Note: Descriptions are shown in the official language in which they were submitted.
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METHODS OF MAKING PLANTS THAT EXHIBIT ENHANCED
DISEASE RESISTANCE
TECHNICAL FIELD
This invention relates to fungal resistance in plants, and more particularly
to
methods of making plants that exhibit enhanced resistance to fungal
infections.
BACKGROUND
Fungal diseases are responsible for damage to many cultivated species. The
amount of damage varies each year, depending on temperature, amount of rain,
and
quantity of inoculum present in fields. In some instances, fungal diseases can
completely
destroy fields, leading to an estimated average loss of yield of 20% of crops
worldwide.
Blackleg, one of the predominant fungal diseases in rape plants, typically
results in losses
of tens of millions of dollars annually. Sclerotinia, another predominant
fungal disease of
C~ucife~ae plants, which includes Brassica plants as well as 400 other species
of plants
including Compositae plants such as sunflower and leguminous plants such as
pea, also
can result in significant economic losses.
Blackleg disease is caused by an Ascomycetes fungus whose perfect or sexual
form is known as Leptosphaef°ia maculans and whose imperfect or asexual
form is known
as Phoma lingam. The sexual form provides the primary inoculum each year and
is
responsible for the high variability of the fungus. L. maculans is in fact a
complex of
species, of which two main groups have been identified, TOX+ and TOX°.
The TOX+
species is aggressive and produces two toxins, sirodesmin and phomalide.
Within the
TOX+ species, several strains or pathogenicity groups (PG) exist. In Europe,
Australia,
and Eastern Canada, PG3 and PG4 are the predominant strains of L. maculans,
while in
western Canada, PG2 is the predominant strain of L. naaculans.
Ascospores on infested canola stubble are the main source of infection by the
virulent (i.e., TOX+) L. maculans. Ascospores are released into the air and
can infect
cotyledons and younger leaves of plant seedling via stomata or wounds. The
initial
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infection is biotrophic, but then becomes necrotrophic and produces asexual
bodies
(pycnidia) in the dead tissue. The spores produced from the pycnidia bodies,
called
pycnidiospores, are believed to spread and infect other leaves of neighboring
plants after
being released by rain splash or other mechanical stress. The consequence of
fungal
invasion of canola plants is the formation of blackened canker in the stem,
hence the
name "blackleg." The blackleg disease can lead to plant death prior to
producing seeds,
and therefore has a major impact on seed yield.
Sclerotinia scle~otiorum, an Asconzycetes fungus, produces white rot on the
plants
it infects. S sclerotioYUm has broad ecological distribution, but is most
common in
temperate regions. S. scle~otio~um produces a fluffy white mass of mycelia on
the
surface of the host and on adjacent soil surfaces. Dense white bodies start
forming within
the mycelia, which becomes black and hard as the fungus matures. The hardened
mycelia
are called sclerotia, which allow the fungus to survive for several years in
soil. When
conditions are suitable, the sclerotia will germinate, and produce either
mycelium, which
1 S can infect plants that are in direct contact with the sclerotia, or spore-
producing apothecia.
Most infections in canola result from air-borne spores produced by apothecia
at the soil
surface. Spores often infect, germinate, and grow in petals and eventually
fall on other
plant tissue. The fungus eventually leads to leaf, stem, and fruit rot. In the
stem, the
fungus will form new sclerotia, which fall to the soil at harvest, thereby
completing the
disease cycle.
Strategies for limiting fungal damage include prophylactic measures such as
crop
rotations or burying of crop debris, fungicide use, and genetic improvement.
Prophylactic
measures, however, are not very effective as fungi can survive for many years
in the soil.
Fungicides can be effective when applied at the appropriate time, but cost
often is high
compared with any gain in yield. Furthermore, genetic improvement in plants
for
resistance to S. sclerotiorum has been limited since only plant species that
exhibit low
tolerance to the fungus have been identified. Thus, a need exists for plants
that are
resistant to such fungi, as well as for methods of improving plant resistance
to fungi.
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SUMMARY
The invention is based on the surprising discovery that decreasing expression
of
the gene encoding delta 12 fatty acid desaturase (fad2) or omega 6 fatty acid
desaturase
(fad6) in plants provides such plants with a significantly enhanced ability to
resist
infection by L. maculans and/or S. sclerotiorum compared with existing natural
resistance. Expression of fad2 or fad6 can be decreased such that Fad2 or Fad6
proteins,
respectively, are reduced at particular locations (e.g., the leaves), at a
particular stage of
development, or upon stimulation by the appropriate environmental conditions.
In one aspect, the invention provides a method for producing a dicotyledonous
plant. Such a method includes introducing a nucleic acid construct into cells
of a plant,
wherein the nucleic acid construct comprises a regulatory element operably
linked to a
delta-12 fatty acid desaturase (fad2) nucleic acid molecule; generating one or
more
progeny plants from the cells; and identifying at least one of the progeny
plants that
exhibits enhanced resistance to Sclerotinia sclenotiof°ufn relative to
a corresponding
control plant.
Representative plants include Brassica plants, Heliarathus annuus plants, and
Cplycirae naax plants. Methods of the invention also can be used to make
hybrid plants
(e.g., Bnassica or Helianthus annuus hybrid plants). Generally, the regulatory
element is
a promoter such as a cruciferin promoter and/or a constitutive promoter. In
some
embodiments, the fad2 nucleic acid molecule includes both fad2F and fad2D
nucleic acid
sequences.
In some embodiments, the fad2 nucleic acid molecule is operably linked to the
regulatory element in sense orientation. Such a molecule can result in co-
suppression of
fad2 nucleic acid sequences in the plant. In another embodiment, a construct
for use in
the methods of the invention can encode a self splicing ribozyme such as the
negative
strand of the satellite RNA from Barley Yellow Dwarf Virus operably linked to
the fad2
nucleic acid molecule.
In another embodiment, the fad2 nucleic acid molecule is operably linked to
the
regulatory element in antisense orientation. In an embodiment, the fad2
nucleic acid
molecule comprises fad2 nucleic acid sequences in sense orientation operably
linked to
fad2 nucleic acid sequences in antisense orientation, wherein the fad2 nucleic
acid
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sequences in sense orientation and the fad2 nucleic acid sequences in
antisense
orientation are on the same strand, wherein the fad2 nucleic acid sequences in
sense
orientation and the fad2 nucleic acid sequences in antisense orientation are
complementary. Such a construct can further include a spacer nucleic acid
sequence,
wherein the spacer nucleic acid sequences operably link the fad2 nucleic acid
sequences
in sense orientation and the fad2 nucleic acid sequences in antisense
orientation, wherein
the spacer nucleic acid sequences are between the fad2 nucleic acid sequences
in sense
orientation and the fad2 nucleic acid sequences in antisense orientation.
Typically, a plant made by a method of the invention produces seeds that
exhibit
an altered oleic acid to linoleic acid ratio relative to seeds produced by the
corresponding
control plant. It is desirable that leaves of the plant exhibit an altered
oleic acid to linoleic
acid ratio relative to leaves from the corresponding control plant.
Methods of the invention can further include producing a plant line from one
or
more of the progeny plants. Methods of the invention also can include
contacting the
progeny plants with a compound selected from the group consisting of salicylic
acid
(SA), j asmonic acid (JA), and ethylene (ET), wherein the contacting is prior
to or after the
identifying.
In some embodiments, the nucleic acid construct further comprises a regulatory
element operably linked to a nucleic acid molecule encoding a KASII
polypeptide, a
nucleic acid molecule encoding a thioesterase polypeptide, or a regulatory
element
operably linked to a nucleic acid molecule encoding an omega 6 fatty acid
desaturase
(fad6). In other embodiments, the methods of the invention further include
introducing a
second nucleic acid construct into the cells, wherein the second nucleic acid
construct
comprises a regulatory element operably linked to a nucleic acid molecule
encoding a
polypeptide selected from the group consisting of thioesterase, 3-ketoacyl
synthase II
(KASII), chitinase, (3-1,3-glucanase, PRS, and PR1.
In another aspect, the invention provides a method for producing a Brassica
plant.
Such a method includes introducing a nucleic acid construct into Brassica
cells, wherein
the nucleic acid construct comprises a regulatory element operably linked to a
delta-12
fatty acid desaturase (fad2) nucleic acid molecule; generating one or more
progeny plants
from the cells; and identifying at least one of the progeny plants that
exhibits enhanced
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resistance to Sclerotinia sclerotiorum and/or Leptosplaae~ia maculans relative
to a
corresponding control plant.
In another aspect, the invention provides a method for producing a Brassica
plant.
Such a method includes providing plant cells comprising a mutation in a fad2
andlor fad6
nucleic acid; generating one or more progeny plants from the cells; and
identifying at
least one of the progeny plants that exhibits enhanced resistance to
Sclerotinia
sclenotionum and/or Leptosphaeria maculans relative to a corresponding control
plant.
In yet another aspect, the invention provides a method for producing a
B~assica
plant. Such a method includes introducing a nucleic acid construct into
Brassica cells,
wherein the nucleic acid construct comprises a regulatory element operably
linked to an
omega 6 fatty acid desaturase (fad6) nucleic acid molecule; generating one or
more
progeny plants from the cells; and identifying at least one of the progeny
plants that
exhibits enhanced resistance to Scle~otinia scle~otiorum and/or Leptosphaeria
rnacularzs
relative to a corresponding control plant.
The invention also features a transgenic plant comprising a nucleic acid
construct.
The construct comprises a regulatory element operably linked to an omega 6
fatty acid
desaturase nucleic acid. The regulatory element confers expression in at least
one
vegetative tissue of the plant, and the plant has enhanced resistance to S.
sclerotioYUm
and/or L. maculans in at least that tissue, relative to the corresponding
resistance in that
tissue of a corresponding control plant. The plant can have enhanced
resistance to S.
scle~otio~um and the regulatory element can confer expression in leaf tissue.
The plant
can be a Bf-assica plant, e.g., a Brassica napus plant. The plant can have
enhanced
resistance to L. maculans and the regulatory element can confer expression in
stem tissue.
The invention also features a traiisgenic plant comprising a nucleic acid
construct.
The construct comprises a regulatory element operably linked to a delta-12
fatty acid
desaturase nucleic acid, and the regulatory element confers expression in at
least one
vegetative tissue of the plant. The plant has enhanced resistance to S.
scle~otiorum andlor
L. maculans in at least that tissue, relative to the corresponding resistance
in that tissue of
a corresponding control plant. The plant can have enhanced resistance to S.
sclerotiorwm
and the regulatory element can confer expression in leaf tissue. The plant can
be a
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Brassica plant, e.g., a Brassica napus plant. The plant can have enhanced
resistance to L.
maculans and the regulatory element can confer expression in stem tissue.
In still another aspect, the invention provides a method for producing a
Br~assica
plant. Such a method includes increasing the ratio of oleic acid to linoleic
acid in
Bnassica cells; generating one or more progeny plants from the cells; and
identifying at
least one of the progeny plants that exhibits enhanced resistance to
Sclef~otinia
sclerotioruna and/or Leptosphaeria maculans. The oleic acid and the linoleic
acid can be
cytosolic or chloroplastic.
In another aspect, the invention provides the use of a nucleic acid construct
comprising a regulatory element operably linked to a fad2 and/or fad6 nucleic
acid
molecule for the enhanced resistance in a plant to S. sclerotioYUm or L.
maculans. The
regulatory element can be a promoter, for example, a cruciferin promoter. In
certain
embodiments, the fad2 and/or fad6 nucleic acids are in sense orientation; in
other
embodiments, the fad2 and/or fad6 nucleic acids are in antisense orientation.
Unless otherwise defined, all technical and scientific terms used herein have
the
same meaning as commonly understood by one of ordinary skill in the art to
which this
invention belongs. Although methods and materials similar or equivalent to
those
described herein can be used to practice the invention, suitable methods and
materials are
described below. All publications, patent applications, patents, and other
references
mentioned herein are incorporated by reference in their entirety. In case of
conflict, the
present specification, including definitions, will control. In addition, the
materials,
methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the
following detailed description, and from the claims.
DESCRIPTION OF DRAWINGS
FIG 1 is a schematic of the pMB 119 construct containing fad2.
DETAILED DESCRIPTION
The invention features methods of making plants that exhibit enhanced
resistance
to fungi, in particular, to S. scler~otiorum and L. maculans. Plants and plant
lines made
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using methods of the invention exhibit an altered ratio of oleic acid (18:1)
to linoleic acid
(18:2), which can be achieved, for example, by decreasing the expression of
the enzymes
involved in the conversion of 18:1 to 18:2 such as fad2 and/or fad6.
Fad2 is also known as omega-6 fatty acid desaturase, cytoplasmic oleic
S desaturase, or oleate desaturase. Fad2 catalyzes the desaturation of
cytosolic oleic acid
(18:1) to linoleic acid (18:2). Desaturation refers to the removal of two
hydrogen atoms
from a molecule, resulting in the formation of a carbon-carbon double bond,
and
generally occurs in plastids and in the endoplasmic reticulum. Specifically,
Fad2
catalyzes the formation of a double bond between carbon positions 6 and 7
(numbered
from the methyl end), i.e., carbons that correspond to positions 12 and 13
(numbered from
the carbonyl carbon) of an 18 carbon-long fatty acyl chain. A microsomal fad2
has been
cloned and characterized (see, for example, Okuley et al., 1994, Plant Cell,
6:147-58).
The nucleotide sequences of higher plant genes encoding microsomal fad2 and
mutants
thereof are described in U.S. Patent Nos. 6,372,965 and 6,342,658.
In B~assica, two major isoforms of Fad2 have been characterized, Fad2D and
Fad2F. Brassica plants having a point mutation in fad2D (e.g., IMC129) exhibit
increased oleic acid content in the seeds, but not in the roots or leaves.
Such plants do not
show any morphological abnormalities. In addition, Br~assica plants containing
a point
mutation in both fad2D and fad2F (e.g., B. yaapus lines Q508 and SQ4275)
exhibit
increased oleic acid in the seeds and roots.
Omega-6 fatty acid desaturase (fad6) is the chloroplastic fad2. Fad6 catalyzes
the
desaturation of chloroplast 16:1 and 18:1 to 16:2 and 18:2, respectively.
Plants deficient
in Fad6 have increased levels of monounsaturated fatty acids and are deficient
in trienoic
fatty acids. Plants carrying a mutation in fad6 are characterized, at low
temperatures, by
leaf chlorosis, a reduced growth rate, and changes in chloroplast morphology
such as a
decrease in size and appressed regions of thylakoid membranes. Fad6 is thought
to be a
transmembrane protein. See, for example, Hitz et al., 1994, Plafat Physiol.,
105:635-641
and GenBank Accession Nos. AF229391, AF229392, and X78311.
In conjunction with decreasing expression of fad2 or fad6, the expression of
other
genes such as those encoding products involved in fatty acid synthesis or host
defense can
be manipulated to further enhance the observed disease resistance. Genes
encoding
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products involved in fatty acid synthesis include, for example, 3-ketoacyl
synthase II
(KASII), and thioesterase. Representative host defense genes include, but are
not limited
to, those encoding chitinase, (3-1,3-glucanase, PRS (e.g., osmotin or osmotin-
like proteins
such as AP24), and PRl . See, for example, PCT Publication No. WO 02/061043.
Additional host defense genes that can be used in the methods of the invention
include
those that encode peroxidases, ribosome inactivating proteins, protease
inhibitors,
defensins, thionins, ribonucleases, polygalacturonase inhibitor proteins,
lipid transfer
proteins, glycine-rich proteins, and extensin or hydroxyproline rich proteins.
3-ketoacyl synthase II (KASII) is involved in the biosynthesis of saturated
fatty
acids in plant chloroplasts. C2 units from aryl thioesters are linked
sequentially,
beginning with the condensation of acetyl Co-enzyme A (CoA) and malonyl-acyl
carrier
protein (malonyl-ACP) to form a C4 acyl fatty acid. This condensation reaction
is
catalyzed by a 3-ketoacyl synthase III (KASIII). The enzyme 3-ketoacyl
synthase I
(K.ASI) catalyzes the stepwise condensation of a fatty acyl moiety (C4 to C
14) with C2
groups and malonyl-ACP to produce a 3-ketoacyl-ACP product that is 2 carbons
longer
than the original substrate (C6 to C16). The last condensation reaction in the
chloroplast,
converting C16 to C18, is catalyzed by KASII. 3-ketoacyl moieties are also
referred to as
13-ketoacyl moieties. Representative KASII sequences include those shown in
GenBank
Accession Nos. AF026149 and U39441, and in Domergue & Post-Beittenmiller,
2000,
Biochern. Soc. Trans., 28:610-3.
A group of enzymes that may regulate the characteristics of plant lipids are
the
acyl-CoA thioesterases (ACHs). Thioesterases hydrolyze an ester bond in long
chain
fatty acyl-CoAs, yielding free fatty acid and CoA. Thus, thioesterases can
thus influence
intracellular levels of free fatty acids and their CoA esters. For example,
when a fatty
aryl group becomes 16 carbons long, a thioesterase enzyme hydrolyzes the fatty
acyl
group, thereby forming free palmitate (palmitoyl-ACP + H20 -~ palmitate + ACP-
SH).
The sequences of representative thioesterases can be found, for example, in
GenBank
Accession Nos. X87842 and AJ488493, as well as U.S. Patent Nos. 5,955,650 and
6,331,664.
Chitinases are endohydrolases that hydrolyze the (3-1,4 bond between the N-
acetylglucosamine residues of chitin, a component of the wall of many
pathogenic fungi.
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Chitinases have been cloned from Arabidopsis, tobacco, bean, cucumber, tomato,
and
bacteria, and the sequences deposited in GenBank (see, for example, GenBank
Accession
No. A16119 for tobacco intracellular chitinase, AB015996 for chiA of Ser~atia
marcescehs, GenBank Accession No. AB026636 for an Arabidopsis thaliana
chitinase,
and GenBank Accession No. AJ301671 for a Nicotihia sylvest~is endochitinase).
See
also, U.S. Patent 5,993,808.
(3-1,3-glucanases are a family of endohydrolases that degrade fragments of
glucan,
another component of the wall of pathogenic fungi. (3-1,3-glucanases have been
cloned
from Arabidopsis, pea, soybean, tobacco, bean, cucumber, tomato, rice, Hevea
(para
rubber), and bacteria. See GenBank Accession Nos. A16121, AB025632, AL353822,
and
D76437 for the nucleic acid and amino acid sequences of glucanases from
tobacco, A.
thaliana, Neurospora crassa, and rice, respectively See also, U.S. Patent Nos.
6,087,560
and 6,066,491.
PRS proteins are thaumatin-like proteins that are part of the osmotin family
of
proteins. Osmotins have an external surface with highly basic residues.
Osmotins are
thought to permeabilize the membrane surface of fungi, resulting in a
modification of the
pH gradient and destabilization of pressure gradients that maintain the tip of
the hyphae in
a tensed state. Consequently, cytoplasmic material is leaked and the hyphae
rupture, or in
the case of spores, the spores lyse. Osmotin or osmotin-like polypeptides have
been
cloned from tobacco, soybean, carrot, cotton, potato, and bean. GenBaiik
Accession Nos.
X65701, AL049500, and D76437 provide the nucleic acid and amino acid sequences
of
osmotin or osmotin-like proteins from tobacco, A. thaliana, and Nicotiana
sylvest~is. See
also, U.S. Patent No. 6,087,161.
Endogenous PRl proteins are highly induced during infection with pathogenic
agents. PRl proteins have been cloned from tobacco, A~abidopsis, and parsley.
See
GenBank Accession Nos. AL031394, X12572, and AI352904 for the nucleic acid and
amino acid sequences of PRl proteins from A. thaliana, parsley, and B. napus,
respectively.
Decreasing expression of fad2 and/or fad6 alone or in combination with
increasing the expression of one or more genes involved in fatty acid
synthesis, or of host
defense genes provides enhanced resistance to S'. sclerotiorum and/or L.
maculans in
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plants. For example, decreasing expression of fad2 and/or fad6 and increasing
the
expression of genes encoding PRS and chitinase can provide enhanced resistance
to
blackleg in a transgenic plant relative to a control plant in which the
expression of fad2 is
not decreased and the amount of such polypeptides is not increased. In
addition,
decreasing expression of fad2 and/or fad6 while increasing the expression of
genes
encoding chitinase, glucanase, and PRS can provide enhanced resistance to S.
sclerotiorum in a plant relative to a control plant.
Nucleic Acid Constructs
Nucleic acid constructs suitable for use in the methods of the invention
include
nucleic acids encoding fad2 or fad6 operably linked to one or more regulatory
elements
such as a promoter. In conjunction with constructs containing fad2 or fad6,
constructs
containing nucleic acids encoding proteins involved in fatty acid synthesis or
host defense
proteins can be used in the methods of the invention to enhance resistance to
S.
scleYOtiorum and/or L. maculates. Standard molecular biology techniques can be
used to
generate nucleic acid constructs.
The expression of fad2 or fad6 can be decreased using co-suppression
technology.
Co-suppression is a reduction in expression of a target gene upon introduction
into a cell
of a nucleic acid that is ultimately transcribed into an mRNA that has
homology with the
target gene's transcript. Therefore, co-suppression of fad2 or fad6 can be
achieved using
a construct that contains a fad2 or fad6 nucleic acid, respectively, operably
linked in sense
orientation to a regulatory element. It is not necessary that the fad2 or fad6
nucleic acid
in the construct be full-length or have 100% homology with the target fad2 or
fad6
nucleic acid, respectively, to be co-suppressed. See, for example, U.S. Patent
Nos.
5,034,323 and 5,231,020 for a description of co-suppression technology.
Expression of fad2 or fad6 also can be decreased using antisense technology.
The
specific hybridization of a fad2 or fad6 antisense molecule with endogenous
fad2 or fad6
nucleic acids, respectively, can interfere with the normal function of the
endogenous
nucleic acid. When the endogenous nucleic acid is DNA, antisense technology
can
disrupt replication and transcription. When the endogenous nucleic acid is
RNA,
antisense technology can disrupt, for example, translocation of the RNA to the
site of
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protein translation, translation of protein from the RNA, splicing of the RNA
to yield one
or more mRNA species, and catalytic activity of the RNA. Antisense technology
can also
facilitate nucleolytic degradation of an endogenous RNA. See, for example,
Brantl, 2002,
Biochim. Biophys. Acta, 1575:15-25 and Sazani et al., 2002, Curr. ~pin.
Botechnol.,
13:46-72. Antisense molecules can be directed at regions encompassing the
translation
initiation or termination codon of fad2 or fad6. Antisense molecules also can
be directed
at the fad2 or fad6 open reading frame (ORF), at the 5' and 3' untranslated
region of fad2
or fad6, and at intron regions and intron-exon junction regions. The
effectiveness of an
antisense molecule to decrease expression of fad2 or fad6 can be evaluated by
measuring
levels of the fad2 or fad6 mRNA or protein, respectively (e.g., by Northern
blotting, RT-
PCR, Western blotting, ELISA, or immunohistochemical staining).
The term "hybridization," as used herein with respect to antisense technology,
means hydrogen bonding, wluch can be Watson-Crick, Hoogsteen, or reversed
Hoogsteen
hydrogen bonding, between complementary nucleotides. It is understood in the
art that
the sequence of a fad2 or fad6 antisense molecule need not be 100%
complementary to
that of its endogenous fad2 or fad6 nucleic acid, respectively, to be able to
hybridize. A
fad2 or fad6 antisense molecule specifically hybridizes to an endogenous fad2
or fad6
nucleic acid, respectively, when (a) binding of the antisense molecule to the
fad2 or fad6
DNA or RNA molecule, respectively, interferes with the normal function of the
fad2 or
fad6 DNA or RNA, respectively, and (b) there is sufficient complementarity to
avoid
non-specific binding of the antisense molecule to non-fad2 or non-fad6
sequences,
respectively, under conditions in which specific binding is desired, i.e.,
under conditions
in which ira vitro assays are performed or under physiological conditions for
ih vivo
assays. In some embodiments, it may be useful to design multiple antisense
molecules
that each hybridize to a different region of fad2 or fad6. In such
embodiments, multiple
antisense molecules can be on the same construct or on different constructs.
RNA interference (RNAi) technology also can be used to decrease expression of
fad2 or fad6. RNAi technology utilizes constructs that produce aberrant RNA
transcripts,
which disrupt transcription and/or translation of the endogenous fad2 or fad6,
respectively. See, for example, U.S. Patent No. 6,506,559; and PCT Publication
Nos.
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WO 99/53050; WO 01/12824; and WO 01129058 for a description of RNAi technology
and its use in decreasing expression of an endogenous nucleic acid.
An RNAi co-suppression construct can include a promoter, a fad2 or fad6
sequence operably linked to the promoter in sense orientation, a nucleic acid
molecule
encoding a self splicing ribozyme operably linked to the fad2 or fad6
sequence,
respectively, and a terminator sequence, operably linked to the self splicing
ribozyme
nucleic acid. The nucleic acid molecule encoding the self splicing ribozyme
can be the
negative strand from the satellite RNA of Barley Yellow Dwarf Virus, for
example.
Alternatively, an RNAi hairpin construct can contain a promoter, a region of
the fad2 or
fad6 sequence operably linked to the promoter in sense orientation, a spacer
nucleic acid
molecule operably linked to the sense fad2 or fad6 sequence, respectively, the
complement of the same region of the fad2 or fad6 sequence, respectively,
operably
linked to the spacer nucleic acid in antisense orientation, and a terminator
sequence
operably linked to the antisense fad2 or fad6 sequence, respectively.
Following
transcription of an RNAi hairpin construct, a double-stranded duplex RNA
(e.g., a
hairpin) is formed between the fad2 or fad6 complementary regions.
The expression of genes that produce proteins involved in fatty acid synthesis
(e.g., KASII, or thioesterases) or host defense (e.g., chitinase, (3-1,3-
glucanase, PRS,
and/or PRl) can be manipulated to further enhance a plant's resistance to
pathogens. For
example, an exogenous nucleic acid encoding a host defense protein can be
operably
linked to a regulatory element and introduced into a plant using transgenic
methods such
as those described herein. The exogenous nucleic acid can be expressed to
produce an
amount of a host defense protein that is in addition to that produced by the
endogenous
nucleic acids. Further by way of example, an exogenous nucleic acid encoding a
protein
involved in fatty acid synthesis operably linked to a regulatory element can
be introduced
into a plant. Expression in plant tissue of such an exogenous nucleic acid can
result in
altered levels of particular fatty acids, thereby enhancing disease
resistance. Altered
levels of fatty acids in the plant tissue can be due to, for example, an
increase in the
amount of such protein (e.g., expression of an additional copy of a nucleic
acid) or a
decrease in the amount of such protein (e.g., by co-suppression of the
exogenous nucleic
acid).
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Regulatory elements typically do not themselves code for a gene product.
Instead,
regulatory elements affect the expression level of the coding sequence.
Suitable
regulatory elements include promoters and enhancers. Regulatory elements can
be
constitutive or inducible, and can be tissue specific (e.g., roots, seeds,
veins, or the like)
or developmental stage specific (e.g., Brassica developmental stages 1, 2, 3,
4, or 5). As
used herein, "constitutive promoter" refers to a promoter that facilitates
expression of a
nucleic acid molecule without significant tissue- or temporal-specificity. An
inducible
promoter refers to a promoter that facilitates expression of a nucleic acid
molecule in
response to a stimulus such as a chemical, or light. Suitable promoters are
known (e.g.,
Weising et al., Anh. Reu Genetics 22:421-478 (1988)). The following are
representative
examples of promoters suitable for use herein: regulatory sequences from fatty
acid
desaturase genes (e.g., Brassica fad2D or fad2F, see PCT Publication No. WO
00/07430);
alcohol dehydrogenase promoter from corn; light inducible promoters such as
the ribulose
bisphosphate carboxylase (Rubisco) small subunit gene promoters from a variety
of
species; major chlorophyll a/b binding protein gene promoters; the 19S or 35S
promoters
of cauliflower mosaic virus (CaMV); hsr203j promoter from tobacco (Ponder et
al., 1994,
PZafZt J., 5:507-21); as well as synthetic or other natural promoters that are
either
inducible or constitutive. See, e.g., U.S. Patent No. 6,087,560. Particularly
useful are
regulatory elements that facilitate expression in vegetative tissues) but
result in little or
no expression in seeds. Such regulatory elements can be operative in
vegetative tissues
such as leaves, both immature and mature, stems, or both leaves and stems.
Such
regulatory elements can confer constitutive expression in a vegetative
tissues) or
inducible expression in a vegetative tissue(s). An example of a vegetative
tissue-specific
regulatory element is the FD sold promoter from Ag~obacterium.
In some embodiments, the regulatory element is a promoter of plastid gene
expression. Non-limiting examples of such a promoter include a 16S ribosomal
RNA
promoter, promoters of the photosynthetic genes rbcL and psbA, as well as the
light
regulated promoter of the psbD operon. See, U.S. Patent No. 5,877,402 for
constructs
suitable for stable transformation of plastids.
In other embodiments, regulatory sequences are seed-specific, i.e., the
particular
gene product is preferentially expressed in developing seeds and expressed at
low levels
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or not at all in the remaining tissues of the plant. Non-limiting examples of
seed-specific
promoters include napin, phaseolin, oleosin, and cruciferin promoters. Further
examples
of suitable regulatory sequences for expression of fad2, fad6, or host defense
genes are
known in the art.
Promoters that result in expression of proteins in different tissues (e.g.,
stems vs.
leaves) may be especially useful in different geographical regions. Sub-
strains of L.
maculans having different degrees of virulence may be present in different
regions of the
world, e.g., PG3 strains in Europe and Australia may have different degrees of
virulence
compared to strains in other geographic regions. In addition, the plant tissue
in which
fungal disease is most commonly observed may vary between different geographic
regions. Thus, promoters can be used that result in expression in those
tissues.
Additional regulatory elements may be useful in the nucleic acid constructs of
the
present invention, including, but not limited to, polyadenylation sequences,
translation
control sequences (e.g., a ribosome binding site), enhancers, introns, and
targeting
sequences (i.e., a sequence targeting to a particular organelle, such as a
plastid). Such
additional regulatory elements may not be necessary for sufficient expression
of fad2 or
fad6 nucleic acids, although they may increase expression by affecting
transcription,
stability of the mRNA, translational efficiency, or the like. Such elements
can be
included in a nucleic acid construct as desired to obtain optimal expression
of fad2 or
fad6 nucleic acids, respectively, in the host cell(s).
An example of a nucleic acid construct encoding fad2 in sense orientation is
shown in FIG 1. The 35S double enhancer promoter (35Sde) was cloned 5' of the
fad2
sequences, and a Nos terminator was cloned into the construct 3' of the fad2
sequence.
One or more additional constructs (e.g., co-suppression, antisense, or RNAi)
can be
generated that contain fad6 nucleic acids, or that contain genes encoding
proteins
involved in fatty acid synthesis or host defense. Nucleic acids encoding such
proteins can
be together on a single construct or each can be on a separate construct.
Decreasing expression of fad2 and/or fad6
Methods of enhancing disease resistance in a plant can be accomplished by
decreasing expression of fad2 and/or fad6. In the context of the present
invention,
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"decreasing expression" means a decrease in the transcription or translation
of a fad2 or
fad6 gene. Expression of fad2 or fad6 can be decreased by mutagenesis, by
transgenics,
by combinations thereof, or by using existing germplasm.
Muta~enesis
S Expression of fad2 and/or fad6 also can be decreased by mutagenesis.
Mutagenesis is the introduction of mutations into nucleic acids, either ih.
vitro or in vivo.
Mutations can be induced in living organisms or in cultured cells by a variety
of
mutagens, including ionizing radiation, ultraviolet radiation, or chemical
mutagens, by
infection with certain viruses which integrate into the host genome, or by the
introduction
of nucleic acids previously mutagenized in vitro. For example, benzo[a]pyrene,
N
acetoxy-2-acetylaminofluorene and aflatoxin B 1 cause GC to TA transversions
in bacteria
and mammalian cells, although benzo[a]pyrene in particular can produce base
substitutions such as AT to TA. N nitroso compounds cause GC to AT
transitions, while
alkylation of the 04 position of thymine results in TA to CG transitions.
1 S Plant cells from Brassica tissue can be mutagenized, for example, by
exposing the
cells to a chemical mutagen, and plants can be regenerated from such cells.
The plant
cells or the regenerated plant can be screened for fatty acid content,
particularly for the
levels of oleic acid and linoleic acid, to identify fad2 or fad6 mutants.
Alternatively, a
mutation can be introduced into a fad2 or fad6 nucleic acid molecule and
introduced
transgenically into a plant.
Trans~enic cells and plants
Transgenic cells and plants in which expression of fad2 or fad6 has been
decreased can be obtained by introducing an appropriate nucleic acid construct
(e.g., a co-
suppression construct, an antisense construct, or an RNAi construct as
described above)
into one or more plant cells and regenerating a plant. A nucleic acid
construct can be
introduced into a plant cell by well-known transformation methods.
Leaves, seeds, or other tissue can be analyzed to identify those plants
containing
the construct or having the desired level of expression of the construct. For
example, the
polymerase chain reaction (PCR) and/or Southern blotting can be used to
determine if
seeds or other tissue contains the nucleic acid construct. Northern and/or
Western blots
can be used to examine expression of the construct.
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Plants used in the methods of the invention can be any dicotyledonous species,
including, for example, Crucifef°ae plants such as Brassica spp. (both
low erucic and high
erucic acid rapeseed). Suitable Brassica species include B. napus, B. juncea,
B. nigra, B.
carinata, B. oleracea, and B. rape. The Brassica variety designated Westar
transformed
with wild type fad2 (Westar/fad2) also can be used in the methods of the
invention. Other
suitable species includes sunflower (Heliantlaus annuus), soybean (Glycine
max), castor
bean (Ricinus communis), peanut (Arachis hypogaea), tomato (Lycopersicoya
esculentuna),
and flax (Linum usitatissimum). In some species, plant lines that exhibit some
degree of
natural disease resistance can be used. In such lines, resistance to blackleg
and/or
Sclerotinia can be improved by decreasing expression of fad2 and/or fad6.
Transformation techniques for use in the invention include, without
limitation,
Agrobacterium-mediated transformation, polyethylene glycol treatment of
protoplasts,
electroporation, and particle gun transformation. Illustrative examples of
transformation
techniques are described in PCT Publication No. WO 99/43202 and U.S. Patent
No.
5,204,253 (particle.gun), and U.S. Patent No. 5,188,958 (Agrobacterium).
Transformation methods utilizing the Ti and Ri plasmids of Agrobacterium spp.
typically
use binary type vectors. Walkerpeach et al., in Plant Molecular Biology
Manual, Gelvin
& Schilperoort, eds., Kluwer Dordrecht, C1:1-19 (1994). If cell or tissue
cultures are
used as the recipient tissue for transformation, plants can be regenerated
from
transformed cultures by techniques known to those skilled in the art. In
addition, various
plant species can be transformed using the pollen tube pathway technique.
A plant described herein may be used as a parent to develop a plant line, or
may
itself be a member of a plant line, i. e., it is one of a group of plants that
display little or no
genetic variation between individuals for a particular trait. Such lines can
be created by
several generations of self pollination and selection for those species
amenable to self
pollination. Vegetative propagation from a single parent using tissue or cell
culture
techniques also can be used. In some embodiments, cytoplasmic male sterility
breeding
systems are used to develop homozygous lines and also can be used to develop
hybrids.
Methods for producing hybrids can be found, for example, in U.S. Patent No.
6,323,392,
and PCT Publication Nos. WO 92/05251 and WO 98/027806. In addition, Ficlc
(1978,
Agronomy, 19:279-337) and Vranceanu & Stoenescu (1971, EuplZytica, 20:536-41)
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describe protocols for hybrid production in sunflower. Additional breeding
techniques to
create plant lines are known in the art.
Transgenic plants can be entered into a breeding program, e.g., to increase
seed, to
introgress the novel constructs) into other lines or species, or for further
selection of
other desirable traits. Desirable traits also can be fixed using di-haploid
methods for
screening and selecting embryos. Additional transgenic plants can be obtained
by
vegetative propagation of a single transgenic plant, for those species
amenable to such
techniques.
Progeny of transgenic plants are included within the scope of the invention,
provided that such progeny exhibit resistance to S. scle~otio~um and/or L.
maculans.
Progeny of an instant plant include, for example, seeds formed on F1, F2, F3,
and
subsequent generation plants, or seeds formed on BC1, BC2, BC3, and subsequent
generation plants. Seeds produced by a transgenic plant can be planted and the
resulting
plants can be bred or otherwise propagated (e.g., selfed) to obtain plants
homozygous for
the construct. Di-haploid plants exhibiting decreased expression of fad2 also
are within
the scope of the invention, and can be generated by methods known in the art.
In some embodiments, a ~transgenic plant expressing fad2 or fad6 from a first
nucleic acid construct can be crossed or mated with a second transgenic plant
expressing
a gene involved in fatty acid synthesis or host defense from a second nucleic
acid
construct to obtain plants having the desired combination of polypeptides and,
thus,
exhibiting the desired level of disease resistance.
Analyzing Plants
Plants that have enhanced resistance to S. scle~otiof~um andlor L. maculans
can be
identified by known techniques, including in vitf~o tests on plant tissue or
field, growth
chamber, or greenhouse tests. For S. sclenotionum, tests for resistance can be
performed
by inoculating stems or detached leaves of plants with S. sclerotiorum
mycelium, then
evaluating any resulting necrosis (e.g., measuring length of necrosis) after a
period of
time sufficient for infection to develop. Blaclcleg resistance can be
evaluated, for
example, by inoculating cotyledons or stems with L. maculans and measuring the
length
of necrosis or mean disease severity (MDS) after a period of time sufficient
for infection
to develop. MDS can be measured on a scale from 0 to 5, with 5 being the
worst.
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Susceptible lines and highly resistant lines are used to obtain control
values. For such
inoculations and evaluations, it is desirable to use a substrain of L.
fnaculans that is
prevalent in the geographic region in which the resulting plants are to be
utilized.
In some embodiments, plants that have enhanced fungal resistance can be
identified by an increased amount of oleic acid in leaf tissue from such
plants. In the case
of Brassica plants, the amount of oleic acid can constitute from about 10% to
about 25%
of the total fatty acid content of the leaf tissue, e.g., about 10% to about
20%, about 11
to about 20%, about 12% to about 20%, about 13% to about 20%, about 14% to
about
20%, about 12% to about 18%, or about 15% to about 18%.
In some embodiments, plants that have enhanced fungal resistance can be
identified by an increased ratio of oleic acid/linoleic acid in leaf tissue
from such plants.
In the case, of Brassica plants, the ratio of oleic acid/linoleic acid can be
from about 1.1
to about 5.0, e.g., about 1.1 to about 4.0, about 1.1 to about 4.0, about 1.2
to about 4.0,
about 1.3 to about 4.0, about 1.4 to about 3.0, or about 1.5 to about 2.5.
In some embodiments, plants that have enhanced fungal resistance can be
identified by an increased amount of oleic acid in stem tissue from such
plants. In the
case of Brassica plants, the amount of oleic acid can constitute from about
20% to about
35% of the total fatty acid content of the stem tissue, e.g., about 20% to
about 30%, about
21% to about 35%, about 22% to about 30%, about 23% to about 30%, about 24% to
about 30%, about 22% to about 25%, or about 15% to about 25%.
A phenotype is considered enhanced when the phenotype of a particular cell,
plant, plant line, or plant population is statistically significantly
different from a
corresponding cell, plant, plant line, or plant population that is a suitable
control, at ap <_
0.05 with an appropriate parametric or non-parametric statistic. A suitable
parametric or
non-parametric statistic includes a Chi-square test, a Student's t-test, a
Mann-Whitney
test, or an F-test. In some embodiments, a difference in phenotype is
statistically
significant at p < 0.01, p < 0.005, or p < 0.001.
In some embodiments, plants that have enhanced fungal resistance can be
identified by in vitro analysis of protein expression levels, evaluation of in
vitro resistance
in plant tissue, and/or in vivo resistance of whole plants in the field.
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Additional Resistance Factors
In addition to the methods described above for decreasing expression of fad2
and/or fad6 using transgenic techniques or mutagenesis, expression of one or
more host
defense genes (exogenous or endogenous) can be induced by compounds such as
salicylic
acid, jasmonic acid, and ethylene to further enhance resistance. Salicylic
acid, jasmonic
acid, and ethylene are endogenous signal molecules that have been implicated
in plant
defense signaling pathways against pathogen infection.
Salicylic acid levels increase locally and systematically in plant tissue
following
pathogen infection, and exogenous application of salicylic acid results in
enhanced
resistance to a broad range of pathogens. Studies have shown that salicylic
acid is
required for the rapid localized activation of the expression of defense
response genes to
produce, for example, PR proteins. Salicylic acid also induced systemic
expression of
defense response genes to produce, for example, PR proteins, which results in
systemic
acquired resistance. An extensive review of the role of salicylic acid in
plant disease
resistance can be found in Dempsey et al. (1999, C~it. Rev Plant Sci., 18:547-
75).
Jasmonic acid, a fatty-acid-derived signaling molecule, is involved in several
aspects of
plant biology including pollen and seed development, and defense against
wounding,
ozone, insect pests and microbial pathogens. Jasmonic acid and/or ethylene can
induce
the production of several PR proteins, including Plant Defensinl.2 (PDF1.2),
Thionin2.1
(THI2.1), Hevein-Like proteins (HEL) and ChitinaseB (CHIB). Experiments
indicated
that nearly half of the genes induced by ethylene were also induced by
jasmonic acid, but
further indicated that jasmonic acid and ethylene were able to independently
regulate
separate sets of genes.
Therefore, plants in which the expression of fad2 and/or fad6 is decreased can
be
treated with one or more of the above-indicated compounds to increase
expression of
genes involved in host defense, thereby further enhancing the resistance to S.
sclerotio~um
and/or L. maculans. In addition, nucleic acids encoding enzymes involved in
the
biosynthesis of such signaling molecules can be introduced into plants using
the methods
and materials described above to increase the amount of salicylic acid in the
plant and
thereby increase expression of genes involved in host defense. See, for
example,
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Verberne et al., 2000, Nat. Biotech., 18:779-83, and Yalpani et al., 2001,
Tlae Plant Cell,
13:1401-9.
The invention will be further described in the following examples, which do
not
limit the scope of the invention described in the claims.
EXAMPLES
Example 1- Materials and Methods
Plant Lines
Westar variety is wild type for both fad2D and fad2F. The IMC129 variety
contains a point mutation in fad2D. The Q508 and SQ4275 lines were derived
from
IMC 129 (i. e., both lines contain the point mutation in fad2D) and each
contains a
different point mutation in fad2F. Q508/fad2 is the Q508 line transformed with
wild type
fad2D under control of the 35S double enhancer promoter (35Sde) (see, for
example, Li
et al., 2001, Plant Sci., 160:877-87 and references therein). The expression
of wild type
fad2D in Q508 plants likely complements both the fad2D and fad2F mutations.
Eyaluation of S. sclerotior~um (Scle~~otinia) Resistance
To test Westar (a susceptible control), IMC129, Q508, SQ4275, and Q508/fad2
lines for Sclerotinia resistance irz vitro, fully expanded young leaves having
a length of
about 10 cm were removed from plants grown in a greenhouse environment and
deposited in a round Petri dish (14 cm diameter) containing agar (7.5 g of
technical grade
agar per liter, 75 ml per dish). AS. scleYOtioYUm mycelial implant (7 mm in
diameter)
was deposited at the tip of the leaf and the dishes were kept at 16°C
in an air-conditioned
room. Humidity was maintained by wrapping the plates in plastic bags. The
length of
necrosis (in cm) along the midrib of the tested leaf was measured four days
later and
compared to the length of necrosis on a control (Westar) leaf. Mycelial
implants were
prepared by growing the fungus on agar-containing water for four days then sub-
culturing
onto potato dextrose agar (PDA) medium for three days. For every line, at
least 4 sets of
5 plants were evaluated using one leaf from each plant (Table II).
Evaluation of Blackle~ Resistance
The Williams test (phoma test on cotyledons) was performed on the Westar (a
susceptible control), IMC129, Q508, SQ4275, Q508/fad2, and Dunkeld (a spring
B.
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napus line that is resistant to the PG3 strain of L. maculans) lines, to
evaluate blackleg
resistance. See Williams & Delwiche, 1979 Cruciferae Conference, Euca~pia,
1980, page
164. Nine plants per line were evaluated for blackleg resistance. The rape
plants were
sown in a flat containing a greenhouse soil mixture and maintained at
25°C until the test
was ready to be performed. The plants were watered and fertilized. Young
leaves were
systematically removed up to the day of the inoculation, which was 10 days
after sowing.
A small hole was made in each cotyledon lobe before inoculation and 10 ~,1 of
a
suspension of pycnidiospores (500,000 spores/ml) were deposited in each hole.
The flats
were placed in cloches for two days. Necrosis diameter was measured two weeks
later.
The blackleg test on stem was performed on 1 month-old plants. A hole was
created in the stem at about 8 cm from the bottom of the plant. Ten ~,l of a
suspension of
pregerminated pycnidiospores (500,000 spores/ml) were deposited in each hole.
Pycnidiospores were obtained by growing the L. maculans PG2 strain on V8
medium
under near-ultraviolet light, recovering the pycnidiospores in sterile water,
and filtering to
remove impurities. Pregerminated pycnidiospores were made by adding glucose to
the
spore suspension at a final concentration of 10 mM. The spores were then pre-
germinated
for 3 days at room temperature. The wound was then covered by an adhesive
bandage
onto which 15.1 of the spore suspension had been applied. The plants were kept
at least
80% humidity for at least the first 4 days after inoculation. The scoring was
performed
one month after inoculation by measuring the length of necrosis on the stem.
Statistical Analysis
The Spectral Analysis Software program (Release 8.02, SAS Institute, Inc.,
Cary,
NC) was used to perform statistical analysis using ANOVA with Duncan grouping
at
95% confidence.
Preparation of Fatty Acid Methyl Esters and Analysis by Capillary GLC
Plant tissue was placed into a 15 ml polypropylene centrifuge tube, dried
overnight at 135°C, minced by shaking with beads, treated with 0.6 ml
of methanol/KOH
solution, mixed on a vortex mixer for 30 sec, and then incubated in a water
bath at 60°C
for 60 seconds. About 4.0 ml of saturated NaCl was added to the tube followed
by 1.0 ml
of iso-octane, mixed on a vortex mixer for 30 sec, and then centrifuged for 5
minutes to
separate and purify the organic layer. Approximately 700 p,1 of the organic
layer
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containing the fatty acid methyl esters was removed from the tube and placed
into a GC
auto-sampler vial. The vial was purged with nitrogen gas to remove the oxygen
and
preserve the sample.
Fatty acid esters were analyzed using American Oil Chemists' Society (AOCS)
Method Ce 1e-91. One ~,1 of each sample was injected into a Hewlett Packard
6890 gas
chromatograph by means of an auto-sampler. A normalized percentage was
calculated
and reported for each fatty acid in the sample.
GC conditions were as follows: Column: 5 m x 0.32 mm DB-Wax (0.5 pm film
thickness); Detector: FID; Inlet temperature: 250°C; Detector
temperature: 250°C; Split
ratio: 100:1; Carrier gas: helium at 30.0 mllmin; and Oven program: 1.0 min at
220°C;
10.0°C/min to 245°C; 3.0 min at 245°C.
pMB 119 Construct
A full-length oleoyl-fatty acid desaturase isoform "D" (fad2D) was amplified
from BYassica raapus genomic DNA (Westar variety) by PCR using primers Fad2D-
ExpS'
having the sequence: 5'-cau cau cau cau aaa aaa aac aac cat ggg tgc agg tgg
aag aat-3'
(SEQ ID NO:1) and Fad2D-Exp3' having the sequence: 5'-acu acu acu acu gtc gac
ata
gaa gag aaa ggt tca g-3' (SEQ ID N0:2). The amplified DNA was ligated into the
pAMPl vector (GIBCO). Subsequently, both strands of the fad2D gene was
sequenced
to confirm fidelity, and then released with SaII digestion. The SaII fragment
containing
the fad2D gene was cloned 3' to the 35Sde promoter at the SaII site in the
p1079 vector.
Next, the DNA region containing the 35Sde-fad2D-Nos terminator cassette was
released
from p1079 by HincdIII and subcloned at the HifadIII site in pBIl21 (Clontech,
Palo Alto,
CA), thereby generating pMB 119. Figure 1 shows a schematic of pMB 119.
Complementation of fad2 Mutations in Q508 Canola Line
The Q508 line contains a point mutation in the fad2D gene and another
different
point mutation in the fad2F gene. This line was transformed with the pMB 119
binary
construct using the Agr~obacteriufn-mediated transformation method. Transgenic
plants
were evaluated for their fatty acid composition (Table I). Results confirmed
that the wild
type fad2D gene was able to complement the fad2 mutation in Q508.
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Table I. Leaf Fatty Acid Composition of Canola Lines
Canola LinesLeaf
Fatty
Acid
Composition
(%)
C16:0 C16:1 C18:0 C18:1C18:2 C18:3
Westar 13.46 0.41 2.81 1.49 13.25 67.56
IMC129 12.54 0.31 1.75 1.39 13.11 70.28
Q508 10.82 0.52 3.42 16.736.77 59.69
SQ4275 11.78 0.44 1.24 11.486.88 67.14
Q508/fad2 12.8 0.46 4.43 3.81 15.67 59.44
Example 2 - Non-Trans~enic Approach To Enhancing Resistance To ScleYOtinia in
Bnassica
IMC129 plants were similar to Westar plants in their resistance to S.
scle~otioruna
infection based on the detached leaf assay (Table II, Table III). Q508 and
SQ4275 lines
displayed enhanced resistance to S. sclerotio~um infection in detached leaves
(Table II,
Table III).
To determine whether the mutations in fad2D and fad2F genes are directly
responsible for the enhanced plant resistance to S. sclerotiorum, the
resistance of
Q508/fad2 to Sclenotinia infection was examined. Q508/fad2 plants show normal
root
growth at low temperatures, and the fatty acid compositions of their seeds,
roots, and
leaves are similar to that of the Westar variety. Q508/fad2 plants, similar to
Westar
variety, did not display enhanced resistance to S. scle~otiorum infection in
leaves (Table
II, Table III). Therefore, the fad2 mutations in Q508 plant are directly
responsible for the
enhanced resistance to S. scleYOtioYUna infection in leaves.
fad2 mutations directly influence the cytosolic levels of 18:1, 18:2, and 18:3
fatty
acids in plant tissues. Thus, the observed plant resistance to S.
sclerotioruna in leaves is
linked to the altered levels of these fatty acids. The most significant change
is in the 18:1
and 18:2 fatty acid levels and their ratio. See Table IV. The high levels of
18:1 fatty acid
and the low levels of 18:2 fatty acid in Q508 and SQ4275 canola lines
correlate with
enhanced disease resistance to S. sclerotio~um infection in leaves (Table II,
Table III).
Conversely, the low levels of 18:1 fatty acid and high levels of 18:2 fatty
acid in 1MC129
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and Q508/fad2 correlate with a level of S. scle~otio~um resistance in leaves
that is similar
to the resistance exhibited by the Westar variety (Table II, Table III).
Table II. Plant Leaf Resistance to Scle~otinia Infection
Canola LinesLesion Length
Relative to
Westar
Westar 100
IMC129 91
Q508 76
SQ4275 75
Q508/fad2 ~ 9~
Table III. Statistical Analysis of Plant Leaf Resistance to S. Sclerotiorum
Infection
Using ANOVA With Duncan Grouping at 95% Confidence
Duncan GroupingsLesion Length LeafCanola
Relative to Westar# Lines
A 100 20 Westar
B 76 24 Q508
B 72 23 SQ4275
aDuncan, 1955, Biometrics, 11:1-42
Table IV. The level of 18:1 and 18:2 fatty acid and their ratio
18:1 18:2 18:1/18:2
Westar 1.5% 13% 0.12
IMC129 1.4% 13% 0.11
Q508 17% 7% 2.47
SQ4275 11.5% 7% 1.67
Q508/fad2D3.8% 16% 0.24
Based on these observations, it is possible to enhance the disease resistance
of
other plant species to necrotrophic pathogen infection, by altering their 18:1
and 18:2
fatty acids level. For example, sunflower leaves are low in oleic acid (~ 3%)
and linoleic
acid (~16%). Therefore, mutagenesis or transformation of sunflower such that
the
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expression of fad2 is decreased in leaf tissue can enhance the resistance of
sunflower
plants to Sclerotihia infections.
Example 3 - Trans eg nic Approach To Enhancing Resistance To Sclef otihia in
Brassica
Canola tissue (Westar variety) was transformed with a construct containing
fad2
in sense orientation under control of the Cruciferin promoter, a Brassica
napus seed
specific promoter, and regenerated into plants (Westar/fad2). Seeds from
Westar/fad2
exhibited co-suppressed fad2 expression as evidenced by the following. The
18:1 and
18:2 fatty acid level in the seeds from Westar/fad2 plants was 85% and 2% of
total fatty
acid content, respectively. Interestingly, when compared to Westar variety,
Westar/fad2
plants also showed an increase in 18:1 content from about 1.5% in Westar
leaves to about
15% in Westar/fad2 leaves, and a decrease in 18:2 content from about 13% in
Westar
leaves to about 8% in Westar/fad2 leaves (Table V). Westar/fad2 plants also
exhibited
enhanced resistance to S. scle~otiorum similar to that exhibited by the Q508
line (Table II,
Table VI).
Table V. Leaf Fatty Acid Composition of Canola Lines
Canola Lines Leaf
Fatty
Acid
Composition
C16:0C16:1C18:0C18:1C18:2C18:3
Westar/fad2 11.170.53 4.01 14.928.21 58.89
Westar 13.460.41 2.81 1.49 13.2567.56
Table VI. Leaf Resistance to S. Sclerotior-um Infection
Canola Lines Lesion Length
Relative to Westar
Westar 100
Westar/fad2 79
Q508 76
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Example 4 - Non-Transgenic Approach To Enhancing Resistance To Blackle~ in
Brassiea
Example 2 describes the creation of several canola lines with mutations in
fad2
genes, and their fatty acid composition in leaves and seeds. These lines were
also tested
for their resistance to L. maculans pathogen, the causal agent of Blackleg
disease using
the cotyledon assay. Plants with enhanced resistance to S. sclerotiorum
infection (Table
II) were also slightly more resistant to L. maculans infection (Table VII).
Table VII. Cotyledons Resistance to L. maculates Infection
Canola Lines Severity of Lesions
Westar 4
IMC 129 4
Q508 3
S Q4275 3
s1 = 1 mm lesion, 2 = 3 mm lesion, 3 = 6 mm
lesion, and 4 = 10 mm lesion
Example 5 - Trans eg nic Approach To Enhancing Resistance To Blackle~ in
Brassica
Example 3 describes the development of Westar/fad2 transgenic plants.
Westar/fad2 plants also were tested for resistance to L. macularas, the causal
agent of
blackleg, using the stem assay. The 18:1 and 18:2 fatty acid levels in
Westar/fad2 seeds
was 85% and 2% of total fatty acid content, respectively. Compared to the
Westar variety,
Westar/fad2 plants showed an increase in 18:1 content of about 6.3% in Westar
stems to
about 26.4% in Westar/fad2 stems, and a decrease in 18:2 content from about
17.5% in
Westar stems to about 7.6% in Westar/fad2 stems (Table VIII).
Interestingly, Westar/fad2 plants with enhanced resistance to S. sclerotiof7cm
infection on the leaf (Table VI) were also slightly more resistant to L.
maculans infection
on the stem (Table IX). The L. maculans resistance observed in Westar/fad2
plants was
statistically significant when compared to the L. maculans resistance observed
in the wild
type Westar plants (LSD = 0.05).
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Table VIII. Stem Fatty Acid Composition of Canola Lines
Canola Lines Stem
Fatty
Acid
Composition
C16:0C16:1C18:0C18:1C18:2C18:3
Westar/fad2 13.640.86 2.0226.437.55 48.40
Westar 17.210.85 4.436.2917.4652.94
Table IX. Stem Resistance to L. maculans Infection
Canola Lines Length of Necrosis Relative
to Westar
Westar 100
Westar/fad2 82
Example 6 - Transgenic Approach to Enhancing Resistance to Scle~otinia in
Sunflower
Sunflower (Helianthus annuus) lines are available that exhibit high oleic acid
in
the seeds (see, for example, Sperling et al., 1990, Z. Natur~forsch, 45:166-
72). The lines
that exhibit high oleic acid in the seeds exhibit an oleic acid content in the
leaves that is
similar to wild type sunflower (see Table X and, for example, Sperling et al.,
1990, Z
NaturfoYSCh, 45:166-72). To generate sunflower plants exhibiting high oleic
acid in their
leaves and/or stem, sunflower plants are transformed with a binary construct
containing a
fad2 nucleic acid under control of the ribulose 1,5-bisphosphate carboxylase
(rbcS)
promoter using a protocol involving a combination of particle bombardment
followed by
Ags-obactey~iuna-mediated transformation (Bidney et al., 1992, Plant Mol.
Biol., 18:301-
13, and Malone-Schoneberg et al., 1994, Plant Sci., 103:199-207).
The resulting plants are analyzed for fatty acid composition as described in
Example l, and compared with the fatty acid composition of wild type sunflower
(Table
X). Those plants exhibiting an increase in 18:1 and a decrease in 18:2 levels
are
evaluated for their resistance to infection by Sclerotinia as described in
Example 1.
ScleYOtifaia resistance in the transgenic sunflower plants is compared to
Sclerotiiz.ia
resistance in wild type sunflower plants.
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Table X. Leaf Fatty Acid Composition of Sunflower
Plant Leaf
Fatty
Acid
Composition
(%)
Variety
C16:0 C16:1 C18:0 C18:1 C18:2 C18:3
Wild 12.7 0.2 0.99 3.37 15.57 63.85
Type
Example 7 - Trans~enic Approach to Enhancing Resistance to Sclerotiraia in
Soybean
Soybean (Glycine max) plants are transformed with a binary construct
containing
a fad2 nucleic acid coding region under control of the rbcS promoter using
Ag~~obacterium-mediated transformation (see, for example, U.S. Patent No.
5,416,011).
The resulting plants are analyzed for fatty acid composition as described in
Example 1. Those plants exhibiting an increase in 18:1 and a decrease in 18:2
levels are
evaluated for their resistance to infection by Scley~otihia as described in
Example 1.
Sclerotinia resistance in the transgenic soybean plants is compared to the
Scle~otinia
resistance exhibited by the wild type soybean plants.
Example 8 - Non-transgenic Approach to Enhancing Resistance to Scle~otiyaia in
Sunflower or Soybean
Mutagenesis, for example, can be used to enhance resistance to Sclerotinia in
sunflower or soybean. Mutagenesis of sunflower or soybean is performed
essentially as
described in U.S. Patent No. 5,668,299 or WO 92/03919. The resulting plants
are
analyzed for fatty acid composition as described in Example l, and compared
with the
fatty acid composition of wild type sunflower (Table X) or wild type soybean.
Those
plants exhibiting an increase in 18:1 and a decrease in 18:2 levels are
evaluated for their
resistance to infection by Scle~otihia as described in Example 1. Scle~otihia
resistance in
the mutagenized sunflower or soybean plants is compared to Scle~otinia
resistance in wild
type sunflower or soybean plants.
Example 9-High Oleic Dihaploid Lines and Sclerotinia Resistance
Leaves were detached from a number of different high-oleic dihaploid canola
lines at a time when the plants were flowering and treated with Scle~otiraia
as described in
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Example 1. The dihaploid canola lines were generated by crossing a high oleic
canola
line (~85%) with a canola line containing typical levels of oleic acid (~55-
60%), and
flowers from the F1 plants were made dihaploid using colchicines. Experiments
were
repeated four times with two pathological tests each. The sizes of the
resulting necrotic
lesions were measured in millimeters (mm) (Table XI). Leaves from the same
plants near
the end of their flowering stage were analyzed for fatty acid composition
(Table XI).
IMC304 and Westar were used as positive and negative controls, respectively.
The coefficient of correlation of Sclerotihia resistance with oleic acid was
Ra=0.3114, and the coefficient of correlation of Scle~otiyaia resistance with
the ratio of
oleic acid to linoleic acid was R2=0.2407.
Table XI. Selerotinia Resistance and Fatty Acid Composition in Dihaploid Lines
sclerotinialeaC16:OC16:1C18:OC18:1C18:218:1/18:2C18:3
necrosis
(mm)
O1ZG.109 24 11.30.4 1.2 13.47.9 1.7 65.0
630022 24 12.20.4 1.6 11.59.7 1.2 63.6
C304 26 11.30.4 1.3 11.19.3 1.2 65.5
630039 27 10.90.4 1.0 12.99.1 1.4 60.7
GA134RT18128 11.30.2 1.5 10.39.4 1.1 65.9
629834 28 12.10.3 1.3 10.69.3 1.1 65.8
629845 29 12.90.4 1.5 13.511.2 1.2 59.0
630042 31 11.40.4 1.2 13.09.8 1.3 62.8
GA134RT67 33 12.60.4 1.5 10.78.0 1.3 65.9
630025 33 11.80.4 1.3 12.28.7 1.4 64.0
GA134RT-4334 12.40.4 1.3 12.47.7 1.6 63.8
630030 34 11.20.4 1.1 13.210.5 1.3 62.2
estar 36 13.50.4 2.8 1.5 13.3 0.1 67.6
630125 36 12.20.3 1.6 8.7 8.1 1.1 64.7
630138 40 12.10.3 1.4 10.110.0 1.0 63.7
630041 43 ~ 12.40.3 1.5 8.1 _ 0.9 67.3
~ ~ 9.3
~
OTHER EMBODIMENTS
It is to be understood that while the invention has been described in
conjunction
with the detailed description thereof, the foregoing description is intended
to illustrate and
not limit the scope of the invention, which is defined by the scope of the
appended claims.
Other aspects, advantages, and modifications are within the scope of the
following
claims.
29
