Control of Tomato Spotted Wilt Virus Using Transgenic Plants that Produce Virus-Specific Monoclonal Antibodies Progress Report — December 1995
Date 12/26/95
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 which
reacts to tomato spotted wilt virus (TSWV).
B. Summary of work
Hybridoma cell lines secreting monoclonal antibodies to TSWV have been
produced. One cell line was selected for the cloning of
the antibody genes. Universal degenerate primers were designed for
PCR amplification of the
variable DNA fragments of heavy and light chains of monoclonal antibodies.
The PCR products were
ligated into one single-chain antibody gene construct and then cloned
into a plasmid vector. The gene
has been subc-loned into a plant transformation vector and transformed
into tobacco Nicotiana
labacian. Preliminary results show that some of the transgenic tobacco
plants were resistant to
systemic infection of TSWV.
C. Results to date
Hybridonia cell lines producing specific monoclonal
antibodies to TSWV have 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. 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. Preliminary results
show that some of the
transgenic tobacco plants developed local lesions on inoculated leaves
without systemic infections on
uninoculated leaves, which suggest the transgenic tobacco plants were
resistant to systemic infection
of TSWV.
D. Future plans covered by the Endowment grant:
The resistant transgenic plants will be
examined in Southern, Northern, and Western blot hybridizations. Gene
segregation of the Fv gene in
the progenies of the transgenic plant lines will be analyzed. The best
lines which show resistance to
TSWV infection will be examined under low and high disease pressures.
E. Anticipated benefits for
floral industry Recent developments in biotechnology have provided
new opportunities to solve
practical agricultural problems. It was recently reported that transgenic
plants expressing a
monoclonal antibody against the coat protein of a tombusvirus have
been produced. 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.
Currently, several other labs are evaluating this antibody-mediated
protection strategy for control of
different plant viruses.
