Development of Resistance to Tomato Spotted Wilt and Similar Viruses inFloral Crops 1996 Proposal
EXECUTIVE SUMMARY
Over the past six years we have been engaged in research designed to characterize
the
tomato spotted wilt-like viruses which infect floral crops. Initial research
led to the discovery
that more than one virus is responsible for this disease in floral crops.
This research resulted
in the development of a serological assay for accurate diagnosing which
is now the industry
standard. Although accurate diagnosis has helped reduce the viruses from
planting stock, tomato
spotted wilt virus (TSWV) and impatiens necrotic spot virus (INSV) will
probably never be
eliminated from the production cycle due to their broad host range and
transmission by thrips
vectors. Thus other measures are necessary to provide adequate levels of
control. During the
last three years in work supported by the Floral Endowment, we focused
our research on the
development of techniques for genetic engineering of resistant plants.
Disease resistance is the
most economical and environmentally-sound approach for the control of plant
diseases; and
genetic engineering has been shown to be an effective strategy for the
development of resistance
to plant viruses. In our work we successfully developed an efficient protocol
for genetically
engineering diverse cultivars of chrysanthemum. We then used this procedure
to develop both
pot and cut-flower chrysanthemum cuitivars (Polaris and Iridon) with a
high level of resistance
to TSWV by genetically engineering them with a gene from the virus. This
technology is now
ready for transfer to the industry.
The overall goal of this proposal is to establish ties with propagators
to make the benefits
of our findings available to the floral industry. The proposal has three
specific objectives: 1)
genetically engineer TSWV resistance into chrysanthemum cultivars of greatest
importance to
the industry, 2) in cooperation with the industry, test the genetically
engineered cultivars under
high disease pressure for disease resistance and horticultural characters,
and 3) examine the plant
and virus factors that affect long-term stability of the resistance.
TOMATO SPOTTED WILT VIRUS RESISTANCE IN CHRYSANTHEMUM
INTRODUCTION
This is a request for a new project whose primary objective is to extend
genetically
engineered virus
resistance for practical application to the industry. For the last several
years,
our laboratories
have been investigating tomato spotted wilt-like viruses which infect floral
and
other crops.
When our research began, it was widely accepted that the disease was caused
by
a single virus,
tomato spotted wilt virus (TSWV) (33, AFE symposium, Sanibel Island). Under
previous funding
from the American Floral Endowment we were the first to recognize that
spotted wilt
was actually caused by a group of related viruses (collectively termed
Tospoviruses)
(18, 19). We
identified a new virus, impatiens necrotic spot virus (INSV), as a major
Tospovirus in
greenhouse-grown floral crops (16, 17, 21). Our research and that of others
in
Europe has now
shown that there are a number of variants and at least two distinct viruses
(5,6,9,12,16,17,31).
Development of antisera for identification of INSV as well as TSWV has
allowed for
accurate diagnosis of these viruses in floral crops.
Control of virus diseases of floral crops usually focuses on use of “clean
stock”, i.e.
propagation
materials that have been indexed and shown to be free of known viruses.
Although
also important
for TSWV and INSV control, use of virus-free propagation material is not
in
itself adequate,
as these viruses are transmitted by an insect vector, thrips. Current control
measures for
Tospoviruses in floral crops focus on early detection and removal of infected
planting material
from production areas as well as preventative measures to control the thrips
vector.
Control of thrips is difficult as these insects have developed resistance
to many
insecticides
and breed successfully in greenhouses all year long. Screening of greenhouses,
isolation of
propagation stock from production areas, eliminating weeds and post-production
reservoir plants
from greenhouses, monitoring of thrips populations and judicious use of
pesticides are
all needed for control.
Use of resistant varieties is the most commonly-used strategy for control
of viruses in
many crop species.
Conventional breeding strategies require the identification of sources
of
disease resistance
genes, a difficult task given the diversity of floral crop species that
are
susceptible
to Tospoviruses. Also, the overriding importance
of, appearance and general
horticultural
traits, the large number of cultivars which are produced per crop, and
the rapid
turnover in
cultivars have made breeding for disease resistance of secondary importance
in floral
crops (28).
However, the development of technologies for gene identification and gene
transfer
into plants
has provided the opportunity for genetically engineering disease resistance
into
horticulturally
desirable cultivars without altering critical quality traits. Further,
extensive
research
with genes from viruses has documented the efficacy of virus nucleocapsid,
or coat
protein
genes in protecting plants against virus infection following transfer and
expression of
these
genes in plants (1, 24). Although the mechanism by which these virus genes
impart
resistance
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 effective for protecting tobacco
against TSWV
(9,20,23).
During the last three years we developed and utilized genetic engineering
techniques to
produce
TSWV-resistant lines of two cultivars of chrysanthemum: Polaris, a highly-susceptible
cut-flower
mum cultivar, and Iridon, a pot mum cultivar. We chose to work with
chrysanthemum
since it is economically important, is highly susceptible to TSWV, and
extensive
information
was available on tissue culture protocols. Our efforts started with the
isolation and
cloning
of the TSWV N gene. We constructed several versions of the TSWV N gene
and tested
these
constructs for their ability to impart resistance to TSWV in tobacco. (23).
Based on the
results
of those experiments, we chose the most effective constructs for our chrysanthemum
studies.
Next, we developed techniques for transfer of the TSWV N gene into chrysanthemum
using
Agrobacterium tumefaciens as a vector. This work started with the screening
of strains
of A.
tumefaciens for their ability to transfer genes to chrysanthemum cells,
and the development
of a chrysanthemum
shoot regeneration protocol appropriate for genetic, engineering of cultivar
Iridon
(32). We later significantly modified our transformation procedure such
that it would
effectively
move genes into both pot and cut-flower chrysanthemum cultivar (27). Using
this
protocol,
we isolated several hundred transgeaic plants of Polaris, and confirmed
that
they contained
the TSWV N gene.
Our most recent efforts have been focused on screening the transformed
chrysanthemum
for resistance
to TSWV. Most of our studies have utilized inoculation with the natural
vector,
the western
flower thrips, Frankliniella occidentalis. We established a thriving thrips
colony
within
an enclosed chamber in a greenhouse. Thrips were allowed to feed on chrysanthemum
and Datura
plants infected with a highly virulent strain of TSWV isolated from chrysanthemum.
Cuttings
from our transgenic plants were then placed in the chamber and monitored
for spotted
wilt infection
by symptom development and serological assays. We identified several lines
which
are immune to systemic infection of the virus even with repeated inoculation
by thrips.
Resistance
of these lines has been shown to be stable through multiple cycles of vegetative
propagation
and through meristem tip culture.
OBJECTIVES AND ANTICIPATED BENEFITS
The overall goal of this proposal is to extend genetically engineered virus
resistance for
practical
application to the industry. This proposal has direct major objectives:
1) Extend our efforts to cultivars of greatest importance to the industry.
We have
successfully
developed a genetic transformation protocol that allows us to transfer
genes into
genetically-diverse
cultivars of chrysanthemum. We will now use our protocol to develop
TSWV resistance
in horticulturally-important cultivars of chrysanthemum.
2) In cooperation with industry, test the transformed, resistant plants
for both resistance
and quality
traits in a commercial setting. The ultimate test of any cultivar 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 cultivar have
not been altered
by the
genetic engineering procedure.
3) Examine factors which will affect stability of resistance in a commercial
setting.
TSWV is
a highly variable virus. Studies with TSWV-resistant cultivars of tomato,
tobacco, and
lettuce
which were developed by conventional breeding have demonstrated that resistance
can
be overcome
by new strains of the virus. We will investigate both plant factors and
virus factors
that impact
stability of resistance to TSWV.
Benefits.
TSWV &
INSV are a widely respected problem in floral crop production in
the United
States and around the world (13,14,21). Stringent, laborious controls aimed
at
removing
infected planting material from propagation and production areas as well
as
preventative
measures to control thrips are both required. Both measures are expensive
in terms
of materials
and labor for implementation. We have developed chrysanthmum cultivars
with
genetically-engineered
resistance to TSWV. Successful deployment of these resistant cultivars
will add
an additional control tool, and reduce the reliance of the floral industry
on the current
expensive
control 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. Successful deployment
will also
pave the
way for using genetic engineering techniques for improvement of floral
crops by genetic
engineering
for horticultural traits other than virus resistance, such as flower color
or delayed
senescence.
MATERIALS AND METHODS
1. Chrysanthemum cultivar transformation. Using the methods recently reported
by our
lab (27,32), we have been successful in transforming cultivars of chrysanthemum
from diverse
genetic backgrounds. For this work we conducted extensive studies on variables
such as explant
source, appropriate strains of Agrobacterium (3, 32), hormones and hormone
inhibitors (4),
temperature, and light intensity and wavelength (29). Regeneration and
transfer protocols
are often highly cultivar-specific, however, our protocol has shown itself
to have broad
applicability. We will now use this protocol to develop TSWV resistance
in horticulturally
important chrysanthemum cultivars. 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 transformed with the TSWV N gene as previously described
(27, 32).
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) (32).
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.
2) In cooperation with industry, test the transformed, resistant plants
for both resistance
and quality
traits in a commercial setting. TSWV is a highly variable virus due in
part to the
fact that its
DNA is partitioned into three different segments (10) and these segments
can
recombine when
multiple isolates infect the same leaf (22,25). We have tested resistance
of our
transgenic plants
by natural inoculation techniques by exposing them to western flower thrips
reared on TSWV-infected
plants. Using this procedure we have only been able to screen for
resistance against
a small number of virus strains, however. There have been problems with
the
development
of resistant strains when TSWV-resistant cultivars of tobacco, tomato and
lettuce,
developed through
conventional breeding, were deployed on a commercial scale (2,8). Studies
with tobacco
genetically engineered for resistance to TSWV have also demonstrated that
strains
can be identified
which are capable of overcoming resistance (25). The true test of any disease-
resistant cultivar
is its response under commercial grower conditions. Thus, in order to define
the spectrum
of resistance and the stability of resistance, we will test our transgenic
plants in
commercial operations
with high disease pressure.
We will also work with representatives of the industry to evaluate possible
changes to
critical quality
traits which may be caused by the tissue culture cycles used in the transformation
procedure. Although
genetic engineering itself has not been shown to alter plant type, tissue
culture and
the natural variation occurring in tissues of vegetatively propagated crops
can lead
to somaclonal
variation and accumulation of deleterious traits. The lines that we have
generated
took normal
under our testing conditions, however, prior to release to the industry,
it is critical
that they also
be tested under commercial conditions for any small, but commercially important
changes that
we have not been able to detect.
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. We have
an excellent
working relationship
with regulatory officials in North Carolina, and anticipate no problems
in
putting together
the necessary documentation.
3) Examine factors which will affect stabiity of resistance in a commercial
setting.
Stability of resistance is impacted by both virus and plant characteristics.
We currently have
ongoing, a basic research program in the lab aimed at defining the genes
in the virus that are
responsible for host range and for disease reaction on resistant plants
(25). These studies should
allow us to define our genetic engineering strategy to obtain broad-spectrum,
stable resistance.
Also, based on extensive research with tobacco, hypotheses have been established
concerning
the genetic and biochemical basis of transgenic resistance, and the cellular
factors needed to
provide broad-spectrum resistance. Thus, during our previous work, we purposefully
transformed plants with versions of the N gene that had been constructed
in different ways; in
particular we have produced plants that produce no, some, or significant
amounts of the
nucleocapsid protein. Testing of our resistant plants developed with these
different gene
constructions will allow us to confirm which strategy is best for practical
TSWV control under
commercial conditions.
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 data
research. The budget requests funds for the salary of a technician needed
to carry out the work,
part-time labor for our greenhouse studies, expendable supplies, and travel
for the cooperative
studies proposed.
LITERATURE CITED
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BUDGET REQUEST
Technical Support
$26,000
Benefits (24.1 %)
$ 6,266
Hourly Labor
$ 2,000
TOTAL SALARIES
$34,266
Expendable Supplies
$ 6,000
Travel
$ 2,000
TOTAL REQUESTED
$42,266
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 sported wilt-like viruses (Tospoviruses).
Members of his research group were responsible for first identifying INSV,
the predomonant
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 time 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.
