Control of Tomato Spotted Wilt Virus Using Transgenic Plants that Produce Virus-Specific Monoclonal Antibodies Progress Report –August 1995
Date Aug. 29, 1995
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 $25,000
Covering Period Jan. 95 to Dec. 95
Anticipated Date of Project Completion/Final Report 1996
Individual(s) Conducting Project:
(List Project Leader First)
John S. Hu - Title Assistant Professor
Telephone Number (808) 956-7281
M. Q. Xu - Title Graduate Assistant
Control of Tomato Spotted Wilt Virus Using Transgenic Plants that Produce Virus-Specific Monoclonal Antibodies
John S. Hu and M. Q. Xu
University of Hawaii
- A. Project Objectives:
- Engineer and express in plants the genes of the monoclonal antibody whichreacts to tomato spotted wilt virus (TSWV).
- B. Summary of Work Conducted:
- Hybridoma cell lines producing specific monoclonal antibodies to TSWV havebeen 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. The PCR products were ligated into one single-chain
antibody gene construct and then cloned into a plant transformation vector.
Transgenic plants have been produced and are being characterized.
- 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 plant transformation vector, and used to transform
tobacco. Hundreds of transgenic plants have been produced. They
contain the single-chain antibody gene and are being characterized for
disease resistance.
- D. Future Plans Covered by the Endowment Grant:
- Transgenic plants will be challenged with TSWV using standard mechanicalinoculation or thrips feeding techniques. Those of them which show virus resistance
will be characterized.
- E. Anticipated Benefits for Floral Industry:
- Recent developments in biotechnology have provided new opportunities to solvepractical agricultural problems. 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.
