Development of a Plant Shoot Temperature Model for Greenhouse Climate Management Progress Report — September 1996
Date Aug. 26, 1996
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. 96 to Dec. 96
Anticipated Date of Project Completion/Final Report 1997
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 Fv antibody gene construct
and transformed into tobacco Nicotiana tabacum. Some of the transgenic
tobacco plants were resistant to systemic infection of TSWV. The transgenic
lines were also characterized by Southern and Northern blot hybridizations.
- C. Results to date:
- Hybridoma 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. Universal degenerate primers were
designed for amplification of variable regions of heavy and light chains
of monoclonal antibodies. PCR products were ligated into one single-chain
antibody gene construct and then cloned into a plant transformation vector,
and used to transform tobacco. Transgenic plants have been produced and
characterized. Several lines showed clear delay in symptom development.
Two of the transgenic tobacco lines were completely resistant to systemic
infection of TSWV. These lines were tested more than five times and
identical results were obtained. Progenies from the two resistant lines
were also resistant to systemic infection of TSWV. Southern and Northern
blot hybridization analyses were done with the transgenic plants. The RNA
expression level of the transgenic plants correlate with the virus resistance
level. Specific antibodies were produced against the single-chain antibody
and will be used in Western blot analysis to examine the expression of
the antibody protein in the transgenic plants.
- D. Future plans covered by the Endowment grant:
- The resistant transgenic plants will be examined in Western blot hybridization
and for resistance to other tospoviruses.
- E. Anticipated benefits for floral industry:
- 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 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.
