989; Hector et al. 1991; Kallerhoff et al. 1990; Kawchuk et al. 1990; Nelson et al. 1987,1988; Powell et al. 1990; Tuner 1987; Van Dun et al. 1988;) . NO apparent negative effects on growth, fertility or physical appearance has been observed in the plants 12 expressing viral coat protein genes. The resistance trait can be stably transmitted over several generations. Practical application of this technology is promising; several transgenic crops have been protected in field experiments under conditions of high infection pressure ( e.g. Nelson et al.1988; Gaser and Fraley, 1989). The coat protein genes have also been introduced into plants in antisense orientation for the purpose of genetically engineering viral resistance (Cuozzo et al. 1988; Hemenway et al. 1988; Kawchuk et a1. 1991; Lindbo and Daugherty 1992; Powell et al. 1989). In all these cases, plants expressing antisense RNA were less susceptible to virus infection than were control plants, but the protection levels were variable. In the case of cucumber mosaic virus (Cuozzo et al. 1988), viral. coat protein.genes were transferred into plants in both orientations for producing sense and antisense RNA. Protection was observed only at 'low inoculum concentration and to a limited extent in transgenic plants expressing antisense ' RNA. In contrast all CP expressing plants were significantly. protected in all inoculum concentrations. Similar results were obtained with potato virus x (Hemenway et al. 1988) and TMV (Powell et al. 1989). However, in plants producing positive- or negative-sense CP RNA of potato leafroll virus (PLRV), both the pattern and level of protection were the same'(Kawchuk et al. 1991). The reason for the difference between PLRV and the ' above viruses is not known, but unlike PVX, CMV or TMV,.which. 13 occur at very high titer, PLRV occurs at low titer and is confined to the phloem of the host plant. Similarly, Lindbo and Daugherty (1992) reported. recently’ that. plant lines expressing either sense defective RNA or antisense transcripts of tobacco etch virus (TEV) CP gene were highly resistant and showed greater protection than the plants producing the full- length CP. Broad spectrum resistances have been observed in CP- mediated protection. Transgenic tobacco plants expressing the 1 coat protein Of TMV or alfalfa mosaic virus have a low but significant degree of protection against other tobamoviruses (Nejida and Beachy 1990), or unrelated viruses such as PVX, PVY, and CMV (Anderson et al. 1989). Tobacco plants accumulating coat protein of the potyvirus soybean mosaic virus (SMV) , a non-pathogen on tobacco, were resistant to infection by two serologically unrelated potyviruses that are pathogens of tobacco, PVY and TEV (Stark and Beachy 1989). A similar result was reported by Ling et al (1991) in tobacco plants expressing papaya ringspot virus CP gene. The degree of heterologous protection could be of considerable agronomic ' benefit by enabling a plant to be protected against many viruses using a limited number of different CP genes. It would be especially useful for resistance to potyviruses, which form the largest, most widely distributed and economically important group of plant viruses (Hollings and Brunt 1981). Many hosts are infected by several different potyviruses. 14 There are several necessary steps to genetically engineer CP-mediated protection in plants (for review see Beachy et al. 1990; Grumet 1990). First, the cDNA that represents the CP Open reading frame (ORF) must be cloned. This is relatively straightforward for viruses with CPs that. are encoded by subgenomic RNAs. However, for viruses such as those in the potyvirus group, were the CP is part of a large polyprotein (Dougherty and Carrington 1988), defining amino terminals of the CP gene can be somewhat more difficult. Second, the CP gene must be identified, sequenced and isolated. Third, the CP ORF must be linked to an appropriate transcriptional promoter, since viral RNAs do not contain promotors capable of transcription in plant chromosomes. The most commonly used promoter for controlling expression of the CP gene in transgenic plants is CaMV35$ promoter, which is a strong, constitutive promoter derived from cauliflower mosaic virus (CaMV) (Guilley et al. 1982). The last step, which can be the most challenging, is the introduction of the gene constructs and regeneration of modified plants. At this time, most examples of resistant plants were produced via Agrobacterium- mediated gene transfer (for detail see first section), largely because these plants were responsive to this transformation system; many other plants including the important vegetable and.Cereal plants are still difficult to be transformed and/or regenerated. For these plants, a model system such as tobacco may be useful for testing CP gene constructs. 15 Although CP-mediated protection has been demonstrated in several virus groups and plant systems, the mechanism responsible for the protection is not yet well understood. The discovery that expression of viral coat protein(CP) in transgenic plants protects these plants against virus infection is in some ways, a natural extension of work by. previous researchers on cross-protection, and it was termed genetically engineered cross-protection (Powell et al. 1986; Turner et al. 1987). For a number of years, agriculturalists have inoculated plants with mild strains of viruses or viroids tijrevent.more virulent strains. from infecting the plant and causing severe disease symptoms. The practice referred to as "cross-protection" has been used to reduce yield loss in some important crops such as tomatoes, potatoes and citrus, due to tomato mosaic virus (ToMV), potato spindle tuber viroid, and citrus tristeza virus respectively (see reviews by Fulton 1986; Urban et al. 1990). There are potential«disadvantages to ' the widespread use of cross protection in agricultural fields. First, the mild strain (protecting strain) might undergo mutation to highly virulent form which could be selected during replication, thereby leading to extensive crop losses rather than protection. Second, the protecting strain might act in synergism with a non-related virus resulting in a disease condition that is more severe than that caused by 'either virus alone. Third, a protecting virus in one plant might be a severe pathogen to another species in field 16 situation. Fourth, the protecting strain may itself cause a small but significant loss in yield (Hamilton 1980). Most or all of these objections, however, could be overcome if cross- protection was engendered in plants as the result of expression of a single viral gene, rather that as a result of infection with an intact virus. A single induced gene is unlike a whole virus, it is not capable of self-replication, multiplying to high levels, or spreading from plant to plant. Sanford and Johnston (1985) predicted that a "normal" host- pathogen relationship can be disrupted if the host organism expresses an essential pathogen derived gene in excess. amounts, at the inappropriate developmental stage or in a dysfunctional form. This may disrupt the normal replicative cycle of the pathogen and result in an attenuated or aborted infection of the host. Several types of experiments indicate that CP gene expression in transgenic plants interferes with an early event in virus infection. Studies with CP expressing plantprotoplasts have yielded important information about early events leading to CP-mediated resistance. Loesch-Fries et. al(1987) reported, that CP expressing ‘protoplasts are resistant to ALMV virus infection, but not to ALMV-RNA. ’ Similarly, Register and Beachy (1988) reported that CP expressing plants and protoplasts were less protected against TMV-RNA, and against TMV virions that were briefly treated at pH 8.0 to swell the virions, than against native TMV virions. 17 According to the current model Of co-translational disassembly for TMV (Wilson 1984; Wilson and Watkins 1986), host.ribosomes bind to the 5’ end Of TMV, and actively uncoat the virus as they proceed to translate the genomic RNA. The results from the above experiments suggested that the coat protein in transgenic plants interferes with the virus life cycle by preventing uncoating of the virus. However, the mechanism of resiStance may not be the same in each case, because the various viruses tested all belong to diverse'virusigroups‘with distinct biologies. For example, transgenic plants expressing the coat protein of potato virus X (PVX) are resistant to infection by RNA as well as whole virion (Hemenway et al. 1988) . One possible explanation for the difference between TMV and PVX may lie in the difference in coating and uncoating mechanisms. There are also examples that argue against a protein-RNA role in resistance. Dun and B01 (1988) reported that, although the CPS encoded by TCM and PBL strains of TRV could encapsidate the RNA molecules of the Other strain, the CP of the TCM strain did not protect plants from infection by the PBL strain. They suggested that it may be due to reduced affinity for RNA or an altered domain involved in a function other than RNA binding. It is also possible that the coat protein interferes at more than one stage in the viral life cycle. The consistent but low level resistance against infection by TMV RNAs in GP expressing' plants (Nelson. et al. 1987), and. protoplasts 18 (Register and Beachy 1988) implies another mechanism of resistance that acts after the uncoating event(s) . It was suggested that TMV CP may play a role in regulating the replication of TMV-RNA (Osbourn et al. 1989). In most CP expressing plants, systemic infection is either prevented or delayed compared.with nontransgenic plants. This could result from interference with the spread of virus from cell to cell in inoculated tissue, the spread from inoculated leaves to uninoculated leaves (Wisniewski et a1. 1990), the egress of virus from the inoculated leaf into the vascular. tissue, movement through the vascular tissue, and/or initiation of infection in other leaves. The results from broad spectrum resistance ‘of plant expressing viral CP genes (Stark and Beachy 1989; Anderson et al. 1989; Nejidat and Beachy 1990; Ling et al. 1991) demonstrated that CP-mediated protection is not a one gene/one virus phenomenon, but rather that the expression of a single CP gene can protect a plant against several different viruses. Plants expressing potyvirus SMV CP were resistant to infection by other two potyviruses, PVY and TEV,’ but not to a tobamovirus, TMV. Protection against ’the heterologous. potyviruses is likely due to amino acid sequence homologies between the "protecting" CP (SMV) and those of the challengers, PVY and TEV (61% and 58% respectively). The failure to protect against TMV may be due to a lack of sequence homology between TMV CP and SMV CP, and/or the, .- 19 difference in life cycle strategies of potyviruses and tobamoviruses. Stark and Beachy (1989) suggested that CP- mediated protection requires structural as well as sequence homology between the protecting and challenging virus CP. This phenomenon might be also true in antisense and sense-defective ‘ CP RNA related protection, but it still needs to be proven. The mechanism of protection in plants expressing antisense or sense defective CP RNA is not clearly defined. However, several possibilities have been prOposed (Cuozzo et al. 1988; Lindbo and Dougherty 1992) . One possibility is that the antisense RNA. may inhibit viral RNA replication and/or translation through the formation of an antisense-sense RNA hybrid. Another’possibility'is that the antisense or sense RNA competes directly with viral proteins(e.g. replicase) or host factors essential for replication of virus. These mechanisms could. depend on expression levels ‘Of antisense RNA. and- inoculum concentrations. Although the reported results demonstrated that the level of antisense CP RNA related protection was variable, the antisense CP RNA was expected to provide greater protection than sense CP transcripts (Nelson et al. 1989), since they could, in theory, hybridize to both sense genomic and subgenomic viral RNAs to interfere with viral replication. To determine the applicability of the antisense RNA approach, further investigation and more tests on different viruses and plants are required. It should be indicated that most mechanistic studies to 20 date have involved TMV-GP, TMV and tobacco and tomato plants; it is very likely that other virus-host-CP combinations will’ be different from TMV system. It is important that other virus groups and different hosts be studied to develop a more complete understanding of mechanisms of resistance, and to improve and extend the CP-mediated resistance. C. POTYVIRUS AND ZUCCHINI YELLOW MOSAIC VIRUS BIOLOGY The potyvirus group is the largest known group of plant viral pathogens, there may be over 100 different members in this group (Hollings and Brunt 1981). Potyviruses have the ability to inflict damage to a wide range of crops around world; it is estimated that one quarter of the world's viruses ' that infect plants come from this group (Hollings and Brunt, 1981; Francki et al., 1985). Frequently, potyviruses are present as part of the natural pathogen population, causing chronic reductions in yield and quality (Lisa and Lecoq, 1984; David and Mizuki, 1986; Nameth et al. 1986). All members of the potyvirus group share common features. The virus particle has a flexuous rod shape and is usually 700-900nm in length and 12-15nm in diameter. The potyvirus genome is a single-stranded, positive sense infectious RNA molecule that is approximately 10,000 nucleotide in length (Allison et al. 1986; Domier et al. 1986). Potyviral RNA' contains a covalently linked protein (VPg) at the 5’ terminus (Murphy et al., 1990) and is polyadenylated at 3' terminus 21 (Hari et al., 1979). Most members of the potyvirus group are transmitted by aphids in a nonpersistent, noncirculative manner, ‘which involves. a ‘viral encoded. helper’ component protein that is thought.to mediate the binding of the virus to the aphid stylet (Thornbury and.Pirone 1983; Berger and.Pirone 1986); transmission in seed or by mites and dodder has been also reported (Hollings and Brunt 1981). Potyviruses can also be mechanically transmitted. Based on a compilation of cell-free expression studies, nucleotide sequence data, and biochemical analysis of gene_ products, the genomic organization of potyviruses has been assessed (Allison et al. 1986; Dougherty and Carrington 1988; Dougherty and Parks, 1991; Marankal and Rhoads, 1991; Verchot et al., 1991). Figure 1 presents the genetic map and putative functions of potyviral proteins. The genes in order from 5"to 3' are: I. P1 protein (31kd), a putative cell to cell.movement protein based on sequence homologies to other viral movement proteins, and a protease that perform the cleavage between P1 and P2 proteins; II. aphid helper component (56kd), that is required for aphid transmission, and is a protease performing the cleavage between P2 and P3 proteins; III. P3 protein (50kd), with unknown function; IV. cytoplasmic inclusion protein (70kd), it has been hypothesized that this protein is involved in replication as a possible RNA helicase based on sequence analysis; in addition, the cytoplasmic - inclusion protein may also be involved in long distance transport and 22 A. Potyvirus genomic nap HEM—J 3 4 5 5 7 8 9 1;; P1 HC P: <21 VP§___NIS._NIJ.__QP__AM 31kb . 56kb 50kb 711m 6kb 49kb 58kb 30kb B. Putative function of potyvirus gene products P1 P1 protein ----------------- Putative cell to cell movement protein, 5' protease HC Helper component protein---Aphid transmission, 5' protease P3 P3 protein ------------------ ?(specific virus-host interaction) CI Cytoplasmic inclution ------- ? (RNA helicase, replication, viral/host protein interactions, long distance transportation) VPg Viral genome-linked -------- RNA stability, replication, infectivity protein NIs Small nuclear inclusion ----- 3' protease protein -NIl Large nuclear inclusion ----- putative RNA-dependent RNA polymerase protein CP Coat protein --------------- Bncapsidation of viral RNA, vector transmission Figure 1. Potyvirus genomic map and putative functions of gene products 23 virus/host interaction; V. genome linked protein (VpG, 6kd),- the ‘VpG 'may involved. in replication, RNA stability‘ and infectivity; VI. small nuclear inclusion protein (49kd), the N-terminal segment is involved in RNA replication, the C- terminal half is associated with a protease responsible for the majority of proteolytic cleavages ; VII. large nuclear inclusion protein (58kd), a putative viral RNA-dependent, RNA polymerase; and finally, VIII. capsid protein (30kd), which encapsidates the viral RNA and participates in aphid transmission. The life cycle of potyviruses starts with transmission of the virus to the host cells. Once inside ‘the host plant, the virus is uncoated. Whether particles are uncoated.prior to, or after, entering the host cell, is not yet clear (Shaw et al. 1985). The single Open reading frame of the viral genome is first translated into a single polyprotein precursor (346kd) (Allison et al., 1986). Rapid autoproteolytic processing occurs after translation, giving rise to. intermediate polyprotein cleavage products and finally the~ eight mature viral proteins. This is accomplished by three proteinases that are encoded by the genome, and autocatalytically released from the polyprotein. These proteins are known as the nuclear inclusion protein (NIa) , the helper component and the P1 proteinases. NIa protease is also- responsible for the subsequent proteolytic events associated with polyprotein processing (Dougherty and Carrington, 1988; . 24 Carrington,1991). Minus RNA strands are synthesized using the viral RNA as template, and then plus strands are replicated from minus strands. The jplus RNA. strands are either translated. or encapsidated. Encapsidation is hypothesized to occur from 5' to 3' end (Allison et al. 1986; Shulka et al. 1988) in a manner similar to another virus group, the potexvirus group (Lok and Abouhaidar 1986). Virus spread from cell to cell is thought to be via the plasmodesmata (Atabekov and Dorokhov 1984). Characteristic inclusion bodies form during infection. The life cycle is completed by subsequent aphid transmisSion of progeny virions to other plants. I Studies of potyviral CPs have shown that both the N and C termini of the potyvirus coat proteins are located on the surface, and that the N termini tend to be most variable (Shukla et al. 1988). Comparisons of sequence data from several potyviral capsid genes (such as Shulkla et al. 1987; Allison.et al. 1985; Ravelonandro et al. 1988; Grumet and Fang 1990) have shown that although external domains vary, the internal domains are conserved among different potyviruses. The conserved domains have been hypothesized to be responsible for protein-protein or protein-RNA binding (Dougherty et al. 1985). Zucchini yellow mosaic virus (ZYMV) is a member of potyvirus group. It is a relatively new, but extremely aggressive and destructive virus. The virus was first 25 described and identified in Italy in 1981 (Lisa et a1); since then it has spread.rapidly throughout Europe, the Middle East, and United States, and several new strains have been identified (Lisa and Lecoq 1984; Davis 1986; Davis and Mizuki 1987). The virus causes a severe disease in many cucurbit species, such as squash, muskmelon and cucumber. Symptoms include mosaic, leaf distortion, stunting, and fruit and seed deformations. It was reported that the disease caused crop losses of 50-100% in individual fields of summer squash and melons from 1982 to 1986 in eastern, southern, midwestern and west coast states of the U.S. (Mcleod et al. 1986; Davis and Miizuki 1987; Nameth et al. 1986; Provvidenti et al. 1984). ZYMV is capable of outcompeting the other two cucurbit potyvirus (PRSV-W and WMV-Z) in establishing infection in rub inoculation (Davis and Mizuki 1987), aphid acquisition (Lecoq D and.Pitrat 1985), and field studies (Alderez 1987). The basis for its success is not yet known. Because of its aggressiveness, virulence and aphid transmission, control of this disease will probably be most effectively Obtained by the development of resistant cultivars (Davis 1986). To genetically engineer plant resistance using the viral coat protein gene, I did the following: (1) developed an efficient Agrobacterium-mediated transformation system. in muskmelon (Fang and Grumet, 1990), (2) sequenced and isolated the ZYMV CP gene (Grumet and Fang, 1990), (3) engineered three versions of CP gene for expression in plants and introduced- 26 them into melon and tobacco plants, and (4) tested the transgenic plants expressing ZYMV CP constructs for protection against ZYMV, PVY and TEV infections. I also developed a simple 'method for purification. of plant genomic DNA ‘to facilitate verification of the inserted gene in transgenic plants (Fang et al., 1992). 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Inheritance of multiple potyvirus resistance from the cucumber TMG-1 line. In Abstracts of 1991 APS Annual Meeting, 566.122 85. Wallrath,M., Gerats,A.G.M., Rogers,S.G., Fraley,R.T., Horsch,R.B. 1986. Chromosomal location of foreign genes in Petunia hybrids. Mol. Gen. Genet. 202:6-15 85. Wilson,T.M.A. 1985. Nucleocapsid disassembly and early gene expression by positive strand.RNA viruses. J. Gen. Virol. 66:1201-1207 CHAPTER ONE AGRQEACTERIUM TQMEEAQLENQ MEDIATED TRANSFORMATION AND REGENERATION OF MUSKMELON PLANTS 39 40 INTRODUCTION One of the most effective means of gene transfer into dicotyledonous plants is to utilize the natural transformation mechanism of Agngagtggium tumefagigns (Fraley et al. 1986). Genes located between the border sequences of the Agzobacterium’s Ti plasmid are inserted into the genome of the ' host. Utilization of this mechanism for gene transfer requires both susceptibility to infection by A. tumefac' s, and the ability to regenerate plants from individual transformed cells via tissue culture. Although many dicots are suitable hosts for Agzabagtezia, only a small number of species have been transformed and regenerated successfully (Gasser and Fraley, 1989). The cucurbit family includes many high value vegetable and fruit crops (cucumbers, melons, squashes). At the time that this work was completed, the only published report of successful transformation and regeneration of a cucurbit species was for cucumber (W M) using the vector A. rhizogenes (Trulson et al. 1986). Several groups have recently reported successful regeneration from muskmelon explants (e.g. Neidz et al. 1989; Dirks and Van Buggenum, 1989). In this chapter, I describe an efficient procedure for the production of transgenic muskmelon (Cugumis EQLQ) plants using the vector A. W. 41 MATERIALS AND METHODS MW: Peeled muskmelon seeds (Hale's Best Jumbo) were sterilized in 15% (v/v) Chlorox (5.25% sodium hypaclorite) with a drop of Tween 20 for ten min. then rinsed three times with sterile distilled water and placed on hormone free MS medium (Murashige and Skoog, 1962) with 0.8% agar. The tissue culture growth room conditions were 25 - 26 C with a 16 h photoperiod provided by cool white flourescent lamps (ca. 2500 lux). The Agggbagtegigm strain was disarmed.A. tumefaciens LBA4404 containing the CIBA-GEIGY binary vector pCIBlO (Rothstein et a1. 1987) with a transferable selectable marker for kanamycin resistance (the neomycin phosphotransferase gene, NPT II) .. Agngagtgzium cultures were grown and maintained an AB medium (Chilton et al. 1984) with 50 mg/l kanamycin. Inoculation and go-cultivation: Excised 4 - 5 day old cotyledons were cut on all edges with a dull scalpel blade,i soaked in a fresh overnight culture of LBA4404 + pCIB10 for 10 ' «- 60 min, blotted dry with sterile filter paper to remove excess bacteria, and then transferred to melon regeneration (MR) medium [MR medium = MS medium with 5 UM IAA, 5 uM BAP, 1 uM ABA and 3% sucrose; Neidz et al. 1989] with or without tobacco (Nigotiang tabacum) nurse cultures for a co- cultivation period of 1 - 7 days. In the experiments where tobacco nurse cultures were used, three m1 of lag stage tobacco suspension cultures grown in MS medium with 2 mg/l . 42 2,4-D and 180 mg/l monobasic potassium phosphate were pipetted onto MR plates three days prior to transformation and kept at 25 - 26 C with 16 h photoperiod. Just before co-cultivatian two pieces of, sterile #3 filter paper were laid over the tobacco cells, explants were put on the paper. n ' _ s ' n: Control melon tissue was tested for sensitivity to kanamycin using MR medium containing 0 - 200 mg/l of kanamycin; based on these results 75 mg/ml was used in transformation experiments. After co-cultivation with Agrgbagteria, explants were washed‘ with sterile distilled. water' to remove excess bacteria, blotted dry, and transferred onto MR medium containing '75 mg/l kanamycin (to select for transformed tissue) and 100 mg/l cefotoxime (to eliminate bacterial carry over). Developing shoots were transferred to hormone free, root inducing medium (MS salts with 3% sucrose, 100 mg/l cefotoxime) with or without 50 mg/l kanamycin. Regenerated plantlets were transplanted to sterile Bacto soil mix as soon as roots appeared and transferred to the greenhouse. an s: Genomic DNA was extracted from young leaf tissue of putative transformed plants using the procedure of Dellaporta et al. (1983). Southern blot analysis was performed according to Maniatis et al. 1982. Random primer Pn-labeled (Feinberg and Vogelstein, 1983), isolated NPT gene was used as probe for hybridization with the genomic DNA. To test for inheritance of the introduced NPT gene, 43 transformed melon plants were either selfed, or crossed to control plants, in the greenhouse. The progeny were examined ' for the NPT gene by dot blot analysis; genomic DNA was extracted from leaves of three week old plants, spotted onto nitrocellulose and probed with labeled NPT fragment. Explants from cotyledons of progeny were also tested for expression of the NPT II gene by culturing on MR medium with kanamycin. 44 RESULTS AND DISCUSSION Won—Wis: Several factors including kanamycin level, Agrgpagtgrium concentration, inoculation time, length of co-cultivation period, and the use of tobacco" nurse cultures uwere tested. to optimize the :melan transformation system. The sensitivity of non-transformed melon tissue to kanamycin ‘was examined using' MR. medium containing 0, 10, 25, 50, 75, 100, or 200 mg kanamycin/l. Normal callus and shoots only developed on medium without kanamycin, some callus and shoots were formed on medium with 10 mg/l. At 25 mg/l a few explants produced slowly growing callus and buds, but no shoots were formed. Kanamycin levels at or above 75 mg/l completely inhibited growth of control explants, and so this level was chosen for routine selection of transformed tissue. Bacterial concentration for inoculation is a critical variable for lettuce transformation (Michelmore et al. 1987), but has little influence on petunia transformation (Horsch et al. 1985). To examine the effect of bacterial titer on gene transfer efficiency for melon, explants were inoculated with concentrations ranging from 105- 10'0 bacteria/ml. Although there ‘was little influence of bacterial titer on transformation and regeneration, the condition of the bacterial culture was very important. Fresh bacteria grown under optimal conditions (overnight from a fresh inoculum culture, 30 C, well-aerated) gave the highest transformation. 45 efficiency. we routinely used a 15 - 24 h culture at a concentration of 107 - 10‘ bacteria/ml. To study the influence of exposure time to bacteria, explants were soaked in bacteria for either 10, 20, 30, or 60 min (Table 1). There was little difference in transformation~ efficiency up to 30 min. A 60 minute exposure reduced efficiency by 40% relative to»10:min exposure, probably due to subsequent bacterial contamination that inhibited explant growth. Ten minute soaks were routinely used. Co-cultivation periods of 1, 3, 5 and 7 days were tested (Table 2). Three days of co-cultivation resulted in the highest percentage of shoot regeneration on kanamycin. With longer periods it was not possible to completely eliminate the Agngagtgria. Tobacco nurse cultures are sometimes used to increase transformation.efficiency (Rogers et al. 1986). The effect of nurse cultures on melon transformation was tested after the‘ optimal inoculation and co-cultivatian times had been determined. In two of four experiments the presence of tobacco nurse cultures resulted in a 10 - 15% higher rate of shoot regeneration, in the other two experiments there was no obvious difference. Since the nurse cultures did not make a large difference in transformation efficiency they were not routinely included. W: Explants were either inoculated with LBA4404 + pCIB10, with LBA4404 without plasmid (no NPT gene), or were not exposed to Agrgpagtggia. Although explants from 46 Table 1. Effect of A. atmefaciens inoculation time on callus and shoot production by melon explants cultured on MR medium with 50 mg-I'l kanamycin. Inoculation Total # Producing Shoots time (min) explants callus (%) produced (%) 10 79 24 (30.4) 8 (10.1) 20 81 21 (25.9) 9 (11.1) 30 77 19 (24.7) 8 (10.3) 60 73 13 (17.8) 4 ($5) Explants were co-cultivated for three days, then transferred to medium with kanamycin, and counted five weeks after inoculation. The data are compiled from two experiments. Table 2. Effect of co-cultivation period on callus and shoot production by melon explants cultured on MR medium with 75 mg-I“ kanamycin. Clo-cultivation (days without Total # Producing Shoots kanamycin) explants callus (%) produced (%) O 117 14 (11.9) 3 (2.6) 1 121 29 (23.9) 11 (9.1) 3 119 48 (40.3) 15 (12.6) S 113 37 (32.7) 12 (10.6) 7 114 17 (14.9) 5 (4.4) Explants for all treatments were inoculated with Agrabacmium for 10 min; counting was done 40 days after inoculation. The data are compiled from three experiments. 47 Fig, 2 Cotyledon explants of melon cultured on medium with 75 mg-I‘l kanamycin. (a) uninoculated control explants; (b) explants inoculated with A. mmefaciens LBA4404 + pCIBlO; (c) control explants inoculated with A. rumefaciens LBA4404 without plasmid. The cultures were photographed three weeks after inoculation. 48 all treatments readily formed callus and shoots on medium without kanamycin, only explants inoculated with LBA4404 + pCIBlO produced callus and regenerated shoots on MR medium with 75 mg/l kanamycin (Figure 2). Buds were produced along the cut edges of the explants 2 - 3 weeks after inoculatiOn. In 2 - 3 more weeks approximately 20 - 30% of the buds developed into shoots that.were large enough to be transferred to rooting medium (this is an ca. 40% reduction relative to control explants without kanamycin). To further select for transformation, 50 mg/l kanamycin was included in the rooting medium. In another 3 - 5 weeks, about 30% of the shoots produced roots (vs. 80% of control shoots rooted on medium without kanamycin) . Plantlets were transferred to soil mix as soon as possible after root initiation, longer growth on medium reduced the survival rate in soil. Although there was some variation in growth rate of the regenerated plants, they appeared to be morphologically normal and were fertile. In other plant transformation systems, antibiotics were reported to affect development of the regenerated plant (Michelmore et al. 1987) and inhibit rooting of the transformed shoot (Jones et al. 1989). Although the reduced rooting observed for melon may be the effect of the antibiotic, the use of the kanamycin in the rooting medium was helpful in reducing the number of putative transgenic, regenerated plants that did not test.pasitive by Southern.blot assay (data not shown). 49 e ' a ses a d ' e nce o a c' s's n : Southern blot analysis was performed to determine if the regenerated plants were transgenic. DNA was isolated from several control melon plants, and from 25 of the ca. 60 regenerated plants that were rooted in. the presence of kanamycin. The NPT probe did not hybridize with DNA from the control plants but did hybridize with DNA from 22 of the 25 kanamycin-resistant regenerated plants. Thus in almost every case there was successful incorporation of the NPT gene into the melon genome. .A Bam HI digest of the DNA from eleven of these plants is shown in Figure 3. Nine were positive for the presence of the the NPT gene; there was no hybridization in the control! lane. Variable hybridization patterns were observed among the NPT- positive plants indicating the expected random integration of the NPT gene into the melon genome. Most transformed plants appreared to contain a single copy of insert DNA (single band), plant #7 may have two or more integrations into its genome (multiple bands). The DNA from several plants was also double-digested with Bam H1 and Bcl I to release an internal fragment from the transferred DNA. In each case, the expected ca. 1.9 kb band was obserVed (data not shown). Four transformants (plant #'s 1, 5, 6, 7) were either. self-pollinated, or backcrossed to non-transformed control plants, to produce the next generation. The progeny were examined for the NPT gene by dot blot analysis; the results 50 A n 1 2345678910117" . - F 0 . .‘ 0 0 Fig. 3 Southern blot hybridization analysis of genomic DNA from putative transgenic melon plants. (A) The DNA was digeSted with Ben: Hi and probed with isolated, 32P-labeled NPT fragnent. Lane :1: one capy reconstruction (50 pg) of 1.9 Itb NPT gene isolated from pClBlO; Lanes 1 - 11: genomic DNA extracted from young leaves of individualiputative transgenic plants. (12 uglane"); Lane m: 12 pg genomic DNA from untreated control melon plant. (8) A subset of the above samples (#5. 6, 7, 9, 10. 11) were doubly digested with Ben: HI and BC! I to release the NPT II gene. Other aspects of DNA preparation and probing were as for A. 51 are summarized in Table 3. The segregation ratios of the progeny of all six families was consistent with predicted Mendelian ratios as tested by X2 analysis. In each case the P values were much greater than the rejection level of P80.05. The progeny from self-crosses of plants 1, 5, and 6, gave the expected 3:1 ratio (NPT positive: NPT negative) for the incorporation of a single gene. Similarly, when plants 1 and - 6 were backcrossed with the parental genotype, the progeny gave the expected 1:1 ratio for single gene incorporation. The 15:1 ratio observed for the progeny of selfed plant #7 implies that two genes were incorporated. In each case, the number of genes determined by progeny analysis was consistent with the observed Southern blot pattern for the original regenerated plants (Figure 3). A leaf callus assay was conducted to determine expression of the kanamycin resistance trait. Young leaves from NPT positive progeny were cultured on MR medium with 75 mg/l kanamycin. Explants from NPT-positive progeny produced callus _ within two weeks; no callus was produced by non-transformed control explants (Figure 4). Thus kanamycin resistance behaved as a dominant trait as reported for other plants (e.g. Chyi et al. 1986, Catlin et al. 1988). W We have successfully transformed and regenerated muskmelon plants using Agrebasterium Lumefasiens and a modified leaf disk procedure. The best conditions in our 52 Table 3. Summary of dot blot data of progeny of transgenic regencrants. NPT" # Plants Probable # of Expected b Family 4» - tested integrations ratios X2” P 1 - self 13 5 18 1 3:1 .073 1.00 1 - cross 9 10 19 1 1:1 .053 11!) 5 - self 13 3 16 1 3:1 330 0.80 6 - self 15 3 18 1 3:1 .667 0.65 6 - cross 9 11 20 1 1:1 - .200 0.90 7 - self 17 1 18 2 15:1 .015 0.75 ‘ Genomic DNA was extracted from leaves of three week old plants and probed with 3zP-labeled NPT fragment. + hybridized to the NPT probe; - did not hybridize. " Based on number of NPT-positive progeny and Southern blot hybridization patterns. ‘ Calculated as X2 = 2: [(lo - el - trifle] using the Yale’s correction factor. Fig, 4 Sample leaf callus assay for expression of the NPT-II gene. Young leaves from control plants (a) or NET positive, F1 progeny 1 (b,c)-were cultured on MR medium with 75 mg-l’1 kanamycin. 54 experiments included the use of 4-5 day old cotyledons soaked for ten min in a fresh overnight culture of A. tumefiagignfi, a three day co-cultivation period, regeneration on MR medium containing 75 mg/l kan, rooting in the presence of 50 mg/l kan and immediate transfer to soil. Efficiency Of transformation and regeneration is 3 - 7% (calculated from initial explant to transgenic plant). Time from initiation of the experiment to plants in the greenhouse is approximately three months. Southern blot data and progeny analysis verify integration of the NPT gene into the melon genome and transmission of the NPT gene to subsequent generations. 55 LITERATURE CITED 1. Catlin D, Ochoa O, McCormick S, Quiros CF (1988) Celery. transformation by Agrobacterium tumefaciens: Cytological and genetic analysis of transgenic plants. Plant Cell Reports 7:100-103 2. Chilton MD, Currier T, Ferrand K, Bendich J, Gordon M, Nester E (1974). Agrobacterium tumefaciens DNA and P58 bacteriophage DNA not detected in crown gall tumors. Proc Natl Acad Sci USA 71:3672-3676 3. Chyi YS, Jargensen AJ, Goldstein D, Tanksley SD, Laaiza- Figueroa F (1986) Locations and stability of Agrobacterium- mediated T-DNA insertions in the Lycopersicon genome. Mol Gen Genet 204:64-69 4. Dellaporta SL, Wood J, Hicks JB (1985) Maize DNA miniprep. In Molecular Biology of Plants. Malmberg, Messing, Sussex (eds.) p. 36-37. 5. Dirks R, Van Buggenum M (1989) In vitro plant regeneration from leaf and cotyledon explants of Cucumis mela L. Plant Cell Report 7:626-627 6. Feinberg AP, Vogelstein B (1983) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132:6-13 7. Gasser CS, Fraley RT (1989) Genetically engineering plants for crop improvement. Science 244:1293-1299 8. Horsch RB, Fry JE, Heffman NL, Eichholtz D, Rogers SG, Fraley RT (1985) A simple and general method for transferring genes into plants. Science 227:1229-1331 9. James DJ, Passey AJ, Barbara DJ, Bevan M (1989) Genetic transformation of apple (Malus pumila Mill) using a disarmed Ti-binary vector. Plant Cell Reports 7:658-661 10. Maniatis T, Fritsch EF, Sambrook J (1982) "Molecular Cloning: A Laboratory Manual" Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11. Michelmore R, Marsh E, Seely S, Landry B (1987) Transformation of lettue (Lactuca sativa) mediated by Agrabacterium tumefaciens. Plant Cell Reports 6:439-442 12. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassay with tobacco tissUe cultures. Physiol Plant 15: 473- -498 56 13. Neidz RP, Smith SS, Dunbar KB, Stephens CT, Murakishi HH (1989) Factors influencing shoot regeneration from catyledanary explants of Cucumis mela. Plant Cell Tissue and Org Cult 18:313-319 14. Rogers SG, Horsch RB, Fraley RT (1986) Gene transfer in plants: production of transformed plants using Ti plasmid vectors. Methods in Enzymology 118:627-640 15. Rothstein SJ, Lahners KN, Lotstein RJ, Carozzi NB, Jayne, SM, Rice DA (1987) Promoter cassettes, antibiotic-resistance genes, and vectors for plant transformation. Gene 53:153-161 16. Trulsan AJ, Simpson RB, Shahin EA (1986) Transformation of cucumber (Cucumis sativa L.) plants with Agrobacterium rhizogenes. Theor Appl Genet 73:11-15 CHAPTER TWO CDNA SEQUENCE ANALYSIS OF THE 3’ TERMINAL REGION OF ZUCCHINI YELLOW MOSAIC VIRUS 57 58 INTRODUCTION Zucchini yellow mosaic virus (ZYMV) , is a relatively new and highly agressive member of the potyvirus group. This virus was first described in Italy and France in 1981 (Lisa at al.), since then it has spread throughout.the world. (Lisa and Lecoq, 1984). It is capable of outcompeting the other two cucurbit potyviruses, the watermelon strain ofpapaya ringspot virus (PRV-W) and watermelon mosaic virus (WMV) in establishing infections in rub inoculation (Davis and Mizuki, 1987), aphid aquisition (Lecoq and Pitrat, 1985), and field studies (Alderez, 1987; Alderez e3; 11., 1985). It also appears to be evolving rapidly. Several new strains have been identified (Lisa and Lecoq, 1984) and a strain capable. of overcoming resistance to the dominant gym gene in muskmelon has been reported (Nameth g; al., 1986). As a group, potyviruses are the most ecOnomically important group of plant viruses; they account for approximately one quarter of all plant viral diseases (Shukla and ward, 1988). Potyvirus virions are flexous filamentous particles 700 - 900 nm long and 11 - 12 nm in diameter (Hollings and Brunt, 1981; Francki Lt a_l., 1985). The genome is a positive sense, polyadenylated, single stranded RNA molecule approximately 10 kb in length. The RNA codes for a single, large polypeptide that is 'subsequently cleaved“ by viral encoded proteases (Allison g; a_., 1986; Domier gt al., 1986; Hellmann gt al., 1988; Carrington and Daugherty, 59 1987). As a first step towards determining whether the coat protein of the potyvirus ZYMV can be used to genetically engineer resistance, a portion of the 3’ end of the ZYMV genome was cloned and sequenced (Grumet and Fang, 1990). This. chapter presents ZYMV cDNA sequence including part of the putative RNA polymerase gene, the‘camplete coat protein gene, and the 3’ untranslated region. The predicted amino acid sequence is compared with other known potyviral sequences. These data show that the ZYMV coat protein has the highly conserved central and C-terminal regions characteristic of the potyvirus group, and an N-terminal region that is unique to ZYMV. The C-terminal regions of several - potyviral polymerase genes were also compared and found to be highly conserved. 60 MATERIALS AND METHODS .‘129-! -e--., 9 i-tl-°.— 01:10. I3._-_rr°' The ConnectiCut strain of ZYMV was kindly provided by Dr. R. Davis and used to rub inoculate zucchini (Qggurbita,pgpg cv.. Black Jack) cotyledons in the greenhouse. Characteristic mosaic symptoms were observed within a week. The virus was verified to be ZYMV by reaction with commercial (AgDia) antibodies to ZYMV. Virus was isolated from infected plant material, RNA preparation and cDNA cloning were performed by Dr. Grumet as descriped in Grumet and Fang (1990). ZYMV cDNA was claned into Bluescript KS+ plasmid (Strategene) and used to transform competent DHSa Eschezichia ggli cells. Clones with inserts ranging from 0.3 - 5.5 kb were obtained. Nestgd dgletions and DNA seggencing. The ca. 1.55 kb_ clone #187 was chosen for sequencing. To facilitate sequencing of both strands, the insert from clone #187 was also cloned into Bluescript KS-. Nested deletions in both directions were performed according to the Strategene protocol based on Yanish-Perron 2; al. (1985). The deletion products were transformed into E. 9911 strain MV1190; clones with inserts of appropriate length to allow for overlapping sequence were used to make singLe stranded DNA using the M13K07 helper phage (Dotto and Zinder, 1984). Eight overlapping clones were chosen for each strand. Dideoxy sequencing was performed according to Sanger (1981) using 61 3SS-dCTP, both strands of the #187 clone were completely sequenced. Sequence analyses were performed using the DNASIS program (Hitachi Software Engineering) and sequence data from the following potyviruses: watermelon mosaic virus 2 (WMV-Z: Yu gt g1., 1989); tobacco etch virus (TEV: Allison g; g1., 1985b; Allison g g1,” 1986); tobacco vein mottling virus (TVMV: Domier g; gl., 1986); potato virus Y (PVY: Shukla g; gl., 1986; Van der Vlugt g; 31., 1989); plum pox virus (va:’ 1., 1988; Maiss g; gl., 1989); and Johnson Ravelonandro g; grass mosaic virus (JGMV: Gough et al., 1987). 62 RESULTS AND DISCUSSIONS The cDNA sequence including a portion of the putative polymerase gene, the full coat protein gene, and the 3' untranslated region (excluding the poly A tail) is shown in Figure 5 along with the predicted amino acid sequence. The 211 nucleotide 3"untranslated.region.iS'within the size range of other potyviral untranslated regions. Possible eukaryotic polyadenylation signals AATAA and TATGT (Zaret and Sherman, 1982) 30 and 90 nucleotides upstream from the poly A tail, respectively, are underlined. A putative AATAA polyadenylation signal has also been noted.for TVMV (Domier gt g1., 1986), but not for the other potyviruses with sequenced 3' terminal regions. However, the possible yeast polyadenylation signal, TATGT, has also been noted for TVMV, TEV and PPV 70-90 nucleotides upstream of the poly A tail (Maiss g; gl., 1989). Since potyviral genome are expressed as polyproteins, the beginning and end of genes occur at protease cut sites (review: Dougherty and Carrington, 1988) . The proposed N-terminal amino acid based on coat protein size, possible protease cut sites [Q/G, Q/S, or Q/A (Domier g; gl., 1986; Dougherty and Carrington, 1988)] and the alignment of the putative replicase.gene (Figure 6-A), is underlined.and marked with a slash (Figure 5). This results in a protein 279 amino acids long, with a predicted Mr of 31,214. Consistent with expression of the potyviral genome as a polyprotein, a single 63 GATCT GATACTTGGAGTCAMGATGAGGATAGCGGGITACITGATMCAIGICATa S7 DLILAVKDEDSGLLONISS TCTITITGCGAACTIGGAGIGMTIATGATTTTTGAGAACGIAGUTAMAGAGM 114 SFGELGLITDFSEIIII‘IE GATCIITGGTTTATGTCGGAGWTGCIAGIIGATGGMTGTAGAIIW 171 DLUFISIIGAILVDGNIIPK GTCGAGAMGAGAGMITGIIIGAATIGTAGAGTGGGAIWITAIG 228 LEGEIIVSILEHDRG Ell CACCGMUGAGGGIAITTGGRTGGGATGATT GAGGQIGGGGGUWG 23$ IIRTEAICAANIEAUGIIIEL T TGGIAGMATCAGAMGTIIIACGIAIGGIIGGIIWGAAGIW 362 LGEIIKFTLUFVEKEEVIE TIGGGAGGGGICGGQAAAGGTGCATAGATAGCTGAGACAGGAGIICGTAAGTTATAI 399 LAALGKAPIIAEIALIKLT ACIGIGIAGGGAGUUTAMAGIGAACIGGCAGGCIACCTMGGIGGATGM ‘56 IDKGAHKGELAIYLGALIIG wrarcntmmmcrmutccrocutmrmcr 513 DIFFEOGDTVNLMGTOPT GTGTGAGATGGTGGAGGIWGACAMGAAGAIW 570 SDAGAIKKDGEDDKGKIK GAGGITACAFCGQGTGAGGTGIGAMM’AGTAGUGGIGIWGAAG 627 0 I SGGGEII AAVIVDK GAT GTGAATGCTGGITCTGATGGMAATTGTGGGGGGICIITMTGACAAAG 684 DVIAGSIIG‘IVPILSKTTK MTGTCAITGCCACGGGTGMAGGAMTGTGAIAGTGGATAIIGAICAITIGGTG 741 KNSLPIVKGNVILDIDHLL GMTATAMCGGGAICAMTTGAGTIATATAAGACACGAGGGIGICAICAGCAGIIG 798 ETKPDGIELYIIIASNGGF GGGTCITGGTTGAACWTTMGAGGGAATATGATITGMCGAGWIGGGA 55 ASUFNGVKTETDLNEGGIG GIT GTMIGMTGGITTGATGGITTGGTGUTTGAMATGGGAGIICAGWII 91 2 VVNNGF WGIEIGTSPDI MT GGAGTGIGGGTTAIGATGGAWGAMTGAGSAASIIGAGIAICGCIIW IIGVUV IDGIE ET LKP ATAGITGMMTGCAMGGGAAGGGTGCGGGAMTMIGGAIGAITTTICAGATGCA 1&6 IVEIAKPTL 01 IIFSD GCGGAGGCAIATATAGAGATGAGAAAI IGCAGAGGCACGATACAIGCGGAGGTATGGT 1m: EAIIE IIAEAPT PIT TTGGT IGGAMRIACGGGATAGGAGIIIAGGAGGAIATGGITTGGATITCTAIGIA 1140 LLIILIDISLAIT F YE GTGMITGIMMGTCGTWGGI GGIGUWIGIMW 1197 VISHIPEIAIEAVAGIK GGTCTTAGGMTGTTTCTTQAGGTIGIIIGGGGIIGATGGMAIGTTGGGAGGAGI 125‘ ALSIVSSILFGLlGIV II AGCWIWWTGUCGTGAIGTTMTAGMACATWIA 131 I SEDIEIIIIAIDV I IIITI. ETAgGTeIGaAT;Q:TG:AGIAMGGGTAGGGGCCIACGTAGGTIAIIGTITCGG I368 TGGCGAGGTMTTCTMTATIIAGGGGTTTATTTGAIATGTIIAGAIIIOGAGAGTG 1‘25 GGGGIGGUGGITIAMKGIWGTIIATQTTAGTIGIGGAGGAGTGGWIAGIGG 1‘82 TTTmTTAGTGTGAGGGTGIGACGAATEGGIGGAGATTAGACTGGGTTTG 1539 GMGGGIW Fig. 5 .The cDNA sequence and predicted amino acid sequence of the terminal 3' l546 nucleotides of ZYMV. The proposed polymerase-coat protein protease cut site is underlined and marked with a slash. The beginning of the conserved trypsin-resistant core protein is marked with a backslash. Possible polyadenylation signal: are underlined. 64 open reading frame continues for the length of the #187 clone, at least 170 amino acids upstream of the putative cut site for the N-terminus of the coat protein. It was not possible to directly determine the N-terminal amino acid of the ZYMV coat protein by chemical sequencing because the protein was N-terminally blocked. This problem has been observed for several other potyviral coat proteins (Shukla g; g1. 1988, Allison g; gl., 1985a; Domier g; g1., 1986), particularly those beginning with S rather than G or A (Shukla g; g1. 1988). The predicted Mr (ca. 31,200) obtained . from the proposed Q/S site is consistent with the value estimated from the SDS-PAGE gel (32,700), but is somewhat smaller than the 36,000 estimate of Lisa g; gl. (1981). Although we cannot be positive about the location of the N- terminus without direct amino acid sequence data, examination of the polymerase gene also aided in assigning the protease cut site. Sequence data from the adjoining putative polymerase gene were aligned (Figure 6-A) with available sequence data from other potyviral polymerases genes beginning at the highly conserved amino acid span, G D D, which is found in viral-encoded polymerases regardless of plant, animal or bacterial origin (Kamer and Argos, 1984). This alignment resulted in assignment of the Q/S dipeptide as the ZYMV polymerase - coat protein junction. 7 The predicted amino acid sequence of the ZYMV coat protein is compared with the published amino acid sequences 65 FKQQSS LFLFMM PREGNR E UDDU L LLM DRIVLI RESHGS PAEESE LXYLDK EDAEEE EPPPDE VHHSK II.I. .umnm IILIL LLVII S L , .mumm UDDD GGGG mwmmmm KRYKVI! G LES—m L L— GLVLMLM SAGMAWW TEEARER IHHHHHH PSSSSSS MMMMMMM TFFFFFF IWUWWWW LLLLLLL PQEEDED KflEWEEE_ STE: k2 CDNDKRN IRiLlRy FITTTI PIERRE QCTSEE mmmmmmm CEIQQRY RVLLLTT IEULLLL_ VKEUEKE BRENNER- DSIHMEE IYYYYYY KPPPPPP GAAAAAA LKKIKKK SGGGGGG EEDQLEQ LEKEAQL AASAAA ALLLLI EQASETE YSNRRSK L Y? Em .nwww.__ PAAESQF RQQQmQ REEEEMT L YVVTFLV RWMUVNE HASKLSQ AYYYYYY IWWFFFF MNKRKRR ARRRRRR AflwIIII VHENEQQ wmmmmmm RFVFMEV RYVRVYD ALNVIIV RNSIDDV SEEEGCD RIGRQEE AIDLEFS DPDFFEM SINEFDM HDINIDA SYNNDLN KDDAQAK KYYLHVY VLFILMM RVAEAME RKEKQEE PLL LTY .mumm.n IERDRAR KEEIAKL VIIULLU NEE-LEE SSINSES ENEDKEQ HISIHUU XGAAAvc EHEIGIS CA G AD A UV D A! SG CEGVAG ADA-IDA DEDPID VRVQIE III-LON GDDCNC SASSGS _W4009m a H HH A V LVE mmmmmmm mmmmmmm the C-terminal region of potyvi f sequence data are as in.Material no acids common to ma mu four or more of the as in List of abbreviations. Comparison of The source a Boxes indicate ami viruses. Abbreviations are replicase genes. mumfim Figure 6-A. 66 of other potyviral coat proteins in Figure 6-B. The ZYMV coat protein shows very strong homology with other potyviral coat proteins in the highly conserved central and carboxy terminal portions of the protein. The N-terminal region is unique. With the exception of 18.6% homology with the other‘ sequenced cucurbit potyvirus coat protein, WMV-z, (if aligned with deletions to maximize homology, or 11.6% if not aligned with deletions), ZYMV shares less than 14% homology with the other potyviral N-terminal regions (Table 4). Overall homology of the amino acid sequence of the ZYMV coat protein with other potyviral coat proteins ranges from 47.5% to 67.1%. This is in agreement with the 38%- 71% range of homologies observed among distinct potyviruses (Shukla and Ward, 1988; Shukla g 11,. , 1989); different strains of the same virus are greater than 90% homologous (Shukla and Ward, 1988; Shukla gt al.. 1989). Potyviral coat proteins can be divided into two general domains. The first region is potyvirus specific (e.g. the N- terminal region of ZYMV shares less than 12% homology with other potyviral N-terminal regions), is subject to removal by limited proteolysis, is externally located, and is the primary antigenic determinant (Shukla and Ward 1989; Dougherty gt 1].. , 1985; Allison gt al. , 1985a). Removal of this region does not interfere with apparent potyviral structure or viral infectivity, and so presumably the RNA binding capacity and coat protein - coat protein interactions involved in viral 67 Al‘IIDIIIVDAGI'I-nfl 'VTA'AATS o IIQ¢8tQ ICCIIL. ACYOQ‘I ICQ13QI IIIVIII YIDIII ITIII. CCIClIA YQBII 8 tAIIOMA QIODI 0 L!!! " Qttlmff III. .6 6.33183 Dill? '88!) 0'05! SKI A .. m ...m It I Tttv tel! '18! QTVQ tQAI Tel. A!!! 9!. ‘ 268 It..‘. . C“K‘O . , o I’IIAI! YIYIYAA I'331‘I tIIILIA IttIVL IMTM “8M1!!! QI‘IQI' ems—mm. mmmmmmm afifilt'A mmmmmmm The Abbreviations sources of sequence data are in material and method. Boxes indicate amino acids common to five or more of the viruses. Figure 6-3. Comparison of potyviral coat protein sequences. are as in Haterial and methods. 68 Table 4. Percentage amino acid sequence homology between Z YM V coat protein and other potyvirus coat protein: Central and Virus‘ Overall N terminus C terminus WMV—Z 67-1 11-6 76-9 PVY-D 59.2 7-0 67-2 TEV-HAT 58-8 4-7 67-7 JGMV 55-0 7-6 65-9 TVMV 50-5 2-3 56-8 PPV 47.5 6.5 61-1 ‘ Abbreviations and sources of sequence data are listed in text. 69 assembly lie downstream within the highly conserved central and C-terminal regions characteristic of potyviral coat proteins (Dougherty gt al., 1985; Shukla and Ward, 1989). The highly conserved core portion of potyviral coat proteins is of interest in attempting to genetically engineer potyviral. resistance. In at least two systems the ability of a viral coat protein to inhibit infection is related to interaction with viral RNA. TMV coat protein is thought to interfere with the TMV uncoating process by interacting with the exposed 5’ end of the viral RNA (Register and Beachy, 1988) . In the QB bacteriophage system, coat protein is. thought to inhibit viral replication by binding to the initiation domain of the replicase gene and thereby interfering with translation of the viral replicase gene (Grumet gt al., 1987). If it is true that the nucleic acid binding domain of potyviruses is located within the highly conserved portion of the protein, and if coat proteins can inhibit viral infection via coat protein - viral RNA interactions, then it may be possible to use a core protein construct to engineer resistance to more than one potyvirus. This possibility will be tested in the work that follows, as well as the possibility that ZYMV coat protein per gg can be. used to confer virus resistance. 7O LITERATURE CITED 1. ALDEREZ, W.C. (1987). Cucurbit potyvirus transmission by alate aphids trapped alive. J. Economic Emtomol. 80:87-92. 2. 2. ALLISON, R.F., DOUGHERTY, W.G., PARKS, T.D., WILLIS, L., JOHNSTON, R.E., KELLY, M. & ARMSTRONG, F.B. (1985a). Biochemical analysis of the capsid gene and capsid protein of tobacco etch virus: N-terminal amino acids are located on the virion's surface. Virol. 147:309-316. 3. ALLISON, R.F., SORENSON, J.C., KELLY, M.E., ARMSTRONG, F.B. & DOUGHERTY, W.G. (1985b). Sequence determination of the capsid protein gene and flanking regions of tobacco etch virus: evidence for synthesis and processing of a polyprotein in potyvirus genome expression. PNAS. U.S.A. 82:3969-3972. 4. ALLISON, R., JOHNSTON, R.E. & DOUGHERTY, W.G. (1986). The nucleotide sequence of the coding region of tobacco etch virus genomic RNA: evidence for the synthesis of a single polyprotein. Virol. 154:9-20. 5. BAULCOMBE, D. (1989). Strategies for virus resistance in 6 plants. Trends in Genetics 5:56-60.- 6 6. DAVIS, R.F. (1986). Partial characterization of zucchini yellows mosaic virus isolated from squash in Turkey. Plant Disease 70:735-738. 7. DAVIS, R.F. & MIZUKI, M.K. (1987). Detection of cucurbit viruses in New Jersey. Plant Disease 71:40644. 8. DOMIER, L.L., FRANKLIN, K.M., SHAHABUDDIN, M., HELLMANN, G.M., OVERMEYER, J.H., HIREMATH, S.T., SLAW, M.F.E., 71 LOMONOSSOFF, G.P., SHAW, J.G. & RHOADS, R.E. (1986). The nucleotide sequence of tobacco vein mottling virus RNA. Nucleic Acids Research14:5417-5430. 9. DOUGHERTY, W.G. & CARRINGTON, J.C. (1988). Expression and. function of potyviral gene products. Ann. Review of Phytopathol. 26:123e143. 10. DOUGHERTY, W.G., WILLIS, L. & JOHNSTON, R.E. (1985). Topographic analysis of tobacco etch virus capsid protein epitopes. Virol. 144:66-72. 11. GOUGH, K.H., AZAD, A.A., HANNA, P.J. & SHUKLA, D.D. (1987). Nucleotide sequence of the capsid and nuclear inclusion protein genes from the Johnson grass strain of sugarcane mosaic virus RNA. J. Gen. Virol. 68:297-304. 12. GRUMET, R. (1989). Genetically engineered plant virus resistance. HortScience 25:508-513. 13. GRUMET, R., SANFORD, J.C. & JOHNSTON, S.A. (1987). Pathogen-derived resistance to viral infection using a negative regulatory molecule. Virol. 161:561-569. 14. GUBLER, U. & HOFFMAN, B.J. (1983). A simple and very efficient method for generating cDNA libraries. Gene 25:263-269. 15. HELLMANN, G.M., THORNBURY D.W., HIEBERT E., SHAW, J.G., PIRONE, T.P., & RHOADS, R.E. (1980). Cell-free translation of tobacco vein mottling virus. Virol. 106:207-216. 16. KAMER, G. & ARGOS, P. (1984). Primary structural comparisons of RNA-dependent polymerases from plant, animal‘ 72 and bacterial viruses. Nucleic Acids Research 12:7269-7281. 1?. LECOQ, H. & PITRAT, M. (1985). Specificity of the helper component-mediated aphid transmission of three potyviruses infecting muskmelon. Phytopathol. 75:890-893. 18. LISA, V., BOCCARDO, G., D’AGOSTINO, G., DELLAVALLE, G. & D'AQUILIO, M. (1981). Characterization of a potyvirus that causes zucchini yellow mosaic. Phytopathol. 571:667-672. 20. LISA, V. & LECOQ, H. (1984). Zucchini yellows mosaic virus. CMI/AAB Descriptions of Plant Viruses, no. 282. 21. MAISS, E., TIMPE, U., BRISSKE, A., JELKMANN, W., CASPER, R., HIMMLER, G., MATTANOVICH, D. & KATINGER, R.W.D. (1989). The complete nucleotide sequence of plum pox virus RNA. J. Gen. Virol. 70:513—524. 22. MANIATIS, T., FRITSCH, E.F. & SAMBROOK, J. (1982). Molecular Cloning: A Laboratory Manual. New York: Cold Spring Harbor Laboratory. 23. PURCIFULL, D., HIEBERT' E. & EDWARDSON, J. (1984a). Watermelon mosaic virus 2. CMI/AAB Descriptions of Plant Viruses, no. 293. 24. PURCIFULL, D., EDWARDSON, J., HIEBERT, E. & GONSALVES D. (1984b). Papaya ringspot virus. CMI/AAB Descriptions of Plant Viruses, no. 292. 25. RAVELONANDRO, M., VARVERI, C., DELBOS, R. & DUNEZ, J. (1988). Nucleotide sequence of the capsid protein gene of plum pox potyvirus. J. Gen. Virol. 69:1509-1516. 26. REGISTER, J. C. & BEACHY, R.N. (1988). Resistance to TMV 73 in transgenic plants results from interference with an early event in infection. Virol. 166:524-532. 27. SANGER, F. (1981). Determination of nucleotide sequences in DNA. Science 214:1205-1210. 28. SHUKLA, D.D. ‘& WARD, C.W. (1989). Identification and classification of potyviruses on the basis of coat protein sequence data and serology. Archives of Viral. 106:171-200. 29. SHUKLA, D.D., INGLIS, A.S., MCKERN, N.M. & GOUGH, H.H. (1986). Coat protein of potyviruses. 2. Amino acid sequence of the coat protein of potato virus Y. Virol. 152:118-125. 30. SHUKLA, D.D., MCKERN, N.M. & WARD, C.W. (1988). Coat protein of potyviruses. 5. Symptomatology, serology, and coat protein sequences of three strains of passionfruit woodiness virus. Archives of Virol. 102:221-232. 31. VAN DER.VLUGT, R., ALLEFS, S., DE HAAN, P. & GOLDBACH, R. (1989). Nucleotide sequence of the 3' terminal region of potato virus Y-N RNA. J. Gen. Virol. 70:229-233. 32. YANISH-PERRON, c., VIERA, J. & MESSING, J. (1985). Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC 19 vectors. Gene 33:103-119. 33. YEH, S.D. & GONSALVES, D. (1985). Translation of papaya ringspot ‘virus RNA in vitro: detection of a possible polyprotein that is processed for caps id protein , cylindrical-inclusion protein, and amorphous-inclusion protein. Virol. 143:260-271. 74 34. YU, M.H., FRENKEL, M.J., MCKERN, N.M., SHUKLA, D.D., STRIKE, P.M. & WARD, C.W. (1989) . Coat protein of potyviruses 6. Amino acid sequences suggest watermelon mosaic virus 2 and ' soybean mosaic virus-N are strains of the same potyvirus. Archives of Viral. 105:55-64. 35. ZARET, K.S. & SHERMAN, F. (1982). DNA sequence required for efficient transcription termination in yeasts. Cell 28:563- 573 . CHAPTER THREE GENETIC ENGINEERING OF THE ZYMV COAT PROTEIN GENE FOR EXPRESSION IN PLANTS 75 76 INTRODUCTION Dramatic progress in the development of plant transformation and regeneration systems as a mechanism for the expression of novel genes has allowed for the genetic engineering of plants for crop improvement. This is most clearly seen by the development of genetically engineered resistance to plant viruses. The resistance was first shown for tobacco mosaic virus(TMV) in tobacco plants (Powell et.a1. 1986); it was found that transgenic tobacco plants expressing the coat protein gene from TMV showed a significant delay in' symptom development after inoculation with.TMV. Coat protein- mediated protection has now been demonstrated for a number of different viruses from at least eight different virus groups, such as the tobamo-, potex-, cucumo-, tobra-, carla-, poty-, and alfalfa mosaic virus groups (for review see Beachy et a1. 1990; Grumet 1990), and more recently the luteovirus group (Kawchuk et al. 1990, 1991). The list is growing rapidly. Another approach has been to use viral antisense genes. In . several cases, the coat protein genes were introduced into. plants in antisense orientation for the purpose of genetically engineering viral resistance, but the protection levels were- different.inmdifferent'viruses and.plants. In some cases there was virtually no protection, in other cases expression of antisense RNA did result in resistance to virus infection. For example, expression of antisense RNA to the AlMV CP gene in transgenic plants did not inhibit viral replication (Van Dun, . 77 1988). Expression of antisense RNA to the CP gene of CMV and PVX result in limited protection at low inoculum concentrations of the corresponding virus (Cuozzo et al., 1988; Hemenway et al., 1988). In contrast, it was recently reported that for plants producing CP RNA of potato leafroll' virus (PLRV), both the pattern and level of protection were the same regardless of‘whether'the positive or negative strand was expressed (Kawchuk et al. 1991). Similarly plant lines expressing either sense defective RNA or antisense transcripts of the potyvirus, tobacco etch virus (TEV) CP gene were highly resistant and showed greater protection than the plants producing the full-length CP (Lindo and Dougherty, 1992). Zucchini yellow mosaic virus (ZYMV) is a very aggressive member of the potyvirus group. Like other potyviruses, the virus particles are flexuous rods of 750 nm in length; the genome consists of a positive sense, single stranded RNA of' about 9.6 kb with a 5’ end genome linked protein (VgP), and a poly (A) tail at the 3’ end. A single open reading frame (ORF) codes for a polyprotein that is proteolytically processed into mature viral gene products (Dougherty and Carrington 1988). The capsid protein is encoded by the sequence present at the 3' end of the large ORF (Allison et al. 1985). To genetically engineer plant resistance to ZYMV and other potyviruses, the ZYMV CP gene has been cloned and the sequence has been determined in several laboratories (Grumet and Fang 1990; Gal-0n et al. 1990; Quemada et al. 1990). The 78 sequence encodes a coat protein with 279 amino acids and a calculated Mr of 31214. The sequence comparison shows that ZYMV shares average 50-60% direct amino acid sequence homology in GP with other potyviruses, the majority of which are located in central and C-terminal regions. In our efforts to engineer resistance, we decided to utilize three versions of the ZYMV CP gene: the full length CP gene, the truncated core portion of the CP gene, and an antisense version of the CP gene. The full length CP gene was used because incorporation and expression of viral capsid protein genes has provided the strongest virus resistance in most reported examples of engineered resistance to date (Beachy et al. 1990). A truncated version of the CP gene fragment (Core), including the highly conserved.center- and carboxyl- terminal regionwwas also used. Since the RNA binding capacity and coat protein- coat. protein interaction involved in 'viral assembly' are contained within the highly conserved core portion of the gene (Dougherty et al., 1983; Shukla and Ward, 1989), the core portion alone might be sufficient to confer resistance. Furthermore, it might be possible that plants expressing the conserved CP gene fragment could be protected from infection by more than one potyvirus. Another potential advantage of using the central and C-terminal region deals with a possible ' environmental concern, in preventing aphid transmission that might result from transencapsidated viruses (Goeferry, 1991). Deleting the amino terminus of the CP would remove a sequence 79 that is essential for aphid transmission (Salomon, 1989). The first example of CP mediated protection using truncated viral CP gene has been reported recently (Lindo and Dougherty, 1992). Plants expressing a truncated TEV CP gene demonstrated high level protection against TEV infection, in fact, the‘ resistance was higher than that of plants expressing full length CP. To express the ZYMV CP gene in plants for the purpose of genetic engineering of resistance to ZYMV and other potyviruses, the intact CP gene sequence and Core fragment were isolated from viral genomic cDNA. Since the ZYMV CP gene is part of a large polyprotein, the CP gene and Core were then given an AUG translational codon to enable translational initiation, and a 5' untranslated region (UTR) was added to facilitate efficient translation of mRNA. The 5' UTR has been verified to be the binding site of proteins and/or ribosomes. during TEV’RNA translation (Carrington and Freed, 1990) and.to be a translational enhancer. Since ZYMV is an RNA virus and does not contain a promoter to initiate transcription, a strong transcriptional promoter, the cauliflower mosaic virus (CaMV) 35$ promoter, was also added. This promoter is highly effective and has been used in most reported genetically engineered virus resistances. The expression of the CP gene in plants also requires a 3'- end sequence that confers termination and polyadenylation (poly A) to the transcripts; in our case the ZYMV 3' poly A tail and CaMV 35$ terminator 80 were used. In this chapter, the construction of the three versions of ZYMV chimeric CP genes and the expression of these genes in transgenic muskmelon and tobacco plants are described. 81 MATERIAL AND METHODS W: The plasmid pTL 37 which contains the TEV 5’ untranslated lead sequence was obtained from Dr. W. Dougherty (Oregon State University). The plasmid pCIB710, which contains the CaMV 35S promoter and the plasmid pCIBlO which includes the T-DNA borders and NPT II gene were provided by CIBA GEIGY Co. A.cDNA clone spanning the 3'-terminal 1,550 nucleotide of ZYMV, which includes the coat protein gene sequence, was cloned as described in chapter 2 A and Grumet and Fang (1990). All recombinant DNA and bacterial manipulations were carried out using standard methods (Maniatis et al. 1982) , unless otherwise indicated. ‘ Restriction enzymes were purchased from Bethesda Research Laboratory or Boeringher Manheim (Life Technologies Inc.) , and were used according to the suppliers' instructions. In vitro mutagenesis was performed using a "Oligonucleotide-directed in vitro mutagenesis system version 2" kit (Amersham ‘ International lnc.), all reaction conditions followed the protocol supplied by the manufacturer. In vitro transcription was performed using the Riboprobe system (Promega Inc.), and. the rabbit. reticulocyte lysate from Promega was used for in vitro translation. Polymerase chain reaction (PCR) was carried out using the GeneAmp PCR Reagent. Kit from .Perkin Elmer Cetus and following the protocol provided by the manufacturer. Elan; transformation: The pCIBlO-derived binary vectors 82 containing ZYMV-GP gene constructs were mobilized from E. coli into the disarmed A. tumefaciens strain LBA4404 (Hoekema et a1. 1983) by tri-parental mating, using the helper plasmid pRK2013 (Comai et al.1983) in E. coli H8101. Cotyledon pieces of muskmelon (Cucumis melo L. cv."Hale’s Best Jumbo") were co- cultivated with A. tumefaciens LBA4404 (Hoekema et a1. 1983) containing pCIBlO-ZYMV CP constructs for three days. Transformed muskmelon plants were regenerated and selected according to Fang and. Grumet (1990) with the following modifications. The transformed cells were selected on 125 mg/l kanamycin in melon regeneration (MR) medium (Neidz et al. 1989) containing 500mg/l carbenicillin. Regenerated shoots were transferred to liquid MS medium containing .150 mg/l kanamycin, 500 mg/l carbenicillin and 0.1 mg/l BAP for two weeks for further selection and elongation. Elongated shoots were transferred to hormone free rooting medium, which contained MS nutrients, 50 mg/l kanamycin, and 400 mg/l . carbenicillin. The rooted plantlets were transplanted into soil and grown in the greenhouse. I Tobacco (N. tabacum cv."Samsun") plants were transformed using the leaf disk protocol of Horsch et al.(1985). Shoots were regenerated on medium consisting of Murashige and Skoog (1962) salt, sucrose (30 g/l), benzyladenine (1 mg/l), naphthalene acetic acid (0.1 mg/l), kanamycin (300 mg/l) and carbenicillin (500 mg/l) . Transformed shoots were subsequently rooted on phytohormone-free medium containing 100 mg/l of 83 kanamycin prior to transferring to soil. Wm: Samples of leaf tissue (20-30 mg) from.kanamycin-resistant regenerated plants were initially screened for expression of NPT II protein using double-antibody sandwich ELISA. NPT II assay kit was purchased from 5 Prime > 3 prime, Inc., and the assay was performed following the instructions supplied by the manufacturer. '5 o 8 ° ts: Genomic DNA. was extracted from young leaf tissue of putatively transformed plants using the procedure of Dellaporta et al. (1985), and purified with the method of Fang et al (1992). The presence of inserted ZYMV CP gene fragments were verified by polymerase. chain reaction (PCR) amplification primers, which anneal to the TEV untranslated region (UTR) 5’ end and ZYMV 3' end respectively. RNA analysis of transgenic plants: Total RNA was isolated from transgenic plants essentially as described bthagy etlal. (1988). The RNA was separated by electrophoresis in 1.8% agarose gel containing formaldehyde, and was transferred to nitrocellulose (Schleicher and Schuell Inc.). The Northern blots were probed with random primer ’32P- labeled ZYMV-CP cDNA fragment isolated from pCIB710-ZCP plasmid using a Random primer DNA labeling kit (United States Biochemical Corp.) t' 's 0 tr se 'c 1 ts: Total soluble protein was extracted from leaf tissue of transgenic plants using the method of (Powell et al., 1986), and the 84 concentration was determined by the method of Bradford (1976) . Protein was separated using a 10% SDS-PAGE gel, with a known amount of purified viral CP as a standard for calculating the expression level of CP in transgenic plants. Electrophoresed proteins were transferred to nitrocellulose. ZYMV CP was detected by rabbit anti-ZYMV CP polyclonal antibodies (Hammar and Grumet, unpublished) and alkaline phosphatase-conjugated goat anti-rabbit secondary antibodies (Sigma). 85 RESULTS o 0 three v s on o c me on To introduce an ATG initiation codon and isolate either the full length CP gene (FL CP) or the potyvirus-conserved central- and carboxyl-terminal region (Core) of the CP gene, in vitro mutagenesis was performed to introduce a Nco I restriction site, which contains an ATG sequence. The specific primers utilized were: (1) For the full length CP, primer RG-4, 5' GAGTG CCTGC CATGG GCATC 3' (position 486 to 505, see Grumet and Fang, 1990) was used. This resulted in three amino acids changes in the sequence, from leucine-glutamine-serine to proline-methionine- alanine. The only change within the CP gene itself is in the first amino acid, from serine to alanine. Both serine and alanine are commonly found in the first position of potyviral coat proteins. (2) For the core version of the gene, primer RG-S, 5' TCCTT GTCCA TGGTG ACAGC 3'(position 610 to 629) was used. This resulted in a single amino acid change from lysine to methionine at the first position within the core portion of the protein. The ZYMV FL CP gene and the core region (840 and 620 nucleotides respectively) were excised from the plasmid ZY187 as Nco I - Pst I fragments (Figure 7),which included the ZYMV 3' untranslated region (226 nucleotide). The fragments were then transferred into the plasmid pTL37, and linked to the.ATG 86 Figure 7.Genetic engineering of the ZYMV CP gene for expression in plants. A Nco I restriction sequence which contains ATG codon was introduced upstream of the CP gene in Bluescript KS derived plasmid pZ187 by in vitro mutagenesis. The CP gene and ZYMV 3'UTR were isolated from plasmid p218? as an Nco I - Pst fragment, and cloned into the plasmid pTL 37 to obtain the TEV 5’UTR. The fragment containing the TEV 5' UTR and ZYMV CP gene cDNA was amplified by PCR using primers that also introduced 891 II restriction sequence to both ends of the amplified fragments. The amplified fragments were then cloned into the Sam HI site of the plasmid pCIB710, between the CaMV 35$ promoter and the CaMV 35$ terminator. The ZYMV CP gene expression cassette was removed from pCIB710 by Xba and Kpn digestion, and inserted into the Agrobacterium binary vector pCIB10 between the left (L) and right (R) T-DNA borders and adjacent to the selectable marker gene NPT II. The pCIB10-ZCP construct was transferred into A. tumafuciens strain LBA4404 by tri-parental mating. The ZYMV Core and ZYMV AS construction followed the same procedure as above, except using truncated ZYMV CP gene or the reverse orientation of the FL CP gene. The restriction sites are:. P, Pst; N, Nco I; B, 391 II; Bm, Bam HI; B/Bm, fusion of 391 II and Ben HI sites; X, Xba; and K, Kpn. CP'T‘DO rum-m mu ’ 22' 5'02! added :7 puncture-n 9"“ PCR eeplieatioe introducing 3911 sites Sign 9"!“- 133 promoter and 358 terminator added Knno-a—i—L E_H Fin-9613710 Cloned into pcxezo NPT adm 1'- Caxv ass Cauv 338 horde: promote: tam-teeter "7 1: border 23 - \\\\ r beam ‘53 2“ i rel-parental mating A . tumcfacicns I ' ts Figure 7. The genetic engineering of ZYMV CP gene for expreSSion in plan 88 immediately following the tobacco etch virus 5’ untranslated region (TEV S’UTR), which is a effective translational enhancer (Carrington and Freed, 1990). ‘ To verify the functionality'of the translational.promotor- CP gene (or Core) construct, the protein products of in vitro transcription and translation were examined for correct size by SDS-PAGE. The expected 30 kd CP and 26 kd Core proteins- were produced (Figure 8). The fragments containing the chimeric'TEV 5’UTR-ZYMV GP (or core)-ZYMV 3’UTR were then amplified and isolated by polymerase chain reaction (PCR) . Two PCR primers were designed for generating Bgl II restriction sites at both ends of the fragments during PCR amplificaation: RG-6: 5’ AGATC TAAAT AACAA ATCTC AACAC 3’ (the 5’ end of TEV UTR), and RG-7: 5' AGATCTCTGC AGCCC TTTTT TTTT (ZYMV 3’ end). The PCR generated fragments were then ligated into the.Bam HI site of p1asmideIB710 (Rothstein et al . 1987) between‘ the cauliflower mosaic virus (CaMV) 358 promoter and the Canl 358 terminator. The resultant clones were analyzed by restriction enzyme digestion to determine the orientation of the inserts with respect to the CaMV 358 promoter and terminator. Constructs containing the FL ZYMV’CP’gene inserted in both sense and antisense orientations were selected to make the sense and antisense RNAs. Three constructs were generated (Figure 9): ZYMV-GP, which contains the FL.CP gene sequence in. 89 97.4— 43.0— 18.4- Figure 8. In vitro transcription and translation of the ZYMV CP and Core genes. ZYMV CP and ZYMV Core cDNA in plasmid pTL 37 were used as the templates for in vitro transcription, and then translated in vitro using rabbit reticulocyte lysate (Promega). The protein products were labeled with 3SS-methionine. Lane A, translational product when ZYMV FL CP RNA was used as template, lane B, the protein product using ZYMV Core RNA as template. 90 l I—le - - ~1—Immsz Figure 9. Three versions of the ZYMV CP gene. The "Coat" construct contains the CaMV 35$ promoter, the TEV 5' UTR, the full length ZYMV CP coding sequence, the ZYMV 3’UTR and the CaMV 35$ terminator. The "Core" construct contains the truncated ZYMV CP central- and carboxyl- terminal region instead of the FL CP. The "Antisense" (AS) construct includes the same components as "Coat" construct, except that the TEV 5' UTR, ZYMV CP coding sequence and 3'UTR are inserted in reverse orientation. 91 the sense orientation; ZYMV-Core, which includes only the 620 nucleotide of the conserved region of the CP gene; and ZYMV- Antisense (AS), which contains the FL CP sequence but in an, antisense orientation. All three constructs contain the CaMV 35$ promoter, TEV 5’ UTR, ZYMV 3' UTR I and CaMV 35$ terminator. These ZYMV CP gene expression cassettes were excised from plasmid pCIB710 by digestion with the restriction enzymes Xba and Kpn, and then ligated into the binary vector pCIBlO (Rothstein et al. 1987), adjacent to the transferable plant- selectable marker for kanamycin resistance, the NOS/NPT II chimeric gene. Both ZYMV gene constructs and NPT II gene were located within left and right A. tumefaciens T-DNA borders. ant nsformatio The A. tumefaciens binary transformation system was used for introducing ZYMV CP constructs into muskmelon and tobacco plants. Kanamycin resistant plants were initially screened for expression of NPT II protein by ELISA. Approximately 45% of the muskmelon plants regenerated in the presence of kanamycin were NPT II test positive. More than 80% of the tobacco regenerants were found to be NPT II positive. Most of the regenerated melon and tobacco plants were healthy, morphologically normal, and produced typical flowers and seeds. No symptoms typical of ZYMV or other viruses, such as mosaic or etching leaves were observed on non-inoculated regenerated plants. 92 The regenerated, NPT-positive melon and tobacco plants were verified for the presence of the inserted ZYMV CP gene sequences by PCR amplification from plant genomic DNA. The expected 1,200 base pair FL CP or 1,000 base pair Core fragments were generated from most (more than 90%) of the 30 tested plants (Figure 10). The transcripts of ZYMV CP constructs were examined by Northern analysis. The results are shown in Figure 11. Hybridization with a labelled ZYMV’ CP fragment revealed strongly hybridizing bands in transgenic melon (M) and tobacco (T) plants (lane M1, M2, M3, T1, T2 and T3), which were each individually transformed with one of the three versions of ZYMV CP gene. The control, vector transformed plants did not give any signal (lane Cs). The estimated size of the specific transcripts produced by plants transformed by FL ZYMV CP gene (both sense and antisense) was 1,200 bases (lane M1, M2, T1 and T2). This compares well with the size of the RNA that was’ expected, which is composed of 150 bases of TEV 5’ UTR, 840 bases of ZYMV FL CP sequence and 226 bases of ZYMV 3' UTR. The ZYMV Core construct transformed plants showed the expected bands of 1,000 bases (lane M3 and T3), including 150 bases of TEV S'UTR, 627 bases of CP sequence and 226 ZYMV 3' UTR. Fourteen of fifteen tested kanamycin resistant, PCR- positive tobacco plants and all eight NPT positive, PCR positive melon plants produced detectable bands of the 93 T P A M C1ABCDEFGHI JKLNCZ Figure 10. PCR amplified ZYMV CP DNA fragments from transgenic tobacco (T) plants, melon (M) plants and plasmid pCIB710 containing ZYMV CP DNA fragments. The samples from left to right are vector transformed tobacco plant (lane C1); lane A and B, FL CP transformed tobacco plants; lanes C and D, Core transformed tobacco plants; lanes E and F, AS tobacco plants; lanes G, H and I, pCIB710 plasmid containing FL CP, AS and Core respectively; lane J, ZYMV AS transformed melon plant; lane K, FL CP melon plant; lane L and N, Core melon plants; and vector transformed melon plant (lane C2). The primers used in PCR were RG-6 and RG-7. The ZYMV FL CP and AS fragments were approximately 1,200 base pairs, and the ZYMV Core fragment was about 1 94 Figure 11. Accumulation of transcripts of CP gene constructs in transgenic plants. The Northern blot was loaded with 10 ug of total RNA isolated from leaves of transgenic melon (M) and tobacco (T) plants. Lane T-l, AS transformed tobacco plant; lane T-2 FL CP tobacco plant; lane T-3 Core tobacco plant; lane T-C, vector transformed control plant; lane M-l AS transformed melon plant, lane M-z FL CP melon plant, lane M-3, Core melon plant, and lane M- C, vector transformed melon plant. The blot was hybridized to 32P- labeled cDNA corresponding to ZYMV CP gene. 95 expected size, even through the expression level was variable (data not shown). These plants had also shown the expected ZYMV CP DNA amplified from total genomic DNA. Expgession of ZYMV coat proteip in tgansfpgmed pigpgs A total of 22 NPT-positive tobacco plants were tested for presence of ZYMV coat protein by Western analysis. .Accumulation of detectable amounts (detection.limit.= 5 ng) of viral protein of the expected size was found in 3 of,8 tobacco plants transformed with the sense full-length CP gene, and 5 of 8 plants transformed with core fragments. None of 8 plants transformed with the ZYMV AS construct produced detectable ZYMV coat protein. The accumulated level of ZYMV coat protein in transgenic tobacco plants ranged from below 0.01% (detection limit) up to 0.05% of total extracted protein. In a total of 13 transgenic melon plants tested by Western analysis, 2 of 5 plants transformed with sense FL CP, and 4 of 5 plants transformed with Core fragment produced detectable amounts of viral protein. The range of protein level was similar to that of the transgenic tobacco plants, from below 0.01% up to 0.05%. No viral protein was found in all three tested melon plants transformed with ZYMV CP AS constructs. Figure 12 shows a western blot containing protein from different transgenic melon plants and control vector- transformed plants. The antiserum to ZYMV CP clearly binds to protein extracted from plants transformed with viral sense cDNA (lanes 5 and 6) and viral Core fragment (lanes 3 and 4), 4".- 96 914-": 43.0- 29.0- 1e4-.~ Figure 12. Detection of ZYMV CP and Core proteins in transgenic melon plants. Total soluble protein was isolated from leaf samples of transgenic plants. 50 ug total protein isolated from each of six individual plants transformed with three versions of ZYMV CP gene constructs was separated on a 10% SDS polyacrylamide gel, transferred to nitrocellulose, and treated with rabbit antibody against ZYMV CP , followed by alkaline phosphatase- conjugated goat anti-rabbit secondary antibody. Lane 1 and 2 contain protein from AS transformed plants R-103 and R-106, lane 3 and 4 are Core plants, Co-310 and Co-323, lane 5 and 6 are FL CP transformed plants, Cp-401 and Cp-207; lane 7 contains 20 ng of purified ZYMV CP; lane 8 is vector transformed plant. The FL CP is approximately 30 kd, the Core protein is about 26 kd. 97 TABLE 5. SEGREGATION RATIO IN R, TRANSGENIC TOBACCO PLANTS Plant line, Total No. NPT+ NPT- +/_Ratio . x5 0? TO-l 33 25 8 3:1 0.011,,3 CP Tl-l 72 54 18 3:1 0.000u CP T5-3 30 22 8 3:1 0.044. C? T8-3 33 24 9 3:1 0.091u CP F-7 51 48 3 15:1 0.013u CP T3-l 22 0 22 — c0 B-7 56 47 9 3:1 2.381" c0 B-2 53 41 12 3:1 0.157n co E-6 22 4 18 - AS R-3 45 35 10 3:1 0.185“ AS A-l 48 44 4 15:1 0.352" AS c-1 45 24 21 - , Plants labeled CP are transformed with the full length CP construct; CO, construct of truncated core portion of the gene; and AS, antisense version of the gene. 2 X22 values calculated as X2 =£[,(Io-eI-‘/2)2/e] using the Yate's correction factor 3 - Ratios not significantly different from.predicted ratios I“by x’ analysis at P+0. 05. 98 but not to the protein from plants transformed with antisense CP cDNA (lanes 1 and 2) or from the control vector transformed plant (lane 8). e t on 8a sis t ns' ted e t e prgpsgenic plants. To study the inheritance of the inserted genes in transgenic plants, progeny of self-fertilized transgenic tobacco plants were analyzed for the presence of the NPT II gene by ELISA. The results of the progeny analysis of transgenic tobacco plants are shown in Table 5. Progeny from.plants CP TO-l, CP Tl-l, CP T5-3, CP T8-3, CO B-2, CO B-7 and AS R-3 segregated with a ratio of 3:1 (NPT + :NPT -), indicating that the NPT genes was inserted at a single locus. The segregation ratio of progeny from plants CP F-7 and.AS A- 1 were 15:1 (NPT +: NPT-), suggesting that the NPT II gene was inserted at two loci. The reason(s) for the aberrant segregation ratios in the progeny from plants CO E-6, AS G-1 and CP T3-1 are unknown. 99 DISCUSSION Three versions of ZYMV CP gene, FL CP, truncated central- and carboxyl- terminal of the CP gene (Core), and AS CP have been constructed and successfully expressed in muskmelon and tobacco plants. ZYMV CP and Core fragments were detected in more than 90% of kanamycin resistant plants by PCR amplification. This indicated that the constructs in the T-DNA region between the right and left borders remained intact during transformation. Northern blot analysis further verified the co-integration of the NPT II gene and the ZYMV CP gene. The Western blots for coat protein products revealed the presence of the 30 kd FL ZYMV CP polypeptide and the 26 kd core protein products in transgenic plants transformed with FL GP or Core constructs, but did not detect any similar protein in AS expressing plants. The northern and western results verified that the engineered ZYMV CP gene constructs were functional for expression in plants. It has been noticed that the protein level of FL GP or Core in different transformants was different, some plants produced higher level of coat protein, which were detected by western analysis, while other plants produced either no protein, or amounts that were below the detection level. On average, levels of protein expression were at least as high, or higher, for Core than FL CP plants. Higher portion of Core plants produced detectable protein than did FL CP plants. The coat protein (or core) in transgenic plants ranged from under 100 0.01% to 0.05% of the total soluble protein. This is within the range of 0.01% to 0.1% of total plant protein, the expression level that has been reported for engineered viral coat protein in different transgenic plants (e.g. Powell et al., 1986; Van Dun et al., 1987). Transgenic melon plants produced comparable amounts of ZYMV CP protein as transgenic tobacco plants. The plant to plant variability for coat protein level maybe caused by positional effects due to insertion in different chromosomal locations. The segregation studies of the progeny of transgenic tobacco plants indicated that the ZYMV CP gene constructs were . transmitted to the next generation. In most lines the segregation ratios of the progeny were 3:1 (NPT positive: NPT negative), indicating the incorporation of a single gene. With these verified transgenic, CP expressing Rb and RI melon and tobacco plants, it was then possible to initiate virus testing for resistance against ZYMV and other potyviruses as described in chapter four. 101 LITERATURE CITED 1. Allison,R.F., Johnston,R. E.,Dougherty,W.G. 1986. The nucleotide sequence of the coding region of tobacco etch virus genomic RNA: evidence for the synthesis of a single polyprotein. Virology 154:9-20 2. Bradford,M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of proteins utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254. 3. Beachy,R.N., Loesch-Fries,s., Tumer, N.E. 1990. Coat protein-mediated resistance against virus infection. Annu. Rev. Phytopathol. 28:451-474 4. Carrington,J.C., and Freed,D.D. 1990. Cap-independent. enhancement of translation by a plant potyvirus 5' nontranslated region. J. Virol. 64:1590-1597. 5. 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Grumet,R. 1990. Genetically engineered plant virus resistance. Hortiscience 25:508-513 12. Grumet, R., Fang,G.-W. 1990. cDNA cloning and sequence analysis of the 3’-terminal region of zucchini yellow mosaic virus RNA. J. Gen. Vriol. 71:1619-1622. 13. Guilley,H., Dudley,R.K., Jonard,G., Balazs,E., Richards,K.E. 1982. Transcription of cauliflower mosaic virus DNA: detection of promoter sequences, and characterization. Cell 30:763-773 14. Hemenway,C., Fang,R.-X., Kaniewski,J.J., Chua,N.-H., Tumer,N.E. 1988. Analysis of the mechanism of protection in transgenic plants expressing the potato virus X coat protein or its antisense RNA. EMBO J. 7:1273-1280 15. Hoekema,A. Hirsch, P.R.Hoykaas,P.J.J. and Schilperoort, R.A. 1983. A binary strategy based on separation of vir and T- region of the Agrobacterium tumefaciens Ti plasmid. Nature 303: 179-180 16. Horsch,R.B. Fry, J.E., Hoffman, N.L., Wallroth, 103 M.,Eichholz, D., Rogers,S. and Fraley,R.T. 1985. 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Maniatis T, Fritsch E.F., Sambrook, J. 1982. "Molecular Cloning: A Laboratory Manual" Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 22. Murashige T. and Skoog, F. 1962. A revised medium for rapid growth and bioassay with tobacco tissue culture. Physiol. Plant 15:473-498 13'" ‘ll: -1_ :- .235 104 23. Nagy,F., Kay, S.A., Chua,N.H. 1988. Analysis of gene expression in transgenic plants. In Plant Molecular Biology Manual. B4 1-29. Gelvin, 8.8. and Schilperoort, R.A. (eds.) Kluwer Academic Publishers, Dordrecht, Belgium. 24. Niedz R.P., Smith S.S., Dunbar K.B., Stephens C.T., Murakishi H.H. 1989. Factors influencing shoot regeneration from cotyledonary explants of Cucumis melo L. Plant Cell Tissue and Organ Cult. 18:313-319. 25. Powell-Abel,P.A., Nelson,R.S., De,B., Hoffman,N., Rogers,S.G. , Beachy, R.N.1986. Delay of disease development in transgenic plants that express the tobacco mosaic virus coat. protein gene. Science 232:738-743 26. Powell,P., Stark,D.M., Sanders,P.R. and Beachy,R.N. 1989. Protection against tobacco mosaic virus in transgenic plants that express TMV antisense RNA. Proc. Natl. Acad. Sci. USA 86:6949-6952 27. Quemada,H., Sieu,L.C., Siemieniak,D.R., Gonsalves,D., Slightom,J.L. 1990. Watermelon mosaic virus II and zucchini yellow mosaic virus: cloning of 3'-terminal regions, nucleotide sequences, and phylogenetic comparisions. J. Gen. Virol. 71:1451-1460. 28. Rothstein,S.J. , Lahners,K.N. , Lotstein,R.J. , Carozzi,N.B. , Jayne, S.M., Rice, D.A. 1987. Promoter cassettes, antibiotic- resistance genes, and vectors for plant transformation. Gene 53: 153-161 29. Stark,D.M. and Beachy,R.N. 1989. Protection against iii. 105 potyvirus infection in transgenic plants: evidence for broad spectrum resistance. BioTechnol. 7:1257-1262 30. Van Dun,C.M.P., Bol,J.F. 1988. Transgenic tobacco plants accumulating tobacco rattle virus coat protein resist infection with tobacco rattle virus and pea early browning virus. Virology 167:649-652 CHAPTER FOUR PLANTS EXPRESSING THE ZYMV COAT PROTEIN GENE CONSTRUCTS ARE PROTECTED AGAINST INFECTION BY ZYMV AND HETEROLOGOUS POTYVIRUSES 106 107 INTRODUCTION Genetically engineered coat protein protection has been used to develop resistance to viruses in several virus groups (for review see Beachy et al., 1990; Grumet 1990). In most of the examples, transgenic plants expressing the CP gene from a given virus were protected against infection by the virus from which the CP gene was isolated (homologous virus). Virus inoculated leaves of the transgenic plants show fewer chlorotic or necrotic lesions relative to control plants, and systemic spread of infection is either prevented, delayed or reduced. CP-mediated protection has also been demonstrated to extend to strains or viruses that are closely related to the virus from which the CP gene was obtained (heterologous viruses). For example, the CP of tobacco mosaic virus (TMV) U1 strain protects against infection by the severe strain of TMV, PV230 (Nelson et al. 1987), and against some other tobamoviruses (Nejidat and Beachy 1990). Expression of the CP genes of the potyviruses soybean mosaic virus (SMV) and papaya ringspot virus (PRV) in transgenic tobacco plants conferred protection against infection by two other potyviruses, tobacco etch virus (TEV) and potato virus Y (PVY, Stark and Beachy, 1989; Ling et al. 1991). Cucurbit plants are subject to severe losses due to infection by three potyviruses, the watermelon strain of PRV (PRV-W), watermelon mosaic virus (WMV) and zucchini yellow mosaic virus (ZXMV). Among these ZYMV is a relatively new but 108 very aggressive member of the potyvirus group that has spread rapidly throughout the world since it was first described in 1981 (Lisa et al. 1981; Davis and Mizuki, 1987). In this. project I sought to engineer resistance to ZYMV, to test for both homologous and heterologous protection, and to gain insight into possible mechanisms of protection by using a set of three different CP-derived constructs. Both melon and tobacco plants were transformed with: (1) the full length ZYMV CP gene; (2) a fragment coding for only the conserved central and C-terminal regions of the protein; and (3) an antisense version of the gene. This chapter describes the effect of these genes on increasing the resistance of transgenic melon and tobacco plants to infection by ZYMV and two heterologous potyviruses, TEV and PVY. 109 MATERIALS AND METHODS 2;§p§;§§ipp_p§_ippppi§; The Connecticut strain of ZYMV was maintained.and increased in zucchini (Cucurbita,pepolcv.IBlack Jack) plants. TEV, TMV and PVY are propagated and maintained in tobacco (Nicotiana tobacum cv. Samsun) plants. Inocula were made from infected zucchini or tobacco plants by homogenizing leaf tissue in 20 mM KPO, buffer, pH 7.0. The homogenate was diluted to a specific concentration (gm leaf tissue : ml buffer volume). PVY, TEV and TMV, were diluted to 1:20, 1:100 and 1:100 respectively. For ZYMV, a 1:75 dilution was used. Elgp§_mg§e;igl: Muskmelon (Cucurbita melo cv. Hale’s Best Jumbo) and tobacco (cv. "Samsun") plants transformed with FL- CP, Core or AS of the ZYMV CP gene were used for the virus infection tests. Rlpdants were produced by self pollinating- Igjplants. To produce a set of replicate individuals for virus testing of the transgenic muskmelon R0 plants, shoots were cut from R0 transgenic plants and rooted in rooting cubes of the Oasis growing medium (Smithers-Oasis Co.), Plants at 20 days after rooting were used for the infection test. The parent plants were used for seed production. Rl melon plants at 16 days after seed germination were inoculated with ZYMV (3-4 leaf stage). R0 tobacco plants with 4-5 leaves, Rl tobacco plants at 22-25 days or 32-35 days after seed germination‘were inoculated with PVY, TEV or TMV. WOW Plants were dusted ‘ with 400 mesh carborundum on the two youngest expanded leaves- 110 and rubbed with viral inocula. The inoculated plants were observed daily for systemic disease symptom development. Leaf tissues were sampled periodically after inoculation to determine virus level in. plants by indirect ELISA. The antibodies used. were: anti-ZYMV antibody raised against ZYMV virion (Hammer and Grumet, unpublished), antibody against PVY (Agdia Inc.), and antibody against the potyvirus group (Agdia Inc.), which was used for detecting TEV. Leaf disk samples were placed in microtiter plates, and then frozen (at -80°C) and thawed twice. The leaf tissue samples were then incubated in coating buffer at 4°C fOr 16 hours. The samples were reacted with 1:1000 (ZYMV or PVY) or 1:100 (TEV) dilution of antibodies for two hours at 37’0 C, prior to the addition of alkaline phosphatase-goat anti-rabbit antibody conjugate (Sigma) at 37° C for another two hours. After washing plates, p-nitrophenyl-phosphate substrate was added and absorbance at 405 nm was monitored using a Datatech plate reader. e ta desi o determi ' otect'o ev tnansggnic plants. To test for protection against ZYMV infection in,I% transgenic melon plants, cuttings from 5-6 independently transformed plants from each group (CP, Core, AS, vector only and non-transformed control) were inoculated with ZYMV by rub inoculation. Symptom development was observed and recorded daily. Samples of the uppermost second and third leaves were collected periodically after inoculation to 111 quantitate virus titer by ELISA. The protection test was performed on two separate sets of independently transformedRo plants from each genotype (a total of 9-16 plants/genotype). The first experiment included vector-only transformed plants as controls in addition to the non-transformed controls. In the resistance test of R1 melon plants, seventeen NPT+ progeny plants from each transgenic line or non-transformed control were inoculated with ZYMV, symptom development was recorded daily. In the heterologous protection tests of transgenic tobacco plants, R0 plants were first screened for resistance against virus infection. Twenty to twenty-five independently transformed plants from.each.group (CP, Core, AS and Controls) were inoculated with PVY. The RO PVY experiments were performed three times, each with different sets of independently transformed plants. A total of 60-70 independent Ro's per constructs were tested. Symptom development was recorded, and virus titer determined by ELISA. Several transgenic plants were allowed to self pollinate to produce progeny. The progeny seedlings of each transgenic line were first verified for the presence of NPT II gene by ELISA (see chapter three). Approximately 10-20 NPT+ plants from each line, 10-20 NPT- plants, and 10-20 non-transformed control plants were inoculated with PVY, TEV or TMV. The PVY and TEV experiments were performed three times, different lines of R1. progeny were included. The TMV experiment was performed once. 112 The number of plants showing disease symptoms was recorded daily. Leaf samples were collected 20 and 50 days after inoculation.and virus titer was determined by ELISA. The virus level in each line was calculated as the mean of the ELISA readings of individual plants in the group. The data for both melon and tobacco plants were analyzed by t-tests and/or~ analysis of variance. 113 RESULTS 1, Ezgtection in transgenic meion plants ngainst zxny infiecpion. Transgenic and control melon plants were inoculated with ZYMV, and monitored daily for the appearance of disease symptoms. All of the non-transformed control plants showed symptom ten days after inoculation (Figure 13). Infected leaves exhibited severe mosaic symptoms (Figure 14), and the growth of infected plants was greatly reduced. All younger leaves and shoots displayed the disease symptoms. ' Symptom development in vector transformed control plants, which had only the NPT II gene, was equivalent to wild type plants in both time to appearance and severity of symptoms (data not shown). Plants expressing ZYMV core protein, showed a 3-10 day delay in symptom appearance, eventually, however, all become infected (Figure 13). The symptoms on the core-protein expressing plants were milder than for the control plants in most cases. Furthermore, after 3-4 weeks, several of the plants appeared to recover; the newly emerging leaves were either asymptomatic or showed very mild symptoms. Similar- results were obtained in both experiments. Five of the six plants expressing ZYMV AS transcripts showed a delay in symptom appearance (3-9 days), the sixth plant, however, did not develop symptoms for at least 90 days post inoculation. The pattern and degree of severity of disease symptoms in three of the five symptom-expressing plants were not 114 INFECTED PLANTS I: it 't : nae-onus: l o +s l J ‘1 L 1 L l I l l l 5 67 8 91011121314161617181020 DAYS POST INOCULM'ION (1:76 INOCULUH) -°- Control + Core 4‘- As -°- GP Figure 13. Zucchini yellow mosaic virus (ZYMV) symptom development in R0 transgenic melon plants. The plants were inoculated with a 1:75 (W/V) homogenate of ZYMV infected leaf tissue. CP, plants transformed with ZYMV FL CP construct; Core, plants transformed with ZYMV Core construct, and AS plants transformed with ZYMV antisense CP construct, the control plants are non-transformed plants. Each genotype includes 6 plants. The symbol used to designate each genotype is shown at the bottom of the frame. 115 Figure 14. Comparison of ZYMV disease symptom in non-transformed control and CP+ transgenic melon plants. Plants were mechanically inoculated with a 1:75 homogenate prepared from leaves of zucchini yellow mosaic virus (ZYMV) infected plant. By three weeks post inoculation, severe classic symptoms (mosaic) of systemic ZYMV infection manifested in the leaf of the control plant (right), whereas the transgenic plant expressing ZYMV CP showed no visible disease symptoms. 116 obviously different from that of the control plants at two weeks post inoculation; two plants, however, showed milder symptoms. With increasing time (greater than three weeks post. inoculation), the symptoms in all four plants were attenuated in younger leaves relative to control plants. In striking contrast to the core protein and antisense expressing plants, all of the plants producing the full length CP, a total of eleven independently transformed R0 plants tested in two experiments showed a much higher protection level against ZYMV infection. In both experiments, symptom appearance in all CP+ plants was delayed for at least 30 days. Eight of the eleven plants did not show any disease symptom for at least 90 days post inoculation, three of the eleven plants produced some very mild, modified symptoms 30 days post inoculation. To compare the extent of virus replication and spread in the transgenic plants expressing constructs of the ZYMV CP gene and the non-transformed control plants, leaf disks were sampled from systemic leaves of each inoculated plant. Virus levels were quantitated by ELISA analysis using antibody against ZYMV. Consistent with the lack of symptoms, the FL CP expressing plants did not accumulate measurable virus levels (Figure 15); the ELISA values were not significantly different from non-inoculated control plants (ANOVA; P=0.05). The virus titer in transgenic plants expressing the Core construct was intermediate to the inoculated controls and the FL CP+ plants. 117 Relative virus level“) 120 ‘°°’ l/ 7 g g ai” /’/’ SO / / i g g ... Z % 5225 Egg; .._ Z Z 55“ ,,-’ 20L % % gfififi z”’ o % Z: 17DPI OSDPI -oConmI Clo? -cone --Control Figure 15. Zucchini yellow mosaic virus (ZYMV) accumulation in R0 transgenic melon plants and non-transformed control plants. The transgenic plants were transformed with full length ZYMV CP gene (FL CP), Core construct (Core) or antisense construct (AS). The plants were mechanically inoculated with a 1:75 homogenate of ZYMV infected tissue. Duplicate leaf disk samples were obtained from upper non-inoculated leaves 17 days post inoculation (DPI) and 45 DPI, and assayed for presence of ZYMV by ELISA as described in Materials and methods. The relative virus titer in non-transformed control plants was defined as 100%. mil . WH i. n “‘4‘: 118 The virus levels 'in the AS plants were highly variable (44% 1 70%) and reflected the variability in the reaction of these plants to infection; e.g. one plant did not have symptoms or. measurable virus levels, another had virus levels as high as controls (data not shown). The difference in virus levels among transgenic melon plants and the control plants persisted throughout the observation period (Figure 15) . At 45 DPI, as at 16 DPI, the mean values for the transgenic groups was significantly less than that of the controls (ANOVA, P=0.05). The resistance observed in the R0 plants was also displayed in their R, progeny. The parental lines (CP 401 and Core 310) used to make R, progeny both had comparable high levels of viral protein expression. All 17 NPT+ progeny from‘ a FL CP line (CP401) were asymptomatic at 25 days post inoculation (Figure 16) , whereas the average time for symptom appearance in control plants is 10 DPI. Several R, Core plants (line 310) showed a delay of several days in symptom appearence, but all of them were infected by 21 DPI. These experiments are still in progress to extend the observation period and test additional R, lines. 2. netggologous protection of transgenic 3,, tobacco pigntg ggaingt PVY infecpi on. To determine if transgenic plants expressing ZYMV CP gene constructs could confer protection against heterologous potyviruses, R0 transgenic tobacco plants ' were inoculated with PVY. Most of the transgenic plants 120 displayed enhanced resistance to PVY infection. Vector transformed controls and wild type plants showed disease symptoms at a similar rate (Table 6-A), all controls were infected 7-9 days after inoculation, depending on the experiment. Transgenic tobacco plants expressing full length CP showed a delay in symptom development from several days to a few weeks when compared to the control plants; a few plants did not exhibit a delay in symptom , whereas several other~ plants were asymptomatic for the whole growth period. A several days delay of symptom appearance was also observed in plants expressing Core or AS. Virus levels in systemic leaves were determined by ELISA assay using antibody against PVY. Those plants transformed with vector only had similar virus titer to the non-transformed controls (Table 6-B). The virus titer for the transgenic plants expressing the different CP constructs, however, was significantly reduced relative to the controls at 25 DPI (Table 6-B). This reduction in virus titer persisted for at least 45 days (data not shown). Those plants that did not become infected had no measurable virus titer;- when several asymptomatic R0 plants were tested as a virus source by rub inoculation, they did not transmit virus. 3. Protection of R, transgenig tgbagco lines agains; EV! injggnign. TheiR,progeny of several transgenic lines were rub inoculated with PVY at 1:20 or 1:100 inoculum, and observed for symptom development. All "Samsun" non-transformed controls became infected within 6 days after inoculation with 1:20 121 TABLE 6-A. va svnpron DEVELOPMENT IN Ro TRANSGENIC TOBACCO PLANTS' , PVY infected plants (%) Genotype # DPI 5 6 7 8 9 10 11 12 .13 14 Control 23 0 26 74 91 100 100 100 100 100 100 Vector only 19 10 67 84 95 100 100 100 100 100 100 OF 25 O O 0 16 78 84 88 88 88 88 Core 22 O O 18 68 73 77 86 100 100 100 AS 23 0 O 0 13 22 69 74 74 83 83 TABLE 6-B. PVY VIRUS TITER IN TRANSGENIC Ro TOBACCO PLANTS AT 25 DAYS POST INOCULATION plants with symptoms] mean relative Genotype total plants Abs amount range Control 16/16 1.012 1.00 0.591-1.373 Vector only 16/16 . 0.914“: 0.90 0.668-1.251 op 21/24 0.344** 0.34 0.750—0.920 - symptoms 3/24 0.088** 0.09 0.075-0.107 + symptoms 21/24 0.379** 0.37 0.101-0.920 Core 13/13 0.685** 10.68 0.328-0.917. AS 20/24 0.219** A 0.22 0.600-0.708 ‘_ symptoms 4/24 0.085** 0.08 0.060-0.110 + symptoms 20/24 0.245** 0.24 0.088-0.708 ' The plants were inoculated with a 1:100 homogenate of PVY infected tissue :Hu.- value not significantly different from control by t- test; **, significantly less than control by t-test, 122 . ‘ L 7 e o s s a name-aun- d-eoee +0104 *0!» -°-v'-r B “SM“W “ c c c ‘ ‘ C I C I . e 3 ‘ . S s 8 e ”MM *0.“ +OH *NH ‘0'“ *UO-Q *UH Figure 17. Potato virus Y (PW) symptom development in tobacco R1 transgenic lines. Percentage of plants showing symptoms at daily intervals post inoculation with a homogenate of 1:20 (W/V) of PV! infected leaf tissue. (A) . Seedlings of transgenic lines of CPTo-l and CPP-l, which were the progeny of the plants transformed with zrnv FL CP construct, and seedlings of non-transformed plant. (B) . R, progeny of the lines transformed with ZYMV Core construct. (C). R, progeny of the lines transformed with ZYMV antisense CP construct. The symbol used to designate each plant line is shown at the bottom of the frame. Results were obtained from the infection of about 20 R, plants from each line. - 123 inoculum (Figure 17). Segregating R, progeny were tested for presence or absence of the introduced NPT gene by NPT II ELISA (see chapter three, Table 5). Those that did not express the NPT II gene showed the same rate of symptom appearance as the wild type controls (data not shown). In the NPT positive individuals that had been transformed with CP constructs, disease symptom appearance was delayed for 2-5 days in the majority of the plants compared to the controls (Figure 17-A), a few plants from the CPF-7 remained asymptomatic for 3-4 weeks. The disease symptoms in most plants were milder, and often younger leaves were devoid of disease symptoms. Virus accumulation as determined by ELISA was correlated with the degree of visual symptoms (Table 7). NPijositi e plants in line CoB-2 and line CoB-7, which were transformed with the ZYMV core gene, also showed a 1-4 day delay in symptom appearance (Figure 17-B); the disease symptoms were milder than for the control plants. Plants expressing antisense CP transcripts, line ASG-l and line ASR-3, displayed a short delay in symptom development,. from 1-2 days (Figure 17-C) , the symptoms were slightly attenuated. The difference in symptoms and virus titer between most transgenic lines and the controls were persistent during the observation period of 50 days post inoculation (data not shown). 5, TEV pnotggni on in trnnsggnic B, pgpggco pignts: The R, transgenic tobacco lines were further tested by inoculation 124 with another potyvirus, TEV. Symptom appearance for most of the transgenic individuals expressing ZYMV CP constructs was delayed for 2-4 days relative to the controls (Figure 18-A). All of the non-transformed control plants and the NPT II negative plants were infected 7 days after inoculation with 1:100 inoculum of TEV, in contrast, only 40-80 % of the transgenic plants within a given line showed diseased symptoms at the same time. Symptoms were milder in most transgenic plants than the control plants. Virus titer was also significantly reduced relative to the controls (Table 7). All of the transgenic lines (CP, Core or AS) displayed similar patterns and protection levels (Figure 18). 5, 21M! CP constructs did not confer pnogection ggains; TM! infegtion. To determine if protection in transgenic plants expressing the different forms of the ZYMV CP gene was specific to members of the potyvirus group, R, progeny were ' challenged with a tobamovirus, TMV. There was no obvious difference between transgenic plants expressing any of three forms of the ZYMV CP gene and the control plants in time to symptom appearance (Figure 18), or symptom severity in systemic leaves post TMV inoculation. 125 elk-fishes.- ”an!” *H *0 ”DI *OF-f OM*-- " 0 O I I q C C I 3 fins-suede- *6” *OI-f *“D'I C m‘mmm see- ‘ ‘ .- .- ..l .I ‘a T S e ? I T .- n annual-une- *e-u --na-a *ase-c Figure 18. Tobacco etch virus (TEV) symptom development in tobacco R1. transgenic lines. Plants were inoculated with a homogenate of 1:100 (W/V) of TEV infected leaf Atissue. (A). Seedlings of transgenic lines of CPTo-l and CPF-l, which were the progeny of the plants transformed with ZYMV FL CP construct, and seedlings of non- transformed plant. (B). Rl progeny of the lines transformed with ZYMV Core construct. (C). R1 progeny of the lines transformed with ZYMV antisense CP construct. The symbol used to designate each plant line is shown at the bottom of the frame. Results were obtained from the infection of about 20 Rl plants from each line. 126 mesons Haunts) ‘20 100 80 60 4O l L 1 1 7 8 9 10 II 12 13 14 oars roar mocuurtou O + Control + CPTl-l + COB-7 ‘9‘ ASH-3 Figure 19. Tobacco mosaic virus (TMV) symptom development in tobacco R1 transgenic lines. Plants were ninoculated with 1:100 (W/V) of TMV infected leaf tissue. The symbol used to designate each plant line is shown at the bottom of the frame. Results were obtained from the infection of about 20 R1 plants from aech line. 127 DISCUSSION These results demonstrate that transgenic melon plants expressing the full length ZYMV coat protein gene are highly resistant to infection by ZYMV. The majority of the CP- expressing R0 melon plants remained asymptomatic for a period of at least two to three months. These plants also had no measurable virus accumulation, while all of the inoculated control plants had a high level of virus. The striking resistance observed in the R0 plants was also evident in their R, progeny. As of 25 days post inoculation the progeny did not exhibit symptoms, whereas the average time to symptom development in the control plants was 10 DPI. 'The R, experiments are still in progress. The potyvirus group is the largest and most agriculturally important group of plant viruses, but at present, reported examples of genetically engineered resistance to potyviruses are limited. Three reports of CP-mediated protection in potyviruses used PVY, TEV and plum pox virus (PPV) CP genes to protect transgenic plants against their corresponding viruses respectively ( Lawson et al., 1990; Lindo and Dougherty 1992; Regner et al., 1992). Our experiments extended genetically engineered virus resistance to an additional economically important potyvirus, ZYMV. Furthermore, with the exception of alfalfa plants that have been engineered for resistance to AlMV, all of the examples of engineered plant virus resistance to date have been limited to Solanaceous species, tobacco, 128 tomato or potato plants. Demonstration of resistance in additional non-Solanaceous species further verifies the general applicability of this approach as a possible method to develop virus resistant plant cultivars. Virus infection tests in transgenic tobacco plants showed that expression of different forms of the ZYMV CP gene also resulted in limited protection against two heterologous, Solanaceous potyviruses, TEV and PVY. Heterologous CP-mediated protection has been reported for two other members of the potyvirus group, SMV and PRV (Stark and Beachy 1989, Ling et al. 1991) against TEV and PVY. Compared to the delay conferred by the SMV and PRV coat proteins, the heterologous protection ' levels in our experiment are not as strong. The differences in effectiveness against heterologous viruses may related to the individual relationships among the different viruses. Despite the limited protection, these results contribute support to the hypothesis that it may be possible to protect against several viruses by using a limited number of different CP genes (Stark and Beachy 1989). Since many crops are often infected by several members of the potyvirus group, this phenomenon would make it much easier to develop resistance to the potyviruses. Of particular interest is to determine whether the ZYMV-CF gene will confer protection against the' other. potyviruses which infect cucurbit plants, e.g. PRV-W and WMV. In all but one case of genetically engineered, coat 129 protein-mediated resistance, full length coat protein genes were used. To gain insight into the mechanism of protection, and to determine whether it would be possible to confer more broad scale protection, we also tested an amino terminal truncated coat protein construct. Potyviral coat proteins are noted to have highly conserved central and carboxyl terminal. regions, the ’trypsin resistant core' portion of the protein, and highly variable amino terminal portions (Shukla and Ward, 1989). Since trypsin treated virion still appear intact in the electron microscope, and are still infectious by rub inoculation, the domains within the CP that are responsible for assembly, CP-RNA interaction and CP-CP interaction, are thought to reside within the core portion of the protein (Shukla et al., 1988; Dougherty et al., 1985). It has been hypothesized for several system that CP-mediated protection involves CP-RNA or CP-CP interaction (Beachy et al., 1990; Grumet, 1990). If these processes are critical for protection against potyvirus infection, then the core portion of the protein would be expected to confer resistance. Interestingly, although the core portion was expressed at levels comparable to the FL CP (Core310 and CP401, see chapter three) and did confer some protection (several days delay and reduced virus titer), it was not as effective as the FL-CP construct which resulted in near immunity. Possibly the core and amino terminus of the protein interfere with ‘virus infection at different stages of the process, or the full 130 length CP may have higher affinity for viral RNA or other CP. molecules than does Core. In the case of protection against the heterologous viruses TEV and PVY, both the FL-CP and Core constructs performed similarly. It may be that the function provided by the core portion or its RNA, which results in a delay in infection and reduction in virus titer, is capable of acting on more than one potyvirus. In contrast, the effect of the amino terminus, which is highly virus specific, may be limited to the virus from which the CP gene was derived. These results are somewhat different from the report by Lindo and Dougherty (1992), which is the only other example using a truncated CP gene to engineer virus resistance. In. their experiments, unlike those with PVY, PPV and ZYMV, the full length TEV CP gene provided little or no protection against TEV infection. On the other hand, tobacco expressing truncated TEV CP were more protected from TEV infection than were transgenic plants expressing the FL TEV CP. The constructs lacking C-terminal amino acids or the constructs lacking both C- and N-terminal amino acids resulted in much better protection against TEV infection than constructs lacking N-terminal amino acids only, which did not show obvious protection against TEV. The reason (s) for the difference between these experiments is unclear. The heterologous protection experiments also give insight into the possible role of the TEV 5’ UTR. All three forms of ZYMV CP gene constructs resulted in relatively similar 131 ‘ protection against both heterologous viruses TEV and PVY. This indicates that the 5’ UTR from TEV may not be a critical factor in conferring protection. Although it may have contributed to protection overall, either by increasing the translational efficiency of the CP constructs, or by interacting with the viral RNA, there did not appear to be a virus specific effect in its interaction with TEV vs. PVY. . Plants expressing antisense RNA of the ZYMV CP plus the TEV 5'UTR resulted in variable protection levels in transgenic melon plants against potyvirus infection. On average the AS constructs were similar to the core constructs, raising the possibility that it was the core RNA rather than the protein that was important. In most virus system example totdate (e.g. CMV, PVX, TMV or AlMV) antisense CP genes have resulted in little or no protection (Cuozzo et al., 1988; Hemenway et al.,1988; Powell et al.,1989; and Van Dun et al., 1988). However, there were two exceptions published recently, where it was hypothesized that it was the RNA that conferred protection. Potato plants expressing antisense CP RNA (including 5' UTR) of potato leafroll virus (PLRV) displayed a high level of resistance to PLRV infection, the pattern and level were similar to plants producing sense RNA (Kawchuk et al. 1991). In transgenic tobacco plants, expression of antisense or translationally-deficientA sense TEV CP RNA resulted in much higher protection against TEV infection than that by expression of the sense version of FL coat protein 132 gene (Lindo and Dougherty, 1992). To understand the mechanism of antisense RNA related protection, and to determine the effectiveness of the strategy, further investigations are required. In summary, we have demonstrated that transgenic melon plants expressing three forms of ZYMV CP gene were protected against ZYMV infection” The best.protection, near'immunity“was observed for melon plants that express the full length ZYMV CP gene and were inoculated with ZYMV. Heterologous protection- against two other potyviruses was also displayed in transgenic tobacco plants expressing ZYMV CP constructs. Melon plants expressing FL CP displayed higher protection levels than Core or AS expressing transgenic plants. To further evaluate the potential usefulness of this resistance in cucurbits, future experiments with the ZYMV CP expressing plants would include test against other ZYMV strains and against other cucurbit potyviruses, tests using aphid inoculation and tests of field performance. 133 LITERATURE CITED 1. Alderz, W.C. 1987. Cucurbit potyvirus transmission by alate ' aphids trapped alive. J. Economic Entomol. 80:87-92. 2. Anderson, B.J., Stark, D.M., Nelson, R.S., Powell, P.A., Tumer, N.E. and Beachy, R.N. 1989. Transgenic plants that express the coat protein genes of TMV and AIMV interfere with disease development of some non-related viruses. Phytopathol. 79:1284-1290. 3. Beachy, R.N., Loesch-Fries, S., Tumer, N.E. 1990. Coat protein mediated resistance against virus in fection. Annu. Rev. Phytopathol. 28:451-474. 4. Carrington, J .C. and Freed, D.D. 1990. Cap-independent enhancement of translation by a plant potyvirus 5’- nonotranslated region. J. Vriol. 64:1590-1597 5. Cuozzo, M., O’connell, K.M., Kaniewski,W., Fang,R.-X., Chua, N.-H., Tumer,N.E. 1988. Viral protection in transgenic tobacco plants expressing the cucumber mosaic virus coat proetin or its antisense RNA. BioTechnol. 6:123-143. 6. Davis, R.F. and Mizaki, M.K. 1985. Detection of cucurbit viruses in New Jersey. Plant disease 71:40-44. 7. Daugherty, W.G., Willis, L. and Johston, R.E. 1985. Topographic analysis of tobacco etch virus capsid protein epitopes. Virol. 144:66-72 8. Grumet, R. 1990. Genetically engineered plant virus . resistance. Hortiscience 25:508-513. 9. Grumet, R. and Fang, G. 1990. cDNA cloning and sequence 134 analysis of the 3'-terminal region of zucchini yellow mosaic virus RNA. J.Gen. Virol. 71:1619-1622. 10. Hemenway, C., Fang, R.-X., Kaniewski, J.J., Chua, N.-H., Tumer, N.E. 1988. Analysis of the mechansim of protection in transgenic plants expressing the potato virus X coat protein or its antisense RNA. EMBO J. 7:1273-1280. 11. Kawchuk, L.M., Martin, R.R., McPherson, J. 1991. Sense and antisense RNA-mediated resistance to potato leafroll virus in russet burbank potato plants. Mol. Plant-Microbe Interact. 4:247-253. 11. Lawson, C., Kaniewiski, W., Haiey, L., Rozman, R., Newell, C., Sanders, P., and Tumer, N.E. 1990. Engineering resistance to mixed virus infection in a commercial potato cultivar: resistance to potato virus x and potato virus Y in transgenic Russet Burbank. BioTechnol. 8:127-134. 12..Lecoq, H. and Pitrat, M. 1985. Specificity of the helper- component-mediated aphid transmission of three potyviruses infecting muskmelon. Phytopathol. 75:890-893. 13. Lindo, J .A. and Dougherty, W.G. 1992. Pathogen-derived resistance to a potyvirus: immune and resistant phenotypes in transgenic tobacco expressing altered forms of a potyvirus coat protein nucleotide sequence. Mol.Plant-Microbe Interact. 5:144-153. 14. Ling, K., Namba, S., Gonsolves, C., Slighton, J.L. and Gonsalves, D. 1991. Protection against detrimental effects of potyvirus infection in transgenic tobacco plants expressing 135 the papaya ringspot virus coat protein gene. BioTechnol. 9:752-758. 10. Loesch-Fries, L.S., Halk, E., Merlo, D., Jarvis, N., Nelson, S. 1987. Expression of alfalfa mosaic virus coat protein. gene and anti-sense cDNA. in transformed. tobacco. tissue. 11. Miller, W.A., Bujarski, J.J., Dreher, T.W. and Hall,T.C. 1986. J. Mol. Biol. 187:537-546. 12. Nejidat, A and Beachy, N. 1990. Transgenic tobacco plants expressing a coat protein gene of tobacco mosaic virus are resistant to some other tobamoviruses. Mol. Plant-Microbe Interactions 3:247-251 13. Nelson, R.S., Powell-Abel, P., and Beachy, R.N. 1987. Lesions and virus accumulation in inoculated transgenic tobacco plants expressing the coat protein gene of tobacco mosaic virus. Virol. 158:126-132. 14. Powell-Abel, P.A., Stark, D.M., Sanders, P.R. and Beachy, R.N. 1989. Protection against tobacco mosaic Vitus in transgenic plants that expree TMV antisense RNA. P.N.A.S. USA 86:6949-6952. 15. Regner, F., Machado, A.C., Machado, M.L.C., Steinkellner, H., Mattanovich, D., Hanzer, K., Weiss, H., and Katinger, H. 1992. Coat protein mediated resistance to Plum Pox Virus in Nicotiana clevelandii and N. benthamiana. Plant Cell Report 11:30-33 1 16. Shulka,D.D., Strike,P.M., Tracy,S.L., Gough,K.H., Harm-ml!— 136 Ward,C.W. 1988. The N- and C-termini of the coat protein of potyviruses are surface located and the N-terminus contains the major virus-specific epitopes. J. Gen. Vio1.69:1497-1503' 17. Shukla, D.D. and Ward, C.W. 1989. Structure of potyvirus coat protein and its application in the taxonomy of the potyvirus group. Adv. In Virus Res. 36:273-314. 18. Stark, D.M. and Beachy, R.N. 1989. Protection against potyvirus infection in transgenic plants: evidence for broat spectrum resistance. BioTechnol. 7: 1257-1262. 19. Van Dan, C.M.P. and 801, J.F. 1988. Transgenic tobacco plants accumulating tobacco rattle virus coat protein resist infection with tobacco rattle virus and pea early browning virus. Virol. 167:649-652. A Quick and Inexpensive Method for Removing Polysaccharides from Plant Genomic DNA ABSTRACT A quick and inerpensive method has been demonstrated to remove polysaccha- ride contamination from plant DNA. Iso- lated plant genomic DNA with polysac- charide contaminants was dissolved in TE (IO M Tris-MCI. pH 7.4. 1 MM ETA) with NaCl ranging flour 0.5-3.0 M. then precipitated with two wltanes of ethanol. Most of the polysaccharides were removed efl'ectt’vely in a single high-salt precipita- tion at 1.0—25 M NaCl. At 3.0 M NaCl. the salt precipitated out of solution. Puri- fied DNA was easily digested by either Hind/ll or EcoRI and was satisfactory as a template for PC R. The results show that high-salt precipitation efi'ectively removed polysaccharides and their inhibitory ef- fects on restriction en:yme and Taq pol- ymerase activity. INTRODUCTION Polysaccharidc contamination is the most common problem affecting plant DNA purity (l0). These carbohydrates can inhibit the activity of many mo- lecular biological enzymes. such as po- lymerases. ligases and restriction en- donucleases (1.9.12.13). and can interfere with concentrating the DNA sample. Unfortunately. most plant DNA isolation and purification methods do not efficiently separate polysaccharides from DNA (3.6.7.10.l2). Phenol-dilato- form extractions are only partially helpful. and the use of CT AB (cetyltri- methylammonium bromide) often re- sults in DNA degradation (unpublished results and personal communications). It was recently reported that Elutip-D (RFC-5 type resin) (Schleicher d: Schuell. Keene. NH) columns can be used to remove polysaccharides from DNA (5). Although effective and sim- ple. this procedure is volume-limited. time-consuming and relatively expen- sive. We sought to adapt the column procedure to a batch method using ion- exchange resins. In doing so. we found that the high-salt buffer used to elute 52 BiaTechniques 137 the DNA from the resin was by itself sufficient to remove the polysaccha- ride. In this repon we show that by disc solving polysaccharide-oontanunated plant DNA samples in TE buffer with high salt (LO-2.5 M NaCl) and then precipitating with ethanol. the polysac- charide remains in solution with the ethanol rather than precipitating with the DNA. This quick and inexpensive methodcanbeusedwith any DNAiso- lation and purification protocol. We tested the efficiency of different con- centrations of salt (NaCl) in separating polysaccharides from DNA samples. The DNA quality was evaluated by re- striction enzyme digestion and by us- ing the DNA as a substrate for PCR. MATERIALS AND METHODS Greenhouse-grown muskmelon (C ttcurnt's melo) and cucumber (C. sa- (it'll!) leaves were chosen for DNA iso- lation because the DNA from these plants is often contaminated with large amounts of polysaccharides. DNA was also isolated from potato (Solanum tu- berosum). soybean (Glycine max) and geranium (Pelargont'um) leaves. Young leaves were quickly frozen in liquid nitrogen and ground to a fine powder in a mortar and pestle. Genomic DNA was isolated according to the prorocol of Dellaporta et al. (4). Isolated DNA was then extracted three times with phenolzchloroformzisoamyl alcohol (24:24: I) and precipitated with two volumes of 95% ethanol. The DNA pellet was redissolved in TE. the appropriate amount of 4 M NaCl was added to give a final concentration ranging from 0.5-3.0 M. and the sam- ple was reprecipitated with ethanol. For effective polysaccharide removal. it was very important to completely dissolve the DNA in TE prior to the ad- dition of salt. Alternatively. NaCl was added to the sample directly after phe- nol extraction dtereby eliminating the need for two ethanol-precipitation steps. The resulting DNA pellet was. washed twice with 75% ethanol and dissolved in TE. Polysaccharide contamination was determined visually (noticeably vis- cous pellets) by refractomeny (ABBE- 3L: Milton Roy. Rochester. NY) or by the phenolosulfunc acid method of Ashwell (2). Pure salmon sperm DNA gave a negligible rtfractornetric read- ing (0.01 for 1 ug DNA/u!) md a negli- gible carbohydrate reading a esti- mated by the phenol-sulfuric acid assay (0% ug/ug DNA). Melon and cucum- ber DNA samples that were precipi- tated with or without high salt (2 M NaCl) were digested with Hindlll and EcoRI and examined on a 0.8% agarose gel. Genomic DNA isolated from aansgenic melon plants (8) was alsousedastemplateforPCR.PCR was performed according to the manu- facmrer's protocol (Perkin Elmer. Nor- walk.Cl')using lOOngofDNAtern- plate for each reaction and two l8-mer primers specific for the neomycin phosphotransferase (NPT) gene [NPT sequencedata from(ll)andananneal- ing temperature of 50°C]. The products were examined on a 1% agarose gel. RESULTS AND DISCUSSION Polysacchan'de contamination is a common problem when isolating DNA from many plant species (10). For ex- ample. a typical DNA sample prepared from young melon leaves. extracted with phenol and chloroform. and pre- cipitated with ethanol was found to contain about 3 ug carbohydrate (as- sayed by the phenol-sulfuric acid method) per ug DNA. This polysac- charide contamination is readily appar- ent by DNA pellets that are extremely viscous. The refracnve index of the polysaccharide-contaminated sample was 4.0 (the refractive index of salmon sperm DNA at a comparable concen- tration [l uyul] was 0.01: i.e.. the DNA itself does not contribute signifi- cantly to the refractive index readings). If the DNA was precipitated in the presence of the proper concentration of salt (NaCl). the polysaccharide re- mained in solution and was discarded with the ethanol supernatant. With in. creasing NaCl concentrations up to 25 M. there was a visible decrease in poly- saccharide content. This was evident as a decrease in tour] dissolved solids as measured by the refractive index (th- urt 1). Although the rtfracuve index varied. the amount of DNA in each Vol. I3. No.1(l992) sample as determined by spectro— photometry and/or ethidium bromide staining was equivalent (approximately I rig/uh. One molar of salt could runove most of the polysaccharide in a single precipitation. For DNA containing very high levels of polyacclnride. however. 1.5 M or 2.0 M NaCI were more effective. Refractometry readings 138 indicated that as much as 90% of the toral dissolved solids were removed by precipitation with l—2.5 M NaCI. Higher salt concentrations resulted in salt precipitation that was difficult to remove. The efficiency of DNA recov- ery was above 90% with all salt levels tested. Salt (2 M NaCl) was also effective in removing polysaccharide from DNA (13:9 Cucumber Agenda Melon Figure I. The effectivenas of different concentrations of salt in removing polysaccharides from melon and cucumber genomic DNA as assayed by refractive index. The refractive index reading of lug/u] salmon sperm is0.0l. 2A MindIII H 123‘ 5678 Ecoltl 9 10 ll 12 ZB C1C21234M de-dongeno-leDNApndpltaud this-salt I-‘mreMCo-p-hond TEl’I'EoIMNaOanECIyJJeILHinfln-catlambdaDNAflmeslmd2.utctttDNAwith ysacdnride ' armed mgenic plantDNA:lmeede4.DNAinl-edfrundte lanelandz.buteontaining ”amides. 54 BioTecllIimea samples of potato. soybean and ger- anium (data not shown). For these spe- cies 3 well a cucumber artd ntelon. procedure was most successful when DNA was prepared from young leaves. In some cases a second precipi- tation with high salt was helpful in re- m0ving additional polysaccharide. 111: quality of DNA following pre- cipitation irt the presence of high salt (TE + 2 M NaCl) was evaluated by di- gestion with the restriction ertzymes re- sulted tn polysaccharide—free DNA tltat was digested completely (thure 2A). Digestion of cucumber DNA showed similar results (data not shown). Pre- cipitation in the presence of high salt also improved the quality of the DNA as a template for PCR. DNA samples isolated from transgenic melon plants were precipitated in TE or TE 4» 2 M NaCl. washed in 70% ethanol and used 3 template for amplifying the intro- duced NPT-coding DNA fragment. Ex- pected fragments were found only in the reaction using high salt precipitated DNA as template (Figure 23). There- fore. precipitation in the presence of high salt is useful in removing polysac- charides that inhibit restriction enzyme and Taq DNA polymerase activity. Overall. the high-salt procedure to re- move polysaccharides from DNA is quick. convenient. inexpensive and is not volume limited. REFERENCES l .Aokl. Y. and II. Koohihra. I972. Irmihitory effects of acid polysaccharides from sea chin errtbryor on RNA polymerase activity Btochim. Biophys. Acta 27233-43. zAahwell. G. I966. New ' ' of sugu you. Methoth Fataymol. 8:85-95 ple.p.3l-33. IICJ. LavaMLGenrm-te itPl-Isl’le- numPrus.NewYork. s.nuhporta.s.l...l. Wood-dJJJiicls. ranovtm Wean- amirwns from DNA. BioTeehtiques I0: I63- I66. Vol. IJ. No. I (I992) 139 6.Doy‘|e.JJ-and.l.l. Doyle. l987. Atapid DNA isolation procedure for small annuities of fresh leaf tissue. mm. Bull. 1m :45. tidal“. M. I975. Purification of DNA by affinity chromatogrwhy: Removal of polysac- chaide m Anal. Biochem. 6.5: 293497. 8.I'-'ang. G. and R. Grumet. I990. Agrohac- tern-r ntmefaciens mediated tramformation ltd regenemion of muskmelon plants. Flatt Cell Rep. 9.!60-I64. Eliot-have. K. and VJ’. lbavadna. I983. Influences of amniotic polysacchuides on DNA synthesis in isolated nuclei and by DNA polymer-me: correlation of observed effeCts with properties of the polysaccharides. Bio- chint Biophys. Acta 740366-475. I0..Vlerray. M5. ltd WI. “aspen. "’80. Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res. 8:432l-3325. ILOka. A. S. I-llroyttkl and T. Minn-u. I98l. Nucleotide seqrrence ot' the kanamycin rests- tartee transpoeon Tn903. J. Mol. Biol. l47: 2l7-226. tantra-m. E. I988. Preparation of genomic DNA from plant tissue. p. 2.3. l-23.3. In FM. Ausubel. R. Brent. R.E. Kingston. D.D. Moore. J.A. Smith. l.G. Seidman and K. Strultl (Eds). Current Protocols in Molecula Biology. Greerte Publishing Associates and Wiley-Interscience. New York. U.Sbloda. M. and K. Manual-Mocha“. l987. Selective inhibition ofDNA polymerase by a polysaccharide purified from slime of Physarum polyeephalum. Biochem. Biophys. Res. Common. [46:6l-66. We would like to thank Dr. Mike Thomashow and Dr. Julia Bell for help/it! reviews d the manuscript. Acknowledg- ment is also made to the Michigan Agricul- tural Erperiment Station for its support of this research. Address correspondence to R. Grumet. HIGH 1 TE UN Ian IV. LIBRARIES “WW UWWWWWWWW 07917622 sm WW 31293 0