Note: Descriptions are shown in the official language in which they were submitted.
CA 02298882 2000-O1-31
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TRANSGENIC PLANTS USING THE TDC GENE FOR CROP IMPROVEMENT
This application claims the benefit of U.S. Provision Application No.
60/054,316, filed July
31, 1997.
BACKGROUND OF THE INVENTION
Field of the Invention
The present invention relates to transgenic plants having surprisingly
improved fungal,
bacterial, and/or nematode disease resistance, wherein the enhanced resistance
arises from enhanced
expression of a tryptophan decarboxylase (TDC) gene.
Summary of the Related Art
Crop losses incurred by diseases and pests have a major negative economic
impact upon all
sectors of agriculture today. Despite attempts to control these losses through
various crop husbandry
techniques (e.g., crop rotation), breeding of resistant cultivars and
application of agrochemicals,
losses remain unacceptably high. As of 1987, approximately 37% of all crops
produced worldwide
are lost to pests such as insects (13%), diseases (12%), and weeds and grasses
(12%). Dependence
1 S on agrochemicals is not only expensive, but it is also detrimental to the
environment and generally
regarded as unhealthy for living creatures. Over the last decade, agricultural
biotechnology has
provided some solutions for disease and insect management. The most intensive
global efforts in
agricultural biotechnology today are directed toward the design and
implementation of effective
transgene-mediated disease and pest resistance strategies.
The tremendous losses incurred by pathogenic fungi on crops worldwide has
fueled the
interest to develop rational and effective anti-fungal strategies in
transgenic plants (for review, see
Cornelissen and Melchers (1993) Plant Physiol. 101, 709-712). One of the
earliest concepts, and
to date, one of the most successful anti-fungal strategies has been to utilize
fungal cell wall-
degrading enzymes to attack an invading pathogen. Proteins that possess
chitinase or [3-glucanase
activity have been purified and characterized from numerous plant, fungal, and
bacterial sources
(Kauffinan et al. (1987) EMBO J. 6, 3209-3212; Legrand et al. (1987) Proc.
Natl. Acad. Sci. USA
84, 6750-6754). The genes that encode for some of these hydrolytic enzymes
have been cloned,
sequenced, and subsequently overexpressed in transgenic plants. The single,
unifying conclusion
that has emerged from these numerous studies is that while modest levels of
fungal protection can
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be afforded by overexpression of these enzymes alone, significantly enhanced
protection can often
be achieved by co-expression of chitinase and ~i-glucanase together. To date,
encouraging results
have been reported in transgenic tomato against Fusarium solani (Jongedijk et
al. (1995) Euphytica
85, 1-8) and in transgenic tobacco against Rhizoctonia solani (Jach et al.
(1995) Plant J. 8, 97-109).
Extensive field testing remains necessary, however, to determine whether these
resistance levels are
sufficient to confer commercially-significant phenotypes.
An alternative anti-fungal technology that has slowly been gaining attention
involves
overexpression of small peptides with antimicrobial activity in transgenic
plants. Some of these
peptides, isolated from non-plant sources such as frog (magainins) and moths
(cecropins), form
amphipathic a-helices upon association with microbial plasma membranes, insert
into the
membranes and interact with one another to initiate pore formation, causing
cell leakage and
eventual cell death (for review, see Boman, H.G. (1991) Cell 6S, 205-207). The
expression of
cecropins in transgenic tobacco, however, has not led to any significant
levels of disease resistance
(Florack et al. (1995) Transgenic Res. 4, 132-141). Other antimicrobial
peptides, termed defensins
(for review, see Broekaert et al. (1995) Plant Physiol. 108, 1353-1358) have
been isolated from
radish (Terras et al. (1992) ,l. Biol. Chem. 267, 15301-15309) and barley
(Mendenez et al. (1990)
Eur. J. Biochem.194, 533-539) and feature a more complex three-dimensional
structure that includes
cysteine-stabilized triple anti-parallel (3 sheets together with an a-helix.
Terras et al. (1995) Plant
Cell 7, 573-588, reported very good levels of protection against infection by
Alternaria in transgenic
tobacco that overexpressed the radish AFP2 protein. A threshold level of AFP2
peptide (which was
not easily obtained) in the transgenic plants was required to detect any
significant level of disease
resistance, however.
Other anti-fungal proteins include ribosome-inactivating proteins (RIP's),
which act by
inhibiting protein synthesis in target cells by a modification of the 28S
rRNA. RIP's do not affect
the ribosomes of the plants in which they are produced, but can be effective
against fungal
ribosomes. A barley RIP 'under control of a wound-inducible promoter was
reported to show
increased resistance to Rhizoctonia solani (Logemann et al. (1992) Biotech 10,
305-308). Finally,
Alexander et al. (1993) Proc.Natl. Acad. USA 90, 7327-7331, demonstrated that
constitutive high-
level expression of tobacco PR-la (pathogenesis-related) protein (of unknown
activity) in transgenic
tobacco resulted in increased resistance to Phytophthora parasitica and
Peronospora tabacina.
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Manipulation of the plant's own defense system -- systemic acquired resistance
(SAR) -- has
drawn tremendous attention in recent years. Activation of this complex network
of defense-related
genes gives rise to protection against a wide range of pathogens, including
bacteria, fungi, and
viruses. Attempts to utilize the entire complex of defense-related genes in
the SAR repertoire
include the two-component system, which has been described by de Wit, P.J.G.M.
(1992) Annul Rev.
Phytopathol. 30, 391-418, the application of chemical inducers of SAR like 2,6-
dichloroisonicotinic
acid (Kessmann et al. (1994) Annul Rev. Phytopathol. 32, 439-459), and the
constitutive expression
of proposed intermediates (e.g., active oxygen species like hydrogen peroxide)
in the signal
transduction pathway for SAR that effectively activates the SAR response (Wu
et al. (1995) Plant
Cell 7, 1357-1368). These approaches hold tremendous potential for conferring
broad spectrum
disease resistance, but with the exception of chemical inducers of SAR; are in
the development phase
and remain largely unproven.
Plants produce a rich diversity of secondary metabolites, which do not seem
necessary for
their basic metabolism, but appear to contribute to their environmental
fitness and adaptability.
These secondary compounds are responsible for aroma (monoterpene indole
alkaloids) and color
{anthocyanins and carotenoids) and are commercial sources of numerous
important pharmaceutical
(alkaloids) and industrial chemicals. Their importance has encouraged
intensive investigation of the
regulation and control of the biosynthetic pathways, and more recently, how
these pathways can be
manipulated through "metabolic engineering," first coined by Bailey, J.E.
(1991) Science 251, 1668-
1675, as "the improvement of cellular activities by manipulation of enzymatic,
transport, and
regulatory functions of the cell with the use of recombinant DNA technology."
The enzymology associated with the biosynthesis of alkaloids has been the
subject of much
study. The tropical plant, Catharanthus roseus (periwinkle), forms a wide
range of terpenoid indole
alkaloids (TIA's), some of which have important medicinal applications. The
leaf derived alkaloids,
vinblastine and vincristine, and the root-derived alkaloids, ajmalicine and
serpentine, are valuable
drugs for treatment of cardiac/circulatory diseases and tumors, respectively.
It is generally accepted
that protoalkaloid production is the first committed step in the TIA pathway
of Catharanthus.
Consequently, tryptophan decarboxylase (TDC), the enzyme that catalyzes this
bridge reaction
between primary (amino acid) and secondary (alkaloid) metabolic pathways, has
drawn much
attention. TDC catalyzes the decarboxylation and conversion of L-tryptophan
into tryptamine.
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Tryptamine and secologanin, another secondary compound, are then condensed to
form strictosidine,
the precursor for all TIA's in Catharanthus.
For microbes and plants alike, the rate-limiting enzyme for tryptophan
biosynthesis has been
identified to be anthranilate synthase (AS), a heterodimeric enzyme composed
of an a and (3 subunit,
which catalyzes the conversion of chorismate to anthranilate. Tryptophan has
been demonstrated
to be a negative feedback regulator of the microbial enzyme. In microbes, when
tryptophan levels
are sufficiently high, tryptophan binds to an allosteric site on the a
subunit, thereby inactivating the
enzyme. Although similar observations have not been made for the plant enzyme,
other studies
suggest that AS is the rate-limiting enzyme for tryptophan biosynthesis. Plant
mutants characterized
by elevated tryptophan levels (~3-fold) have been found to contain amino acid
changes in their
anthranilate synthase coding regions.
More recently, biochemical studies with the two purified anthranilate synthase
a subunits
(ASaI and ASa2) from Ruta graveolens, and molecular analysis of the genes that
encode these
proteins have provided some new insight into the complexities of tryptophan
biosynthesis. R.
graveolens, like C. roseus, is a medicinal plant which produces a large number
of tryptophan-derived
monoterpenoid indole alkaloids. Bohlmann et al (1996) Plant Physiol. 111, 507-
514, separately
purified the ASaI and ASa2 subunits from E. coli strains designed to
overexpress the two proteins.
They noted that while the ASa2 enzyme was completely feedback inhibited by ~10
mM tryptophan,
the activity of the ASa 1 enzyme was essentially unaffected by tryptophan
levels approaching I00
mM. They further demonstrated that ASaI (but not ASa2) transcript levels and
enzyme activity
were dramatically induced in Ruta cultures that had been exposed to fungal
elicitors. They
concluded that ASaI and ASa2 participate equally in the housekeeping role of
maintaining
appropriate tryptophan levels. However, upon exposure to fungal elicitors that
induce the TIA
pathway, the tryptophan feedback-resistant ASaI becomes responsible for
supplying the copious
amounts of tryptophan that will be required for conversion into tryptamine and
ultimately, indole
alkaloids. Thus, the tryptophan-resistant ASal becomes a specific enzyme for
secondary
metabolism.
In 1988, De Luca et al. (1988) Plant Physiol. 86, 447-450, noted that TDC
enzymatic
activity was highly regulated in germinating seedlings, supporting similar
observations made in cell
suspension cultures. Later, De Luca et al. (1989) Proc. Natl. Acad. USA 86,
2582-2586, described
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the cloning and characterization of a TDC cDNA clone from Catharanthus
seedlings. The
availability of the TDC gene as a nucleic acid probe has helped to elaborate
the developmental and
environmental cues that affect TDC expression during plant growth and in cell
suspensions. For
example, Pasquali et al. (1992) Plant Mol. Biol. 18, 1121-1131, reported that
TDC steady-state
transcript levels were most abundant in roots, moderately abundant in leaves
and barely detectable
in the flowers and stems of 3-month-old Catharanthus plants. They further
demonstrated that TDC
expression in cell suspension cultures was down-regulated by addition of
auxin, but strongly induced
by treatment with fungal elicitors. This report, along with others (Roewer et
al. ( 1992) Plant Cell
Rep. 11, 86-89; Berlin et al. (1993) Transgenic Res. 2, 336-3444; Goddijn et
al. (1992) Plant Mol.
Biol. 18, 1113-1120; and Goddijn et al. (1995) Transgenic Res. 4, 315-323),
helped establish the
importance of TDC as the first committed step in TIA biosynthesis.
Given its pivotal role in TIA biosynthesis in plants, efforts were initiated
to overexpress TDC
in transgenic plants for the purpose of increasing tryptamine levels. Songstad
et al. (1990) Plant
Physiol. 94, 1410-1413, placed the TDC cDNA under control of the strong,
constitutive cauliflower
mosaic virus (CaMV) 35S RNA promoter and introduced this transgene into
tobacco. Transgenic
tobacco lines were recovered which accumulated up to 250 times their normal
levels of tryptamine
{in excess of 1 mg/gram fresh weight [gfw]) with no apparent deleterious
effect on plant growth and
development.
In contrast to tobacco, transgenic canola, which overexpressed the same TDC
transgene,
exhibited a very striking phenotype. Chavadej et al. (1994) Proc. Natl. Acad.
USA 9I, 2166-2170,
reported that their best transgenic canola line never expressed >9% of the TDC-
specific activity and
only accumulated 2% of the tryptamine found in the most active transgenic
tobacco line. Despite
the relatively low TDC activity and tryptamine levels, the pool of available
soluble tryptophan for
tryptophan-derived indole glucosinolate production was so depleted that
glucosinolate levels were
reduced in all tissues, especially in mature seeds where glucosinolates
accumulated to only 3% of
that which were found in non-transformed seeds. Thus, TDC effectively out-
competed the indole
glucosinolate biosynthetic enzymes for available tryptophan by diverting it to
the production of
tryptamine.
In TDC-expressing potato, tryptamine accumulation was found to be very tissue
specific
(Yao et al. (1995) Plant Cell 7, 1787- 1799). While tryptamine accumulated to
high levels in the
leaves of transgenic potato plants, tryptamine was undetectable in the tubers
and was only detected
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after wounding or fungal elicitor treatment. The re-direction of tryptophan
into tryptamine resulted
in a dramatic decrease in the levels of soluble tryptophan, phenylalanine, and
phenylalanine derived
phenolic compounds, including chiorogenic acid, the major soluble phenolic
ester in potato tubers.
This 2-3-fold reduction in phenoiic esters led to reduced synthesis of
polyphenolic compounds like
lignin because of a limited supply of phenolic monomers. This in turn led to
altered cell wall
structures in the tubers, which proved to increase susceptibility of the
tubers to infection by
Phytophthora infestans.
Thomas et al. (1995) Plant Physiol. 109, 717-720, extended the observations
made by
Songstad and co-workers in TDC-expressing tobacco when they reported that
tryptamine-
accumulating tobacco plants adversely affected whitefly development and
reproduction. Bemisia
tabaci, the sweet potato whitefly, was used to test the effects of tryptamine
on insect feeding and
development. Whitefly emergence tests revealed that pupae emergence (to
adulthood) on TDC-
expressing plants was typically reduced 3 to 7-fold relative to control
plants. They speculated that
tryptamine may exert its anti-whitefly effects) during either larval and pupal
development and/or
adult selection of a leaf for feeding and oviposition.
Pasquali et al., supra, and Roewer et al. (1992) Plant Cell Rep. 11, 86-89,
noted that
enzymes in the Catharanthus TIA biosynthetic pathway, including TDC, were
coordinately induced
upon exposure to fungal elicitors. A short report by Miyagawa et al ( 1994)
Biosci. Biotech.
Biochem. 58, 1723-1724, detected the presence of fluorescent compounds
deposited at the site of
infection by powdery mildew in barley. One of these compounds, tryptamine, was
suggested to be
acting as an anti-fungal agent.
However, this collection of observations answered neither the question of
whether
tryptamine, normally only a precursor in the TIA biosynthetic pathway, could
itself contain broad-
spectrum anti-fungal activity, nor what levels could be attained in planta to
be sufficient to confer
enhanced resistance to infection by fungal pathogens. To date, the answers to
these questions have
remained elusive.
SUMMARY OF THE INVENTION
We have surprisingly discovered that tryptamine can effectively inhibit in
vitro and in vivo
growth of both phytopathogenic bacteria and fungi. Furthermore, tryptamine is
shown herein to
inhibit phytopathogenic nematodes. As a result, the present invention affords
constitutive expression
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in plants of the gene encoding tryptophan decarboxylase (TDC), the enzyme that
converts tryptophan
to tryptamine, to confer enhanced resistance to infection to a broad spectrum
of phytopathogenic
fungi, bacteria, and nematodes. Similarly the AS gene can boost this pathway,
making more
tyrptamine.
The present invention further provides a gene construct containing a promoter
and a DNA
sequence encoding a protein with tryptophan decarboxylase activity and/or AS
activity.
The invention also provides transgenic plants and multicellular plant tissue
having enhanced
fungal and/or bacterial disease resistance andlor nematode resistance, wherein
the enhanced
resistance is a result of expression of a TDC andlor AS transgene.
The foregoing merely summarizes certain aspects of the invention and are not
intended, nor
should they be construed, as limiting the invention in any manner. All
patents, patent applications,
and publications are hereby incorporated by reference in their entirety.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. Chimeric (A) E35S::TDC::nos and (B) UBQ3::TDC::nos constructs. A DNA
fragment containing the duplicated enhancer region, promoter, transcription
initiation site and 5'
untranslated region (UTR) from the CaMV 35S RNA region was fused to the 5' UTR
of the TDC
cDNA from plasmid pTDCS. A second DNA fragment containing the promoter,
transcription
initiation site and S' UTR from the Arabidopsis thaliana UBQ3 gene was fused
to the 5'UTR of the
TDC cDNA from plasmid pTDCS. A DNA fragment containing the polyA addition
signal from the
nopaline synthase gene (nos) is responsible for transcript maturation at the
3' end in both transgenes.
Figure 2. Flowers from tryptamine-accumulating petunias show enhanced
resistance to
infection by Botrytis cinerea. Newly opened flowers from greenhouse petunias
(3 flowers/line) were
detached, placed in water and then inoculated with a freshly prepared Botrytis
spore suspension (in
0.005% Tween 20 to discourage spore clumping) containing 0.25 x 103 or 1 x 103
spores/ml. After
the flowers were sprayed to the point of runoff with an atomizer, the
inoculated flowers were placed
in a humidity chamber and incubated at room temperature for 4 days. Disease
progression was
monitored daily (up to 97 h) using a disease rating scale ranging from 0 to 5
where 0 = uninfected;
1= 1-15 small, necrotic lesions; 2 =15-30 larger, necrotic lesions; 3 =
lesions begin to coalesce; 4
= lesions almost fully coalesced; and 5 collapsed flower.
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DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present inventors have surprisingly discovered (as is demonstrated in the
Examples,
infra) that enhanced TDC expression can confer on plants resistance to a broad
spectrum of both
bacterial, fungal, and nematode phytopathogens. In accordance with this
discovery, the present
invention comprises methods and nucleic acid constructs for enhancing
resistance in plants to
phytopathogenic bacteria, fungi, and nematodes by transforming plant cells
with the gene coding for
tryptophan decarboxylase (TDC). Although the invention is not limited by any
theory of action, we
believe that enhanced TDC expression results in enhanced phytopathogenic
resistance through
increased levels of tryptamine and/or alkaloids produced therefrom. The
invention further comprises
transgenic plant tissue thereby produced.
In a frst aspect, the invention provides nucleic acid constructs comprising
the TDC gene.
Any plant TDC gene can be used. Upon transformation of plant cells, these
constructs are useful
for conferring enhanced fungal, bacterial, and/or nematode resistance to a
wide variety of plants and
against a broad spectrum of phytopathogenic fungi, bacteria, and nematodes.
In another embodiment of this aspect, the invention provides nucleic acid
constructs
comprising an AS {preferably ASal) and a TDC transgene. Any plant AS gene can
be used.
Conferral of resistance to bacterial, fungal and nematode phytopathogens by
overexpression of
tryptamine (and/or alkaloids produced therefrom) can be enhanced by ensuring a
constant, sufficient
supply of its precursor, tryptophan. Published observations on the
anthranilate synthase a subunits
(ASaI and ASa2) indicate that constitutive expression of the Ruta ASal subunit
in heterologous
plants can circumvent the normal regulatory mechanisms in transformed cells
and lead to elevated
tryptophan levels. We have found that co-transformation with ASal and ASa2 led
to enhanced
levels of tryptophan and an increased amount of tryptamine. Co-introduction of
constitutively-
expressed AS {preferably ASaI) and TDC transgenes should provide increased
tryptophan levels for
immediate conversion into tryptamine. A single plasmid harboring both
constitutively-expressed
transgenes can be viewed as portable expression cassette for increasing
tryptamine levels in virtually
any plant. Alternatively, an AS transgene could be used by itself.
In plants, tryptophan biosynthesis is believed to reside exclusively in the
plastid; no cytosolic
localization is implicated. This conclusion has been solidified by the
observation that all the genes
in the tryptophan biosynthesis pathway that have been cloned to date encode
proteins that contain
a recognizable transit peptide at their N-terminus to direct the proteins to
the organelle. No gene has
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thus far been cloned which lacks this feature, thus bolstering the argument
that tryptophan
biosynthesis is exclusively plastid-localized. Therefore, expression of the
TDC gene in the plastid
will result in better substrate availability (as well as higher TDC activity
as is generally associated
with plastid gene expression), which should lead to higher levels of
tryptamine accumulation.
Accordingly, the nucleic acid constructs according to this aspect of the
invention will further
comprise targeting regions at the 3' and 5' ends, which regions target the
constructs according to the
invention to the plant nucleus or plastid.
In this aspect of the invention, any plant promoter can be operatively linked
to the TDC
and/or ASa 1 genes. In a preferred embodiment, the constructs according to
this aspect of the
invention will preferably be operatively linked to the UBQ3, UBQ10, CaMV 35S
RNA, or the
enhanced version of the CaMV 35S RNA (E35S) promoter. In another preferred
embodiment, the
constructs further comprise a pUC-based vector containing the 3' flanking
region of the nopaline
synthase gene (hos) from Agrobacterium tumefaciens. The promoters of the UBQ3
and UBQIO
genes, members of the polyubiquitin gene family in Arabidopsis thaliana, have
been described by
Norns et al., Plant Mol. Biol. 21:8995-8906 (1993).
Where more elegant control of tryptamine production within the plant is
desirable, one or
both of the transgenes can be fused and thereby operationally linked to tissue-
specific promoters.
For example, in petunia, one or both of the transgenes can be fused to petal-
specific promoters to
confer resistance to infection by Botrytis cinerea.
Constructs according to this aspect of the invention can also comprise a
selectable marker
gene, expression of which by the transformed cell enables one to identify and
isolate the transformed
cell or cells from amongst other cells. Any plant selectable marker gene known
in the art can be
used in the present invention. In a preferred embodiment, the selectable
marker gene is the nptII
(neomycin phosphotransferase II) or hph (hygromycin phosphotransferase) gene.
Cells expressing
these preferred selectable marker genes are resistant to kanamycin (for
nuclear nptIl transformation)
and hygromycin (for nuclear transformation) or glyphosate (for plastid hph
transformation) and can
be selected for by exposing cells subject to transformation (by, e.g.,
biolistic delivery) to media
containing the minimum level of kanamycin, hygromycin, or glyphosate that kill
untransformed
cells. Of course, the agent to which transformed cells are subject for
selection purposes should
correspond to the selectable marker gene employed in the transformation.
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The sequence and structure of the constituent elements of the nucleic acid
constructs
according to this aspect of the invention are publicly available, and
constructs according to this
aspect of the invention can be made by routine, art recognized techniques.
Exemplary methods are
described, e.g., in Example l, infra.
In a second aspect, the invention provides methods for enhancing the
resistance of plants to
phytopathogenic fungi, bacteria, and nematodes. In a preferred embodiment, the
method comprises
transforming plant tissue with a construct according to the first aspect of
the invention. In another
embodiment, when co-transformation with both the TDC and ASa 1 genes is
desired, the two genes
can be on separate expression vectors and co-transformed simultaneously or
sequentially.
Transformation can be accomplished in either the nucleus or the plastid, as
determined by
the targeting regions of the nucleic acid construct. Details of plastid
transformation can be found,
e.g., in co-pending international application PCT/US98/**** (WO 99/*****),
entitled, "Improved
Plastid Transformation Of Higher Plants And Production Of Transgenic Plants
With Herbicide
Resistance," filed July 23, 1998, and U.S. Application Serial No. 08/899,061,
filed July 23, 1997.
Any of the numerous methods for transformation can be used, e.g.,
Agrobacterium (for
nuclear transformation), PEG treatment, electroporation, and biolistic
delivery. Preferably, biolistic
delivery is employed.
In a third aspect, the invention provides transgenic plant tissue that
expresses the TDC gene
and/or the ASal gene. As used herein, "plant tissue" includes a plant cell or
cells, multicellular
plant tissue, and whole plants. Transgenic plants according to this aspect of
the invention can be
made according to the second aspect of the invention using nucleic acid
constructs according to the
first aspect of the invention. When cells (e.g., cell suspensions or calli) or
plant tissue samples (e.g.,
leaf or other plant parts) are transformed and selected, whole plants can be
obtained using standard,
art recognized methods of culturing and growth.
All plants are suitable for transformation with the TDC gene according to the
invention.
Agricultural and horticultural plants are of considerable importance, but many
are susceptible to
microbial infection, requiring extensive application of chemicals for disease
control. A reduction
in chemical applications because of increased plant resistance is always
desirable for worker safety
and environmental reasons.
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The present invention will now be illustrated by examples that are provided
solely for
purposes of illustration and are not intended, nor should they be construed to
be limiting in any way.
Those skilled in the art will appreciate that variations and modifications of
the following can be
made without exceeding the scope or spirit of the invention.
EXAMPLES
Example 1
Construction of TDC- and/or AS-containing constructs
To increase tryptamine production throughout the entire plant, the TDC gene
was preferably
placed under the control of two very active, constitutively expressed
promoters. The CaMV 35S
RNA promoter from the CaMV genome is a very well-characterized promoter for
expression of
transgenes in both dicotyledonous and monocotyledonous plants. The enhanced
version (Kay et al.,
(1987) Science 236, 1299-1302,) of the CaMV 35S RNA promoter (E35S) was
preferably used. A
second promoter, UBQ3, is derived from Arabidopsis thaliana and normally
directs expression of
a member of the polyubiquitin gene family. The present inventors have
determined that this
promoter directs high levels of reporter gene expression throughout the entire
plant in transgenic
petunias.
Both promoters were moved into a pUC-based vector already containing the 3'
flanking
region of the nopaline synthase gene (nos) from Agrobacterium tumefaciens.
This DNA sequence
element possesses the recognition site for polyA addition to the transcript.
Also already resident on
this plasmid was a multi-cloning site region containing a number of unique
restriction enzyme sites
for insertion of additional DNA sequence elements. The CaMV 35S and UBQ3
promoters were
moved into this plasmid (while maintaining a number of unique restriction
sites between the
promoter and the nos 3' sequence element) to create plasmids pSANl4 and
pSAN151, respectively.
The TDC gene was originally cloned from Catharanthus roseus, or vinca (De Luca
et al.,
( 1989), supra. A 1.75 kbp fragment containing the full-length cDNA was cloned
from a cDNA
library by immunodetection methods. The TDC cDNA contains an open reading
frame coding for
a protein of 500 amino acids, corresponding to a molecular mass of ~56 kDa.
The cDNA from
plasmid pTDCS (De Luca et al., 1989, supra) was removed by digestion with Pst
I and Xho 1, the
single-strand overhangs at each end removed by treatment with T4 DNA
polymerase, and cloned
into the Sma I sites of plasmids pSANl4 and pSANI S 1 to create plasmids
pSAN213 and pSAN247,
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respectively (Figure 1). This effectively fused the 5' UTR region of the TDC
gene to the S' UTR
regions of the transcripts of the gene promoters. Since these constructs were
to be introduced via
particle bombardment, there was no direct requirement to transfer them to a T-
DNA-based plant
vector like pBINl9. Also, since the TDC-containing plasrnids were to be co-
bombarded with a
second plasmid containing a plant selectable marker gene like nptll (neomycin
phosphotransferase
I 1 ) or hph (hygromycin phosphotransferase), no marker gene was added to the
TDC-containing
plasmid.
Several gene constructs containing the ASal gene alone or in combination with
the TDC
gene were constructed. Construct pSAN368 contains the ASaI gene driven by the
UBQ10 promoter
and, constructs pSAN369 and pSAN310 contained in addition to the ASa1 gene the
TDC gene
driven by the UBQ3 pormoter.
Example 2
Transformation of Petunia with TDC gene
The TDC transgenes were introduced into petunia by particle bombardment.
Briefly, two
plasmids, one containing the TDC transgene and the second containing an nptll
transgene, were co-
precipitated in equivalent amounts onto ~ 1 pm M-10 tungsten particles. The
DNA coated particles
were then bombarded into petunia leaf explants that had been placed on a
nutrient agar media. After
two successive bombardments, the leaf explants were allowed to recover on the
nutrient media for
four to five days. After recovery, the leaf explants were cut into small
pieces and placed onto
selective agar media containing kanamycin. Kanamycin-resistant transformed
shoots were
regenerated and shoots were transferred to fresh media and rooted in the
absence of kanamycin.
Small leaf samples were excised from in vitro-maintained plantlets and cell-
free extracts
prepared for HPLC analysis of tryptophan and tryptamine content. In
untransformed petunia
plantlets that were maintained under in vitro conditions, tryptamine levels
were approximately 4 ~,g
per gram fresh weight (pg/gfw) of leaf tissue. In TDC-expressing petunias,
transgenic lines
accumulated in excess of 300 mg/gfw, approaching 100-fold over endogenous
levels.
Example 3
Inhibition of fungal pathogens by arvptamine
isolates of several phytopathogenic fungi were grown on nutrient agar media
under
conditions that encouraged sporulation. Conidiospore suspensions were prepared
from Fusarium
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solani, Fusarium graminearum, Thielaviopsis basicola, and Botrytis cinerea.
For Phytophthora
parasitica zoospores were prepared from liquid culture. For Rhizoctonia solani
R2, a preparation
of mycelial fragments (avg. length of 4 cells) substituted for the spores. In
a 96-well microtiter plate,
1 x 103 spores (except where noted), either germinated or non-germinated, in
50 lcl 0.005% Tween
20 were added to 50 p.l aliquots of serially-diluted tryptamine prepared in
potato dextrose broth.
Microtiter plates were covered and incubated at room temperature with gentle
shaking for 24-48 h.
After 48 h, the lowest concentration of tryptamine which inhibited all fungal
growth was designated
the minimum inhibitory concentration (MIC ) value. In some cases, spores were
pre-incubated to
allow germination to occur before exposure to tryptamine. The results are
shown in Table 1.
Table 1
Tryptamine minimum inhibitory concentration (MIC) values for fungal
phytopathogens
Phytopathogen MIC9 (mg/ml)
Phytophthora parasitica
(vinca)
non-germinated (1 x 102) 0.25
germinated (1.5 x 102) 0.25-0.50
Phytophthora parasitica
(petunia)
non-germinated NAb
germinated (2 x 10z) 0.25-0.50
Fusarium solani
non-germinated > 1.00
germinated 0.25-0.50
Fusarium graminearum
non-germinated >0.50
germinated NA
Thielaviopsis basicola
non-germinated 0.25
germinated 0.25
Botrytis cinerea
non-germinated 0.50
germinated NA
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Phytopathogen MICa (mg/mI)
Rhizoctonia solani R2
mycelial fragments (1 x >I.00
102)
a MIC: minimum tryptanune concentration required to inhibit all fungal growth
after 48 hours.
b not assayed
As can be observed in Table 1, non-germinated spores from the vinca isolate of
Phytophthora parasitica were inhibited by 0.25 mglml tryptamine. Germinated
spores from both
Phytophthora isolates were sensitive to 0.25-0.50 mg/ml tryptamine. Similar
results were observed
for Thielaviopsis basicola (germinated and non-germinated spores). Regarding
the Fusarium
species, non-germinated spores from F. graminearum were somewhat sensitive to
tryptamine while
those from F. solani were considered insensitive. However, germinated spores
from F. solani were
sensitive. Also, growth from nongerminated spores of Botrytis cinerea were
inhibited by 0.50
I O mg/ml tryptamine. Rhizoctonia solani mycelial fragments were unaffected by
1 mg/ml tryptamine.
In general, nearly all the fungi exhibited some level of sensitivity to
treatment with tryptamine.
Example 4
Fungicidal and Fungistatic activity of tryptamine
To determine whether tryptamine's mode of action was fungicidal or
fungistatic, Botrytis
cinerea spores were exposed to toxic levels of tryptamine for various times,
then diluted to sub-lethal
concentrations to permit growth. If the spores now germinated and grew, then
tryptamine's effect
was considered to be fungistatic. If no growth was observed, the effect was
concluded to be
fungicidal.
In a 96-well microliter plate, 1 x 10~ Botrytis spores (in 50 ~.1 0.005% Tween
20) were
treated with serial dilutions of tryptamine. The experiment was set up with
three treatments. One
treatment was incubated at room temperature and the wells were scored for
growth after 48 h and
96 h. The second treatment was incubated at room temperature for 22 h, the
wells scored for growth,
then diluted 4-fold with 0.5X potato dextrose broth before continuing
incubation for another 48 h
and a final reading for growth. The third and last treatment was incubated at
room temperature for
46 h, the wells scored for growth, then diluted 4-fold with 0.5X potato
dextrose broth before
continuing incubation for another 48 h and a final reading for growth.
As can be observed in Table 2, Botrytis spores maintained in 0.50 mg/mI
tryptamine
throughout the entire experiment (growth noted at 46 and 94 h) showed
essentially no growth while
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all growth was inhibited at 1 mg/ml. Botrytis spores exposed to I mg/ml
tryptamine for 22 h showed
no growth, but when diluted to 0.25 mg/ml, germinated and grew normally over
the next 48 h.
Similarly, spores exposed to 2 mg/ml for 22 h showed no growth, but when
diluted to 0.5 mg/ml,
germinated and grew, albeit relatively poorly. These results indicated that
exposure to tryptamine
concentrations as high as 2 mg/ml for 22 h was not toxic to Botrytis. Spores
exposed to 0.5 mg/ml
tryptamine for 46 h showed little growth, but when diluted to 0.12 mg/ml, grew
normally. However,
spores exposed to I mg/ml and 2 mg/ml tryptamine showed no growth after 46 h,
and after 4-fold
dilution, either grew very poorly (1 mg/ml reduced to 0.25 mg/ml} or not at
all (2 mg/ml reduced
to 0.5 mg/ml) over the next two days. These results suggested that tryptamine
was indeed killing
the Botrytis spores. Thus, it appears as though tryptamine can act as both a
fungistatic and
fungicidal compound (at least against Botrytis), being fungicidal only when
the concentration and
exposure period are high enough and long enough, respectively.
Table 2
T'ryptamine acts primarily as a fungistatic agent
against Botrytis cinerea rather than a fungicidal one
Initial Duration Reduced Duration
Concentration (hours) Growth' Concentration (hours) Growth
(pg/mi)
0.00 46 + NA 48 +
0.12 46 + NA 48 +
0.25 46 + NA 48 +
0.50 46 +I- NA 48 +/-
1.00 46 - NA 48 -
2.00 46 - NA 48
0.00 22 + 0.00 48 +
0.12 22 + p,03 4g +
0.25 22 + 0.06 48 +
0.50 22 +/- 0.12 48 +
1.00 22 - 0.25 48 +
2.00 22 - 0.50 48 +/-
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Initial Duration Reduced Duration
Concentration (hours) Growth' Concentration (hours) Growth
(pg/ml)
0.00 46 + 0.00 48 +
0.12 46 + 0.03 48 +
0.25 46 + 0.06 48 +
0.50 46 +/- 0.12 48 +
1.00 46 - 0.25 48 +/-
2.00 I 46 I - ~ 0.50 I 48 j
where + represents normal growth; +~- represents dramatically reduced growth;
and - represents no visible growth.
b not applicable
Example 5
Inhibition of phytobacteria by tryptamine
In a series of experiments similar to those described in Example 3, a
collection of
phytopathogenic bacteria were tested for their sensitivity to tryptamine.
Cultures of Agrobacterium
tumefaciens, Erwinia amylovora, Erwinia carotovora, Pseudomonas cichorii,
Pseudomonas
syringae, and Xanthomonas campestris were grown to saturation density. After
dilution and growth
for 7-8 h, cells from log-phase cultures were treated with tryptamine. Log-
phase cells were grown
in LB broth at 28°C (ODs9o = 0.05 -0.15) and 50 ~tl of cells added to
50 ~.l of serial dilutions of
tryptamine made in LB broth. Microtiter plates were incubated overnight at
28°C. The following
day, the lowest concentration of tryptarnine which inhibited all bacterial
growth was recorded as the
minimum inhibitory concentration (MIC) value. Table 3 shows the results.
Table 3
Tryptamine minimum inhibitory concentration (MIC) values for bacterial
phytopathogens
Phytopathogen MICe (mg/ml)
Agrobacterium tumefacien0.50-1.00
Erwinia amylovora >1.00
Erwznia carotovora >1.00
Pseudomonas cichorii0.25-0.50
Pseudomonas syringae0.50-1.00
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Phytopathogen MICa (mg/mI)
Xanthomonas campestris0.25-0.50
a MIC: minimum tryptamine concentration required to inhibit all bacterial
growth after 24 hrs.
P. cichorii and X. campestris cells exhibited greatly reduced growth in the
presence of 0.25 mg/ml
tryptamine, and no growth at 0.5 mg/ml. A. tumefaciens and P. syringae cells
showed dramatically
reduced growth at 0.5 mg/ml and were completed inhibited by 1 mg/ml. In
contrast, both Erwinia
strains grew, albeit more slowly, in the presence of 1 mg/ml tryptamine and
were considered to be
relatively resistant. Overall, the results in Table 3 clearly support the
notion that tryptamine
possesses anti-bacterial properties. Taken together with the in vitro anti-
fungal results presented in
Examples 3 and 4, tryptamine exhibits impressive broad spectrum, of
antimicrobial properties.
Example 6
Tryptamine tissue distribution and tryptophan dependence in transgenic petunia
Three TDC-expressing transgene petunia lines were produced and carefully
evaluated for
their tryptamine levels and disease resistance characteristics. Petunia line
PE-261 contains the TDC
gene under control of the E35S promoter while lines PE-310 and PE-312 contain
the TDC gene
under control of the UBQ3 promoter. Tryptamine levels were measured in the
leaf tissue of both
in vitro and greenhouse-grown plants. Leaf tissue from in vitro and greenhouse
plants and petal
tissue from greenhouse plants (all ~10-20 mgs fresh weight) were excised and
homogenized in 0.2
ml cold 50 mM Na3P04, pH 7.4 buffer. Cell debris was removed by centrifugation
at 10,000 x g for
5 min at 4 °C. The cleared supernatant was moved to a fresh tube and
assayed for tryptamine
content by HPLC using a fluorescence detector (excitation at 278 nm and
emission at 360 nm).
Known amounts of authentic tryptamine (Sigma) were chromatographed to
construct a standard
curve for the determination of tryptamine levels in the experimental samples.
The results are
presented in Table 4.
Table 4
Tryptamine accumulation in TDC-expressing petunias
Tr'YPtamine,
pg/gfw'
Line ~
Leaf, Tissue Leaf, GreenhousePetal, Greenhouse
culture
V26 4 <1.4 6
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Tryptamine, pg/gfw
Li -
ne Leaf, Tissue Leaf, GreenhousePetal, Greenhouse
culture
PE-261 310 80 310
PE-310 215 58 592
PE-312 141 42 478
~g tryptamma per gram fresh weight of tissue
As can be seen, under tissue culture conditions, the tryptamine levels ranged
from 141-310 pg/gfw.
However, under greenhouse conditions, the levels decreased approximately 4-
fold for all three lines
to 42-80 ~g/gfw. It is especially worth noting that this decrease can most
likely be attributed to a
reduction in the level of available tryptophan in the cell. The levels of
tryptophan in the leaves of
the untransformed V26 control and each of the transgenic lines dropped ~4-fold
when plants were
transferred to the greenhouse. In all three transgenic lines, the ratio of
tryptamine to tryptophan
content in the cells remained unchanged. These results strongly support the
idea that tryptophan
availability may be a crucial limiting factor in the production of tryptamine
within the leaf.
As further evidence that tryptophan availability is rate-limiting for the in
planta production
of tryptamine in the transgenic petunias, the following experiment was
devised. Stem cuttings from
an untransformed V26 plant and line PE-261 were placed into tubes containing
either water or 1
mg/ml tryptophan, and then incubated at room temperature for three days to
permit uptake. After
3 days, leaf tissue samples were assayed for tryptophan and tryptamine levels.
The results are
presented in Table 5.
Table 5
Tryptamine production in TDC-expressing petunias is limited by tryptophan
availability
Line Treatment Tf'YPtophan, T~-yptamine, p,glgfw
,uglgfw
V26 Water 42 2,g
Tryptophan18,026 3.3
PE-261 Water 11 81
PE-261 Tryptophan2,785 2,263
As can be observed in Table 5, the V26 control samples in either water or
tryptophan accumulated
the same low level of tryptamine (3-4 mglgfw), even though tryptophan levels
were dramatically
higher in the tryptophan-treated sample (nearly 70-fold higher). Line PE-261
in water showed the
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expected levels of tryptophan and tryptamine (81 mg/gfw). However, when PE-261
was supplied
with exogenous tryptophan, tryptamine levels soared to 2,263 mg/gfw, a 28-fold
increase over the
water treated sample. Interestingly, the level of tryptophan in the tryptophan-
treated PE-261 line
remained 7-fold below the V26 sample treated with tryptophan suggesting that
the conversion of
tryptophan to tryptamine was extremely efficient and rapid. Taken together,
these results provided
convincing evidence that the in plants production of tryptamine by TDC is
limited by tryptophan
availability.
Example 7
Transgenic TDC-expressing Petunia resistance to powdery mildew
Greenhouse-grown petunia lines PE-261, PE-310, PE-312 and an untransformed V26
control
were inoculated on their leaf surface with a 5 ~.1 droplet containing 103
powdery mildew spores. The
third leaf {from the top) on each of five shoots of a single plant was
inoculated. Each line was
represented by four plants (for a total of 20 inoculation sites). Disease
progression was monitored
for approximately two weeks by recording the percentage of inoculated sites
infected and measuring
the diameter of the fungal colony with calipers to calculate their size
(area). The results are
displayed in Table 6.
Table 6
Line Disease incidencesDisease severityb
V26 100 100
PE-261 30 6
PE-310 36 11
PE-312 55 12
a percent infecrion of inoculated sites relative to an untransformed control
assigned a value of 100
b relative colony size (area) compared to an untransforcned control assigned a
value of 100
In all three transgenic lines, disease incidence was reduced compared to
inoculated,
untransformed controls. The incidence of disease was reduced 2-fold for PE-312
and approximately
3-fold for lines PE-261 and PE-310. The reduction in disease incidence
correlated well with the
tryptamine levels in the leaf {lowest in PE-312, 42 ~.g/gfw, and highest in PE-
261, 80 Itg/gfw).
Moreover, disease severity was also reduced compared to the untransformed
control line. For lines
PE-310 and PE-312, disease severity was reduced approximately 9-fold while
disease severity for
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PE-261 was reduced nearly 17-fold. Taken together, these data strongly support
the argument that
tryptamine-accumulating petunias show enhanced resistance to infection by
powdery mildew. It is
especially worth noting that reductions in both disease incidence and severity
correlated extremely
closely with tryptamine amounts in the leaf tissue of the three different
lines. It should also be
mentioned that the in vitro sensitivity of powdery mildew to tryptamine
remains unknown since this
pathogen is an obligate parasite and cannot be assayed by our usual method.
Example 8
Transgenic TDGexpressing Petunia resistance to Botrytis cinerea
We also evaluated whether the flower petals from the TDC-expressing petunia
Lines showed
increased resistance to infection by Botrytis cinerea. Flower petals were
measured for their
tryptamine content and shown to contain between 300 and 600 ug/gfw tryptamine.
The high levels
of tryptamine in the petals is directly attributable to two factors, a higher
concentration of available
tryptophan and promoter activity. The CAMV 35S promoter is known to be
extremely active
throughout the entire petunia flower. Moreover, we have discovered that the
UBQ3 promoter is even
more active than the 35S promoter in petunia flowers, especially in a mature
flower which is fully
opened.
To assess Botrytis resistance in the petal, newly opened flowers from
greenhouse plants were
detached and placed into tubes containing water. A Botrytis spore suspension
of 250 spores/ml was
sprayed to the point of runoff and the inoculated flowers placed into a
humidity chamber at room
temperature. The rate of disease progression was then followed for 4-5 days
and petals scored on
a scale of 0 (uninfected) to 5 (flower collapse). Within 25 h, the
untransformed control petals began
to show necrotic lesions caused by Botrytis. These lesions rapidly expanded so
that by 97 h, most
flowers had totally collapsed. In contrast, the three transgenic lines showed
only mild levels of
infection (small, isolated lesions) after the 4-day period. At higher inoculum
levels (103 spores/ml),
the V26 control and PE-261 lines showed equal susceptibility to infection by
Botrytis. However,
lines PE-3I0 and PE-312 still exhibited greatly reduced rates of infection by
Botrytis. After 97 h,
PE-310 flowers still showed only a small number of isolated lesions. Disease
symptoms were only
slightly more advanced in PE312 flowers as the lesions were more numerous and
larger.
As previously noted in the powdery mildew experiments, there was a tight
correlation
between tryptamine levels and the degree of Botrytis resistance achieved. The
strongest (PE310) and
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weakest (PE-261) Botrytis-resistant lines contained the highest (592 wg/gfw)
and lowest (310
p.g/gfw) amounts of tryptamine, respectively. Moreover, it now appears that
the in vitro studies
presented in Example 3 provided meaningful results as it was predicted that
500 p.g/ml tryptamine
would be required to inhibit Botrytis (compare to the petal values of 310-592
ug/gfw). Taken
together with the powdery mildew results, a very unifying and consistent
picture emerges that shows
that tryptamine can confer enhanced levels of disease resistance against
fungal pathogens, and that
the degree of resistance achieved is directly dependent upon the in planta
tryptamine concentration.
Example 9
Sensitivity of nematodes to tryptamine
Experiments similar to those described in Example 3 were designed to test the
effect of
tryptamine on phytopathogenic nematodes. Larvae of the Root Knot nematode
(Meloidogyne hapla)
were exposed to various concentrations of tryptamine to test their
sensitivity. A suspension of
nematodes was prepared by extracting them from soil around heavily infected
tomato plants. In a
24-well plate, 100-150 root-knot nematodes were added in a volume of 285 pl,
next 215 pI of
tryptamine solution was added to each well. The final concentrations of
tryptamine were based on
a 500 p.I volume. Microtiter plates were covered and incubated at 25 C with
gentle shaking for 48
h. At 48 h the number of mobile and immobile nematodes in each well was
counted in a counting
chamber using a dissecting microscope.
As can be observed in Table 7, tryptamine is very active against the root-knot
nematodes.
Nematodes were immobilized at 3I wg/ml, a concentration readily achieved in
transgenic plants (see
Example 6).
Table 7
Number
Tr of nematodes
tamine conc
(m
l
l
yp
. Mobile Immobile
g
m
)
Experiment 1
0 110 1
0 100 3
0.125 17 118
0.250 7 170
0.500 6 177
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Number
of nematodes
1.0 2 181
Experiment 2
0 140 3
0 139 2
0.016 100 4
0.031 6 113
0.062 7 87
0.125 I 0 145
To determine if TDC-expressing plants are resistant to nematodes, leaf disks
of transgenic
petunia lines were inoculated with the foliar nematode Aphelenchoides
fragariae. Briefly, leaf disks
(2.1 cm diameter) were cut from surface sterilized greenhouse grown leaves of
the transgenic V26
petunia lines (PE-310 and PE-312) and the non-transgenic control. Disks were
placed onto wet filter
paper in a mufti-well dish (1 disk/well) and then inoculated with a drop
containing 50 A. fragariae
nematodes that had been raised on a sterile tobacco callus culture. Inoculated
disks were incubated
for eighteen days at mom temperature and then the number of nematodes per well
was quantified.
Although, there was an increase in nematode population in each well, the two
transgenic lines (PE-
310, PE-312) showed only a slight increase (from 50 to 74 or 67 respectively)
while the non-
transgenic control increased four fold (from 50 to 194).
We have shown that two unrelated plant pathogenic nematode species are
sensitive to
tryptamine either by direct contact in solution (M. hapla) or by feeding on
transgenic petunia that
overexpresses TDC (A. fragariae). These species were selected solely on the
basis of being readily
available and should not represent a biased sample. We conclude that
tryptamine is a generally
effective nematicide. Therefore, TDC transgenic plants of any species
overexpressing the TDC
and/or AS gene should be resistant to nematodes, as long as the target tissue
has adequate amounts
of tryptamine. In the plants we have tested, tryptophan and tryptamine levels
are low in the roots.
Therefore, to protect roots from nematodes it may be necessary to use an AS
gene to boost
tryptophan and tryptaxnine levels in roots. In the case of root pathogenic
nematodes such as
Meloidogyne sp. and Heterodera sp., they induce special plant structures for
feeding. These
specialized feeding cells act as plant metabolic sinks. These specialized
feeding cells may have
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enough tryptophan for the TDC gene to produce a concentration of tryptamine
that is efficacious to
root feeding nematodes, even without the booster affect of an AS gene.
Example 10
Botrytis cinerea resistance in other transgenic plant species
Several other plant species (poinsettia cv. Angelika, geranium Designer
Scarlet, lisianthus
and another bedding plant) were transformed with the TDC gene. A measurable
increase in
tryptamine was found in transgenic poinsettia and geranium lines. Transgenic
poinsettia lines
demonstrated resistance to Botrytis cinerea in a leaf disk assay. Briefly,
twelve leaf disks (8 mm in
diameter) from each tissue culture-maintained transformed lines (and an
untransformed control) were
punched out with a cork borer and placed onto moistened Whatman 3M paper
inside a sterile plastic
bioassay dish. A freshly-prepared suspension ofBotrytis spores (103 spores in
2.5 ul) was then
pipetted onto the leaf disk surface. The humidity chamber was sealed and the
leaf disks left at 20°C
to permit disease development. For the next 3-14 days (timeline is species-
dependent), disease
progression was monitored and recorded as percentage of leaf disks infected.
Transgenic geranium lines demonstrated resistance to Botrytis cinerea
infection when
flowers were inoculated.
In at least one transgenic plant species we observed increased tryptophan and
tryptamine
concentration when the ASITDC combination was used.
23
