Development of a Plant Shoot Temperature Model for Greenhouse Climate Management 1993 Proposal
Antibodies
Tomato spotted wilt virus (TSWV) is one of the most devastating diseases
on floricultural and vegetable crops in Hawaii and worldwide (Ie, 1970).
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 is reasonable to
think that plants producing antibodies to a virus would be resistant to
infection by that virus. 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 objectives for the period of the proposed
work are to:
1) Clone the genes of the Ig gamma and kappa protein chains of the monoclonal
antibody which reacts to TSWV.
2) Sequence and engineer the genes and subclone the genes into expression
vectors.
The long term goal of this research is to control TSWV using transgenic
plants that produce TSWV-specific monoclonal antibodies.
The antibody-mediated protection principle that I am proposing to test
may be generally applicable to plant viruses. This research might thus
establish a more broadly applicable approach for controlling plant viruses.
Technically, it may be an easier approach for controlling plant viruses
than coat protein-mediated protection. For example, a major segment of
the genes for the Ig gamma heavy chain and Ig kappa light chain protein
subunits are conserved and their sequence known. This makes engineering
of the cDNA relatively straight forward. Production of monoclonal antibodies
to plant viruses are routine and the mRNAs for the Ig gamma and kappa proteins
are produced in abundant amounts, which makes relatively easy to isolate
and characterize.
Since TSWV has a very wide host range, infecting 192 dicotyledonous
species in 33 families and eight monocotyledonous species in 5 families
(Cho et al., 1987). 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.
Part B. Detailed Proposal:
1. Introduction and background information
Tomato spotted wilt virus (TSWV) causes devastating diseases on floricultural
and vegetable crops in Hawaii and worldwide (Ie, 1970). For instance, TSWV
causes 50-100% crop losses in chrysanthemum production in Hawaii (Hu, personal
observation). The virus has a very wide host range, infecting 192 dicotyledonous
species in 33 families and eight monocotyledonous species in 5 families
(Cho et al., 1987). The virus particle is isometric, about 70-90 nm in
diameter and is bound by a membrane. It has one nucleoprotein and three
glycoproteins. The virus has a multipartite single stranded RNA genome
composed of three RNA species (de Haan et al., 1989). TSWV is the sole
member of the tomato spotted wilt virus group (Tospovirus). It is the only
plant virus which is transmitted in a persistent manner by several species
of thrips. The virus is very difficult to control (le, 1970).
2. Review of significant literature
Recent developments in biotechnology have provided new opportunities
to solve practical agricultural problems. Genetic engineering offers new
approaches to produce virus resistant varieties. Coat protein-mediated
protection has been shown to be a promising weapon for the control of many
plant virus diseases (e.g. Cuozzo et al., 1988; Hemenway et al., 1988;
Powell-Abel et al., 1986). Several laboratories are using this approach
to control TSWV in tomato, potato, etc. (Dr. Gonsalves, Dr. German, personal
communication).
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. Hiatt and colleagues (1989)
isolated mRNAs from mouse hybridoma cells secreting monoclonal antibodies,
and produced cDNAs of mRNAs that code for the immunoglobulin (Ig) light
(kappa) and heavy (gamma) protein subunits of an antibody that binds a
low molecular weight phosphonate ester. These cDNAs were inserted separately
into the genome of tobacco plants via Agrobacterium tumefaciens. Remarkably,
some progenies resulting from crosses between selected transgenic plants
that expressed either light or heavy chain proteins produced significant
amounts of functional antibodies. Antibodies from these transgenic plants
had similar binding capacities for the phosphonate esters as those produced
by mouse hybridoma cells. This is the first case where functional antibodies
have been produced by a plant.
3. Objectives
It is reasonable to think that plants producing antibodies to a virus
would be resistant to infection by that virus. 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. Although
there is no published information on this subject for plant viruses, clearly,
this is indeed a rational approach for controlling plant viruses through
biotechnological methods.
The objectives for the period of the proposed work are to:
1) Clone the genes of the Ig gamma and kappa protein chains of the monoclonal
antibody.
2) Sequence and engineer the genes and subclone the genes into expression
vectors.
The long term goal of this research is to control TSWV using transgenic
plants that produce TSWV-specific monoclonal antibodies.
The antibody-mediated protection principle that I am proposing to test
may be generally applicable to plant viruses. This research might thus
establish a more broadly applicable approach for controlling plant viruses.
Technically, it may be an easier approach for controlling plant viruses
than coat protein-mediated protection. For example, a major segment of
the genes for the Ig gamma heavy chain and Ig kappa light chain protein
subunits are conserved and their sequence known. This makes engineering
of the cDNA relatively straight forward. Production of monoclonal antibodies
to plant viruses are routine and the mRNAs for the Ig gamma and kappa proteins
are produced in abundant amounts, which makes relatively easy to isolate
and characterize. Since TSWV has a very wide host range. 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.
4. Materials and methods
Part 1: Cloning of the Antibody Genes.
Hybridoma cell lines producing specific monoclonal antibodies to TSWV
have been made (Hsu et al. 1990; Cho, personal communication). The cell
lines are maintained in the Monoclonal Antibody Facilities at University
of Hawaii at Manoa. These monoclonal antibodies have different specificities,
some react with a few TSWV isolates, others react with many TSWV isolates;
some react with the nucleoprotein of TSWV, others react with the glycoproteins
of TSWV. The monoclonal antibodies have been tested in ELISA and Western
blot analysis. One cell line (TSWV-MAb8C4D6), which has broad specificity
to TSWV isolates and reacts to the nucleoprotein of TSWV (Hu and Wang,
unpublished data), has been selected for the cloning of the antibody genes.
RNAs were isolated from the cell line (Chomczynski and Sacchi, 1987)
and purified using oligo-dT columns to obtain enriched preparations of
polyadenylated mRNAs. Specific oligonucleotide probes to the conserved
regions of the genes have been made (Hu and Wang, unpublished data). The
probes have been used in Northern blot hybridization analysis to examine
these RNAs. Full length RNAs 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. Standard cloning
procedures were followed (Hu et al., 1991). Clones in the cDNA library
is being identified by colony hybridization using the same oligonucleotide
probes described above.
Part II. Engineering of the antibody genes.
Clones reacting to specific probes for the conserved regions will be
checked for size and the full length clones will be sequenced. The validity
to our clones will be verified by checking the sequences against published
sequences of the conserved and the leader sequence regions. The full length
cDNAs will be subcloned into expression vectors and the gene products monitored
by Western blot using antibodies to Ig gamma and kappa proteins.
5. Facilities and equipment available
My laboratory is equipped for recombinant DNA and virology research.
Available equipment includes electrophoresis and sequencing apparati, a
high-speed centrifuge, an ultracentrifuge, a fractionator, a SpeedVac,
three microcentrifuge, a refrigerator, a walk-in cold room, -20′C and -80′C
freezers, a PCR DNA amplification temperature cycler, a microplate reader,
and a separate room for work with radioactive isotopes. Also available
for use are a scintillation counter and a transmission electron microscope
in the building, and a Dupont Biolistic device (gene gun) at the nearby
Hawaiian Sugar Planters’ Association in Aiea. The adjacent Biotechnology
Facility of the University of Hawaii is equipped to handle peptide sequencing
and oligo primer synthesis. The Monoclonal Antibody Center in Microbiology
Department takes care monoclonal antibody production and cell line maintenance.
6. Literature cited:
Cho, J.J., Mau. R.F.L., Mitchell, W.C., Gonsalves, D., and Yudin, L.S.
1987. Host list of tomato spotted wilt virus (TSWV) susceptible plants.
Univ. Hawaii Coll. Trop. Agric. Hum. Resour. Res. Ext. ser. 078. l2pp.
Chomczynski, P. and Sacchi, N. 1987. Single-step method of RNA isolation
by acid guanidinium thiocyante-phenol-chloroform extraction. Anal. Bio.
162:156- 159. Cuozzo, M., O’Connell, K.M., Kaniewski, W., Fang, R.X., Chua,
N.H., and Turner, N.E. 1988. Viral protection in transgenic tobacco plants
expressing the cucumber mosaic virus coat protein or its antisense RNA.
Bio/Technology 6:549-557. de Haan, P., Wagemakers, L., Peters, D., and
Goldbach, R. 1989. Molecular cloning and terminal sequence determination
of the S and M RNAs of tomato spotted wilt virus. J. Gen. Virol. 70:3469-3473.
Hiatt, A., R. Cafferkey, and K. Bowdish. 1989. Production of antibodies
in transgenic plants. Nature 342: 76-78. Hemenway, C., Fang, R. X., Kaniewski,
W.K., Chua, N.H., and Turner, N.E. 1988. Analysis of the mechanism of protection
in transgenic plants expressing the potato virus X coat protein or its
antisense RNA. EMBO J. 7:1273-1280. Hsu, H.T., Wang, Y.C., Lawson, R.H.,
Wang, M., and Gonsalves, D. 1990. Splenocytes of mice with induced immunological
tolerance to plant antigens for construction of hybridoma secreting tomato
spotted wilt virus-specific antibodies. Phytopathology 80: 158-162.
Hu, J.S., Siemieniak, D.R., Slightom, J.L., and Gonsalves, D. 1991.
Molecular cloning, sequencing, and identificaton of squash mosaic virus
coat protein genes. J. Gen. Virol. (Submitted). Ie, T.S. 1970. Tomato spotted
wilt virus. No. 39 in: Descriptions of Plant Viruses, Common. Mycol. Inst./Assoc.
Appl. Biol., Surrey, England. Powell-Abel, P.P., Nelson, R.S., De, B.,
Hoffmann, N., Rogers, S.G., Fraley, R.T., and Beachy, R.N. 1986. Delay
of disease development in transgenic plants that express the tobacco mosaic
virus coat protein gene. Science 232:738-743.
7. Detailed budget
A. Salaries and Wages
Research Assistant $ 5,000
B. Materials and Supplies
Biotechnology Center $ 1,500 Monoclonal Antibody Center $ 1,000 Chemicals,
enzymes $ 3,000 Isotopes $ 1,000 General supplies $ 1,000
C. Travel $ 2,500
D. Total Cost $ 15,000
Part C. Project Leader Qualification:
I have previously used nearly all of the techniques involved in this
project to clone and characterize the coat protein genes of squash mosaic
virus (Hu et al. 1991). In addition, similar recombinant DNA techniques
and plant gene transfer systems are being used in collaborative research
with Dr. A. Kuehnle, Department of Horticulture, University of Hawaii,
on coat protein-mediated protection with cymbidium mosaic virus and odontoglossum
ringspot virus.
