Control of Tomato Spotted Wilt Virus Using Transgenic Plants that Produce Virus-Specific Monoclonal Antibodies Progress Report — March 1993
Date Feb. 24, 1993
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. ‘93 to Dec. ‘93
Anticipated Date of Project Completion/Final Report 2/96
Individual(s) Conducting Project:
(List Project Leader First)
Dr. John S. Hu - Title Assistant Professor
Telephone Number (808) 956-7281
Mr. Z.C. Wu
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) Clone the genes of the Ig gamma and kappa protein chains of the monoclonalantibody which reacts to TSWV.
- 2) Sequence and engineer the genes and subclone the genes into expressionvectors.
- B. Summary of Work Conducted:
- MAbs 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. 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. Specific clones identified
in colony hybridization have been sequenced. Tobacco plant tissue culture system
(transformation and regeneration) has been developed.
- C. Results to Date:
- Hybridoma cell lines producing specific monoclonal antibodies to TSWV havebeen made. One cell line (TSWV-MAb 8C4D6), which has broad specificity to
TSNW 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. Specific clones identified in colony hybridization
have been sequenced. Tobacco plant tissue culture system
(transformation and regeneration) has been developed.
- D. Future Plans Covered by the Endowment Grant:
- Further characterize the specific clones. Clones containing the complete genesof gamma 1 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 break-through 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 TSNW could be introduced into many
floricultural crops, for control of this devastating virus disease.
