Control of Tomato Spotted Wilt Virus Using Transgenic Plants that Produce Virus-Specific Monoclonal Antibodies Progress Report — September 1993
Date 8/23/93
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 1996
Individual(s) Conducting Project:
(List Project Leader First)
Dr. John S. Hu - Title Assistant Professor
Telephone Number (808) 956-7281
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) Clone the genes of the Ig gamma and kappa protein chains of the monoclonal
antibody which reacts to TSWV.
vectors.
analysis. 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 characterized.
been 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. 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. The clones were verified by checking the sequences against
published sequences of the conserved and the leader sequence regions.
Specific primers were designed to amplify some frangments of the
genes in polymerase chain reaction to obtain full length clones.
chain and the leader sequences will be engineered for transformation work.
floricultural 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 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. 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.
