Control of Tomato Spotted Wilt Virus in Floral Crops 1993 Proposal
SUMMARY
The past 5 years we have been engaged in research designed to
characterize the tomato
spotted wilt-like viruses which infect floral and other agricultural
crops, This research has lead
to the realization that more than one virus is responsible for
this disease in floral crops and that
the predominant TSWV-Iike virus in floral crops is almost never
found in other crops. As part of
this research we developed the serological assays for accurate
diagnosis which is now the
industry standard. Although this has helped reduce the viruses
from planting stock they will
probably never be eliminated from the production cycle and thus
other measures are, necessary
to provide adequate levels of control to allow sensitive crops
such as gloxinia and impatiens to I I
be efficiently produced. Recently we have begun studies to use
genetically engineered cross-
protection to introduce selected viral genes into plants to impart
resistance to these viruses.
These genes have been tested in the model systems Nicotiana tabacum
and Nicotialla
benthamiana for their ability to impart resistance. We have begun
studies to develop procedures
for their introduction into floral crops. Due to the availability
of tissue culture information, we
began our investigation with chrysanthemum. We recently (June
1992) reported our
regeneration and transformation protocols for chrysanthemum at
the American Association of
Horticultural Science Symposium on “In Vitro Culture and Horticultural
Breeding”. In addition
to being an economically important crop, we believe that chrysanthemum
may be used as the
experimental model for transformation of floral crops. That is,
certain chrysanthemum cultivars
have been shown to be amenable to these procedures and thus will
allow us to test novel ideas in
an efficient way. Although, the focus of this proposal is to
extend our findings to other floral
crops, we intend to continue our research on chrysanthemum in
order to develop this as a test
system for floral crops.Our primary objective is the develop transformation protocols
for gloxinia and then
impatiens for the initial purpose of introducing resistance to
TSWV and similar viruses. In
addition, we intend to develop the chrysanthemum system into
a model for transformation of
floral crops. These protocols will not only be applicable for
introduction of virus resistance, but
may also be used to introduce genes for other horticultural traits.
Introduction
This is a new project whose primary objective is to introduce
newly identified 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 practical
means of control for these viruses.
Under our recent grant we identified the new virus, INSV as the
predominant Tospovirus
in greenhouse grown floral crops (17, 18, 22). We were then able
to characterize this virus and
compare it to the common isolates of TSWV. We then began isolating
the viral gene which,
when introduced into the, plant, imparts resistance to that virus
(19). Although this approach has
shown promise for other viruses for about 6 years (1, 23), it
was only within the past year that
several groups have shown that it will be effective for Tospoviruses(
8, 21, Daub & Moyer,
Unpublished), Recently, we have made significant progress towards
applying this to floral
crops. We have successfully developed a transformation procedure
for the chrysanthemum cv.
Iridon which will allow the introduction of TSWV resistance genes
and presumably other genes
into that crop (30,31). The development of TSWV resistant chrysanthemum
is currently
underway.
The goal of the proposed research is to extend this form of resistance
to other floral
crops which are particularly susceptible to TSWV and INSV. We
intend to continue refining
the chrysanthemum system as it can be used as a model system
to test new ideas for floral crops;
however, primary emphasis will be on extending the project to
include gloxinia and then to
double flowered impatiens. The research has three components
which are common to each of
the crops: 1. Development of regeneration protocols for each
crop (target cultivars) that will
allow regeneration from small pieces of tissue such as leaf discs.
2. Development of procedures
for introduction of the gene so that it will be stably incorporated
into the plants genome (the
transformation procedure). 3. Evaluation of plants for virus
resistance and cultivar
characteristics. Progress on transformation of chrysanthemum,
although not yet routine, has
progressed remarkably fast. The progress was due in part to the
years of experience in mum
tissue culture which enabled us to start essentially at stage
2. Progress on gloxinia and impatiens
will necessarily be slower in that studies will be initiated
at stage 1.
LITERATURE REVIEW AND WORK IN PROGRESS
Tomato spotted wilt virus is now a widely respected problem in
ornamental crop
production (14, 15, 22), 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.
Our goal is to develop
resistant varieties in the most sensitive crops which will reduce
the reliance on these expensive
measures. Since 1986 there has been considerable interest in
a form of resistance which is
based oil introducing genes from the virus into plants (1, 23).
The presence of these genes
confers varying degrees of resistance in the plant to natural
infection by the virus. This
phenomenon is now well documented and in the process of being
used for many viruses,
including TSWV (1, 8, 21). We have studies underway to expand
these preliminary reports and
have confirmed that this strategy of resistance does indeed work
for TSWV (for details of
progress see below). The resistance has been referred to as “pathogen
derived resistance” (25) or
more specifically “capsid protein mediated resistance” (1). The
process involves isolating the
specific viral gene and then introducing it into plants using
gene transfer technology (genetic
engineering).
When our research began on TSWV it was widely accepted that this
unusual virus was
unique, that is that there was only one virus causing the problem
(32, AFE symposium, Sanibel
Island). Our research here in the United States and that of others
in Europe has shown that there
are two viruses that have been well characterized and there are
other variants as well
(5, 6, 11, 17, 18, 29). As mentioned above, this group of related
viruses is now referred to
collectively as the Tospoviruses. Most of our virus research
on identification and
characterization of tospoviruses has been conducted in support
of developing resistance to these
viruses. We were the first to recognize the existence of a 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)
(19,20). A similar virus has
been isolated in Europe and similarly assigned to INSV (5) We
have isolated the nucleocapsid
(N) gene from this virus to be used in our resistance studies
(19). We have shown how the N
protein accumulates in infected cells, along with other viral
coded proteins (29). These findings
were consistent with recent findings for TSWV (16). We have also
located and characterized
other INSV genes (20). The characterization of these other genes
was undertaken to provide the
opportunity to utilize other genes on the INSV genome as potential
sources of resistance. These
studies were conducted not only to satisfy scientific curiosity
but also for patent purposes. At tile
present time; however, the focus of the resistance effort revolves
around the N gene of TSWV
and INSV.
Due to the importance of the N gene for resistance as well as
its importance as a determinant of
classifying Tospoviruses we are currently conducting detailed
investigations of the variability of
the N gene between tile different isolates of Tospoviruses. We
have developed a panel of
monoclonal antibodies which provide a measure of relatedness
of the various Tospoviruses (11).
One of these monoclonal antibodies reacts not only to both TSWV
and INSV, but also to several
unclassified Tospoviruses isolated in Europe and South America
as well as Peanut Bud Necrosis
virus found in India, Thus, this monoclonal antibody has significant
implications towards
simplifying current serological testing procedures as well as
providing an increased ability to
detect unrecognized viruses. Efforts are continuing to monitor
Tospoviruses in floral crops.
Current Status of Ornamental Regeneration and Transformation
Ornamental crops pose a challenge 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. Ample evidence was presented documenting
the successful isolation
of transformed plants, however the methods used to generate these
plants were often considered
proprietary.
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. However, protocols developed for one
cultivar can often be adapted for
others (see “work in progress” below for an example from our
work on chrysanthemum). The
goal of our work will be to begin to define conditions required
for regeneration and
transformation of important ornamental species such that these
protocols can serve as a
foundation for others who wish to adapt these methods to cultivars
of interest. Our focus will be
on crops susceptible to TSWV and INSV (chrysanthemum, gloxinia,
double flowered impatiens)
so that we can begin immediately toward the development of resistance
in these crops.
Work in Progress. We have successfully developed shoot regeneration
protocols for
three cultivars of chrysanthemum (Iridon, Hekla, and Polaris)
(30) using modifications of a
protocol provided by Dr. R. Trigiano (University of Tennessee).
All protocols utilize leaf
explant tissue cultured on Murashige and Skoog (MS) rnedium supplemented
with 11.5 M
Indoleacetic acid (IAA). Optimum concentrations of benzyladenine
(BA) ranged from 0.5 to 5
M depending on tile cultivar. In addition, regeneration of Hekla
requires transfer to hormone-
free medium after two weeks, and Polaris requires the presence
of 20 M CoCl2, transfer to
hormone-free medium when shoot primordia first appear, and exclusion
of blue wavelengths of
light during shoot formation.
Initial efforts for developing transformation protocols centered
on the identification of
strains of Agrobacterium tumefaciens which were virulent oil
the three chrysanthemum cultivars.
The three cultivars did not differ significantly from each other
in their response to the three
strains tested (ACH5, A281, CHRY5), but there were differences
between strains. CHRY5 and
A281 were significantly more virulent (91 - 94% infection) as
compared to 7% for ACH5.
A successful transformation protocol was developed for the cultivar
Iridon using a
disarmed vector strain derived from strain A281 and 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.
Genetically-engineered Resistance to TSWV
Two papers have recently been published documenting the efficacy
of the TSWV coat
protein gene in protection of tobacco against TSWV (8, 21). We
have confirmed these studies in
our laboratory. Tobacco was transformed with the coat protein
gene, and transformants were
identified which were protected against infection by TSWV. Resistance
was stably inherited
through a seed cycle, and progeny of highly-resistant plants
are being analyzed further.
Our current efforts are aimed at the development of alternate
constructs of the TSWV
coat protein gene. In addition to the full-length constructs
of the N gene for both TSWV and
INSV we have also made constructs which carry sequences outside
the N gene coding region, a
complementary construct as well as five other partial N gene
constructs to test for antisense
activity. These genes are being put into Nicotiana tabacum to
test for levels of resistance against
the virus from which the constructs were made as well as to test
against examples of the TSWV-
like variants. This is a very important aspect of the testing
in that these multi-isolate tests will
give some indication of how well the resistance can be expected
to hold up under commercial
production conditions. Although we are continuing to develop
and test new strategies, we have
constructs for both TSWV and INSV which should provide some level
of protection. This,
construct development is not a major portion of this research.
Rather it is the deployment of the
resistance genes we currently have.
Based on success with the tobacco system, we have begun transforming
TSWV
constructs into the chrysanthemum cv. Iridon (31). To date over
50 transformed plants have
been recovered and the presence of the N gene confirmed. Plants
are currently being
vegetatively propagated, and inoculation studies will begin soon.
OBJECTIVES
The objectives of this proposal are two-fold:
1. To use chrysanthemum as a model system to investigate strategies
of coat-protein-
mediated cross protection for the development of TSWV and INSV-resistant
ornamental crops.
2. To develop regeneration and transformation protocols for target
cultivars of two other
TSWV-susceptible ornamental crops, gloxinia and double flowered
impatiens. Once protocols
are developed, these crops will also be transformed with the
viral gene constructs.
MATERIALS AND METHODS
1. TSWV-resistant chrysanthemum. Iridon will be the cultivar
used for these studies
due to its ease of regeneration and transformation. Iridon will
be transformed with gene
constructs previously demonstrated to impart resistance. 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 ftom the original transformants will
be tested for stability of the gene,
virus resistance and varietal characteristics.
Resistance to the viruses in the transformed plants will be conducted
by artificial
inoculation of the transformed plants with a representative sample
of virus isolates. Our initial
efforts will utilizing mechanical inoculation techniques which
are well established. Although
transmission efficiency is somewhat low for chrysanthemum as
compared to other crops,
attempts are being rnade to improve the inoculation efficiency.
We also have established a
collaborative effort with entomologists at North Carolina State
University who have experience
with thrips transmission. Following approbate controlled tests,
the plants expressing resistance
will be introduced into commercial growing conditions for further
resistance testing.
Our overall goal is to confirm the efficacy of coat-protein-mediated
transformation for
protection of chrysanthemum against TSWV, and to develop a model
system for testing various
genes in floral crops. Information on the efficacy of different
gene constructs will allow us to
choose the best construct for use in gloxinia and double flowered
impatiens.
2. Regeneration and transformation of gloxinia and double-flowered
impatiens. Work
on these crops will follow the strategy used in our lab for chrysanthemum
(30). First, we will
develop protocols for regeneration which are appropriate for
Agrobacterium-mediated
transformation (i.e. regeneration frorn cut edges of leaf or
stem explants). Second, we will
determine which strains of Agrobacterium are most virulent on
these species. Third, we will use
the results of those studies to develop a transformation protocol.
With both crops we will initially start with several cultivars
in order to identify those that
are most amenable to in vitro culture. There are published reports
of successful regeneration of
shoots from pedicel segments and leaf disks of gloxinia (2,13,24).
Our initial efforts will be to
start with those procedures and modify them as necessary. If
necessary, procedures used for in
vitro culture of african violets (28) will also be tried. Published
reports on impatiens in ratio
culture have been limited primarily to reports of shoot tip micropropagation
(12,27) which may
not be directly applicable to regeneration from leaf or stem
exploits. Our initial strategy will
thus be to try a wide range of hormones, hormone concentrations,
and shifts in concentrations
during culture. Other factors which we will test include temperature,
light intensity and
wavelengths (26), and the role of hormone inhibitors (4).
Tests for virulence of different strains of Agrobacterium tumefaciens
will be done using
protocols used in our lab on chrysanthemum. Bacterial cultures
will be injected into stems of
plants as well as being applied to wounds on leaves. Inoculation
sites will be observed for tumor
formation. In addition, virulence will be quantified by treating
leaf disk explants with bacteria
and plating them on hormone-free medium. Callus development in
the absence of externally-
supplied hormones indicates the successful infection and transfer
of tumor genes (3). As with
the regeneration studies we will test a number of cultivars to
determine those susceptible to our
Agrobacterium strains.
Once regeneration protocols and strain specificity have been determined,
we will develop
transformation protocols. We will utilize the best cultivars
and disarmed versions of the
appropriate Agrobacterium strains which we have available. Initial
efforts will utilize a vector
plasmid carrying marker genes for kanamycin resistance and GUS
expression. Some of the
parameters which will be investigated include bacterial concentration,
time between wounding
(cutting) of explants and infection, appropriate co-culture media,
and the use of vir gene
inducers. Transformants will be selected on kanamycin-containing
medium and tested for levels
of GUS expression. Once successful transformants have been obtained,
we will switch to using
the appropriate TSWV constructs as determined from our studies
with chrysanthemum.
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. It
will however, be necessary to replace such items when their useful
life expectancey is exceeded
and to make necessary repairs. It will also be necessary to purchase
small items such as
electrophoresis apparatus which is included in the supplies section.
LITERATURE CITED
1. Beachy, R. N., Loesch-Fries, S., and Turner, N. E. 1990. Coat-protein
mediated resistance against virus
infection. Annual Review of Phytopathology 28:451-474.
2. Bigot, C. 1974. Obtention de plantes entieres a partir de
pcdoncules floraux de Gloxinia hybrida. cultives in
vitro. Z. Pflanzenphysiol. Bd. 73:178-183.
3. Bush, A. L. and S. G. Pueppke. 1991. Cultivar-strain specificity
between Chrysanthemum morifolium and
Agrobacterium tumefaciens. Physiol. Molec. Plant Pathol. 39:309-323.
4. Chraibi, K. M., A. Latche, J. P. Roustan, and J. Fallot. 1991.
Stimulation of shoot regeneration from
cotyledons of Helianthus annus by the ethylene inhibitors, silver
and cobalt. Plant Cell Rept. 10:204-207.
5. de Avila, A. C., de Haan, P., Kitajima, E. W., Kormelink,
R., Resende, R. dde O., Goldbach, R., Peters, D.
1992. Characterization of a distinct isolate of tomato spotted
wilt virus (TSWV) from Impatiens sp. in the
Netherlands. J. Phytopathology 134:133-151.
6. de Avila, A. C., Huguenot, C., Resende, R., de O., Kitajima,
E. W., Goldbach, R. W., and Peters, D. 1990.
Serological differentiation of 20 isolates of tomato spotted
wilt virus. Journal of General Virology 71:2801-2807.
7. de Haan, P., Wagemakers, L., Peters, D., and Goldbach, R.
1990. The S RNA segment of tomato spotted wilt
virus has an ambisense character. Journal of General Virology
71:1001-1007.
8. Gielen, J. J. L., deHaan, P., Kool, A. J., Peters, D., van
Grinsven, M. Q. J. M., and Goldbach, R. W. 1991.
Engineered resistance to tomato spotted wilt virus, A negative-strand
RNA virus. Bio/Technology 9:1363-1367,
9. German, T., Ullman, D., and Moyer, J. W. 1992. Tospoviruses:
Diagnosis, Molecular Biology, Phylogeny and
Vector Relationships. Annual Review of Phytopathology. In Press.
10. Golelmboski, D. B., Lomonossoff, G. P., and Zaitlin, M. 1990.
Plants transfomed with a tobacco mosaic virus
nonstructural gene sequence are resistant to the virus. Proc.
Natl. Acad. Sci. USA 87:63311-6315.
11. Hall, J. M. and Moyer, J. W. 1992. Epitope analysis of the
N protein from serologically distinct Tospoviruses.
Annual Meeting of American Society of Virology. July 1992.
12. Han, K., and L. C. Shephens. 1987. Growth regulators affect
in vitro propagation of two interspecific
impatiens hybrids. Sci. Hort. 32307-313.
13. Johnson, B. B. 1979. In Vitro propagation of gloxinia from
leaf explants. HortSci. 13:149-150.
14. Jones, R. K. and Moyer, J. W. 1986. Tomato spotted wilt virus
in gloxinia in North Carolina. N. C. Flower
Growers Bulletin 30:11-13.
15. Jones, R. K. and Moyer, J. W. 1987. Exacum, a new host for
Tomato spotted wilt virus. North Carolina
Flower Growers Bullefin 31:1-2.
16. Kormelink, R., Kitajima, E. W., deHaan, P., Zuidema, D.,
Peters, D., Goldbach, R. 1991. the nonstructural
protein (NSs) encoded by the ambisense; S RNA segment of tomato
spotted wilt virus is associated with fibrous
structures in infected cells. Virology 181:459-468.
17. Law, M. D. and Moyer, J. W. 1989. Physicochemical analysis
of a serologically distinct tomato spotted wilt
virus strain. Phytopathology 79:1157.
18. Law, M. D. and Moyer, J. W. 1990. A tomato spotted wilt-like
virus with a serologically distinct N protein.
Journal of General Virology 71:933-938.
19. Law, M. D., Speck, J., and Moyer, J. W. 1991. Nucleotide
sequence of the 3′noncoding region and N gene of
the S RNA of a serologically distinct tospovirus. Journal of
general Virology. 72:2597-2601.
20. Law, M. D., Speck, J. and Moyer J. W. 1992, The M RNA of
Impatiens necrotic spot Tospovirus
(Bunyaviridae) has an ambisense genomic organization. Virology
188:732-74 1.
21. MacKenzie. D. J. and P. I. 1992. Resistance to Tomato spotted
wilt virus infection in transgenic tobacco
expressing tile viral nucleocapsid gene. Molecular Plant-Microbe
Interactions 5:34-40.
22. Moyer, J. W. and Jones, R. K. 1991. Tomato spotted wilt virus.
Ball Redbook. Ball Publications.
23. Powell-Abel, P., Nelson, R. S., De, B., Noffman, 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.
24. Raman. K. 1977. Rapid multiplication of Streptocurpus and
gloxinia from in vitro cultured pedicel segments.
Z. Pflanzenphysiol. Bd. 83:411-418.
25. Sanford, J. C. and Johnston, S. A. 1985. the concept of pathogen
derived resistance: Deriving resistance genes
from the parisites own genome. J. Theor. Biol. 113:395-405.
26. Stasinopoulos, T. C., and Hangarter, R. P. 1990. Preventing
photochemistry in culture media by long-pass
light filters alters growth of cultured tissues. Plant Physiol.
93:1365-1369.
27. Stephens, L. C., S. L. Krell, and J. L. Weigle. 1985. In
vitro propagation of java, new guinea, and java x new
guinea impatiens. HortSci. 20:362-363.
28. Torres, K. C. 1983. Tissue Culture Techniques for Horticultural
Crops, Chpt 6. Van Nostrand Reinhold, NY.
29. Urban, L. A. Huang, P.Y. and Moyer, J. W. 1991. Cytoplasmic
inclusions in cells infected with isolates of L
and I serogroups of tomato spotted wilt virus. Phytopathology
81:525-529.
30. Urban, L. A., J. M. Sherman, J. W. Moyer, and M. E. Daub.
1992. Regeneration and Agrobacterium-mediated
transformation of chrysanthemum. Poster presented at the International
Society of Horticultural Science
Symposium on “In Vitro Culture and Horticultural Breeding”, Baltimore,
MD, Julie 1992.
31. Urban, L.A., J. Speck, J.W. Moyer, and M.E. Daub. 1992. Transformation
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nucleocapsid gene of tomato spotted wilt virus. Phytopathology
82 (In Press) (Abstract).
32. Wang, M., and Gonsalves, D. 1990. ELISA detection of various
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specific antisera to structural proteins of the virus. Plant
Disease 74:154-158.
BUDGET
Technical Support 24000 25000 26000
(23.9% FICA, INs, etc) 5472 5700 5928
Total Salaries 29472 30700 31928
Supplies 6264 6275 6286
TOTAL REQUESTED 35736 36975 38214
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 reserach at UC-Davis prior to coming to NCSU. He has
17 years experience in working
with viruses of vegetatively propagated crops. He has 6 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 providing antisera to
the industry for diagnosis and clean-stock programs. His research
includes both applied and
basic aspects of plant virology as evidenced by his balance of
publications.
Margaret E. Daub is an associate professor of plant pathology
at North Carolina State
University. She received a Ph.D. in plant pathology from the
University of Wisconsin-Madison
and did postdoctoral work in plant tissue culture at Michigan
State University. She has had 13
years experience working with all aspects of plant in vitro culture
and transformation, and has
written a number of 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.
