Control of Tomato Spotted Wilt Virus in Floral Crops 1995 Proposal
DEVELOPMENT OF RESISTANCE
TO TOMATO SPOTTED WILT AND OTHER SIMILAR
VIRUSES IN FLORAL CROPS
INTRODUCTION
This is a request for continuation of funding of a project whose primary
objective is
to introduce genes for resistance to tomato spotted wilt virus (TSWV) and
impatiens
necrotic spot virus (INSV; formerly TSWV-I) into selected floral crops.
The entire group
of tomato spotted wilt-like viruses are now known collectively as the Tospoviruses.
During
the past five years we have been investigating the Tospoviruses which infect
floral and other
crops. The American Floral Endowment has been a primary supporter of this
research. We
feel very fortunate to have been able to increase the level of understanding
to the point that
we are now ready to extend this research directly to the development of
resistant cultivars
and thus provide a more efficient means of control for these viruses.
Under previous funding from the American Floral Endowment we identified
the new
virus, INSV, as the predominant Tospovirus in greenhouse-grown floral crops
(14,15,19).
When our research began on spotted wilt, it was widely accepted that the
disease was caused
by a single virus (29, AFE symposium, Sanibel Island). Our research and
that of others in
Europe has now shown that there are a number of variants and at least two
distinct viruses
(4,5,7,10,14,15,26). We were the first to recognize the existence of the
second Tospovirus
(17). This virus was provisionally designated TSWV-I, but we have shown
that it is
sufficiently distinct to be a separate virus and named it impatiens necrotic
spot virus (INSV)
(16,17). Development of antisera for identification of INSV has allowed
for accurate
diagnosis of this virus in floral crops.
Our research on identification and characterization of Tospoviruses has
allowed us to
use genetic engineering techniques to develop resistance to these viruses.
We have isolated
the nucleocapsid (or “N”) gene from both viruses (16). Extensive research
with similar
genes from other viruses has documented the efficacy of nucleocapsid or
coat protein genes
in protecting plants against virus infection following transfer and expression
of these genes in
plants (1, 21). Although the mechanism involved in virus protection is
not understood,
extensive laboratory and field trials with crop plants transformed with
such genes has
documented the high levels of resistance achieved through this approach.
Several groups
have shown that this approach is also effective for protecting tobacco
against TSWV
(7,18,20). During the last two years we have made significant progress
towards applying
this technique to floral crops.
Ornamental crops pose a challenge for the development of resistance through
genetic engineering both because of the diversity of species and cultivars
available as well as
a lack of extensive studies on tissue culture protocols. At an international
symposium on in
vitro culture of horticultural crops held in Baltimore in June, 1992, only
15 of the 83
presentations given dealt specifically with ornamental crops. More troubling,
perhaps, is the
lack of information provided in many of these presentations on regeneration
and
transformation protocols. In order for both the public and private sector
to make advances in
the improvement of ornamental crops through genetic engineering, there
needs to be an
information base from which to proceed. In all crops that have been studied,
both shoot
regeneration and transformation are highly cultivar-specific. We have chosen
to use
chrysanthemum as a model system for floral crop transformation since it
has been the most
extensively studied.
In our previous work we successfully developed shoot regeneration protocols
for three
cultivars of chrysanthemum (27) using modifications of a protocol provided
by Dr. R.
Trigiano (University of Tennessee). Initial efforts for developing transformation
protocols
centered on the identification of strains of Agrobacterium tumefaciens
which were virulent on
the three chrysanthemum cultivars. A successful transformation protocol
was then developed
for the cultivar ‘Iridon’ using a disarmed vector strain carrying a vector
plasmid with two
marker genes, one for kanamycin resistance and the other for B- glucuronidase
(GUS)
activity. Kanamycin-resistant plants were regenerated, rooted, and transferred
to soil in the
greenhouse. Plants were also shown to have high levels of GUS activity,
thus confirming
stable transformation.
During the last year we have refined and modified our transformation procedure
and
have determined that it is effective for moving genes into both pot and
cut flower mums.
We
have also constructed several versions of the TSWV nucleocapsid gene and
have tested
these constructs for their ability to impart resistance to TSWV in tobacco
(20). Based on the
results of those experiments, we chose the most effective constructs and
successfully
transformed them into chrysanthemum. Using both mechanical and thrips inoculation
with a
highly virulent chrysanthemum isolate of TSWV, we have identified plants
which show a
high level of virus resistance.
Due to the importance of the N gene for resistance as well as its importance
as a
determinant of classifying Tospoviruses, we are also currently conducting
detailed
investigations of the variability of the N gene between the different isolates
of Tospoviruses.
We have developed a panel of monoclonal antibodies which provide a measure
of relatedness
of the various Tospoviruses (10). One of these monoclonal antibodies reacts
to both TSWV
and INSV. Thus, this monoclonal antibody has potential for simplifying
current serological
testing procedures as well as providing an increased ability to detect
unrecognized viruses.
OBJECTIVES
AND ANTICIPATED BENEFITS
The overall objective of our current research is to use chrysanthemum as
a model
system to investigate the efficacy of transforming plants with the virus
nucleocapsid gene for
the development of TSWV and INSV-resistant ornamental crops. We have three
specific
objectives for the coming year:
1) Determine the efficacy of our transformation protocol on a broader range
of
chrysanthemum cultivars. If the protocol has broad applicability, it will
pave the way for
using genetic engineering techniques for improvement of diverse cultivars.
Such efforts are
not limited to introduction of virus resistance. Given the rapid progress
in identifying and
isolating genes from plants and other organisms, such efforts will also
allow for introduction
of genes for other horticultural traits such as flower color or delayed
senescence.
2) Identify highly resistant plants of both pot and cut flower mums, and
test them for
resistance against diverse virus isolates. Successful deployment of the
resistant transgenic
plants will require that they express resistance to a broad range of virus
isolates found in
commercial settings. Some of the different constructions of the nucleocapsid
gene used to
transform the plants were designed to provide broad-spectrum resistance.
All transformed
plants will be tested against a diversity of isolates. Those with the broadest
resistance will
be used for objective 3.
3) To begin to test the transformed, resistant plants for both resistance
and quality
traits in a commercial setting. The ultimate test of any variety is its
performance in a
commercial setting. Tests are needed both to measure the stability and
efficacy of the
resistance to virus isolates and inoculum load present under commercial
conditions. It is also
necessary to determine that critical quality characteristics of the variety
have not been altered
by the genetic engineering procedure.
Benefits. TSWV & INSV are a widely respected problem in floral crop
production
(11, 12, 19). Stringent, laborious controls aimed at removing infected
planting material from
propagation and production areas as well as preventative measures to control
thrips (the
primary means of spread of this virus in floral crops) are both required.
Both measures are
expensive in terms of materials and labor for implementation. Using chrysanthemum
as a
model, our goal is to develop resistant varieties, thus reducing the reliance
of the floral
industry on these expensive measures as well as reduce sources of virus
inoculum. Success
with chrysanthemum will provide an excellent test case for extending this
technology to other
highly susceptible floral crop species such as gloxinia and impatiens.
MATERLALS AND METHODS
1. Chrysanthemum cultivar transformation. Using modifications of the methods
recently reported by our lab (27), we have been successful in transforming
both a pot and cut
flower mum variety. For this work we conducted extensive studies on variables
such as
explant source, appropriate strains of Agrobacterium (2, 27), hormones
and hormone
inhibitors (3), and temperature and light intensity and wavelength (24).
Regeneration and
transformation protocols are often highly cultivar-specific, however, our
success with two
genetically different varieties suggest that our protocol may have broad
applicability. If so,
such a protocol will allow for the efficient use of genetic engineering
technology for
chrysanthemum improvement. We will work with representatives of the floral
industry to
identify key varieties which differ in important horticultural traits.
Explant tissue from these
varieties will be tested for transformation ability. Transformed plants
will be selected on
kanamycin-containing medium, and the presence of the TSWV gene confirmed
by Southern
blot analysis and/or polymerase chain reaction (PCR). For constructs which
produce
proteins, levels of protein expression will be assayed by serological techniques
(Western
blots). Plants vegetatively propagated from the original transformants
will be tested for
stability of the gene, virus resistance and varietal characteristics. Success
in this work will
provide information needed to determine the practicality of genetic engineering
for
chrysanthemum improvement.
2) Test resistant plants against diverse virus isolates. Resistance to
the viruses in the
transformed plants will be conducted by both thrips and mechanical inoculation
techniques
with a representative sample of virus isolates. Transmission efficiency
using mechanical
inoculation is somewhat low for chrysanthemum as compared to other crops,
and we are
investigating strategies to improve the inoculation efficiency. Both temperature
and virus
isolate have proved critical in successful mechanical inoculation. Inoculum
is produced from
plants of Nicotiana benthamiana systemically infected with virus. Infected
leaves are ground
in buffer, and plants are inoculated by rubbing the inoculum onto leaves
which have been
dusted with carborundum to wound the leaves. Thrips inoculation is conducted
by rearing
thrips on chrysanthemum plants infected with the virus. Young cuttings
of transformed
plants are placed around the inoculum plants for five weeks, after which
they are sprayed to
eradicate the thrips and observed for the development of symptoms. This
procedure has
proved to be highly effective in screening of our transgenic. plants. Quantitative
determination of virus resistance is determined by measuring virus titre
in the upper leaves of
the plant using a serological assay (ELISA).
3) Test transformed plants in a commercial setting. Plants found to carry
high
levels of resistance to a broad range of isolates will need to be tested
in a commercial
setting. We will work with Dr. R. K. Jones, our department’s extension
specialist on
ornamental crops, and with representatives from industry to identify settings
and experiments
for evaluating both virus resistance and possible alterations in horticultural
traits of our most
promising transgenic plants. These studies will require approval from State
and Federal
regulatory agencies, as such a test involves release of genetically engineered
plants.
Discussions with Federal officials have indicated that a full application
will be necessary, as
the nucleocapsid gene of these viruses is not included in the exemptions
granted to
experiments conducted with transgenic plants which express “conventional”
virus capsid
protein genes. We have an excellent working relationship with regulatory
officials in North
Carolina, and anticipate no problems in putting together the necessary
documentation.
Facilities and Equipment.
We currently have all of the major equipment items, growth rooms and transfer
hoods
for the tissue culture and plant transformation research. In addition we
have adequate
greenhouse space. We also have all of the major items needed for the virology
portion of
the research. The budget requests funds for the salary of a technician
needed to carry out
the work. Also a budget for expendable supplies is included.
LITERATURE CITED
1. Beachy, R. N., Loesch-Fries, S., and Turner,
N. E. 1990. Ann. Rev.
Phytopathology 28:451-474.
2. Bush, A. L. and S. G. Pueppke. 1991. Physiol.
Molec. Plant Pathol. 39:309-323.
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J. Fallot. 1991. Plant Cell Rept.
10:204-207.
4. de Avila, A. C., de Haan, P., Kitajima, E. W.,
Kormelink, R., Resende, R. dde O.,
Goldbach, R., Peters, D. 1992. J. Phytopathology 134:133-151.
5. de Avila, A. C., Huguenot, C., Resende, R.,
de O., KitaJima, E. W., Goldbach, R.
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D., van Grinsven, M. Q. J. M.,
and Goldbach, R. W. 1991. Bio/Technology 9:1363-1367.
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Ann. Rev. Phytopathology
30:315-348.
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Virology. July 1992.
11. Jones, R. K. and Moyer, J. W. 1986. N. C. Flower
Growers Bulletin 30:11-13.
12.
Jones, R. K. and Moyer, J. W. 1987. North Carolina Flower Growers Bulletin
31:1-2.
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Law, M. D., Speck, J., and Moyer, J. W. 1991. J. Gen. Virology 72:2597-2601.
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Law, M. D., Speck, J. and Moyer J. W. 1992. Virology 188:732-741.
18.
MacKenzie, D. J. and Ellis, P. J. 1992. Molec. Plant-Microbe Interact.
5:34-40.
19.
Moyer, J. W. and Jones, R. K. 1991. Ball Redbook. Ball Publications.
20.
Perez, R. S. M. Geske, J. Speck, P. Reece, J. W. Moyer, and M. E. Daub.
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Daub. 1993. Phytopathology 83: 1355 (Abstract).
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BUDGET REQUEST
Technical Support
$26,000
Benefits (24.1%)
$ 6,266
Total Salaries
$32,266
Supplies
$ 5,000
TOTAL REQUESTED
$37,266
PROJECT LEADER QUALIFICATIONS
James W. Moyer is a professor of plant pathology at North Carolina State
University.
He received his Ph. D in plant pathology at The Pennsylvania State University
in 1975 and
did post-doctoral research at the University of California at Davis prior
to coming to NCSU.
He has had 18 years experience in working with viruses of vegetatively
propagated crops and
seven years experience investigating tomato spotted wilt-like viruses (Tospoviruses).
Members of his research group were responsible for first identifying INSV,
the predominant
virus problem in floral crops, and for providing antisera to the industry
for diagnosis and
clean-stock programs. His research includes both applied and basic aspects
of plant virology.
Margaret E. Daub is a professor of plant pathology at North Carolina State
University. She received a Ph.D. in plant pathology from the University
of Wisconsin at
Madison and did postdoctoral work in plant tissue culture at Michigan State
University. She
has had 15 years experience working with all aspects of plant in vitro
culture and
transformation, and has written several review articles on this subject.
Her research has
included in vitro selections of callus and protoplast cultures for phytotoxin
resistance, somatic
hybridization for transfer of resistance genes in plants, somaclonal variation
for improved
disease resistance, and plant transformation for pathogen gene expression.
