Note: Descriptions are shown in the official language in which they were submitted.
CA 02614353 2008-01-07
WO 2007/006079 PCT/AU2006/000952
-1-
MANAGEMENT OF PLANT PATHOGENS
BACKGROUND OF THE INVENTION
FIELD OF THE INVENTION
The present invention relates generally to a multi-faceted approach to the
control of plant
patliogens including plant pests. More particularly, the present invention
relates to plants
such as crop plants genetically modified to produce at least two pesticidal or
pestistatic
agents which in combination provide the plant with enhanced resistance or
reduced
susceptibility to plant pests. The present invention fu.rther provides genetic
constructs
encoding the pesticidal or pestistatic agents and compositions comprising the
agents. An
integrated plant pest management strategy is also part of the present
invention.
DESCRIPTION OF THE PRIOR ART
Bibliographic details of the publications referred to in this specification
are also collected
at the end of the description.
Reference to any prior art in this specification is not, and should not be
taken as, an
acknowledgment or any form of suggestion that this prior art forms part of the
common
general knowledge in any country.
Plant pathogens including insect pests and fungi are a major contributor to
losses in
agricultural crops. For example, the European corn borer alone is estimated to
cost the
corn growing industry up to a billion dollars a year in the United States in
lost production
and control measures. One particularly important agricultural crop is cotton.
The cotton
industry is a rapidly growing cash crop in many countries and in Australia,
for example,
approximately 200,000 bales of cotton are spun each year. Of these, 94% are
sent for
export. However, cotton plants are susceptible to a wide range of pests and
the cost of
controlling pest infestation in cotton in Australia alone is in the tens of
millions of dollars.
CA 02614353 2008-01-07
WO 2007/006079 PCT/AU2006/000952
-2-
Traditionally, the primary metliod of controlling plant pests such as insects
is the use of
varying spectrum chemical insecticides and fungicides. However, for
environmental,
regulatory and health reasons, the use of chemicals on crops is being
discouraged. In fact,
many broad spectrum chemicals have been banned from use or their use severely
limited to
particular crops or in particular agricultural regions. There is an urgent
need to develop
alternative strategies to the control and management of crop pests including
insects and
fungi.
One approach is the use of more natural agents. One such agent is a serine
proteinase
inhibitor.
Several members of the families Solanaceae and Fabaceae accumulate serine
proteinase
inhibitors in their storage organs and in leaves in response to wounding
(Brown and Ryan,
Biochemistry 23:3418-3422, 1984; Richard Phytochemistry 16:159-169, 1977). The
inhibitory activities of these proteins are directed against a wide range of
proteinases of
microbial and animal origin, but rarely against plant proteinases (Richardson,
1977 supra).
It is believed that these inhibitors are involved in protection of the plants
against pathogens
and predators. In potato tubers and legume seeds, the inhibitors can comprise
10% or more
of the stored proteins (Richardson, 1977 supra), while in leaves of tomato and
potato
(Green and Ryan, Science 776-777, 1972), and alfalfa (Brown and Ryan, 1984
supra),
proteinase inhibitors can accumulate to levels of 2% of the soluble protein
within 48 hours
of insect attack, or other types of wounding (Brown and Ryan, 1984 supra;
Graham et al,
Plants 169:399-405, 1986). High levels of these inhibitors (up to 50% of total
soluble
protein) are also present in unripe fruits of the wild tomato, Lycopersicon
peruvianum
(Pearce et al, Planta 175:527-531, 1988).
There are two families of serine proteinase inhibitors in tomato and potato
(Brown and
Ryan, 1984 supra). Type I inhibitors are small proteins (monomer Mr 8100)
which inhibit
chymotrypsin at a single reactive site (Melville and Ryan, Archives of
Biochemistry and
Biophysics 138:700-702, 1970; Plunkett et al, Arch Biochem Biophys 213:463-
472, 1982).
Inhibitors of the type II family generally contain two reactive sites, one of
which inhibits
CA 02614353 2008-01-07
WO 2007/006079 PCT/AU2006/000952
-3-
chymotrypsin and the other trypsin (Bryant et al, Biochemistry 15:3418-3424,
1976;
Plunkett et al, 1982 supra). The type II inhibitors have a monomer Mr of
12,300 (Plunkett
et al, 1982 supra). Proteinase inhibitor I accumulates in etiolated tobacco
(Nicotiana
tabacum) leaves (Kuo et al, Arch Biochem Biophys 230:504-510, 1984), and
elicitors from
Phytophthora parasitica var. nicotianae were found to induce proteinase
inhibitor I
accumulation in tobacco cell suspension cultures (Rickauer et al, Plant
Physiol 9:1065-
1070, 1989).
The serine proteinase inhibitor precursor from tobacco, NaPI, has been
particularly well
characterized and used in pest control (See United States Patent Nos.
6,031,087, 6,440,727,
6,451,573 and 6,261,821 all of which are incorporated herein by reference).
Certain species of microorganisms are also known to possess pathogenocidal
activity
against a broad range of pests including Lepidoptera, Diptera, Coleptera,
Hemiptera and
others. Bacillus thuringiensis and Bacillus papilliae are two particularly
useful biological
control agents. Pesticidal activity is concentrated in parasporal crystalline
protein
inclusions although pesticidal proteins have also been isolated from the
vegetative growth
stage of Bacillus. Several genes encoding pesticidal proteins have been cloned
and
characterised (See United States Patent Nos. 5,366,982 and 5,840,868).
Corn and cotton plants have been genetically engineered to produce pesticidal
6-
endotoxins and crytoxins from B. thuringiensis (Aronson Cell Mol Life Sci
59(3):417-425,
2002; Schnepf et al, Microbiol Mol Biol Rev 62(3):775-806, 1998).
Despite the success of biological control agents, there is always a danger of
resistance
developing. There is also some variability associated with the cytotoxicity of
a single
agent.
There is a need to develop alternative strategies for pest control in plants.
CA 02614353 2008-01-07
WO 2007/006079 PCT/AU2006/000952
-4-
SUMMARY OF THE INVENTION
Throughout this specification, unless the context requires otherwise, the word
"comprise",
or variations such as "comprises" or "comprising", will be understood to imply
the
inclusion of a stated element or integer or group of elements or integers but
not the
exclusion of any other element or integer or group of elements or integers.
In accordance with the present invention, a multi-faceted approach is provided
for pest
control in plants, and in particular crop plants. In this approach, modules of
pesticidal or
pestistatic agents derived from biological sources are combined in plants or
in
compositions. At least one agent is a serine proteinase inhibitor or a
precursor thereof, and
at least one other agent acts in a manner different to a proteinase inhibitor.
Generally, at
least one agent is a Bt protein such as a Cry family protein a Vegetative
Insecticidal
Protein (VIP) or is a defensin like molecule. In a preferred embodiment, the
serine
proteinase inliibitor is from the Solanaceae family such as but not limited to
NaPI from
tobacco. Genetic material encoding the pesticidal or pestistatic agents is
generally
introduced into a desired plant which is then rendered genetically capable of
producing the
pesticidal or pestistatic agents. In combination, the two or more pesticidal
or pestistatic
modules confer enhanced, increased, more stable, more efficacious, greater
pesticidal or
pestistatic activity, broader spectrum and/or longer lasting resistance or
reduced
susceptibility to pest infestation. By "pest" includes any plant pathogen or
insect or any
agent which carriers a serine proteinase. Examples of plant pests include
fungi, yeasts, and
insects. Insects groups particular contemplated by the present invention
include such as
Helicoverpa, Leptiderpa, Coleoptera, Diptera, Orthoptera, Diatraea and
Spocloptera.
Accordingly, one aspect of the present invention provides a plant genetically
modified to
produce at least two pesticidal or pestistatic agents wherein at least one
agent is a serine
proteinase inhibitor or precursor thereof wherein in precursor form, comprises
at least three
monomers wherein at least one monomer is a trypsin inhibitor and at least one
other
monomer is a chymotrypsin inhibitor and wherein at least one other agent is
not a serine
proteinase inhibitor.
CA 02614353 2008-01-07
WO 2007/006079 PCT/AU2006/000952
-5-
More particularly, the present invention provides a genetically modified plant
or its
progeny resulting from self-crossing, back-crossing or crossing with another
plant, said
plant comprising at least two pesticidal and/or pestistatic agents wherein at
least one of
said agents results from the genetic modification or crossing wherein at least
one agent is a
serine proteinase inhibitor or precursor thereof from the Solanaceae family
wherein in
precursor form, it comprises at least three monomers wherein at least one
monomer is a
trypsin inhibitor and at least one monomer is a chymotrypsin inhibitor and
wherein at least
one other agent is a non-serine proteinase inhibitor, wherein said plant
exhibits resistance
or reduced susceptibility to a pest.
In a preferred embodiment, the serine proteinase inhibitor or precursor is
from tobacco and
is referred to as NaPI. In another preferred embodiment, the other agent is an
endotoxin
such as a Bt protein including a Cry family protein, Cry, VIP or is a defensin
or a
component thereof. Reference to a defensin includes its N-terminal portion,
acidic tail
and/or a central portion alone or fused to a heterologous protein.
Accordingly, another aspect of the present invention provides a genetically
modified plant
or its progeny resulting from self-crossing, back-crossing or crossing with
another plant,
said plant comprising at least two pesticidal and/or pestistatic agents
wherein at least one
of said agents results from the genetic modification or crossing wherein at
least one agent
is the NaPI precursor or a monomer thereof or a recombinant or derivative
thereof and
wherein at least one other agent is a non-serine proteinase inhibitor wherein
said plant
exhibits resistance or reduced susceptibility to a pest.
Another aspect of the present invention contemplates a genetically modified
plant or its
progeny resulting from self-crossing, back-crossing or crossing with another
plant, said
plant comprising at least two pesticidal and/or pestistatic agents wherein at
least one of
said agents results from the genetic modification or crossing wherein at least
one agent is a
serine proteinase inhibitor or precursor thereof from the Solanaceae family
wherein in
precursor form, it comprises at least three monomers wherein at least one
monomer is a
CA 02614353 2008-01-07
WO 2007/006079 PCT/AU2006/000952
-6-
trypsin inhibitor and at least one monomer is a chymotrypsin inhibitor and
wherein at least
one other agent is a non-serine proteinase inhibitor, wherein said plant
exhibits resistance
or reduced susceptibility to a pest except and wherein at least one other
agent is a Bt family
protein such as a Cry family protein or defensin. Examples of Cry proteins
include Cry9,
Cry2, Cry3, CrylAc, VIP.
More particularly, the present invention is directed to a genetically modified
plant or its
progeny resulting from self crossing, back crossing or crossing with another
plant, said
plant comprising at least two pesticidal and/or pestistatic agents wherein at
least one of
said agents results from the genetic modification or crossing wherein at least
one agent is
NaPI precursor or a monomer thereof or a recombinant or derivative thereof and
wherein
at least one other agent is a non-serine proteinase inhibitor wherein said
plant exhibits
resistance or reduced susceptibility to a pest except wherein the least one
other agent is
selected from the list comprising a Bt protein and a defensin.
Examples of Bt crystal proteins as described in Hofte and Whiteley Microbial
Reviews
53:242-255, 1989.
The present invention further provides two or more genetic constructs encoding
the at least
two pesticidal or pestistatic agents or a single construct encoding the at
least two agents.
In addition, compositions capable of being applied to crops, leaves, seeds,
flowers or roots
such as by spray, paint, powder or micronized vapour and comprising the at
least two
agents are contemplated by the present invention.
Parts, reproductive parts, fruits, seeds, flowers and harvested portions of
crops genetically
modified to produce the at least two pesticidal or pestistatic agents also
form part of the
present invention.
CA 02614353 2008-01-07
WO 2007/006079 PCT/AU2006/000952
-7-
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 is a graphical representation of a mass of larvae surviving on a diet
of cotton leaf
discs coated with CrylAc. The control leaf discs were from cv Coker; the test
leaf discs
were from cv Coker homozygous for the NaPI gene. (Note: Leaves were selected
to give
only a small reduction in weight of larvae.)
Figure 2 is a graphical representation of a mass of larvae surviving on a diet
of cotton leaf
discs coated with CrylAc. The control leaf discs were from cv Coker; the test
leaf discs
were from cv Coker homozygous for the NaPI gene.
Figure 3 is a graphical representation of mortality of larvae after feeding on
a diet of
cotton leaves coated with CrylAc. The control leaf discs were from cv Coker;
the test leaf
discs were from cv Coker expressing the NaPI gene.
Figure 4 is a graphical representation showing the average weight of larvae
from day 11 to
day 12 (approximately 20 hours). Error bars represent standard error of the
mean.
Figure 5 is a graphical representation showing the mean increase in larval
mass from day
11 to day 12. Error bars represent standard error of the mean.
CA 02614353 2008-01-07
WO 2007/006079 PCT/AU2006/000952
-8-
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a multi-faceted approach to pest control in
plants, and in
particular crop plants.
Reference to a "multi-faceted approach" means the use of two or more agents in
combination to effect a pesticidal or pestistatic outcome on a targets pest or
group of pests
on commencement of or during its infestation or attempted infestation of a
plant. The
multi-faceted approach is modular in design in that each pesticidal or
pestistatic agent is
considered a modular component. Different modular components are used in the
overall
pest management system.
By "pestistatic" is meant the reduction in growth or reproduction or
colonization ability of
a pest without necessitating death of the pest. Whilst pesticidal activity is
preferred, a
pestistatic effect is acceptable and forms part of the present invention.
As used herein" pesticidal activity" includes "insecticidal activity" when the
target pest is
an insect or "fungicidal activity" when the target pest is a fungus or
"bacteriocidal activity"
when the target pest is a bacterium or "nematodicidal activity" when the
target pest is a
nematode. These terms refer to the activity of an agent, also referred to as a
"pesticidal
modular component" against a pest. The activity is conveniently measured in
terms of the
effect on pest mortality, pest weight loss, pest repellency and other
behavioural,
physiological and/or physical changes of the pest including ability to digest
protein, the
ability to grow, maintain itself and/or reproduce after exposure to the agent.
Accordingly,
the agent has an adverse effect on pest fitness as determined by at least one
measurable
parameter. In accordance with the present invention, at least two agents have
an effect on
pest fitness which may be additive or synergistic or otherwise complementary.
Accordingly, the at least two agents may exhibit the same effect on the
measurable
parameter of pest fitness or the combined effect may differ to the individual
effects of each
agent. In addition, at least one agent may exhibit pesticidal activity and
another agent may
CA 02614353 2008-01-07
WO 2007/006079 PCT/AU2006/000952
-9-
exhibit pestistatic activity. Preferably, however, both agents or the combined
effect of the
agents is pesticidal.
The agents are proteinaceous in nature when they are produced by plant cells
but may be
chemically modified when incorporated into a pesticidal or pestistatic
composition. The
agents may, therefore, be peptides, polypeptides or proteins as described
herein or
chemical analogs or chemical mimetics or functional equivalents.
As indicated above, the agents exhibit an effect on pests which effect may be
referred to as
an "impact". Examples of impacts including changes in feeding ability or
habits or
capacity, ability to undergo development changes, cytotoxicity, growth
retardation,
reduction in reproductivity, effects on eggs or larvae, and mortality or
morbidity of the
pest. In addition, the efficacy of the pesticidal or pestistatic activity can
be assess by the
effect on the crop plant e.g. growth yeild, fewer pests, reduced pathology.
The subject agents are provided to or in a plant in a "pesticidal effective
amount" or in a
"pestistatic effective amount". These amounts connotes the quantity of agents,
separately
or in combination, to provide individual or overall pesticidal or pestistatic
activity or other
appropriate impact.
Reference herein to a plant includes a monocotyledonous plant or a
dicotyledonous plant
and the plant may be generated from genetically transformed callus or tissue
or it may be a
progeny of the transformed plant or a cross between the transformed plant or
its progeny
and a non-transformed plant. As used herein, the term plant includes plant
cells, plant
protoplasts, plant cell tissue cultures from which plants can be regenerated,
plant calli,
plant clumps, and plant cells that are intact in plants or parts of plants
such as embryos,
pollen, ovules, seeds, leaves, flowers, branches, fruit, kernels, ears, cobs,
husks, stalks,
roots, root tips, anthers, and the like.
Examples of plants of interest include, but are not limited to, corn (Zea
mays), Brassica sp.
(e.g., B. napus, B. rapa, B. juncea), particularly those Brassica species
useful as sources of
CA 02614353 2008-01-07
WO 2007/006079 PCT/AU2006/000952
-10-
seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale
cereale), sorghum
(Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetum
glaucum),
proso millet (Panicum miliaceuni), foxtail millet (Setaria italica), finger
millet (Eleusine
coracana), sunflower (Helianthus annuus), safflower (Carthamus tinctorius),
wheat
(Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum),
potato
(Solanuni tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium
barbadense,
Gossypium hirsutum), sweet potato (Ipomoea batatus), cassaya (Manihot
esculenta),
coffee (Coffea spp.), coconut (Cocos nucifef a), cotton pineapple (Ananas
comosus), citrus
trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana
(Musa spp.),
avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango
(Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew
(Anacardium
occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus),
sugar
beets (Beta vulgaris), sugarcane (Saccharum spp.), oats, barley, vegetables,
ornamentals,
and conifers.
Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g., Lactuca
sativa),
green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas
(Lathyrus spp.),
and members of the genus Cucumis such as cucumber (C. sativus), cantaloupe (C.
cantalupensis), and musk melon (C. melo). Ornamentals include azalea
(Rhododendron
spp.), hydrangea (Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis),
roses (Rosa
spp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias (Petunia
hybrida), carnation
(Dianthus caryophyllus), poinsettia (Euphorbia pulcherrima), and
chrysanthemum.
Conifers that may be employed in practicing the present invention include, for
example,
pines such as loblolly pine (Pinus taeda), slash pine (Pinus elliotii),
ponderosa pine (Pinus
ponderosa), lodgepole pine (Pinus contorta), and Monterey pine (Pinus
radiata); Douglas-
fir (Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitka spruce
(Picea
glauca); redwood (Sequoia sempervirens); true firs such as silver fir (Abies
amabilis) and
balsam fir (Abies balsamea); and cedars such as Western red cedar (Thuja
plicata) and
Alaska yellow-cedar (Chamaecyparis nootkatensis). Plants of the present
invention include
crop plants (for example, cotton, corn, alfalfa, sunflower, Brassica, soybean,
cotton,
safflower, peanut, sorghum, wheat, millet, tobacco, etc.), such as corn and
soybean plants.
CA 02614353 2008-01-07
WO 2007/006079 PCT/AU2006/000952
-11-
Turfgrasses include, but are not limited to: annual bluegrass (Poa annua);
annual ryegrass
(Lolium multiflorum); Canada bluegrass (Poa compressa); Chewings fescue
(Festuca
rubra); colonial bentgrass (Agrostis tenuis); creeping bentgrass (Agrostis
palustris);
crested wheatgrass (Agropyron desertorum); fairway wheatgrass (Agropyron
cristatum);
hard fescue (Festuca longifolia); Kentucky bluegrass (Poa pratensis);
orchardgrass
(Dactylis glonaerata); perennial ryegrass (Lolium per=enne); red fescue
(Festuca rubra);
redtop (Agrostis alba); rough bluegrass (Poa trivialis); sheep fescue (Festuca
ovina);
smooth bromegrass (Bromus inermis); tall fescue (Festuca arundinacea); timothy
(Phleum
pratense); velvet bentgrass (Agrostis, canina); weeping alkaligrass
(Puccinellia distans);
western wheatgrass (Agropyron smithii); Bermuda grass (Cynodon spp.); St.
Augustine
grass (Stenotaphrum secundatum); zoysia grass (Zoysia spp.); Bahia grass
(Paspalum
notatum); carpet grass (Axonopus affinis); centipede grass (Eremochloa
ophiuroides);
kikuyu grass (Pennisetum clandesinum); seashore paspalum (Paspalum vaginatum);
blue
gramma (Bouteloua gracilis); buffalo grass (Buchloe dactyloids); sideoats
gramma
(Bouteloua curtipendula).
Plants of interest include grain plants that provide seeds of interest, oil-
seed plants, and
leguminous plants. Seeds of interest include grain seeds, such as corn, wheat,
barley, rice,
sorghum, rye, millet, etc. Oil-seed plants include cotton, soybean, safflower,
sunflower,
Brassica, maize, alfalfa, palm, coconut, flax, castor, olive etc. Leguminous
plants include
beans and peas. Beans include guar, locust bean, fenugreek, soybean, garden
beans,
cowpea, mungbean, lima bean, fava bean, lentils, chickpea, etc.
Preferred plants contemplated herein include cotton, sweet corn, tomato,
tobacco, piniento,
potato, sunflower, citrus, plums, sorghum, leeks, soybean, alfalfa, beans,
pidgeon peas,
chick peas, artichokes, curcurbits, lettuce, Dianthus, geraniums, cape
gooseberry, maize,
flax and linseed, lupins, broad beans, garden peas, peanuts, canola,
snapdragons, cherry,
sunflower, pot marigolds, Helichrysum, wheat, barley, oats, triticale,
carrots, onions,
orchids, roses and petunias.
CA 02614353 2008-01-07
WO 2007/006079 PCT/AU2006/000952
-12-
The embodiments of the present invention may be effective against a variety of
pests. For
purposes of the present invention, pests include, but are not limited to,
insects, fungi,
bacteria, nematodes, acarids, protozoan pathogens, animal-parasitic liver
flukes, and the
like. Pests of particular interest are insect pests, particularly insect pests
that cause
significant damage to agricultural plants. The term "insect pests" as used
herein refers to
insects and other similar pests such as, for example, those of the order Acari
including, but
not limited to, mites and ticks. Insect pests of the present invention
include, but are not
limited to, insects of the order Lepidoptera, e.g. Achoroia grisella, Acleris
gloverana,
Acleris variana, Adoxophyes orana, Agrotis ipsilon, Alabama argillacea,
Alsophila
pometaria, Amyelois transitella, Anagasta kuehniella, Anarsia lineatella,
Anisota
senatoria, Antheraea pernyi, Anticarsia gemmatalis, Archips sp., Argyrotaenia
sp., Athetis
mindara, Bombyx mori, Bucculatrix thurberiella, Cadra cautella, Choristoneura
sp.,
Cochylls hospes, Colias eurytheme, Corcyra cephalonica, Cydia latifers eanus,
Cydia
pomonella, Datana integerrima, Dendrolimus sibericus, Desmiafeneralis,
Diaphania
hyalinata, Diaphania nitidalis, Diatraea grandiosella, Diatraea saccharalis,
Ennomos
subsignaria, Eoreuma loftini, Esphestia elutella, Erannis tilaria, Estigmene
acrea, Eulia
salubricola, Eupocoellia ambiguella, Eupoecilia ambiguella, Euproctis
chrysorrhoea,
Euxoa messoria, Galleria mellonella, Grapholita molesta, Harrisina americana,
Helicoverpa subflexa, Helicoverpa zea, Heliothis virescens, Hemileuca oliviae,
Homoeosoma electelluna, Hyphantia cunea, Keiferia lycopersicella, Lambdina
fi.scellaria
fiscellaria, Lambdina fi`scellaria lugubrosa, Leucoma salicis, Lobesia
botrana, Loxostege
sticticalis, Lymantria dispar, Macalla thyrisalis, Malacosoma sp., Mamestra
brassicae,
Mamestra configurata, Manduca quinquemaculata, Manduca sexta, Maruca
testulalis,
Melanchrapicta, Operophtera brumata, Orgyia sp., Ostrinia nubilalis,
Paleacrita vernata,
Papilio cresphontes, Pectinophora gossypiella, Phryganidia califoNnica,
Phyllonorycter
blancardella, Pieris napi, Pieris rapae, Plathypena scabra, Platynota
flouendana,
Platynota stultana, Platyptilia carduidactyla, Plodia interpunctella, Plutella
xylostella,
Pontia protodice, Pseudaletia unipuncta, Pseudoplasia includens, Sabulodes
aegrotata,
Schizura concinna, Sitotroga cerealella, Spilonta ocellana, Spodoptera sp.,
Thaurnstopoea
pityocampa, Tinsola bisselliella, Trichoplusia hi, Udea rubigalis, Xylomyges
curiails, and
CA 02614353 2008-01-07
WO 2007/006079 PCT/AU2006/000952
-13-
Yponomeuta padella.
Also, the embodiments of the present invention may be effective against insect
pests,
including but not limited to insects selected from the orders Diptera,
Hyrnenopter=a,
Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthroptera, Thysanoptera,
Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera, Coleoptera, etc.,
particularly
Lepidoptera. Insect pests of the invention for the major crops include, but
are not limited
to: Maize: Ostrinia nubilalis, European corn borer; Agrotis ipsilon, black
cutworm;
Helicoverpa zeae, corn earworm; Spodoptera frugiperda, fall armyworni;
Diatraea
grandiosella, southwestern corn borer; Elasmopalpus lignosellus, lesser
cornstalk borer;
Diatraea saccharalis, surgarcane borer; western corn rootworm, e.g.,
Diabrotica virgifera
virgifera; nortllern corn rootworm, e.g., Diabrotica longicornis barberi;
southern corn
rootworm, e.g., Diabrotica undecimpunctata howardi; Melanotus spp., wireworms;
Cyclocephala borealis, northern masked chafer (white grub); Cyclocephala
immaculata,
southern masked chafer (white grub); Popillia japonica, Japanese beetle;
Chaetocnema
pulicaria, corn flea beetle; Sphenophorus maidis, maize billbug; Rhopalosiphum
maidis,
corn leaf aphid; Anuraphis rnaidiradicis, corn root aphid; Blissus leucopterus
leucopterus,
chinch bug; Melanoplus femurrubrum, redlegged grasshopper; Melanoplus
sanguinipes,
migratory grasshopper; Hylemya platura, seedcorn maggot; Agromyza parvicornis,
corn
blotch leafminer; AnaphothNips obscrurus, grass thrips; Solenopsis milesta,
thief ant;
Tetranychus urticae, two spotted spider mite; Sorghum: Chilo partellus,
sorghum borer;
Spodoptera frugiperda, fall armyworm; Helicoverpa zea, corn earworm;
Elasmopalpus
lignosellus, leser cornstalk borer; Feltia subterranea, granulate cutworm;
Phyllophaga
crinita, white grub; Eleodes, Conoderus, and Aeolus spp., wireworms; Oulema
melanopus,
cereal leaf beetle; Chaetocnema pulicaria, corn flea beetle; Sphenophorus
maidis, maize
billbug; Rhopalosiphum maidis; corn leaf aphid; Sipha flava, yellow sugarcane
aphid;
chinch bug, e.g., Blissus leucopterus leucopterus; Contarinia sorghicola,
sorghum midge;
Tetranychus cinnabarinus, carmine spider mite; Tetranychus urticae, two-
spotted spider
mite; Wheat: Pseudaletia unipunctata, army worm; Spodoptera frugiperda, fall
armyworm; Elasmopalpus lignosellus, lesser cornstalk borer; Agrotis
orthogonia, pale
western cutworm; Elasmopalpus lignosellus, lesser cornstalk borer; Oulema
melanopus,
CA 02614353 2008-01-07
WO 2007/006079 PCT/AU2006/000952
-14-
cereal leaf beetle; Hypera punctata, clover leaf weevil; southern corn
rootworm, e.g.,
Diabrotica undecirnpunctata howardi; Russian wheat aphid; Schizaphis graminum,
greenbug; Macrosiphum avenae, English grain aphid; Melanoplusfemurrubrum,
redlegged
grasshopper; Melanoplus differentialis, differential grasshopper; Melanoplus
sanguinipes,
migratory grasshopper; Mayetiola destructor, Hessian fly; Sitodiplosis
mosellana, wheat
midge; Meronayza anaericana, wheat stem maggot; Hylemya coarctata, wheat bulb
fly;
Frankliniella fusca, tobacco thrips; Cephus cinctus, wheat stem sawfly; Aceria
tulipae,
wlieat curl mite; Sunflower: Cylindrocupturus adspersus, sunflower stem
weevil;
Smicronyx fulus, red sunflower seed weevil; Smicronyx sordidus, gray sunflower
seed
weevil; Suleima helianthana, sunflower bud moth; Homoeosoma electellum,
sunflower
moth; Zygogramma exclamationis, sunflower beetle; Bothyrus gibbosus, carrot
beetle;
Neolasioptera murtfeldtiana, sunflower seed midge; Cotton: Heliothis
virescens, tobacco
budworm; Helicoverpa zea, cotton bollworm; Spodoptera exigua, beet armyworm;
Pectinophora gossypiella, pink bollworm; boll weevil, e.g., Anthonomus
grandis; Aphis
gossypii, cotton aphid; Pseudatomoscelis seriatus, cotton fleahopper;
Trialeurodes
abutilonea, bandedwinged whitefly; Lygus lineolaris, tarnished plant bug;
Melanoplus
femurrubrum, redlegged grasshopper; Melanoplus differentialis, differential
grasshopper;
Thrips tabaci, onion thrips; Franklinkiella fusca, tobacco thrips; Tetranychus
cinnabarinus, carmine spider mite; Tetranychus urticae, two-spotted spider
mite; Rice:
Diatraea saccharalis, sugarcane borer; Spodoptera ftugiperda, fall armyworm;
Helicoverpa zea, corn earworm; Colaspis brunnea, grape colaspis; Lissorhoptrus
oryzophilus, rice water weevil; Sitophilus oryzae, rice weevil; Nephotettix
nigropictus, rice
leafhoper; chinch bug, e.g., Blissus leucopterus leucopterus; Acrosternum
hilare, green
stink bug; Soybean: Pseudoplusia includens, soybean looper; Anticarsia
gemmatalis,
velvetbean caterpillar; Plathypena scabra, green cloverworm; Ostrinia
nubilalis, European
corn borer; Agrotis ipsilon, black cutworm; Spodoptera exigua, beet armyworm;
Heliothis
virescens, tobacco budworm; Helicoverpa zea, cotton bollworm; Epilachna
varivestis,
Mexican bean beetle; Myzus persicae, green peach aphid; Empoasca fabae, potato
leafliopper; Acrosternum hilare, green stink bug; Melanoplus femurrubrum,
redlegged
grasshopper; Melanoplus differentialis, differential grasshopper; Hylemya
platura,
seedcorn maggot; Sericothrips variabilis, soybean thrips; Thrips tabaci, onion
thrips;
CA 02614353 2008-01-07
WO 2007/006079 PCT/AU2006/000952
-15-
Tetranychus turkestani, strawberry spider mite; Tetranychus urticae, two-
spotted spider
mite; Barley: Ostrinia nubilalis, European corn borer; Agrotis ipsilon, black
cutworm;
Schizaphis graminum, greenbug; chinch bug, e.g., Blissus leucopterus
leucopterus;
Acrosternuna hilare, green stink bug; Euschistus servus, brown stink bug;
Jylemya platura,
seedcorn maggot; Mayetiola destructor, Hessian fly; Petrobia latens, brown
wheat mite;
Oil Seed Rape: Vrevicoryne brassicae, cabbage aphid; Phyllotreta cruciferae,
crucifer flea
beetle; Phyllotreta striolata, striped flea beetle; Phyllotreta nemorum,
striped turnip flea
beetle; Meligethes aeneus, rapeseed beetle; and the pollen beetles Meligethes
rufimanus,
Meligethes nigrescens, Meligethes canadianus, and Meligethes viridescens;
Potato:
Leptinotarsa decemlineata, Colorado potato beetle.
Furthermore, embodiments of the present invention may be effective against
Hemiptera
such as Lygus hesperus, Lygus lineolaris, Lygus pratensis, Lygus rugulipennis
Popp, Lygus
pabulinus, Calocoris norvegicus, Orthops compestris, Plesiocoris rugicollis,
Cyrtopeltis
modestus, Cyrtopeltis notatus, Spanagonicus albofasciatus, Diaphnocoris
chlorinonis,
Labopidicola allii, Pseudatomoscelis seriatus, Adelphocoris rapidus,
Poecilocapsus
lineatus, Blissus leucopterus, Nysius ericae, Nysius raphanus, Euschistus
servus, Nezara
viridula, Eurygaster, Coreidae, Pyrrhocoridae, Tinidae, Blostomatidae,
Reduviidae, and
Cimicidae. Pests of interest also include Araecerus fasciculatus, coffee bean
weevil;
Acanthoscelides obtectus, bean weevil; Bruchus rufmanus, broadbean weevil;
Bruchus
pisorum, pea weevil; Zabrotes subfasciatus, Mexican bean weevil; Diabrotica
balteata,
banded cucumber beetle; Cerotoma trifurcata, bean leaf beetle; Diabrotica
virgifera,
Mexican corn rootworm; Epitrix cucumeris, potato flea beetle; Chaetocnema
confinis,
sweet potato flea beetle; Hypera postica, alfalfa weevil; Anthonomus
quadrigibbus, apple
curculio; Sternechus paludatus, bean stalk weevil; Hypera brunnipennis,
Egyptian alfalfa
weevil; Sitophilus granaries, granary weevil; Craponius inaequalis, grape
curculio;
Sitophilus zeamais, maize weevil; Conotrachelus nenuphaN, plum curculio;
Euscepes
postfaciatus, West Indian sweet potato weevil; Maladera castanea, Asiatic
garden beetle;
Rhizotrogus naajalis, European chafer; Macrodactylus subspinosus, rose chafer;
Tribolium
confusum, confused flour beetle; Tenebrio obscurus, dark mealworm; Tribolium
castaneum, red flour beetle; Tenebrio molitor, yellow mealworm.
CA 02614353 2008-01-07
WO 2007/006079 PCT/AU2006/000952
-16-
Nematodes include parasitic nematodes such as root-knot, cyst, and lesion
nematodes,
including Heterodera spp., Meloidogyne spp., and Globodera spp.; particularly
members
of the cyst nematodes, including, but not limited to, Heterodera glycines
(soybean cyst
nematode); Heterodera schachtii (beet cyst nematode); Heterodera avenae
(cereal cyst
nematode); and Globodera rostochiensis and Globodera pailida (potato cyst
nematodes).
Lesion nematodes include Pratylenchus spp.
The present invention is directed to a pest management system for plants which
is multi-
faceted in the sense that it involves two or more pesticidal and/or
pestistatic agents
generally designed to exhibit a combined impact on plant pests. At least one
agent is a
serine proteinase inhibitor and least one agent is a non-serine proteinase
inhibitor (i.e. it
acts via a mechanism different to the inhibition of serine proteinase
activity). The multi-
faceted system may employ both agents simultaneously or sequentially. In
either event,
this is described as the agents operating in "combination".
Accordingly, one aspect of the present invention provides a genetically
modified plant or
its progeny resulting from self-crossing, back-crossing or crossing with
another plant, said
plant comprising at least two pesticidal and/or pestistatic agents wherein at
least one of
said agents results from the genetic modification or crossing wherein at least
one agent is a
serine proteinase inhibitor or precursor thereof from the Solanaceae family
wherein in
precursor form, it comprises at least three monomers wherein at least one
monomer is a
trypsin inhibitor and at least one monomer is a chymotrypsin inhibitor and
wherein at least
one other agent is a non-serine proteinase inhibitor, wherein said plant
exhibits resistance
or reduced susceptibility to a pest.
The serine proteinase inhibitor is preferably from tobacco and may comprise in
precursor
form at least three monomers, or four monomers, or five monomers, or six
monomers, or
seven monomers, or eight monomers wherein at least one monomer inhibits
trypsin and at
least one monomer inhibits chymotrypsin.
CA 02614353 2008-01-07
WO 2007/006079 PCT/AU2006/000952
-17-
The term "precursor" as used herein is not intended to place any limitation on
the utility of
the precursor molecule itself or a requirement that the molecule first be
processed into
monomers before proteinase inhibitory activity is expressed. The precursor
molecule has
proteinase inhibitory activity and the present invention is directed to the
precursor and to
the individual monomers of the precursor.
In a most preferred embodiment, the proteinase inhibitor or its precursor is
from Nicotiana
alata (NaPI) as described in International Patent Application No.
PCT/AU93/00659 [WO
94/13 8 10] which is incorporated herein by reference.
Accordingly, another aspect of the present invention provides a genetically
modified plant
or its progeny resulting from self-crossing, back-crossing or crossing with
another plant,
said plant comprising at least two pesticidal and/or pestistatic agents
wherein at least one
of said agents results from the genetic modification or crossing wherein at
least one agent
is the NaPI precursor or a monomer thereof or a recombinant or derivative
thereof and
wherein at least one other agent is a non-serine proteinase inhibitor wherein
said plant
exhibits resistance or reduced susceptibility to a pest.
The preferred at least one other agent is a toxin including an endotoxin such
as derived
from a microorganism or fungus. Particularly useful examples include toxins
derived from
Bacillus species. Reference to a toxin includes a peptide, polypeptide or
protein exhibiting
pesticidal or pestistatic activity but which does not inhibit solely a serine
proteinase. It
may inhibit a serine proteinase only if it also exhibits another mode of
pesticidal or
pestistatic activity. Particularly useful toxins are Bt-related toxins from B.
thuringiensis
such as the Cry family of endotoxins. Examples of these endotxins include
Cry9, Cry2s,
Cry3s or CrylAc family of endotoxins.
The term "Cry9 family" is used herein to refer to nucleotide or amino acid
sequences
which share a high degree of sequence identity or similarity to previously
described wild
type Cry9 or Cry9D sequences. Bt toxin or endotoxin is intended to include the
broader
class of Cry toxins found in various status of B. thuringiensis and includes
such toxins, as
CA 02614353 2008-01-07
WO 2007/006079 PCT/AU2006/000952
-18-
CrylAc, Cryls, Cry2s and Cry3s. Examples of suitable toxins can be found in US
Patent
Application Nos. 2005/0138685, 2004/0016020 and 2004/0221333 which are each
incorporated herein by reference.
Other suitable toxins include vegetative insecticidal protein (VIP) and a
defensin. Suitable
defensins are described in International Patent Application No.
PCT/AU2004/000524
which is incorporated herein by reference.
According, another aspect of the present invention contemplates a genetically
modified
plant or its progeny resulting from self-crossing, back-crossing or crossing
with another
plant, said plant comprising at least two pesticidal and/or pestistatic agents
wherein at least
one of said agents results from the genetic modification or crossing wherein
at least one
agent is a serine proteinase inhibitor or precursor thereof from the
Solanaceae family
wherein in precursor form, it comprises at least three monomers wherein at
least one
monomer is a trypsin inhibitor and at least one monomer is a chymotrypsin
inhibitor and
wherein at least one other agent is a non-serine proteinase inhibitor, wherein
said plant
exhibits resistance or reduced susceptibility to a pest except and wherein at
least one other
agent is selected from the list comprising a Bt protein, a Cry9 family
protein, a Cry2
family protein or Cry3 family protein, Cry1AC, a VIP and a defensin.
More particularly, the present invention is directed to a genetically modified
plant or its
progeny resulting from self crossing, back crossing or crossing with another
plant, said
plant comprising at least two pesticidal and/or pestistatic agents wherein at
least one of
said agents results from the genetic modification or crossing wherein at least
one agent is
NaPI precursor or a monomer thereof or a recombinant or derivative thereof and
wherein
at least one other agent is a non-serine proteinase inhibitor wherein said
plant exhibits
resistance or reduced susceptibility to a pest except wherein the least one
other agent is
selected from the list comprising a Bt protein including a Cry family protein,
a VIP and a
defensin.
CA 02614353 2008-01-07
WO 2007/006079 PCT/AU2006/000952
-19-
Reference herein to NaPI, proteinase inhibitors, Bt, Cry9, CrylAc, Cryls,
Cry2s, Cry3s,
VIP and defensin and the like include derivatives and fragments and otherwise
modified
forms. Examples of modified forms include mutagenized forms such as mutants
with
altered substrate specificity or which exhibit greater stability or which
direct the agent to a
plant cell organelle such as a vacuole.
Serine Protease inhibitors such as NaPIs are known to act directly on the gut
proteases of
the digestive system of lepidopteran pests. A complex is formed between the
specific
inhibitor (either a trypsin or chymotrypsin inhibitor) and the protease
(either a trypsin or
chymotrypsin). These complexes are then excreted in the frass. The effect is
to diminish
the digestive capacity of the insect for protein and to deplete the protein
reserves of the
insect larvae.
Protease inhibitors may also affect the innate immunity system of insects. For
this effect to
occur, the inhibitors must move into the haemolymph where they can complex
with the
proteases in the cascades of the innate immunity system. Normally there is
very restricted
access of foreign molecules to this fundamental immune system of insects. An
aspect of
our invention is to provide conditions which allow enhanced access of PIs to
the innate
immunity system.
As indicated above, the present invention extends to parts of the genetically
modified
plants or,their progeny which include seeds, pollen, leaves, flowers, stigmas,
stems, roots,
root hairs, bark and apical meristems. Parts also include root stock or other
form of
commercially exploitable plant parts.
The present invention further provides nucleic acid molecules encoding the
pesticidal or
pestistatic agents. The nucleic acid molecules may be in a single construct,
in multiple
constructs or may be in situ in a plant cell genome. Conveniently, the nucleic
acid
molecules form a genetic composition. The nucleic acid molecules may also be
part of
beads such as beads used in biolistic DNA transfer.
CA 02614353 2008-01-07
WO 2007/006079 PCT/AU2006/000952
-20-
Accordingly, another aspect of the present invention provides a genetic
composition
comprising at least one construct encoding a serine proteinase inhibitor or a
precursor form
thereof wherein in precursor form it comprises at least three monomers wherein
at least
one monomer is a trypsin inhibitor and at least one other monomer inhibits
chymotrypsin
and at least one construct encodes a non-serine proteinase inllibitor.
Generally, the nucleic acid molecules are operably linked to one or more
promoters.
The promoter may regulate the expression of the nucleotide sequence encoding
the agent,
or differentially with respect to cell, the tissue or organ in which
expression occurs or, with
respect to the developmental stage at which expression occurs, or in response
to external
stimuli such as physiological stresses, or pathogens, or metal ions, amongst
others.
Preferably, the promoter is capable of regulating expression of a nucleic acid
molecule in a
plant cell, tissue or organ, at least during the period of time over which the
nucleotide
sequence encoding the agent is expressed therein.
Plant-operable promoters are particularly preferred for use in the constructs
of the present
invention. Examples of suitable promoters include pCaMV 35S (Fang et al, Plant
Cell
1:141-150, 1989), PGEL1 (Hajdukiewicz et al, Plant Mol Biol 25:989-994, 1994),
class III
chitinase (Samac and Shah, Plant Cell 3:1063-1072, 1991), pin2 (Keil et al,
EMBO J
8:1323-1330, 1989), PEP carboxylase (Pathirana et al, Plant J 12:293-304,
1997; MAP
kinase (Schoenbeck et al, Molec Plant-Microbe Interact, 1999), MSV (Legavre et
al, In:
Vth International Congress of Plant Molecular Biology, Singapore, 1997), pltp
(Hsu et al,
Plant Sci 143:63-70, 1999), pmpi (Cordero et al, In: General Meeting of the
International
Program on Rice Biotechnology of the Rockefeller Foundation, Malacca,
Malaysia, 1997)
or glutamin synthase (Pujade-Renaud et al, Plant Physiol Biochem 35:85-93,
1997).
In the present context, the terms "in operable connection with" or "operably
under the
control" or similar shall be taken to indicate that expression of the nucleic
acid molecule is
CA 02614353 2008-01-07
WO 2007/006079 PCT/AU2006/000952
-21 -
under the control of the promoter sequence with which it is spatially
connected; in a cell,
tissue, organ or whole plant.
A number of promoters can be used in the practice of the invention. The
promoters can be
selected based on the desired outcome. The nucleic acids can be combined with
constitutive, tissue-preferred, inducible, or other promoters for expression
in the host
organism. Suitable constitutive promoters for use in a plant host cell
include, for example,
the core promoter of the Rsyn7 promoter and other constitutive promoters
disclosed in WO
99/43838 and U.S. Pat. No. 6,072,050; the core CaMV 35S promoter (Odell et al,
Nature
313:810-812, 1985); rice actin McElroy et al, Plant Cell 2:163-171, 1990);
ubiquitin
(Christensen et al, Plant Mol Biol 12:619-632, 1989 and Christensen et al,
Plant Mol Biol
18:675-689, 1992); pEMU (Last et al, TheoN Appl Genet 81:581-588, 1991); MAS
(Velten
et al, EMBO J 3:2723-2730, 1984); ALS promoter (U.S. Pat. No. 5,659,026), and
the like.
Other constitutive promoters include, for example, those discussed in U.S.
Pat. Nos.
5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463;
5,608,142;
and 6,177,611.
Depending on the desired outcome, it may be beneficial to express the gene
from an
inducible promoter. Of particular interest for regulating the expression of
the nucleotide
sequences of the present invention in plants are wound-inducible promoters.
Such wound-
inducible promoters, may respond to damage caused by insect feeding, and
include potato
proteinase inhibitor (pin II) gene (Ryan Ann Rev Phytopath 28:425-449, 1990;
Duan et al,
Nature Biotechnology 14:494-498, 1996); wunl and wun2, U.S. Pat. No.
5,428,148; winl
and win2 (Stanford et al, Mol Gen Genet 215:200-208, 1989); systemin (McGurl
et al,
Science 225:1570-1573, 1992); WIP1 (Rohmeier et al, Plant Mol Biol 22:783-792,
1993;
Eckelkamp et al, FEBS Letters 323:73-76, 1993); MPI gene (Corderok et al,
Plant J
6(2):141-150, 1994); and the like, herein incorporated by reference.
Additionally, pathogen-inducible promoters may be employed in the methods and
nucleotide constructs of the present invention. Such pathogen-inducible
promoters include
those from pathogenesis-related proteins (PR proteins), which are induced
following
CA 02614353 2008-01-07
WO 2007/006079 PCT/AU2006/000952
-22-
infection by a pathogen; e.g., PR proteins, SAR proteins, beta-l,3-glucanase,
chitinase, etc.
See, for example, Redolfi et al, Meth JPlant Pathol 89:245-254, 1983; Uknes et
al, Plant
Cell 4:645-656, 1992; and Van Loon Plant Mol Virol 4:111-116, 1985. See also
WO
99/43819, herein incorporated by reference.
Of interest are promoters that are expressed locally at or near the site of
pathogen
infection. See, for example, Marineau et al, Plant Mol Biol 9:335-342, 1987;
Matton et al,
Molecular Plant-Microbe Interactions 2:325-331, 1989; Somsisch et al, Proc
Natl Acad
Sci USA 83:2427-2430, 1986; Somsisch et al, Mol Gen Genet 2:93-98, 1988; and
Yang
Proc Natl Acad Sci USA 93:14972-14977, 1996. See also, Chen et al, Plant J
10:955-966,
1996; Zhang et al, Proc Natl Acad Sci USA 91:2507-2511, 1994; Warner et al,
Plant J
3:191-201, 1993; Siebertz et al, Plant Cell 1:961-968, 1989; U.S. Pat. No.
5,750,386
(nematode-inducible); and the references cited therein. Of particular interest
is the
inducible promoter for the maize PRms gene, whose expression is induced by the
pathogen
Fusarium moniliforme (see, for example, Cordero et al, Physiol Mol Plant Path
41:189-
200, 1992).
Chemical-regulated promoters can be used to modulate the expression of a gene
in a plant
through the application of an exogenous chemical regulator. Depending upon the
objective,
the promoter may be a chemical-inducible promoter, where application of the
chemical
induces gene expression, or a chemical-repressible promoter, where application
of the
chemical represses gene expression. Chemical-inducible promoters are known in
the art
and include, but are not limited to, the maize In2-2 promoter, which is
activated by
benzenesulfonamide herbicide safeners, the maize GST promoter, which is
activated by
hydrophobic electrophilic compounds that are used as pre-emergent herbicides,
and the
tobacco PR-la promoter, which is activated by salicylic acid. Other chemical-
regulated
promoters of interest include steroid-responsive promoters (see, for example,
the
glucocorticoid-inducible promoter in Schena et al, Proc Natl Acad Sci USA
88:10421-
10425, 1991 and McNellis et al, Plant J 14(2):247-257, 1998 and tetracycline-
inducible
and tetracycline-repressible promoters (see, for example, Gatz et al, Mol Gen
Genet
227:229-237, 1991, and U.S. Pat. Nos. 5,814,618 and 5,789,156), herein
incorporated by
CA 02614353 2008-01-07
WO 2007/006079 PCT/AU2006/000952
- 23 -
reference.
Tissue-preferred promoters can be utilized to target enhanced pesticidal
protein expression
within a particular plant tissue. Tissue-preferred promoters include those
discussed in
Yamamoto et al, Plant J 12(2):255-265, 1997; Kawamata et al, Plant Cell
Physiol
38(7):792-803, 1997; Hansen et al, Mol Gen Genet 254(3):337-343, 1997; Russell
et al,
Transgenic Res 6(2):157-168, 1997; Rinehart et al, Plant Physiol 112(3):1331-
1341, 1996;
Van Camp et al, Plant Physiol 112(2):525-535, 1996; Canevascini et al, Plant
Physiol
112(2):513-524, 1996; Yamamoto et al, Plant Cell Physiol 35(5):773-778, 1994;
Lam
Results Probl Cell Differ 20:181-196, 1994; Orozco et al, Plant Mol Biol
23(6):1129-1138,
1993; Matsuoka et al, Proc Natl Acad Sci USA 90(20):9586-9590, 1993; and
Guevara-
Garcia et al, Plant J 4(3):495-505, 1993. Such promoters can be modified, if
necessary, for
weak expression.
Leaf-preferred promoters are known in the art. See, for example, Yamamoto et
al, 1997
supra; Kwon et al, Plant Physiol 105:357-67, 1994; Yamamoto et al, 1994 supra;
Gotor et
al, Plant J 3:509-18, 1993; Orozco et al, 1993 supra and Matsuoka et al, 1993
supra.
Root-preferred or root-specific promoters are known and can be selected from
the many
available from the literature or isolated de novo from various compatible
species. See, for
example, Hire et al, Plant Mol Biol 20(2):207-218, 1992 (soybean root-specific
glutainine
synthetase gene); Keller and Baumgartner Plant Cell 3(10):1051-1061, 1991
(root-specific
control element in the GRP 1.8 gene of French bean); Sanger et al, Plant Mol
Biol
14(3):433-443, 1990 (root-specific promoter of the mannopine synthase (MAS)
gene of
Agrobacterium tumefaciens); and Miao et al, Plant Cell 3(1):11-22, 1991 (full-
length
cDNA clone encoding cytosolic glutamine synthetase (GS), which is expressed in
roots
and root nodules of soybean). See also Bogusz et al, Plant Cell 2(7):633-641,
1990, where
two root-specific promoters isolated from hemoglobin genes from the nitrogen-
fixing
nonlegume Parasponia andersonii and the related non-nitrogen-fixing nonlegume
Trema
tomentosa are described. The promoters of these genes were linked to a(3-
glucuronidase
reporter gene and introduced into both the nonlegume Nicotiana tabacum and the
legume
CA 02614353 2008-01-07
WO 2007/006079 PCT/AU2006/000952
-24-
Lotus cof-niculatus, and in both instances root-specific promoter activity was
preserved.
Promoters of ro1C and rolD root-inducing genes of Agrobacterium rhizogenes
rnay also be
used (see Limerick Plant Science 79(1):69-76). Teeri et al, EMBO J 8(2):343-
350, 1989)
describe gene fusion to lacZ to show that the Agrobacteriuna T-DNA gene
encoding
octopine synthase is especially active in the epidermis of the root tip and
that the TR2'
gene is root specific in the intact plant and stimulated by wounding in leaf
tissue, an
especially desirable combination of characteristics for use with an
insecticidal or larvicidal
gene. The TR1' gene fused to nptII (neomycin phosphotransferase II) showed
similar
characteristics. Additional root-preferred promoters include the VfENOD-GRP3
gene
promoter (Kuster et al, Plant Mol Biol 29(4):759-772, 1995); and rolb promoter
(Capana et
al, Plant Mol Biol 25(4):681-691, 1994. See also U.S. Pat. Nos. 5,837,876;
5,750,386;
5,633,363; 5,459,252; 5,401,836; 5,110,732; and 5,023,179.
"Seed-preferred" promoters include both "seed-specific" promoters (those
promoters active
during seed development such as promoters of seed storage proteins) as well as
"seed-
germinating" promoters (those promoters active during seed germination). See
Thompson
et al, BioEssays 10:108, 1989, herein incorporated by reference. Such seed-
preferred
promoters include, but are not limited to, Ciml (cytokinin-induced message);
cZ19B1
(maize 19 kDa zein); and milps (myo-inositol-1-phosphate synthase) (see U.S.
Pat. No.
6,225,529, herein incorporated by reference). Gamma-zein and Glob-1 are
endosperm-
specific promoters. For dicots, seed-specific promoters include, but are not
limited to, bean
0-phaseolin, napin, (3-conglycinin, soybean lectin, cruciferin, and the like.
For monocots,
seed-specific promoters include, but are not limited to, maize 15 kDa zein, 22
kDa zein, 27
kDa zein, g-zein, waxy, shrunken 1, shrunken 2, globulin 1, etc. See also WO
00/12733,
where seed-preferred promoters from endl and end2 genes are disclosed; herein
incorporated by reference. A promoter that has "preferred" expression in a
particular tissue
is expressed in that tissue to a greater degree than in at least one other
plant tissue. Some
tissue-preferred promoters show expression almost exclusively in the
particular tissue.
Where low level expression is desired, weak promoters will be used. Generally,
the term
"weak promoter" as used herein refers to a promoter that drives expression of
a coding
CA 02614353 2008-01-07
WO 2007/006079 PCT/AU2006/000952
-25-
sequence at a low level. By low level expression at levels of about 1/1000
transcripts to
about 1/100,000 transcripts to about 1/500,000 transcripts is intended.
Alternatively, it is
recognized that the term "weak promoters" also encompasses promoters that
drive
expression in only a few cells and not in others to give a total low level of
expression.
Where a promoter drives expression at unacceptably high levels, portions of
the promoter
sequence can be deleted or modified to decrease expression levels.
Such weak constitutive promoters include, for example the core promoter of the
Rsyn7
promoter (WO 99/43838 and U.S. Pat. No. 6,072,050), the core 35S CaMV
promoter, and
the like. Other constitutive promoters include, for example, those disclosed
in U.S. Pat.
Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680;
5,268,463;
5,608,142; and 6,177,611; herein incorporated by reference.
The construct preferably contains additional regulatory elements for efficient
transcription,
for example, a transcription termination (or terminators) sequence.
The term "terminator" refers to a DNA sequence at the end of a transcriptional
unit which
signals termination of transcription. Terminators are 3'-non-translated DNA
sequences
generally containing a polyadenylation signal, which facilitates the addition
of
polyadenylate sequences to the 3'-end of a primary transcript. Terminators
active in plant
cells are known and described in the literature. They may be isolated from
bacteria, fungi,
viruses, animals and/or plants or synthesized de novo.
As with promoter sequences, the terminator may be any termination sequence
which is
operable in the cells, tissues or organs in which it is intended to be used.
Examples of terminators particularly suitable for use in the synthetic genes
of the present
invention include the SV40 polyadenylation signal, the HSV TK polyadenylation
signal,
the CYCl terminator, ADH terminator, SPA terminator, nopaline synthase (NOS)
gene
terminator of Agrobacterium tumefaciens, the terminator of the cauliflower
mosaic virus
(CaMV) 35S gene, the zein gene terminator from Zea mays, the Rubisco small
subunit
CA 02614353 2008-01-07
WO 2007/006079 PCT/AU2006/000952
-26-
gene (SSU) gene terminator sequences, subclover stunt virus (SCSV) gene
sequence
terminators, any rho-independent E. coli terminator, or the lacZ alpha
terminator, amongst
others.
In a particularly preferred embodiment, the terminator is the SV40
polyadenylation signal
or the HSV TK polyadenylation signal which are operable in animal cells,
tissues and
organs, octopine synthase (OCS) or nopaline synthase (NOS) terminator active
in plant
cells, tissue or organs, or the lacZ alpha terminator which is active in
prokaryotic cells.
Those skilled in the art will be aware of additional terminator sequences
which may be
suitable for use in performing the subject invention. Such sequences may
readily be used
without any undue experimentation.
Means for introducing (i.e. transfecting or transforming) cells with the
constructs are well-
known to those skilled in the art.
The constructs described supra are capable of being modified further, for
example, by the
inclusion of marker nucleotide sequences encoding a detectable marker enzyme
or a
functional analogue or derivative thereof, to facilitate detection of the
synthetic gene in a
cell, tissue or organ in which it is expressed. According to this embodiment,
the marker
nucleotide sequences will be present in a translatable format and be
expressed. In addition,
transport sequences may be included to direct one or more agents to particular
plant
organnelles.
Those skilled in the art will be aware of how to produce the constructs
described herein
and of the requirements for obtaining the expression thereof, when so desired,
in a specific
cell or cell-type under the conditions desired. In particular, it will be
known to those skilled
in the art that the genetic manipulations required to perform the present
invention may
require the propagation of a genetic construct described herein or a
derivative thereof in a
prokaryotic cell such as an E. coli cell or a plant cell or an animal cell.
CA 02614353 2008-01-07
WO 2007/006079 PCT/AU2006/000952
-27-
The constructs of the present invention may be introduced to a suitable cell,
tissue or organ
without modification as linear DNA, optionally contained within a suitable
carrier, such as
a cell, virus particle or liposome, amongst others. To produce a genetic
construct, a nucleic
acid is inserted into a suitable vector or episome molecule, such as a
bacteriophage vector,
viral vector or a plasmid, cosmid or artificial chromosome vector which is
capable of being
maintained and/or replicated and/or expressed in the host cell, tissue or
organ into which it
is subsequently introduced.
Accordingly, a further aspect of the present invention provides a genetic
construct which at
least comprises a genetic element encoding one or both pesticidal or
pestistatic agents as
herein described and one or more origins of replication and/or selectable
marker gene
sequences.
Usually, an origin of replication or a selectable marker gene suitable for use
in bacteria is
physically-separated from those genetic sequences contained in the genetic
construct
which are intended to be expressed or transferred to a plant cell, or
integrated into the
genome of a plant cell.
As used herein, the term "selectable marker gene" includes any gene which
confers a
phenotype on a cell on which it is expressed to facilitate the identification
and/or selection
of cells which are transfected or transformed with a genetic construct of the
invention or a
derivative thereof.
Suitable selectable marker genes contemplated herein include the ampicillin-
resistance
gene (Amp'), tetracycline-resistance gene (Tc% bacterial kanamycin-resistance
gene
(Kan'), the zeocin resistance gene (Zeocin is a drug of the bleomycin family
which is trade
mark of InVitrogen Corporation), the AURI-C gene which confers resistance to
the
antibiotic aureobasidin A, phosphinothricin-resistance gene, neomycin
phosphotransferase
gen (nptII), hygromycin-resistance gene, (3-glucuronidase (GUS) gene,
chloramphenicol
acetyltransferase (CAT) gene, green fluorescent protein-encoding gene or the
luciferase
gene, amongst others.
CA 02614353 2008-01-07
WO 2007/006079 PCT/AU2006/000952
-28-
Preferably, the selectable marker gene is the nptII gene or Kan' gene or green
fluorescent
protein (GFP)-encoding gene.
Those skilled in the art will be aware of other selectable marker genes useful
in the
performance of the present invention and the subject invention is not limited
by the nature
of the selectable marker gene.
The present invention extends to all genetic constructs essentially as
described herein,
which include further genetic sequences intended for the maintenance and/or
replication of
said genetic construct in prokaryotes or eukaryotes and/or the integration of
said genetic
construct or a part thereof into the genome of a eukaryotic cell or organism.
Standard methods may be used to introduce the constructs into the cell, tissue
or organ, for
example, liposome-mediated transfection or transformation, transformation of
cells with
attenuated virus particles or bacterial cells, cell mating, transformation or
transfection
procedures known to those skilled in the art.
Additional means for introducing recombinant DNA into plant tissue or cells
include, but
are not limited to, transformation using CaC12 and variations thereof, direct
DNA uptake
into protoplasts, PEG-mediated uptake to protoplasts, microparticle
bombardment,
electroporation, microinjection of DNA, microparticle bombardment of tissue
explant or
cells, vacuum-infiltration of tissue with nucleic acid, or in the case of
plants, T-DNA-
mediated transfer from 4grobactef ium to the plant tissue.
For microparticle bombardment of cells, a microparticle is propelled into a
cell to produce
a transformed cell. Any suitable ballistic cell transformation methodology and
apparatus
can be used in performing the present invention. Exemplary apparatus and
procedures are
disclosed by Stomp et al, (U.S. Patent No. 5,122,466) and Sanford and Wolf
(U.S. Patent
No. 4,945,050). When using ballistic transformation procedures, the genetic
construct may
incorporate a plasmid capable of replicating in the cell to be transformed.
CA 02614353 2008-01-07
WO 2007/006079 PCT/AU2006/000952
-29-
Examples of microparticles suitable for use in such systems include 1 to 5 m
gold
spheres. The DNA construct may be deposited on the microparticle by any
suitable
technique, such as by precipitation.
The methods of the invention involve introducing a polypeptide or
polynucleotide into a
plant. "Introducing" is intended to mean presenting to the plant the
polynucleotide or
polypeptide in such a manner that the sequence gains access to the interior of
a cell of the
plant. The methods of the invention do not depend on a particular method for
introducing a
polynucleotide or polypeptide into a plant, only that the polynucleotide or
polypeptides
gains access to the interior of at least one cell of the plant. Methods for
introducing
polynucleotide or polypeptides into plants are known in the art including, but
not limited
to, stable transformation methods, transient transformation methods, and virus-
mediated
methods.
"Stable transformation" is intended to mean that the nucleotide construct
introduced into a
plant integrates into the genome of the plant and is capable of being
inherited by the
progeny thereof. "Transient transformation" is intended to mean that a
polynucleotide is
introduced into the plant and does not integrate into the genome of the plant
or a
polypeptide is introduced into a plant.
Transformation protocols as well as protocols for introducing nucleotide
sequences into
plants may vary depending on the type of plant or plant cell, i.e., monocot or
dicot,
targeted for transformation. Suitable methods of introducing nucleotide
sequences into
plant cells and subsequent insertion into the plant genome include
microinjection
(Crossway et al, Biotechniques 4:320-334, 1986), electroporation (Riggs et al,
Proc Natl
Acad Sci USA 83:5602-5606, 1986), Agrobacterium-mediated transformation (U.S.
Pat.
Nos. 5,563,055 and 5,981,840), direct gene transfer (Paszkowski et al, EMBO J
3:2717-
2722, 1984), and ballistic particle acceleration (see, for example, U.S. Pat.
Nos. 4,945,050;
5,879,918; 5,886,244; and 5,932,782; Tomes et al, Plant Cell, Tissue, and
Organ Culture:
Fundamental Methods, 1995; and McCabe et al, Biotechnology 6:923-926, 1988);
and
CA 02614353 2008-01-07
WO 2007/006079 PCT/AU2006/000952
-30-
Lecl transformation (WO 00/28058). For potato transformation see Tu et al,
Plant
Molecular Biology 37:829-838, 1998 and Chong et al, Transgenic Research 9:71-
78, 2000.
Additional transformation procedures can be found in Weissinger et al, Ann Rev
Genet
22:421-477, 1988; Sanford et al, Particulate Science and Technology 5:27-37,
1987
(onion); Christou et al, Plant Physiol 87:671-674, 1988 (soybean); McCabe et
al,
Bio/Technology 6:923-926, 1988 (soybean); Finer and McMullen In Vitro Cell Dev
Biol
27P:175-182, 1991 (soybean); Singh et al, Theor Appl Genet 96:319-324, 1998
(soybean);
Datta et al, Biotechnology 8:736-740, 1990 (rice); Klein et al, Proc Natl Acad
Sci USA
85:4305-4309, 1988 (maize); Klein et al, Biotechnology 6:559-563, 1988
(maize); U.S.
Pat. Nos. 5,240,855; 5,322,783 and 5,324,646; Klein et al, Plant Physiol
91:440-444, 1988
(maize); Fromm et al, Biotechnology 8:833-839, 1990 (maize); Hooykaas-Van
Slogteren et
al, Nature (London) 311:763-764, 1984; U.S. Pat. No. 5,736,369 (cereals);
Bytebier et al,
Proc Natl Acad Sci USA 84:5345-5349, 1987 (Liliaceae); De Wet et al, The
Experimental
Manipulation of Ovule Tissues, 197-209, 1985 (pollen); Kaeppler et al, Plant
Cell Reports
9:415-418, 1990 and Kaeppler et al, Theor Appl Genet 84:560-566, 1992 (whisker-
mediated transformation); D'Halluin et al, Plant Cell 4:1495-1505, 1992
(electroporation);
Li et al, Plant Cell Reports 12:250-255, 1993 and Christou and Ford Annals of
Botany
75:407-413, 1995 (rice); Osjoda et al, Nature Biotechnology 14:745-750, 1996
(maize via
Agrobacterium tumefaciens); all of which are herein incorporated by reference.
In a further embodiment of the present invention, the genetic constructs
described herein
are adapted for integration into the genome of a cell in which it is
expressed. Those skilled
in the art will be aware that, in order to achieve integration of a genetic
sequence or genetic
construct into the genome of a host cell, certain additional genetic sequences
may be
required. In the case of plants, left and right border sequences from the T-
DNA of the
Agrobacterium tumefaciens Ti plasmid will generally be required.
The present invention further extends to an isolated cell, tissue or organ
comprising the
constructs or parts thereof. The present invention extends further to
regenerated tissues,
organs and whole organisms derived from said cells, tissues and organs and to
propagules
and progeny thereof as well as seeds and other reproductive material.
CA 02614353 2008-01-07
WO 2007/006079 PCT/AU2006/000952
-31-
For example, plants may be regenerated from transformed plant cells or tissues
or organs
on hormone-containing media and the regenerated plants may take a variety of
forms, such
as chimeras of transformed cells and non-transformed cells; clonal
transformants (e.g. all
cells transformed to contain the expression cassette); grafts of transformed
and
untransformed tissue (e.g. a transformed root stock grafted to an
untransformed scion in
citrus species). Transformed plants may be propagated by a variety of means,
such as by
clonal propagation or classical breeding techniques. For example, first
generation (or Tl)
transformed plants may be selfed to give homozygous second generation (or T2)
transformed plants, and the T2 plants further propagated through classical
breeding
techniques.
The present invention contemplates any other DNA sequence differing in its
codon usage
but encoding the same protein or a similar protein with substantially the same
pesticidal or
pestistatic activity, can be constructed, depending on the particular purpose.
It has been
described in some prokaryotic and eucaryotic expression systems that changing
the codon
usage to that of the host cell is desired for gene expression in foreign hosts
(Bennetzen &
Hall, J Biol Chem 257:3026, 1982; Itakura, Science 198:1056-1063, 1977). Codon
usage
tables are available in the literature (Wada et al, Nucl Acids Res 18:2367-
1411, 1990;
Murray et al, Nucleic Acids Research 17:477-498, 1989) and in the major DNA
sequence
databases. Accordingly, synthetic DNA sequences can be constructed so that the
same or
substantially the same proteins are produced. It is evident that several DNA
sequences can
be devised once the amino acid sequence of the instant agents proteins of this
invention.
Such other DNA sequences include synthetic or semi-synthetic DNA sequences
that have
been changed in order to inactivate certain sites in the gene, e.g. by
selectively inactivating
certain cryptic regulatory or processing elements present in the native
sequence, or by
adapting the overall codon usage to that of a more related host organism,
preferably that of
the host organism in which expression is desired. Synthetic DNA sequences
could also be
made following the procedures described in EP 0 385 962, EP 0 618 967, or EP 0
682 115.
Small modifications to a DNA sequence such as described above can be routinely
made by
CA 02614353 2008-01-07
WO 2007/006079 PCT/AU2006/000952
-32-
PCR-mediated mutagenesis (Ho et al, Gene 77:51-59, 1989; White et al, Trends
in Genet
5:185-189, 1989). New synthetic or semi-synthetic genes can be made by
automated DNA
synthesis and ligation of the resulting DNA fragments.
To prevent or delay the development of resistance by pests, it is preferred to
also express in
the same plant host, preferably a transgenic plant, the other protein or
protein complex,
which has a different mode of action, and a high toxicity to the same pest
targeted by the
first agent (e.g. serine proteinase inhibitor) when produced in a transgenic
host, preferably
a plant. Suitable candidates to be combined with the serine proteinase
inhibitor include the
mature VIP1Aa protein when combined with the mature VIP2Aa or VIP2Ab protein
of
PCT publication WO 96/10083 in case these VIP proteins have a different mode
of action
compared to the serine proteinase inhibitors; the corn rootworm toxins of
Photorhabdus or
Xenorhabdus spp., e.g., the insecticidal proteins of Photorhabdus luminescens
W-14 (Guo
et al, J Biol Chem 274:9836-9842, 1999); the CryET70 protein of WO 00/26378;
the
insecticidal proteins produced by Bt strains PS80JJ1, PS149B1 and PS167H2 as
described
in WO 97/40162, particularly the about 14 kD and about 44 kD proteins of Bt
strain
PS149B1; the Cry3Bb protein of U.S. Pat. No. 6,023,013; protease inhibitors
such as the
N2 and Rl cysteine proteinase inhibitors of soybean (Zhao et al, Plant Physiol
111:1299-
1306, 1996) or oryzastatine such as rice cystatin (Genbank entry S49967), corn
cystatin
(Genbank entries D38130, D10622, D63342) such as the corn cystatin expressed
in plants
as described by Irie et al, Plant Mol Biol 30:149-157, 1996). Also included
herein are all
equivalents and variants, such as truncated proteins retaining insecticidal
activity, of any of
the above proteins.
DNA of the encoding serine proteinase inhibitor genes of the subject
invention, can be
ligated in suitable expression vectors and transformed in E. coli, and the
clones can then be
screened by conventional colony immunoprobing methods (French et al, Anal
Biochem
156:417-423, 1986) for expression of the toxin with monoclonal or polyclonal
antibodies
raised against proteinase inhibitor. Also, the DNA can be ligated in suitable
Bt shuttle
vectors (Lereclus et al, Bio/Technology 10:418, 1992) and transformed in a
crystal minus
Bt-mutant. The clones are then screened for production of ISP proteins (by SDS-
PAGE,
CA 02614353 2008-01-07
WO 2007/006079 PCT/AU2006/000952
-33-
Western blot and/or insect assay).
The genes encoding the invention can be sequenced in a conventional manner
(Maxam and
Gilbert Methods in Enzymol 65:499-560, 1980; Sanger Proc Natl Acad Sci USA
74:5463-
5467, 1977) to obtain the DNA sequence. Sequence comparisons indicated that
the genes
are different from previously described genes encoding proteins secreted
during the
vegetative growth phase of Bacillus or other bacterial species and Bacillus
thuringiensis
crystal proteins with activity against Coleoptera (Crickmore, et al,
Microbiology and
Molecular Biology Reviews 62:807-813, 1998; WO 98/44137, WO 94/21795, WO
96/10083, WO 00/09697, WO 9957282, and WO 9746105).
A pesticidal or pestistatic composition of the subject invention can also be
formulated in a
conventional manner using the microorganisms transformed with the genes, or
preferably
their respective proteins or pesticidally or pestistatically effective
portions thereof as an
active ingredient, together with suitable carriers, diluents, emulsifiers
and/or dispersants
(e.g., as described by Bernhard and Utz, An Environmental Biopesticide: Theory
and
Practice 255-267, 1993). This pesticidal or pestistatic composition can be
formulated as a
wettable powder, pellets, granules or dust or as a liquid formulation with
aqueous or non-
aqueous solvents as a foam, gel, suspension, concentrate, etc. Known
microorganisms
include cells of Pseudomonas or other bacteria that serve to encapsulate the
proteins in a
stable environment prior to application to the insects. Also included in the
invention is a
product comprising the agents described herein as a combined preparation for
simultaneous, separate or sequential use to protect corn plants against corn
rootworms,
particularly such product is an insecticidal composition or a transgenic corn
plant.
A method for controlling pests in accordance with the subject invention can
comprise
applying (e.g., spraying), to a locus (area) to be protected, an pesticidal or
pestistatic
effective amount of the agents or host cells transformed with the gene of the
subject
invention. The locus to be protected can include, for example, the habitat of
the insect
pests or growing vegetation or an area where vegetation is to be grown.
CA 02614353 2008-01-07
WO 2007/006079 PCT/AU2006/000952
-34-
To obtain the agents , cells of the recombinant hosts expressing the agents
can be grown in
a conventional manner on a suitable culture medium and the protein can then be
obtained
from the medium using conventional means. The agent can then be separated and
purified
by standard techniques such as chromatography, extraction, electrophoresis, or
the like.
The protease-resistant toxin form can then be obtained by protease, e.g.
cysteine or serine
protease, digestion of the protein.
While the invention does not depend on a particular biological mechanism for
increasing
the resistance of a plant to a plant pest, expression of the nucleotide
sequences of the
invention in a plant can result in the production of the pesticidal or
pestistatic proteins of
the invention and in an increase in the resistance of the plant to a plant
pest. The plants of
the invention find use in agriculture in methods for impacting plant pests.
Certain
embodiments of the invention provide transformed crop plants, such as, for
example,
cotton plants, which find use in methods for impacting insect pests of the
plant.
A "subject plant or plant cell" is one in which genetic alteration, such as
transformation,
has been effected as to a gene of interest, or is a plant or plant cell which
is descended
from a plant or cell so altered and which comprises the alteration. A
"control" or "control
plant" or "control plant cell" provides a reference point for measuring
changes in
phenotype of the subject plant or plant cell.
A control plant or plant cell may comprise, for example: (a) a wild-type plant
or cell, i.e.,
of the same genotype as the starting material for the genetic alteration which
resulted in the
subject plant or cell; (b) a plant or plant cell of the same genotype as the
starting material
but which has been transformed with a null construct (i.e., with a construct
which has no
known effect on the trait of interest, such as a construct comprising a marker
gene); (c) a
plant or plant cell which is a non-transformed segregant among progeny of a
subject plant
or plant cell; (d) a plant or plant cell genetically identical to the subject
plant or plant cell
but which is not exposed to conditions or stimuli that would induce expression
of the gene
of interest; or (e) the subject plant or plant cell itself, under conditions
in which the gene of
interest is not expressed.
CA 02614353 2008-01-07
WO 2007/006079 PCT/AU2006/000952
-35-
As indicated above, one of skill in the art will readily acknowledge that
advances in the
field of molecular biology such as site-specific and random mutagenesis,
polymerase chain
reaction methodologies, and protein engineering techniques provide an
extensive
collection of tools and protocols suitable for use to alter or engineer both
the amino acid
sequence and underlying genetic sequences of proteins of agricultural
interest.
Thus, the Cry9 family proteins of the invention may be altered in various ways
including
amino acid substitutions, deletions, truncations, and insertions. Methods for
such
manipulations are generally known in the art. For example, amino acid sequence
variants
of the pesticidal or pestistatic proteins can be prepared by introducing
mutations into a
synthetic nucleic acid (e.g., DNA molecule). Methods for mutagenesis and
nucleic acid
alterations are well known in the art. For exainple, designed changes can be
introduced
using an oligonucleotide-mediated site-directed mutagenesis technique. See,
for example,
Kunkel Proc Natl Acad Sci USA 82:488-492, 1985; Kunkel et al, Methods in
Enzymol
154:367-382, 1987; U.S. Pat. No. 4,873,192; Walker and Gaastra, Techniques in
Molecular Biology 1983, and the references cited therein.
The mutagenized Cry9 family nucleotide sequences of the invention may be
modified so as
to change about 1, 2, 3, 4, 5, 6, 8, 10, 12 or more of the amino acids present
in the primary
sequence of the encoded polypeptide. Alternatively, even more changes from the
native
sequence may be introduced such that the encoded protein may have at least
about 1% or
2%, or about 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, or even about 13%,
14%,
15%, 16%, 17%, 18%, 19%, or 20%, 21%, 22%, 23%, 24%, or 25%, 30%, 35%, or 40%
or
more of the codons altered, or otherwise modified compared to the
corresponding wild-
type protein. In the same manner, the encoded protein may have at least about
1% or 2%,
or about 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, or even about 13%, 14%,
15%,
16%, 17%, 18%, 19%, or 20%, 21%, 22%, 23%, 24%, or 25%, 30%, 35%, or 40% or
more
additional codons compared to the corresponding wild-type protein. It should
be
understood that the mutagenized Cry9 family nucleotide sequences of the
present invention
are intended to encompass biologically functional, equivalent peptides which
have
CA 02614353 2008-01-07
WO 2007/006079 PCT/AU2006/000952
-36-
pesticidal activity, such as an improved pesticidal activity as determined by
antifeedant
properties against fall armyworm larvae. Such sequences may arise as a
consequence of
codon redundancy and functional equivalency that are known to occur naturally
within
nucleic acid sequences and the proteins thus encoded.
One of skill in the art would recognize that amino acid additions and/or
substitutions are
generally based on the relative similarity of the amino acid side-chain
substituents, for
example, their hydrophobicity, charge, size, and the like. Exemplary amino
acid
substitution groups that take various of the foregoing characteristics into
consideration are
well known to those of skill in the art and include: arginine and lysine;
glutamate and
aspartate; serine and threonine; glutamine and asparagine; and valine,
leucine, and
isoleucine.
In certain embodiments the nucleic acid sequences of the present invention can
be stacked
with any combination of polynucleotide sequences of interest in order to
create plants with
a desired phenotype. For example, the polynucleotides of the present invention
may be
stacked with any other polynucleotides encoding polypeptides having pesticidal
and/or
insecticidal activity, such as other B. thuringiensis toxic proteins
(described in U.S. Pat.
Nos. 5,366,892; 5,747,450; 5,736,514; 5,723,756; 5,593,881; and Geiser et al,
Gene
48:109, 1986), pentin (described in U.S. Pat. No. 5,981,722) and the like. The
combinations generated can also include multiple copies of any one of the
polynucleotides
of interest. The polynucleotides of the present invention can also be stacked
with any other
gene or combination of genes to produce plants with a variety of desired trait
combinations
including but not limited to traits desirable for animal feed such as high oil
genes (e.g.,
U.S. Pat. No. 6,232,529); balanced amino acids (e.g. hordothionins (U.S. Pat.
Nos.
5,990,389; 5,885,801; 5,885,802; and 5,703,049); barley high lysine
(Williamson et al, Eur
J Biochem 165:99-106,1987; and WO 98/20122) and high methionine proteins
(Pedersen
et al, JBiol Chem 261:6279, 1986; Kirihara et al, Gene 71:359, 1988; and
Musumura et al,
Plant Mol Biol 12:123, 1989); increased digestibility (e.g., modified storage
proteins (U.S.
Application Ser. No. 10/053,410, filed Nov. 7, 2001); and thioredoxins (U.S.
Application
Ser. No. 10/005,429, filed Dec. 3, 2001)), the disclosures of which are herein
incorporated
CA 02614353 2008-01-07
WO 2007/006079 PCT/AU2006/000952
-37-
by reference.
The polynucleotides of the present invention can also be stacked with traits
desirable for
disease or herbicide resistance (e.g., fumonisin detoxification genes (U.S.
Pat. No.
5,792,931); avirulence and disease resistance genes (Jones et al, Science
266:789, 1994;
Martin et al, Science 262:1432, 1993; and Mindrinos et al, Cell 78:1089,
1994);
acetolactate synthase (ALS) mutants that lead to herbicide resistance such as
the S4 and/or
Hra mutations; inhibitors of glutainine synthase such as phosphinothricin or
basta (e.g., bar
gene); and glyphosate resistance (EPSPS gene and GAT gene as disclosed in U.S.
application Ser. No. 10/004,357; and 10/427,692); and traits desirable for
processing or
process products such as high oil (e.g., U.S. Pat. No. 6,232,529); modified
oils (e.g., fatty
acid desaturase genes (U.S. Pat. No. 5,952,544; WO 94/11516)); modified
starches (e.g.,
ADPG pyrophosphorylases (AGPase), starch synthases (SS), starch branching
enzymes
(SBE) and starch debranching enzymes (SDBE)); and polymers or bioplastics
(e.g., U.S.
Pat. No. 5,602,321; beta-ketothiolase, polyhydroxybutyrate synthase, and
acetoacetyl-CoA
reductase (Schubert et al, J Bacteriol 170:5837-5847, 1988) facilitate
expression of
polyhydroxyalkanoates (PHAs)), the disclosures of which are herein
incorporated by
reference. One could also combine the polynucleotides of the present invention
with
polynucleotides providing agronomic traits such as male sterility (e.g., see
U.S. Pat. No.
5,583,210), stalk strength, flowering time, or transformation technology
traits such as cell
cycle regulation or gene targeting (e.g. WO 99/61619; WO 00/17364; WO
99/25821), the
disclosures of which are herein incorporated by reference.
These stacked combinations can be created by any method including but not
limited to
cross breeding plants by any conventional or TopCrosse methodology, or genetic
transformation. If the traits are stacked by genetically transforming the
plants, the
polynucleotide sequences of interest can be combined at any time and in any
order. For
example, a transgenic plant comprising one or more desired traits can be used
as the target
to introduce furtller traits by subsequent transformation. The traits can be
introduced
simultaneously in a co-transformation protocol with the polynucleotides of
interest
provided by any combination of transformation cassettes. For example, if two
sequences
CA 02614353 2008-01-07
WO 2007/006079 PCT/AU2006/000952
-38-
will be introduced, the two sequences can be contained in separate
transformation cassettes
(trans) or contained on the same transformation cassette (cis). Expression of
the sequences
can be driven by the same promoter or by different promoters. In certain
cases, it may be
desirable to introduce a transformation cassette that will suppress the
expression of the
polynucleotide of interest. This may be combined with any combination of other
suppression cassettes or overexpression cassettes to generate the desired
combination of
traits in the plant. It is further recognized that polynucleotide sequences
can be stacked at a
desired genomic location using a site-specific recombination system. See, for
example,
W099/25821, W099/25854, W099/25840, W099/25855, and W099/25853, all of which
are herein incorporated by reference.
Compositions of the invention find use in protecting plants, seeds, and plant
products in a
variety of ways. For example, the compositions can be used in a method that
involves
placing an effective ainount of the pesticidal or pestistatic composition in
the environment
of the pest by a procedure selected from the group consisting of spraying,
dusting,
broadcasting, or seed coating.
Before plant propagation material (fruit, tuber, bulb, corm, grains, seed),
but especially
seed, is sold as a commercial product, it is customarily treated with a
protectant coating
comprising herbicides, insecticides, fungicides, bactericides, nematicides,
molluscicides, or
mixtures of several of these preparations, if desired together with further
carriers,
surfactants, or application-promoting adjuvants customarily employed in the
art of
formulation to provide protection against damage caused by bacterial, fungal,
or animal
pests. In order to treat the seed, the protectant coating may be applied to
the seeds either by
impregnating the tubers or grains with a liquid fomlulation or by coating them
with a
combined wet or dry formulation. In addition, in special cases, other methods
of
application to plants are possible, e.g., treatment directed at the buds or
the fruit.
The plant seed of the invention comprising the nucleotide sequences encoding
the agents
may be treated with a seed protectant coating comprising a seed treatment
compound, such
as, for example, captan, carboxin, thiram, methalaxyl, pirimiphos-methyl, and
others that
CA 02614353 2008-01-07
WO 2007/006079 PCT/AU2006/000952
-39-
are commonly used in seed treatment. In one embodiment within the scope of the
invention, a seed protectant coating comprising a pesticidal composition of
the invention is
used alone or in combination with one of the seed protectant coatings
customarily used in
seed treatment.
In the present invention, a composition includes whole organisms, cells,
spore(s),
pesticidal or pestistatic protein(s), pesticidal or pestistatic component(s),
pest-impacting
component(s), mutant(s), living or dead cells and cell components, including
mixtures of
living and dead cells and cell components, and including broken cells and cell
components
or an isolated pesticidal or pestistatic protein can be formulated with an
acceptable carrier
into a pesticidal composition(s) that is, for example, a suspension, a
solution, an emulsion,
a dusting powder, a dispersible granule, a wettable powder, and an
emulsifiable
concentrate, an aerosol, an impregnated granule, an adjuvant, a coatable
paste, and also
encapsulations in, for example, polymer substances.
Such compositions disclosed above may be obtained by the addition of a surface-
active
agent, an inert carrier, a preservative, a humectant, a feeding stimulant, an
attractant, an
encapsulating agent, a binder, an emulsifier, a dye, a UV protectant, a
buffer, a flow agent
or fertilizers, micronutrient donors, or other preparations that influence
plant growth. One
or more agrochemicals including, but not limited to, herbicides, insecticides,
fungicides,
bactericides, nematicides, molluscicides, acaracides, plant growth regulators,
harvest aids,
and fertilizers, can be combined with carriers, surfactants or adjuvants
customarily
employed in the art of formulation or other components to facilitate product
handling and
application for particular target pests. Suitable carriers and adjuvants can
be solid or liquid
and correspond to the substances ordinarily employed in formulation
technology, e.g.,
natural or regenerated mineral substances, solvents, dispersants, wetting
agents, tackifiers,
binders, or fertilizers. The active ingredients of the present invention are
normally applied
in the form of compositions and can be applied to the crop area, plant, or
seed to be treated.
For example, the compositions of the present invention may be applied to grain
in
preparation for or during storage in a grain bin or silo, etc. The
compositions of the present
invention may be applied simultaneously or in succession with other compounds.
Methods
CA 02614353 2008-01-07
WO 2007/006079 PCT/AU2006/000952
-40-
of applying an active ingredient of the present invention or an agrochemical
composition
of the present invention that contains at least one of the pesticidal proteins
produced by the
bacterial strains of the present invention include, but are not limited to,
foliar application,
seed coating, and soil application. The number of applications and the rate of
application
depend on the intensity of infestation by the corresponding pest.
Suitable surface-active agents include, but are not limited to, anionic
compounds such as a
carboxylate of, for example, a metal; a carboxylate of a long chain fatty
acid; an N-
acylsarcosinate; mono or di-esters of phosphoric acid with fatty alcohol
ethoxylates or salts
of such esters; fatty alcohol sulfates such as sodium dodecyl sulfate, sodium
octadecyl
sulfate or sodium cetyl sulfate; etlioxylated fatty alcohol sulfates;
ethoxylated alkylphenol
sulfates; lignin sulfonates; petroleum sulfonates; alkyl aryl sulfonates such
as alkyl-
benzene sulfonates or lower alkylnaphtalene sulfonates, e.g., butyl-
naphthalene sulfonate;
salts of sulfonated naphthalene-formaldehyde condensates; salts of sulfonated
phenol-
formaldehyde condensates; more complex sulfonates such as the amide
sulfonates, e.g., the
sulfonated condensation product of oleic acid and N-methyl taurine; or the
dialkyl
sulfosuccinates, e.g., the sodium sulfonate of dioctyl succinate. Non-ionic
agents include
condensation products of fatty acid esters, fatty alcohols, fatty acid amides
or fatty-alkyl-
or alkenyl-substituted phenols with ethylene oxide, fatty esters of polyhydric
alcohol
ethers, e.g., sorbitan fatty acid esters, condensation products of such esters
with ethylene
oxide, e.g., polyoxyethylene sorbitar fatty acid esters, block copolymers of
ethylene oxide
and propylene oxide, acetylenic glycols such as 2,4,7,9-tetraethyl-5-decyn-4,7-
diol, or
ethoxylated acetylenic glycols. Examples of a cationic surface-active agent
include, for
instance, an aliphatic mono-, di-, or polyamine such as an acetate,
naphthenate or oleate; or
oxygen-containing amine such as an amine oxide of polyoxyethylene alkylamine;
an
amide-linked amine prepared by the condensation of a carboxylic acid with a di-
or
polyamine; or a quaternary ammonium salt.
Examples of inert materials include but are not limited to inorganic minerals
such as
kaolin, phyllosilicates, carbonates, sulfates, phosphates, or botanical
materials such as
cork, powdered corncobs, peanut hulls, rice hulls, and walnut shells.
CA 02614353 2008-01-07
WO 2007/006079 PCT/AU2006/000952
-41-
The compositions of the present invention can be in a suitable form for direct
application
or as a concentrate of primary composition that requires dilution with a
suitable quantity of
water or other diluant before application. The pesticidal concentration will
vary depending
upon the nature of the particular formulation, specifically, whether it is a
concentrate or to
be used directly. The composition contains 1 to 98% of a solid or liquid inert
carrier, and 0
to 50% or 0.1 to 50% of a surfactant. These compositions will be administered
at the
labeled rate for the commercial product, for example, about 0.01 lb-5.0 lb.
per acre when
in dry form and at about 0.01 pts.-10 pts. per acre when in liquid form.
Insect pests may be tested for pesticidal or pestistatic activity of
compositions of the
instant invention in early developmental stages, e.g., as larvae or other
immature forms.
The insects may be reared in total darkness at from about 20 C. to about 30 C.
and from
about 30% to about 70% relative humidity. Bioassays may be performed as
described in
Czapla and Lang JEcon Entomol 83(6):2480-2485, 1990. Methods of rearing insect
larvae
and performing bioassays are well known to one of ordinary skill in the art.
A wide variety of bioassay techniques are known to one skilled in the art.
General
procedures include addition of the experimental compound or organism to the
diet source
in an enclosed container. Pesticidal activity can be measured by, but is not
limited to,
changes in mortality, weight loss, attraction, repellency and other behavioral
and physical
changes after feeding and exposure for an appropriate length of time.
Bioassays described
herein can be used with any feeding insect pest in the larval or adult stage.
The present invention is further described by the following non-limiting
Examples.
CA 02614353 2008-01-07
WO 2007/006079 PCT/AU2006/000952
-42-
EXAMPLE 1
NaPI
A bioassay was developed based on feeding larvae of H. arnaigera on cotton
leaves
homozygous for NaPI and coated with varying concentrations of CrylAc. The NaPI
leaves were specifically selected to give a small reduction in weight of the
larvae. (In
previous bioassays, without CrylAc, leaves have been used which give a greater
effect on
larval growth and development.)
Preparation of Leaves
The leaves were cut into discs using a hole puncher and coated with CrylAc
solutions by
immersing in the test solution for 5 minutes. The discs were removed from the
solution
with tweezers and then air dried on plastic mesh (approx. 30 min). When there
was no
visible moisture on the leaf disc surface, the discs were transferred to wells
of a 24 well
plate.
The wells contained filter paper moistened with water. Untransformed leaf
material of cv
Coker was used in control experiments.
Handling of Eggs
H. armigera eggs (supplied by Department of Primary Industry & Fisheries,
Indooroopilly,
Queensland, Australia) were delivered by air freight to the laboratory and
incubated (18-
20 C depending on developmental stage) to the brown egg stage (pre hatching).
Eggs were
suspended in polyacrylate (Aquakeep polyacrylate, 1 mg/ml H20) and one egg
placed in
each well containing a leaf disc. Single eggs were then transferred to
individual wells.
The wells were covered with perforated Mylar film and incubated at 25 in a
controlled
temperature cabinet with lights (16 hr light, 8 hr dark). The larvae were
removed at day 6
or 7 and individually weighed.
CA 02614353 2008-01-07
WO 2007/006079 PCT/AU2006/000952
- 43 -
Preparation of CrylAc solution
CrylAc crystal protein/spore mix (10 mg from CSIRO, Canberra, Australia) was
dissolved
in 0.9 ml of 50mM Na2 C03, (pH 10.5). Bovine Pancreatic Trypsin (Sigma T-8642
Type
8) stock 0.75 mg/ml was prepared in the same buffer and 0.1 ml of stock added
to the
crystal protein spore mix and incubated overnight at room temperature. The
solution was
centrifuged (14,000 rpm) for 5 minutes and the supernatant collected and
stored at -70 C.
The concentration of activated CrylAc protein was estimated as 2.5 mg/ml on
the basis of
50% starting material being CrylAc, and 50% of this being the final activated
material.
Estimated molecular weights protoxin 133 kD: activated toxin 66 kD. Dilutions
were
prepared in 0.03% v/v Triton Ag 98 as wetting agent.
A suitable assay for CrylAc is described in Liao et al, Jlnvertebrate Pathol
80:55-63.
Experiment 1- Examination of the effect on mass of larvae
In this experiment, sub-lethal concentrations of CrylAc were selected. The
focus was to
examine the effect on mass of the larvae. The second fully expanded leaf (from
growing
tip) of glasshouse grown plants was used for disc preparation. Experiment
duration was 7
days.
Results
The egg hatch was 70-100%; 12 eggs per test (1 egg/well). The mass of
surviving larvae is
shown in Figure 1. There was insignificant mortality.
It appears that both CrylAc and NaPI contribute to the decrease in weight of
the larvae
with increasing concentrations of CrylAc. (The result for cv Coker at 10 ug
Cry 1 Ac seems
anomalous.
CA 02614353 2008-01-07
WO 2007/006079 PCT/AU2006/000952
-44-
Experiment 2 - Effects of high concentration of CryIAc
This experiment was designed to achieve mortality by using higher
concentrations of
CrylAc. Higher numbers of eggs were used. For Day 1, the third fully expanded
leaf was
used. For Day 4, additions of leaf material of the second fully expanded leaf
was used.
Experiment duration was 6 days.
Results
The egg hatch was 50-83%; 40 eggs per test (1 egg/well). The mass of surviving
larvae is
shown in Figure 2. The mortality is shown in Figure 3.
This experiment is essentially the same as experiment 1 except that the
concentrations of
CrylAc are increased over the range 50 to 250 ug/ml. These concentrations
result in
significant mortality, and thus the data presented refers to a subset of the
original sample
that have survived at each CrylAc concentration.
The difference in mass between the larvae feeding on Coker or NaPI leaves is
essentially
the same at each concentration of CrylAc over the range 50-250 ug/ml.
With no added CrylAc, there is not significant difference in mortality. At
each
concentration of CrylAc there is a significant enhancement of mortality in
leaves
expressing NaPI over that obtained with Coker control material.
In no case, in any of the experiments, did expression of the NaPI gene
diminish the effect
of CrylAc.
CA 02614353 2008-01-07
WO 2007/006079 PCT/AU2006/000952
- 45 -
Experiment 3 - Bioassay with third instar larvae
The aim of this experiment was to determine the effect of Bt toxin (CrylAc)
and NaPI on
3rd to 4th instar H. armigera larvae.
Larvae (48) were grown for 11 days on cotton leaves cv Coker before being
transferred to
new plates containing fresh leaf discs. The 48 larvae were separated into 4
treatments of 12
larvae.
The four treatments were as follows:
- Untransformed Coker 315 leaves
- Transgenic NaPI leaves
- Untransformed Coker leaves coated with 100 ug/mL CrylAc
- Transgenic NaPI leaves coated with 100 ug/mL CrylAc
The larvae were grown for a further 20 hours at 25 C. Larval weight was
recorded at days
7, 11 and 12.
Experimental details
Two, 24 well plates were used. Single H. armigera eggs, suspended in
Polyacrylate (lg/L),
were placed in each well. Initially one leaf disc was placed in each well.
Further discs were
added when required.
Coker leaves at positions 3, 4 and 5 were used for the first 11 days. The
leaves were
harvested, cut into discs and pooled before they were added to the wells. Only
leaves from
position 2 or 3 were used for the treatments.
CA 02614353 2008-01-07
WO 2007/006079 PCT/AU2006/000952
-46-
Results
All H. armigera eggs used in the assay hatched (Table 1).At day 11 the larvae
were
weighed and then fed the test diets. About 20 hours later (day 12) the larvae
were weighed
again (Table 1, Figure 4).
Table 1. Results of H. arnaigera bioassay using 3ra instar larvae
CrylAc concentration 0 ug/mL 100ug/mL
C N C N
No eggs 12 12 12 12
Hatched larvae 12 12 12 12
Surviving larvae 11 11 12 12
Average weight (mg) day 11 64.6 65.3 67.1 69.0
Average weight (mg) day 12 95.2 91.0 81.4 74.5
1o increase in mass day 11-12 47% 39% 21% 8%
C- untransforined Coker leaves, N- transgenic leaves expressing NaPI
There was a small difference in average weight gained between the larvae fed
on control
Coker leaves and the transgenic NaPI expressing leaves over the 20 hours. The
control
larvae increased their weight by an average of 48% while the NaPI fed larvae
increased
their weight by an average of 41% (Table 1, Figure 5). Note that the average
weight of
larvae at day 11 was slightly different for each test group (Table 1) and this
must be taken
into account in the calculations. Since the larvae were all fed the same diet,
this difference
is due to natural variation of the larvae.
There was a greater difference in weight when the larvae were fed CrylAc. The
CrylAc
fed larvae only increased their weight by an average of 21% (Table 1, Figure
5).
CA 02614353 2008-01-07
WO 2007/006079 PCT/AU2006/000952
-47-
Finally, larvae fed transgenic leaves coated with Cry1Ac only increased their
weight by an
average of 7%. It is interesting to note that 3 out of the 121arvae actually
lost weight or did
not increase in weight (Table 1, Figure 5).
These results suggest that the combination of NaPI and CrylAc increases the
effect in
diminishing larval growth. These results are in line with those obtained from
Examples 1
and 2 in which freshly hatched larvae were allowed to feed for 7 days on test
leaves.
CA 02614353 2008-01-07
WO 2007/006079 PCT/AU2006/000952
-48-
EXAMPLE 2
Properties of midgut membrane
When H arnaigera larvae are fed a diet containing NaPI or Bt proteins, such as
CrylAc,
there are different effects on the gut epithelial cells.
The presence of Bt in the diet of lepidopteran pests results in a swelling of
the columnar
cells and a proliferation of stem cells at the base of the epithelial layer.
Ultimately cells in
the epithelial layer burst when exposed to Bt and related bacterial toxins. In
contrast, the
presence of NaPIs in the diet causes a limited swelling of cells in the
epithelial layer
without bursting. There is some associated movement of material into the
intercellular
spaces. When the diet contains both Bt and NaPI, there is an extensive and
more rapid
breakdown of the epithelial cell structures. The intercellular spaces are more
distended and
there is enhanced access of material to the haemolymph. Material which
penetrates through
to the haemolyinph may then come into contact with proteases involved in the
innate
immunity response. In particular, when PIs gain access to the haemolymph they
can
interfere with the protein cascades essential to this innate immunity
response. The ability
of the insect gut to regenerate after damage from either toxin is reduced.
Those skilled in the art will appreciate that the invention described herein
is susceptible to
variations and modifications other than those specifically described. It is to
be understood
that the invention includes all such variations and modifications. The
invention also
includes all of the steps, features, compositions and compounds referred to,
or indicated in
this specification, individually or collectively, and any and all combinations
of any two or
more of said steps or features.
CA 02614353 2008-01-07
WO 2007/006079 PCT/AU2006/000952
-49-
BIBLIOGRAPHY
Aronson Cell Mol Life Sci 59(3):417-425, 2002
Bennetzen & Hall, JBiol Chem 257:3026, 1982
Bernhard and Utz, An Environmental Biopesticide: Theory and Practice 255-267,
1993
Bogusz et al, Plant Cell 2(7):633-641, 1990
Brown and Ryan, Biochemistry 23:3418-3422, 1984
Bryant et al, Biochemistry 15:3418-3424, 1976
Bytebier et al, Proc Natl Acad Sci USA 84:5345-5349, 1987
Canevascini et al, Plant Physiol 112(2):513-524, 1996
Capana et al, Plant Mol Biol 25(4):681-691, 1994
Chen et al, Plant J 10:955-966, 1996
Chong et al, Transgenic Research 9:71-78, 2000
Christensen et al, Plant Mol Biol 12:619-632, 1989
Christensen et al, Plant Mol Biol 18:675-689, 1992
Christou et al, Plant Physiol 87:671-674, 1988
Christou and Ford Annals of Botany 75:407-413, 1995
CA 02614353 2008-01-07
WO 2007/006079 PCT/AU2006/000952
-50-
Cordero et al, In: General Meeting of the International Program on Rice
Biotechnology of
the Rockefeller Foundation, Malacca, Malaysia, 1997
Cordero et al, Physiol Mol Plant Path 41:189-200, 1992
Corderok et al, Plant J 6(2) :141-150, 1994
Crickmore, et al, Microbiology and Molecular Biology Reviews 62:807-813, 1998
Crossway et al, Biotechniques 4:320-334, 1986
Czapla and Lang JEcon Entomol 83(6):2480-2485, 1990
Datta et al, Biotechnology 8:736-740, 1990
De Wet et al, The Experimental Manipulation of Ovule Tissues:197-209, 1985
D'Halluin et al, Plant Cell 4:1495-1505, 1992
Duan et al, Nature Biotechnology 14:494-498, 1996
Eckelkamp et al, FEBS Letters 323:73-76, 1993
Fang et al, Plant Cell 1:141-150, 1989
Finer and McMullen In Vitro Cell Dev Biol 27P:175-182, 1991
French et al, Anal Biochem 156:417-423, 1986
Fromm et al, Biotechnology 8:833-839, 1990
Gatz et al, Mol Gen Genet 227:229-237, 1991
CA 02614353 2008-01-07
WO 2007/006079 PCT/AU2006/000952
-51 -
Geiser et al, Gene 48:109, 1986
Gotor et al, Plant J 3:509-18, 1993
Graham et al, Plants 169:399-405, 1986
Green and Ryan, Science 776-777, 1972
Guevara-Garcia et al, Plant J 4(3):495-505, 1993
Guo et al, JBiol Chem 274:9836-9842, 1999
Hajdukiewicz et al, Plant Mol Biol 25:989-994, 1994
Hansen et al, Mol Gen Genet 254(3):337-343, 1997
Hire et al, Plant Mol Biol 20(2):207-218, 1992
Ho et al, Gene 77:51-59, 1989
Hofte and Whiteley Microbial Reviews 53:242-255, 1989
Hooykaas-Van Slogteren et al, Natzire (London) 311:763-764, 1984
Hsu et al, Plant Sci 143:63-70, 1999
Irie et al, Plant Mol Biol 30:149-157, 1996
Itakura, Science 198:1056-1063, 1977
Jones et al, Science 266:789, 1994
Kaeppler et al, Plant Cell Reports 9:415-418, 1990
CA 02614353 2008-01-07
WO 2007/006079 PCT/AU2006/000952
-52-
Kaeppler et al, Theor Appl Genet 84:560-566, 1992
Kawamata et al, Plant Cell Physiol 38(7):792-803, 1997
Keil et al, EMBO J8:1323-1330, 1989
Keller and Baumgartner Plant Cell 3(10):1051-1061, 1991
Kirihara et al, Gene 71:359, 1988
Klein et al, Biotechnology 6:559-563, 1988
Klein et al, Plant Physiol 91:440-444, 1988
Klein et al, Proc Natl Acad Sci USA 85:4305-4309, 1988
Kunkel et al, Methods in Enzymol 154:367-3 82, 1987
Kunkel Proc Natl Acad Sci USA 82:488-492, 1985
Kuo et al, Arch Biochem Biophys 230:504-510, 1984
Kuster et al, Plant Mol Biol 29(4):759-772, 1995
Kwon et al, Plant Physiol 105:357-67, 1994
Lam Results Probl Cell Differ 20:181-196, 1994
Last et al, TheoN Appl Genet 81:581-588, 1991
Leach and Aoyagi (1991)
Legavre et al, In: Vth International Congress of Plant Molecular Biology,
Singapore, 1997
CA 02614353 2008-01-07
WO 2007/006079 PCT/AU2006/000952
-53-
Lereclus et al, Bio/Technology 10:418, 1992
Li et al, Plant Cell Reports 12:250-255, 1993
Liao et al, Jlnvertebrate Pathol 80:55-63
Limerick Plant Science 79(1):69-76
McCabe et al, Bio/Technology 6:923-926, 1988
McCabe et al, Biotechnology 6:923-926, 1988
McElroy et al, Plant Cell 2:163-171, 1990
McGurl et al, Science 225:1570-1573, 1992
McNellis et al, Plant J 14(2):247-257, 1998
Marineau et al, Plant Mol Biol 9:335-342, 1987
Martin et al, Science 262:1432, 1993
Matsuoka et al, Proc Natl Acad Sci USA 90(20):9586-9590, 1993
Matton et al, Molecular Plant-Microbe Interactions 2:325-331, 1989
Maxam and Gilbert Methods in Enzymol 65:499-560, 1980
Melville and Ryan, Archives of Biochemistry and Biophysics 138:700-702, 1970
Miao et al, Plant Cell 3(1):11-22, 1991
Mindrinos et al, Cell 78:1089, 1994
CA 02614353 2008-01-07
WO 2007/006079 PCT/AU2006/000952
-54-
Murray et al, Nucleic Acids Research 17:477-498, 1989
Musumura et al, Plant Mol Biol 12:123, 1989
Odell et al, Nature 313:810-812, 1985
Orozco et al, Plant Mol Biol 23(6):1129-1138, 1993
Osjoda et al, Nature Biotechnology 14:745-750, 1996
Paszkowski et al, EMBO J 3:2717-2722, 1984
Pathirana et al, Plant J 12:293-304, 1997
Pearce et al, Planta 175:527-531, 1988
Pedersen et al, JBiol Chem 261:6279, 1986
Plunkett et al, Arch Biochem Biophys 213:463-472, 1982
Pujade-Renaud et al, Plant Physiol Biochem 35:85-93, 1997).
Redolfi et al, Meth JPlant Pathol 89:245-254, 1983
Richard Phytochemistry 16:159-169, 1977
Rickauer et al, Plant Physiol 9:1065-1070, 1989
Riggs et al, Proc Natl Acad Sci USA 83:5602-5606, 1986
Rinehart et al, Plant Physiol 112(3):1331-1341, 1996
Rohmeier et al, Plant Mol Biol 22:783-792, 1993
CA 02614353 2008-01-07
WO 2007/006079 PCT/AU2006/000952
-55-
Russell et al, Transgenic Res 6(2):157-168, 1997
Ryan Ann Rev Phytopath 28:425-449, 1990
Samac and Shah, Plant Cell 3:1063-1072, 1991
Sanford et al, Particulate Science and Technology 5:27-37, 1987
Sanger et al, Plant Mol Biol 14(3):433-443, 1990
Sanger Proc Natl Acad Sci USA 74:5463-5467, 1977
Schena et al, Proc Natl Acad Sci USA 88:10421-10425, 1991
Schnepf et al, Microbiol Mol Biol Rev 62(3):775-806, 1998
Schoenbeck et al, Molec Plant-Microbe Interact, 1999
Schubert et al, JBacteriol 170:5837-5847, 1988
Siebertz et al, Plant Cell 1:961-968, 1989
Singh et al, Theor Appl Genet 96:319-324, 1998
Somsisch et al, Mol Gen Genet 2:93-98, 1988
Somsisch et al, Proc Natl Acad Sci USA 83:2427-2430, 1986
Stanford et al, Mol Gen Genet 215:200-208, 1989
Teeri et al, EMBO J8(2):343-350, 1989
Thompson et al, BioEssays 10:108, 1989
CA 02614353 2008-01-07
WO 2007/006079 PCT/AU2006/000952
-56-
Tomes et al, Plant Cell, Tissue, and Organ Culture: Fundamental Methods, 1995
Tu et al, Plant Molecular Biology 37:829-838, 1998
Uknes et al, Plant Cell 4:645-656, 1992
Van Camp et al, Plant Physiol 112(2):525-535, 1996
Van Loon Plant Mol Virol 4:111-116, 1985
Velten et al, EMBO J 3:2723-2730, 1984
Wada et al, Nucl Acids Res 18:2367-1411, 1990
Walker and Gaastra, Techniques in Molecular Biology 1983
Warner et al, Plant J 3:191-201, 1993
Weissinger et al, Ann Rev Genet 22:421-477, 1988
White et al, Trends in Genet 5:185-189, 1989
Williamson et al, Eur JBiochem 165:99-106,1987
Yainamoto et al, Plant Cell Physiol 35(5):773-778, 1994
Yamamoto et al, Plant J 12(2):255-265, 1997
Yang Proc Natl Acad Sci USA 93:14972-14977, 1996
Zhang et al, Proc Natl Acad Sci USA 91:2507-2511, 1994
Zhao et al, Plant Physiol 111:1299-1306, 1996
