Control of Tomato Spotted Wilt Virus Using Transgenic Plants that Produce Virus-Specific Monoclonal Antibodies Progress Report — December 1994
Date 12/9/94
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 $ $6,500
Covering Period Jan.94 to Dec.94
Anticipated Date of Project Completion/Final Report 96
Individual(s) Conducting Project:
(List Project Leader First)
John S. Hu - Title Assistant Professor
Telephone Number (808) 956-7281
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:
- Engineer and express the genes of the Ig gamma and kappa protein chains of themonoclonal antibody which reacts to tomato spotted wilt virus (TSWV).
- B. Summary of Work Conducted:
- A panel of monoclonal antibodies specific to TSWV have been produced. Onehybridoma cell line (TSWV-MAb 8C4D6) has been Selected for the cloning of the
antibody genes. Complementary DNAs were produced to these RNAs using oligo
(dT) as a primer and reverse transcriptase. Universal degenerate primers were
designed for amplification of variable regions of heavy and light chains of monoclonal
antibodies. PCR products were examined in Southern blot hybridization and were
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 for
transformation into tobacco.
- C. Results to Date:
- Hybridoma cell lines producing specific monoclonal antibodies to TSWV havebeen made. 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. 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. Universal degenerate
primers were designed for amplification of variable regions of heavy and light chains
of monoclonal antibodies. Specific cDNA, which were made to TSWV-MAb mRNA,
were used as templates in PCR using the universal primers. PCR products were
examined in Southern blot hybridization and were 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.
- D. Future Plans Covered by the Endowment Grant:
- The gene will be subcloned into a plant transformation vector pBI121 andtransformed into tobacco plants. Transgenic plants will be challenged with TSWV
using standard mechanical inoculation or thrips feeding techniques. Further
support in 1995 will be needed to complete the project.
- E. Anticipated Benefits for Floral Industry:
- Recent developments in biotechnology have provided new opportunities tosolve 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 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 data show a delay in symptom
development and reduction on virus replication suggesting a 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. The long term goal of this research is to control TSWV using transgenic
plants that 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.
