Development of a Plant Shoot Temperature Model for Greenhouse Climate Management Progress Report — September 1992
Date Aug. 24, 1992
Title of Project Control of Tomato Spotted Wilt Virus Using Transgenic Plants that Produce
Virus-Specific Monoclonal Antibodies
Institution where work is being conducted University of Hawaii
Amount of Endowment Grant $ 10,000
Covering Period Jan. 1992 to Dec. 1992
Anticipated Date of Project Completion/Final Report 1996
Individual(s) Conducting Project:
(List Project Leader First)
Dr. John S. Hu - Title Assistant Professor
Telephone Number (808) 945-7231
Mr. Z. C. Wu - Title Graduate Assistant
Control of Tomato Spotted Wilt Virus Using Transgenic Plants That Produce Virus-Specific Monoclonal Antibodies
John S. Hu and Z. C. Wu
University of Hawaii
- A. Project Objectives:
- 1) Select a hybridoma cell line producing a MAb to the nucleoprotein of TSWV.
- 2) Clone the MAb genes.
- B. Summary of Work Conducted:
- MAb’s specific to TSWV have been characterized in ELISA and Western blotanalysis. One hybridoma cell line (TSWV-MAb 8C4D6) has been selected for the
cloning of the antibody genes. RNAs have been isolated from the cell line for cloning
of the antibody heavy chain and light chain genes. Full length RNAs that code for the
Ig gamma and kappa proteins were used for cloning. Complementary DNAs were
produced to these RNAs using oligo-dT as a primer and reverse transcriptase. A
cDNA library was obtained. Specific clones are being characterized and sequenced.
- C. Results to Date:
- Hybridoma cell lines producing specific monoclonal antibodies to TSWV havebeen made. These monoclonal antibodies have different specificities, some react with
a few TSWV isolates, others react with many TSWV isolates; some react with the
nucleoprotein of TSWV, others react with the glycoproteins of TSWV. The
monoclonal antibodies have been tested in ELISA and Western blot analysis. One cell
line (TSWV-MAb8C4D6), which has broad specificity to TSWV isolates and reacts to
the nucleoprotein of TSWV, has been selected for the cloning of the antibody genes.
It has been determined that the monoclonal antibody has a gamma 1 heavy chain and
kappa light chain. RNAs have been isolated from the cell line for cloning of the
antibody heavy chain and light chain genes. Specific oligonucleotide probes to the
conserved regions of the genes have been made. Full length mRNAs that code
for the Ig gamma and kappa proteins were used for cloning.
Complementary DNAs were produced to these mRNAs using oligo-dT
as a primer and reverse transcriptase. A cDNA library was obtained.
Specific clones are being characterized and sequenced. Plant tissue
culture work (transformation and regeneration) was initiated.
- D. Future Plans Covered by the Endowment Grant:
- Further characterize and sequence the specific clones. Clones containing thecomplete genes of gamma I heavy chain or kappa light chain and the leader
sequences will be selected for future engineering and transformation work. The final
goal of this research is to control TSWV using transgenic plants that produce TSWV-
specific monoclonal antibodies.
- E. Anticipated Benefits for Floral Industry:
- Tomato spotted wilt virus (TSWV) is one of the most devastating diseases onfloricultural and vegetable crops in Hawaii and worldwide. 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 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. It is reasonable to
think that plants producing antibodies to a virus would be resistant to infection by that
virus. 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.
- The long term goal of this research is to control TSWV using transgenic plantsthat produce TSWV-specific monoclonal antibodies. Since TSWV has a very wide
host range, infecting 192 dicotyledonous species in 33 families and eight
monocotyledonous species in 5 families. 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.
