Development of a Plant Shoot Temperature Model for Greenhouse Climate Management 1995 Proposal
that Produce
Tomato spotted wilt virus (TSWV) is one of the most devastating diseases
on floricultural and vegetable crops in Hawaii and worldwide (Ie, 1970).
This virus disease is very difficult to control. Recent developments in
biotechnology have provided new opportunities to solve practical agricultural
problems. Genetic engineering offers new approaches to produce virus resistant
varieties. A scientific breakthrough has presented a new possibility for
controlling plant virus diseases through the use of transgenic plants that
produce antibodies to specific plant viruses. It was recently reported
that transgenic plants expressing a monoclonal antibody against the coat
protein of a Tombusvirus have been produced (Tavladoraki et al. 1993).
Their preliminary data show a delay in symptom development suggesting a
possible role of the antibodies in plant protection.
Supported in part by the American Floral Endowment, we have obtained
the single- chain antibody gene from hybridoma cells which secrete specific
monoclonal antibodies to TSWV. The gene has been subcloned into a plant
transformation vector and used to transform tobacco.
The long term goal of this research is to control TSWV using transgenic
plants that produce TSWV-specific monoclonal antibodies. The objectives
for the period of the proposed work are to: 1) Development of transgenic
tobacco plants, and 2) Evaluation of the transgenic plants in greenhouse
and in the field.
Since TSWV has a very wide host range, infecting 192 dicotyledonous
species in 33 families and eight monocotyledonous species in 5 families
(Cho et al., 1987). If this approach works, the specific genes that encode
monoclonal antibodies to TSWV could be introduced into many floricultural
crops, for control of this devastating virus disease.
2) Introduction and Literature Review
Tomato spotted wilt virus (TSWV) causes devastating diseases on floricultural
and vegetable crops in Hawaii and worldwide (Ie, 1970). For instance, TSWV
causes 50- 100% crop losses in chrysanthemum production in Hawaii (Hu,
personal observation). The virus has a very wide host range, infecting
192 dicotyledonous species in 33 families and eight monocotyledonous species
in 5 families (Cho et al., 1987). TSWV is the type member of the tomato
spotted wilt virus group (Tospovirus). It is transmitted in a persistent
manner by several species of thrips. The virus is very difficult to control
(Ie, 1970).
Recent developments in biotechnology have provided new opportunities
to solve practical agricultural problems. Genetic engineering offers new
approaches to produce virus resistant varieties. Coat protein-mediated
protection has been shown to be a promising weapon for the control of many
plant virus diseases (e.g. Cuozzo et al., 1988; Hemenway et al., 1988;
Powell-Abel et al., 1986). Several laboratories are using this approach
to control TSWV in tomato and other crops. (e.g. Pang et al. 1993; Kim
et al. 1994).
A recent scientific breakthrough has presented a new possibility for
controlling plant virus diseases through the use of transgenic plants that
produce antibodies to specific plant viruses (Hiatt et al., 1989). It was
reported that transgenic plants expressing a monoclonal antibody against
the coat protein of a Tombusvirus have been produced (Tavladoraki et al.
1993). Their preliminary data show a delay in symptom development suggesting
a possible role of the antibodies in plant protection. The antibody molecules
may bind to the nucleoproteins to prevent uncoating in the early stage
of infection, or bind to the nucleoprotein molecules to prevent assembly
of virions in the later stages of virus replication. Such a system would
be analogous, in a general way, to the common antibody defense system in
animals.
Supported in part by the American Floral Endowment, hybridoma cell lines
producing specific monoclonal antibodies to TSWV have been produced. Universal
degenerate primers were designed for PCR amplification of the variable
regions of heavy and light chains of monoclonal antibodies. Specific cDNAs,
which were made to TSWV- MAb mRNA, were used as templates in PCR using
universal primers. PCR products were examined in Southern blot hybridization
and found to be specific to the TSWV-MAb gene. The PCR products were ligated
into one single-chain antibody gene construct and then cloned into a plasmid
vector. The gene has been subcloned into a plant transformation vector
and used to transform tobacco.
3) Objectives & Anticipated Benefits
The long term goal of this research is to control TSWV using transgenic
plants that produce TSWV-specific monoclonal antibodies.
The objectives for the period of the proposed work are to:
1) Development of transgenic tobacco plants.
2) Evaluation of the transgenic plants in greenhouse and in the field.
The antibody-mediated protection principle may be generally applicable
to plant viruses. This research might thus establish a more broadly applicable
approach for controlling plant viruses. Since TSWV has a very wide host
range. If this approach works, the specific genes that encode monoclonal
antibodies to TSWV could be introduced into many floricultural crops, for
control of this devastating virus disease.
4) Materials and Methods
Objective 1: Development of transgenic tobacco plants in greenhouse.
The single-chain antibody gene construct was cloned into a plant transformation
vector and used to transform tobacco by Agrobacterium tumefaciens -mediated
transformation system (An, 1986). Antibiotic-resistant tissue were induced
to regenerate shoots and roots. Several hundreds of transformed tobacco
plants have been obtained. Results from preliminary experiments show that
100% of transformed plants tested contained the NPT II gene. Leaves from
the transformed plants are being examined for integration of the genes
using Southern blot hybridization. Production of RNA messages for the genes
in the transgenic plants will be tested using Northern blot hybridization.
Because we do not have antibodies to monitor the expression of the single-chain
antibody in transgenic plants, we plan to use the in vitro-expressed single-chain
antibody as an immunogen to produce polyclonal antibodies. The single-chain
antibody gene have been subcloned into in vitro expression plasmid vector
pET-24d. The single-chain antibody was expressed in E. coli, purified,
and injected into a rabbit to produce antiserum, which will be used in
ELISA and Western blot assays to monitor the expression of the single-chain
antibody in transgenic plants.
Objective 2: Evaluation of the transgenic plants in greenhouse and
in thefield.
Based on experiences on production of resistant transgenic plants for
control of plant viruses using viral genes, it is important to screen large
numbers of transgenic plants to obtain plants which are highly resistant
to virus infection. The hundreds of transgenic plant lines will be inoculated
with TSWV mechanically and using viruliferous thrips in greenhouse. Those
of the lines which show high resistance to TSWV infection will be examined
in the field experiments under low and high disease presures.
5) Literature cited:
Cho, J.J., Mau, R.F.L., Mitchell, W.C., Gonsalves, D., and Yudin, L.S.
1987. Host list of tomato spotted wilt virus (TSWV) susceptible plants.
Univ. Hawaii Coll.Trop. Agric. Hum. Resour. Res. Ext. ser. 078. 12pp.
Chomczynski, P. and Sacchi, N. 1987. Single-step method of RNA isolation
by acid guanidinium thiocyante-phenol-chloroform extraction. Anal. Bio.
162: 156-159.
Cuozzo, M., O’Connell, K.M., Kaniewski, W., Fang, R.-X., Chua, N.-H.,
and Turner, N.E. 1988. Viral protection in transgenic tobacco plants expressing
the cucumber mosaic virus coat protein or its antisense RNA. Bio/Technology
6:549- 557. de Haan, P.,
Wagemakers, L., Peters, D., and Goldbach, R. 1989. Molecular cloning
and terminal sequence determination of the S and M RNAs of tomato spotted
wilt virus. J. Gen. Virol. 70:3469-3473.
Pang, S. Z. et al. 1993. Different mechanisms protect tansgenic tobacco
against tomato spotted wilt and impatiens necrrotic spot Tospoviruses.
Bio/Technology 11: 819- 824.
Hiatt, A., R. Cafferkey, and K. Bowdish. 1989. Production of antibodies
in transgenic plants. Nature 342: 76-78.
Hemenway, C., Fang, R.-X., Kaniewski, W.K., Chua, N.-H., and Turner,
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.
Hsu, H.T., Wang, Y.C., Lawson, R.H., Wang, M., and Gonsalves, D. 1990.
Splenocytes of mice with induced immunological tolerance to plant antigens
for construction of hybridoma secreting tomato spotted wilt virus-specific
antibodies. Phytopathology 80: 158-162.
Hu, J. S., Pang, S. Z., Nagpala, P. G., Siemieniak, D. R., Slightom,
J. L. and Gonsalves. D. 1993. The coat protein genes of squash mosaic virus:
cloning, sequence analysis, and expression in tobacco protoplasts. Archives
of Virology 130:17-31.
Ie, T.S. 1970. Tomato spotted wilt virus. No. 39 in: Descriptions of
Plant Viruses, Common. Mycol. Inst./Assoc. Appl. Biol., Surrey, England,
Kim, J. W., Sun, S. S., and German, T. 1994. Disease resistance in tobacco
and tomato plants transformed with the tomato spotted wilt virus nuclecapsid
gene. Plant Dis. 78: 615-621.
Powell-Abel, P.P., Nelson, R.S., De, B., Hoffmann, N., Rogers, S.G.,
Fraley, R.T., and Beachy, R.N. 1986. Delay of disease development in transgenic
plants that express the tobacco mosaic virus coat protein gene. Science
232:738-743.
Tavladoraki, P. et al. 1993. The engineering of “phytoantibodies” for
virus protection in transgenic plants. in International Symposium “Molecular
genetics of plant-microbe interactions”. Rutgers, New Jersey, April 21-24.,
1993.
6) Budget
A. Salaries and Wages Graduate
Research Assistant $ 17,000
B. Materials and Supplies
Chemicals, enzymes, media $ 2,500
Isotopes $ 1,000
Greenhouese and Field Experiments $ 1,500
General supplies $ 1,000
C. Travel $ 2,000
D. Total Cost $ 25,000
7) Leader Qualifications
Similar recombinant DNA techniques and plant gene transfer systems are
being used in collaborative research with Dr. A. Kuehnle, Department of
Horticulture, University of Hawaii, on coat protein-mediated protection
with cymbidium mosaic virus, odontoglossum ringspot virus, and banana bunchy
top virus.
My laboratory is equipped for recombinant DNA and virology research.
Available equipment includes electrophoresis and sequencing apparati, a
high-speed centrifuge, an ultracentrifuge, a fractionator, a SpeedVac,
three microcentrifuge, a refrigerator, a walk-in cold room, -20C and -80C
freezers, a PCR DNA amplification temperature cycler, a microplate reader,
and a separate room for work with radioactive isotopes. Also available
for use are a scintillation counter and a transmission electron microscope
in the building, and a Dupont Biolistic device (gene gun) at the nearby
Hawaiian Sugar Planters’ Association in Aiea. The adjacent Biotechnology
Facility of the University of Hawaii is equipped to handle peptide sequencing
and oligo primer synthesis. The Monoclonal Antibody Center in Microbiology
Department takes care monoclonal antibody production and cell line maintenance.
Transgenic plants will be maintained in USDA-certified containment greenhouses
that are available at the University of Hawaii.
