Development of Resistance to Tomato Spotted Wilt and Similar Viruses inFloral Crops 1998 proposal
Executive Summary
This proposal is to complete the third year of a project designed to improve resistance to TSWV in floral crops. Chrysanthemum is being used as the model to test different strategies. We have eompleted objective two, which was to submit transformed cultivars for testing. These have now been tested for trueness to type with some of the lines passing that test. Last year the project was delayed based on concerns related to commercialization. The project was reinitiated with a primary goal being to replace elements of our transformation system that were subject to restrictive patents or licenses. Although a promoter was identified that has considerable merit, both for being available and to provide additional levels of resistance, several other significant modifications would be required. Our investigations uncovered other obstacles to creating a vector, which would be free of all restrictive patents. It is our opinion based on experience with this project and others to initiate a project to develop a strategy for the commercialization of floral crops improved utilizing plant transformation technology. We have discussed this goal in the proposal along with the other objectives of the proposal, which are to increase the durability of the resistance and to test the alternative promoter as a means of enhancing virus resistance.
Tomato Spotted Wilt Virus Resistance in Floral Crops Introduction and Literature Review. The original objectives of this proposal (August 1996 July 1999) were to focus on chrysanthemum as a model crop for the introduction of TSWV resi stance into floral crops. Research ft3nded in our previous proposal, prior to the initiation of this proposal, resulted in significant progress in introducing resistance to tomato spotted wilt virus (TSWV) into chrysanthemum and in extending those findings to a range of chrysanthemum cultivars by developing a more widely applicable transformation protocol for the crop ~rban et al, 1994; Sherman et al 1998a Th Press; Sherman et al 1998b, In Press). As originally proposed in Tomato spotted wilt and Impatiens necrotic spot tospoviruses (TSWV & INSV, respectively) remain significant threats to the floral crop industry. At the recent (January, 1998) retreat on TSWV at North Carolina State University both viruses were cited as significant problems and that with the increasing prevalence of TSWV in field crops, that the risk posed by these viruses to floral crops will continue.
Control of viruses in general and TSWVIINSV in particular relies on use of propagation material free of these viruses, insect vector control, scouting of production facilities for the virus and the insect vector, and the use of resistant cultivars. Where virus pressure is particularly high, it is necessary to avoid ultrasensitive crops such as Gloxinia. Resistance to TSWV has been difficult to obtain, particularly resistance that is stable. TSWV has long been recognized for its ability to overcome resistance est, 1967. The recent development ofplantkansformation protocols for ornamental crops has greatly enhanced our opportunity to introduce resistance from a variety of sources, especially that resistance which is derived from the pathogen itself such as capsid protein resistance. In our other research, we have begun to identify mechanisms used by TSWV to overcome resistance (Qiu et al, accepted with revision; Hoffman et al in preparation). In this research, we have shown that even the capsid protein based resistance may be overcome. These findings, as well as those of others (e.g. Prins et al, 1997), suggest that other parts of the viral genome should be included to enhance the resistance.
As per the renegotiated objectives of this proposal, the focus of the current year was on replacing the 35 5 promoter patented by Monsanto. We agreed and an individual was ultimately hired (Feb 1997) to begin construction of a vector. Additionally during the past year we have been engaged in collaboration with the technology transfer at NCSU in attempting to assess the current status of our ability to commercialize transgenic plants. Additional elements which should be replaced include the Nos terminator and the NPT selectable marker. The Agrobacterium-mediated transformation strategy is also patented with several patents being relevant. The alternative biolistic transformation strategy is also covered by patents and licenses. Considerable time has been spent during the past year gathering information useflil in developing a strategy which would lead to commercial izable plants that could overcome concerns raised by restrictive patents, recognizing that no strategy will be free of all patents and licenses. Examples of this are the possible use of the ocs/mas ’super promoter’ ~i et al 1995) for virus resistance. Selection markers as alternatives to NPT II, are already available and in fact have been necessary in our other research on New Guinea Impatiens. In summary, it is not yet possible to design a vector free of all licensing concerns, it is possible to begin testing alternative elements which could be incorporated into a new strategy that will minimize risk associated with licensing
Objectives and Anticipated Benefits:
Objectives:
The objectives for the final year of this project are designed to bring research initiated under the original and revised objectives to a reasonable conclusion. The two research objectives identified for the coming year are a necessary extension of the original objectives and will provide useflil information to the industry. They are: 1) Investigate other regions of the TSWV genome as sources of resistance as a means of increasing the durability of resistance and 2) Evaluate a novel promoter, an element of the transformation vector, required in transformation to improve effectiveness of resistance. These two objectives are designed to extend our previous research. As indicated in the original proposal we intend to extend the initial findings that demonstrated, that the nucleocapsid gene from TSWV would impart resistance, to include a gene from another part of the genome. We have selected the NSm gene that has been shown to impart resistance ~rins et al, 1997). We do not know if the NSm gene, when combined with the nucleocapsid gene, will impart a more stable form of resistance. However, it is clear that total reliance on the nucleocapsid gene will result in resistance being overcome. The third objective is to develop an informed strategy for ftiture projects for floral crop improvement that employs plant transformation technology.
Benefits:
TSWV/INSV remain a serious threat to floral crop production. Resistance is an essential strategy for controlling viruses. Dependence on the other elements of control, that include clean stock programs, scouting and insect vector control will continue to require expensive inputs for labor and insecticides. Resistance is missing from the control strategy in floral crops. Development ofthis resistance is now a two-fold process: Identification and testing of the appropriate genetic strategy and now as we come nearer to deployment, the adoption of a strategy for commercialization. While plant patents (patented varieties) has become a part of doing business, the maze of technology patents is a relatively new hurdle for the floral crop industry as well as Land Grant Institutions. There is little doubt now that the technology provides the opportunity for significant impact in crop improvement as evidenced by the progress made in cotton, potato, corn and other crops. However, it is becoming increasingly not made to 1) inform the entire enterprise (industry leaders and researchers) regarding questions related to commercializing cultivars developed with patented technology and 2) and develop a strategy for acquisition of the technology. While this is still an evolving process, it is time to inform the participants and to develop a strategy that might be followed by individual companies and researchers as well as guide AFE/)(Sin establishing ~nding priorities. It is far too complex and time intensive an issue to leave to each investigator or to be resolved annually by an ever changing research committee. Licenses, patents and commercialization are issues that must become part of the planning process, but should not consume inordinate resources or inhibit initiative that is more appropriately applied to providing solutions to constraints of the floral crop industry.
Materials and Methods:
Objective I will test the added benefit of combining the NSm gene together with the N gene from TSWV to inhibit the ability of the virus to overcome the resistance provided by the N gene alone. We will use the widely adapted transformation protocol (Sherman et al, 1998a In Press) to introduce the N and Nsm genes into chrysanthemum ‘Polaris’. We will use an N gene construct used in our previous experiments and will produce and Nsm gene as previously described ~rins et al, 1997). Resulting transformants will be tested by mechanical inoculation with isolates used in previous trials (Sherman et al 1998b In Press). In addition, we will use two resistance-breaking isolates. One is a reassortant isolated in our greenhouse experiments (Qiu et al, 1998 accepted with revision) and another isolate shown to overcome the SW-S gene in tomato, TSWV resistance in pepper and N gene resistance (Hoffman et al In preparation). Plants will be tested at optimal temperature for TSWV infection (20-25 C). Transformation will be confirmed by selection on antibiotic resistant media, PCR of tissue for the transgene, Southern analysis and assays for residual Agrobacterium tumefaciens.
Objective 2 will test the efficacy of the ocs/mas ’superpromoter’ (Ni et al, 1995) as a promoter to drive the viral transgene that imparts resistance. We have obtained the promoter in the vector pEl 120. The construct was obtained from S B Gelvin, the originator and NCSU has approval to use this promoter for research purposes. We will use a modified version of the construct, replacing the GUS gene with the TSWV N gene. Transformation will be monitored as described above. In addition, we will conduct Northern analyses in comparative trials with our standard constructs driven by the CaMV 35s promoter. Both vectors are based on the pBI 101 vector.
Objective 3 addresses the increasingly important issue of commercialization and the role of AFE/~n this process. I am proposing that during the next year the Research Committtee and the Board begin a dialogue which will include outside experts who deal with the issue. Examples would include technology transfer administrators from Universities as well as consultants that advise corporations on these issues. A brief document should be created describing the issues facing researchers and producers related to development and deployment of transgenic plants. Upon completion of this phase of the project, fi~irther discussions can be held to develop policy regarding support of research utilizing transgenic plants. Possible outcomes might be the development of a research project to develop technology free of patents, as has been suggested. Alternatively, concerted efforts might be made to obtain existing technology for use by researchers or to have commercial interests actually perform transformations in cooperation with researchers. Other alternatives will emerge through the information gathering process.
New Literature
Daub, M. E., Jones, R. J., and Moyer, J. W. 1996. Biotechnical approaches for virus resistance in floral crops. Pp.335-351, in Biotechnology of Ornamental Plants, Eds Geneve, r. L., Preece, J. E. and Merkie, S. ~ CAB International Wallingford.
Daughtery, M. L., Jones, R. K., Moyer, J. W., Daub, M. E. and Baker, 3. R. 1997. tospoviruses strike the greenhouse industry: INSV has become a major pathogen on flower crops. Plant Disease ~eature article) 81:1220-1230.
Hoffman, K, Qiu, W. P, and Moyer, J. W. Reassortants of tomato spotted wilt tospovirus (TSWV) overcoming host- and pathogen-mediated resistance in tomato and tobacco. In Preparation for submission to MPMI.
Ni, M, Cui, D., Einstein, 3., Narasimhulu, S., Vergara, C. E., and Gelvin, S. B. 1995. Strength and tissue specificity of chimeric promoters derived from the octopine and mannopine synthase genes. The Plant Journal 7:661-676
Prins M, Kikkert M, Ismayadi C, de Graauw W, de Haan P, Goldbach R Characterization ofRNA-mediated resistance to tomato spotted wilt virus in transgenic tobacco plants expressing’NS~) gene sequences. Plant Mol Biol. 1997 Jan; 33(2): 235-243.
Qiu, W. P., Geske, S. M., Hickey, C. M. and Moyer, 3. W. Tomato spotted wilt Tospovirus genome reassortment and genome specific adaptation. Virology (Accepted with revision)
Sherman, J. M., Moyer, J. W. and Daub, M. E. 1998 A regeneration and Agrobacterium mediated transformation system for genetically diverse Clrrysanthemeum cultivars. J. Amer. Soc Hort Sci. 123: In press.
Sherman, J. M., Moyer, J. W. and Daub, ME. 1998b Tomato spotted wilt virus resistance in Chrysanthemum expressing the viral nucleocapsid gene. Plant Disease 82: In Press.
Urban, L. ~, Sherman, 3. M., Moyer, J. W. Moyer, Daub, M. E. 1994. High frequency shoot regeneration and Agrobacterium-mediated transformation of chrysanthemum (Dendranthema grandiflora)
MOYER & DAUB TSWV RESISTANCE BUDGET REQUEST
Technical Support $26,000
Benefits (24.2% + .8.25%hourly) $ 6,457
Hourly Labor $ 2,000
TOTAL SALAJUES $34,457
Expendable Supplies Travel $ 5,809 $ 2,000
TOTAL REQUESTED $42,266
PROJECT LEADER QUALIFICATIONS
James W. Moyer is a professor of plant pathology at North Carolina State University. He received his Ph. D in plant pathology at The Pennsylvania State University in 1975 and did post-doctoral research at the University of California at Davis prior to coming to NCSU. He has had 18 years experience in working with viruses of vegetatively propagated crops and seven years experience investigating tomato spotted w~t-like viruses (Tospoviruses). Members of his research group were responsible for first identifying INSV, the predominant virus problem in floral crops, and for providing antisera to the industry for diagnosis and clean-stock programs. His research includes both applied and basic aspects of plant virology.
Margaret B. Daub is a professor of plant pathology at North Carolina State University. She received a Ph.D. in plant pathology from the University of Wisconsin at Madison and did postdoctoral work in plant tissue culture at Michigan State University. She has had 15 years experience working with all aspects of plant in vitro culture and transformation, and has written several review articles on this subject. Her research has included in vitro selections of callus and pro toplast cultures for phytotoxin resi~tance, somatic hybridization for transfer of resistance genes in plants, somaclonal variation for improved disease resistance, and plant transformation for pathogen gene expression.
