U.S. patent application number 09/798373 was filed with the patent office on 2001-09-20 for methods for making male-sterile plants.
This patent application is currently assigned to Pioneer Hi-Bred International, Inc.. Invention is credited to Briggs, Steven P., Gray, John, Hu, Gongshe, Johal, Gurmukh S..
Application Number | 20010023501 09/798373 |
Document ID | / |
Family ID | 22133981 |
Filed Date | 2001-09-20 |
United States Patent
Application |
20010023501 |
Kind Code |
A1 |
Johal, Gurmukh S. ; et
al. |
September 20, 2001 |
Methods for making male-sterile plants
Abstract
Compositions and methods for enhancing disease resistance to a
pathogen in a plant are provided. Methods of the invention comprise
stably transforming a plant with an antisense nucleotide sequence
for a gene encoding an enzyme in the C-5 porphyrin metabolic
pathway and operably linking said antisense sequence to a
pathogen-inducible promoter, such that invasion of a cell by a
pathogen elicits a hypersensitive-like response that results in
confinement of the pathogen to cells of initial contact.
Transformed plants and seeds are provided. Nucleotide sequences
encoding a wild-type maize urod gene useful in the present
invention and the amino acid sequence for the protein encoded
thereby are provided. These compositions are also useful for
regulating cell death in specifically targeted tissues. A maize
lesion mimic, dominant mutant phenotype, designated Les22, and the
molecular basis for its manifestation are also provided.
Inventors: |
Johal, Gurmukh S.;
(Johnston, IA) ; Briggs, Steven P.; (Del Mar,
CA) ; Gray, John; (Toledo, OH) ; Hu,
Gongshe; (Albany, CA) |
Correspondence
Address: |
ALSTON & BIRD LLP
PIONEER HI-BRED INTERNATIONAL, INC.
BANK OF AMERICA PLAZA
101 SOUTH TYRON STREET, SUITE 4000
CHARLOTTE
NC
28280-4000
US
|
Assignee: |
Pioneer Hi-Bred International,
Inc.
|
Family ID: |
22133981 |
Appl. No.: |
09/798373 |
Filed: |
March 2, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09798373 |
Mar 2, 2001 |
|
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09260843 |
Mar 2, 1999 |
|
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60076754 |
Mar 4, 1998 |
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Current U.S.
Class: |
800/286 |
Current CPC
Class: |
C12N 15/8263 20130101;
C12N 15/8243 20130101; C12N 15/8261 20130101; C12N 15/8242
20130101; C12N 15/8289 20130101; Y02A 40/146 20180101; C12N 9/88
20130101; C12N 15/8282 20130101 |
Class at
Publication: |
800/286 |
International
Class: |
C12N 015/82 |
Claims
What is claimed is:
1. A method for producing male sterile plants, said method
comprising transforming said plant with a DNA construct comprising
an antisense nucleotide sequence for a gene encoding an enzyme in
the C-5 porphyrin pathway of said plant operably linked to a stamen
promoter that drives expression in a plant cell, and regenerating
stably transformed plants wherein said gene in the C-5 porphyrin
pathway is selected from the group consisting of: (a) the
nucleotide sequence set forth in SEQ ID NO: 1; (b) a nucleotide
sequence having 90% sequence identity to the sequence of SEQ ID NO:
1; (c) a nucleotide sequence having 80% sequence identity to the
sequence of SEQ ID NO: 1; and, (d) a nucleotide sequence that
encodes the amino acid sequence set forth in SEQ ID NO: 2.
2. The method of claim 1, wherein said antisense nucleotide
sequence comprises at least 20 nucleotides.
3. A transformed plant having stably integrated into its genome a
DNA construct comprising an antisense nucleotide for a gene
encoding an enzyme in the C-5 porphyrin pathway of said plant
operably linked to a stamen-preferred promoter that drives
expression in a plant cell, wherein said gene is selected from the
group consisting of: (a) the nucleotide sequence set forth in SEQ
ID NO: 1; (b) a nucleotide sequence having 90% sequence identity to
the sequence of SEQ ID NO: 1; (c) a nucleotide sequence having 80%
sequence identity to the sequence of SEQ ID NO: 1; and, (d) a
nucleotide sequence that encodes the amino acid sequence set forth
in SEQ ID NO: 2.
4. The plant of claim 3, wherein said antisense nucleotide sequence
comprises at least 20 nucleotides.
5. The plant of claim 3, wherein said plant is male-sterile.
6. The plant of claim 4, wherein said plant is male-sterile.
7. The plant of claim 3, wherein said plant is a monocotyledonous
plant.
8. The plant of claim 7, wherein said monocotyledonous plant is a
maize plant.
9. The plant of claim 5, wherein said plant is a monocotyledonous
plant.
10. The plant of claim 8, wherein said monocotyledonous plant is a
maize plant.
11. Transformed seed of the plant of claim 3.
12. Transformed seed of the plant of claim 5.
13. Transformed seed of the plant of claim 9.
14. Transformed seed of the plant of claim 10.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of U.S. Provisional
Application Ser. No. 60/076,754, filed Mar. 4, 1998 and is a
Divisional of Ser. No. 09/260,843, filed Mar. 2, 1999, the
disclosures of which are herein incorporated by reference.
FIELD OF THE INVENTION
[0002] The invention relates to the genetic manipulation of plants,
particularly to transforming plants with nucleotide sequences that
regulate cell death and enhance disease resistance.
BACKGROUND OF THE INVENTION
[0003] A host of cellular processes enable plants to defend
themselves from disease caused by pathogenic agents. These
processes apparently form an integrated set of resistance
mechanisms that is activated by initial infection and then limits
further spread of the invading pathogenic microorganism.
[0004] Subsequent to recognition of a potentially pathogenic
microbe, plants can activate an array of biochemical responses.
Generally, the plant responds by inducing several local responses
in the cells immediately surrounding the infection site. The most
common resistance response observed in both nonhost and
race-specific interactions is termed the "hypersensitive response"
(HR). In the hypersensitive response, cells contacted by the
pathogen, and often immediately adjacent cells, rapidly collapse
and dry in a necrotic fleck. Other responses include the deposition
of callose, the physical thickening of cell walls by lignification,
and the synthesis of various antibiotic small molecules and
proteins. Genetic factors in both the host and the pathogen
determine the specificity of these local responses, which can be
very effective in limiting the spread of infection to localized
lesions.
[0005] The hypersensitive response in many plant-pathogen
interactions results from the expression of a resistance (R) gene
in the plant and a corresponding avirulence (avr) gene in the
pathogen. The resistance gene in the plant and the avirulence gene
in the pathogen often conform to a gene-for-gene relationship. That
is, resistance to a pathogen is only observed when the pathogen
carries a specific avirulence gene and the plant carries a
corresponding or complementing resistance gene. Hence, there is a
specificity requirement to bring about enhanced disease resistance
using the avr::R gene-for-gene hypersensitive response.
[0006] Many environmental and genetic factors cause general leaf
necrosis in maize and other plants. In addition, numerous recessive
and dominant genes cause the formation of discrete or expanding
necrotic lesions of varying size, shape, and color (see, for
example, Wolter et al. (1993) Mol. Gen. Genet. 239:122; Dietrich et
al. (1994) Cell 77:565; Greenberg et al. (1994) Cell 77:551).
Because lesions of some of these mutants resemble those associated
with known diseases of maize, these genetic defects have been
collectively called disease lesion mimics.
[0007] Lesion mimic mutations of maize have been shown to be
specified by more than forty independent loci. It is intriguing
that more than two thirds of these disease lesion mimic mutations
display a partially dominant, gain-of-function inheritance, making
it the largest class of dominant mutants in maize. These lesion
mimic plants produce discrete disease-like symptoms in the absence
of any invading pathogens.
[0008] Despite the availability of the large number of lesion mimic
mutations in plants, the mechanistic basis and significance of this
phenomenon, and the wild-type function of the genes involved, are
poorly understood. The expression of most, if not all, lesion
mimics is developmentally programmed and is easily affected by
genetic background. One nearly ubiquitous feature of most mimics is
the death of afflicted tissues, the extent of which is often
enhanced by intense light, making it likely that reactive oxygen
species are involved in the etiology of lesion mimics (see, for
example, Johal et al. (1995) BioEssays 17:685; Dangl et al. (1996)
Plant Cell 8:1793). In fact, superoxide has been shown to be
responsible for the expression of lesions in the Arabidopsis lsd1
mutant (Jabs et al. (1996) Science 273:1853). The existence of both
determinant and propagative lesion type mimics suggests that cell
death is either initiated precociously or is contained inadequately
in these mutants. Since cell death in plants, like in animals, has
relevance to development, differentiation, and maintenance, lesion
mimics afford an excellent model for understanding how cell death
is regulated and executed in plants. Recently, genes for three
mimics from three plant species have been cloned (Buschges et al.
(1997) Cell 88:695-705; Dietrich et al. (1997) Cell 88:685-694;
Gray et al. (1997) Cell 89:25-31). As expected from their recessive
loss-of-function phenotypes, they all appear to encode cell death
suppressible functions that are unique to plants.
[0009] While it is relatively straightforward to comprehend the
nature of the defect in a recessive loss-of-function mutation, it
is often not possible to predict from the phenotype what the
mechanistic basis of a dominant mutation might be. One such maize
dominant mutation is Les22 (previously designated Les*-2552), which
is characterized by the formation of discrete, tiny whitish gray
bleached or necrotic spots on leaf blades that partly resemble
hypersensitive response lesions in appearance. Like most lesion
mimics of maize, the expression of Les22 lesions is cell
autonomous, developmentally dictated, and light-dependent.
[0010] Cell death and lesion formation during the expression of
disease mutant mimics is frequently mediated by oxygen free
radicals, which also mediate cell death and lesion formation during
the hypersensitive response associated with gene-for-gene
specificity of plant-pathogen interactions. The molecular basis for
this similarity can be used to genetically engineer plants for
enhanced disease resistance.
SUMMARY OF THE INVENTION
[0011] Compositions and methods for creating or enhancing disease
resistance to a pathogen in a plant are provided. The methods
comprise genetically engineering a plant to initiate a nonspecific
hypersensitive-like response upon pathogenic invasion of a plant
cell. More particularly, the invention discloses methods for stably
transforming a plant with an antisense nucleotide sequence for a
gene involved in regulation of the C-5 porphyrin metabolic pathway.
The antisense sequence is operably linked to a pathogen-inducible
promoter. Expression of the antisense nucleotide sequence in
response to pathogenic invasion of a cell effectively disrupts
porphyrin metabolism of the transformed plant cell of the present
invention. As a result, photosensitive porphyrins accumulate,
leading to a hypersensitive-like response within the invaded cell
and development of a localized lesion wherein the spread of the
pathogen is contained.
[0012] Transformed plants and seeds, as well as methods for making
such plants and seeds are additionally provided.
[0013] Nucleotide sequences encoding a wild-type maize urod gene
useful in the present invention and the amino acid sequence for the
protein encoded thereby are provided. These compositions are also
useful for regulating cell death in specifically targeted
tissues.
[0014] A maize lesion mimic, dominant mutant phenotype, designated
Les22, and the molecular basis for its manifestation are also
provided.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 shows the area of pathogen-associated necrotic tissue
on lesion-expressing leaves, as well as those that at the time of
inoculation were free of Les22 lesions, and corresponding tissue of
wild-type sibs ten days following inoculation of leaf tissue with
C. heterostrophus (Drechs.) spores. Results shown represent the
mean .+-.SEM for two inoculations per tissue type per plant for a
total of four plants per genotype.
[0016] FIG. 2 shows the effect of Les22 and position of leaf tissue
on levels of free and total salicylic acid in uninoculated plants.
Leaves of Les22 are compared to wild-type leaves. Results shown
represent the mean .+-.SEM for four determinations per tissue type
pooled from three plants.
DETAILED DESCRIPTION OF THE INVENTION
[0017] The present invention is drawn to compositions and methods
for creating or enhancing disease resistance in a plant.
Accordingly, the methods are also useful in protecting plants
against pathogens. By "disease resistance" is intended that the
plants avoid the disease symptoms that are the outcome of
plant-pathogen interactions. That is, pathogens are prevented from
causing plant diseases and the associated disease symptoms. The
methods of the invention can be utilized to protect plants from
disease, particularly those diseases that are caused by plant
pathogens.
[0018] Pathogens of the invention include, but are not limited to,
viruses or viroids, bacteria, insects, fungi, and the like. Viruses
include tobacco or cucumber mosaic virus, ringspot virus, necrosis
virus, maize dwarf mosaic virus, etc. Specific fungal and viral
pathogens for the major crops include: Soybeans: Phytophthora
megasperma fsp. glycinea, Macrophomina phaseolina, Rhizoctonia
solani, Sclerotinia sclerotiorum, Fusarium oxysporum, Diaporthe
phaseolorum var. sojae (Phomopsis sojae), Diaporthe phaseolorum
var. caulivora, Sclerotium rolfsii, Cercospora kikuchii, Cercospora
sojina, Peronospora manshurica, Colletotrichum dematium
(Colletotichum truncatum), Corynespora cassiicola, Septoria
glycines, Phyllosticta sojicola, Alternaria alternata, Pseudomonas
syringae p.v. glycinea, Xanthomonas campestris p.v. phaseoli,
Microsphaera diffusa, Fusarium semitectum, Phialophora gregata,
Soybean mosaic virus, Glomerella glycines, Tobacco Ring spot virus,
Tobacco Streak virus, Phakopsora pachyrhizi, Pythium
aphanidermatum, Pythium ultimum, Pythium debaryanum, Tomato spotted
wilt virus, Heterodera glycines Fusarium solani; Canola: Albugo
candida, Alternaria brassicae, Leptosphaeria maculans, Rhizoctonia
solani, Sclerotinia sclerotiorum, Mycosphaerella brassiccola,
Pythium ultimum, Peronospora parasitica, Fusarium roseum,
Alternaria alternata; Alfalfa: Clavibater michiganese subsp.
insidiosum, Pythium ultimum, Pythium irregulare, Pythium splendens,
Pythium debaryanum, Pythium aphanidermatum, Phytophthora
megasperma, Peronospora trifoliorum, Phoma medicaginis var.
medicaginis, Cercospora medicaginis, Pseudopeziza medicaginis,
Leptotrochila medicaginis, Fusarium, Xanthomonas campestris p.v.
alfalfae, Aphanomyces euteiches, Stemphylium herbarum, Stemphylium
alfalfae; Wheat: Pseudomonas syringae p.v. atrofaciens, Urocystis
agropyri, Xanthomonas campestris p.v. translucens, Pseudomonas
syringae p.v. syringae, Alternaria alternata, Cladosporium
herbarum, Fusarium graminearum, Fusarium avenaceum, Fusarium
culmorum, Ustilago tritici, Ascochyta tritici, Cephalosporium
gramineum, Collotetrichum graminicola, Erysiphe graminis f.sp.
tritici, Puccinia graminis f.sp. tritici, Puccinia recondita f.sp.
tritici, Puccinia striiformis, Pyrenophora tritici-repentis,
Septoria nodorum, Septoria tritici, Septoria avenae,
Pseudocercosporella herpotrichoides, Rhizoctonia solani,
Rhizoctonia cerealis, Gaeumannomyces graminis var. tritici, Pythium
aphanidermatum, Pythium arrhenomanes, Pythium ultimum, Bipolaris
sorokiniana, Barley Yellow Dwarf Virus, Brome Mosaic Virus, Soil
Borne Wheat Mosaic Virus, Wheat Streak Mosaic Virus, Wheat Spindle
Streak Virus, American Wheat Striate Virus, Claviceps purpurea,
Tilletia tritici, Tilletia laevis, Ustilago tritici, Tilletia
indica, Rhizoctonia solani, Pythium arrhenomannes, Pythium
gramicola, Pythium aphanidermatum, High Plains Virus, European
wheat striate virus; Sunflower: Plasmophora halstedii, Sclerotinia
sclerotiorum, Aster Yellows, Septoria helianthi, Phomopsis
helianthi, Alternaria helianthi, Alternaria zinniae, Botrytis
cinerea, Phoma macdonaldii, Macrophomina phaseolina, Erysiphe
cichoracearum, Rhizopus oryzae, Rhizopus arrhizus, Rhizopus
stolonifer, Puccinia helianthi, Verticillium dahliae, Erwinia
carotovorum pv. carotovora, Cephalosporium acremonium, Phytophthora
cryptogea, Albugo tragopogonis; Corn: Fusarium moniliforme var.
subglutinans, Erwinia stewartii, Fusarium moniliforme, Gibberella
zeae (Fusarium graminearum), Stenocarpella maydi (Diplodia maydis),
Pythium irregulare, Pythium debaryanum, Pythium graminicola,
Pythium splendens, Pythium ultimum, Pythium aphanidermatum,
Aspergillus flavus, Bipolaris maydis O, T (Cochliobolus
heterostrophus), Helminthosporium carbonum I, II & III
(Cochliobolus carbonum), Exserohilum turcicum I, II & III,
Helminthosporium pedicellatum, Physoderma maydis, Phyllosticta
maydis, Kabatiella-maydis, Cercospora sorghi, Ustilago maydis,
Puccinia sorghi, Puccinia polysora, Macrophomina phaseolina,
Penicillium oxalicum, Nigrospora oryzae, Cladosporium herbarum,
Curvularia lunata, Curvularia inaequalis, Curvularia pallescens,
Clavibacter michiganense subsp. nebraskense, Trichoderma viride,
Maize Dwarf Mosaic Virus A & B, Wheat Streak Mosaic Virus,
Maize Chlorotic Dwarf Virus, Claviceps sorghi, Pseudonomas avenae,
Erwinia chrysanthemi pv. zea, Erwinia carotovora, Corn stunt
spiroplasma, Diplodia macrospora, Sclerophthora macrospora,
Peronosclerospora sorghi, Peronosclerospora philippinensis,
Peronosclerospora maydis, Peronosclerospora sacchari, Sphacelotheca
reiliana, Physopella zeae, Cephalosporium maydis, Cephalosporium
acremonium, Maize Chlorotic Mottle Virus, High Plains Virus, Maize
Mosaic Virus, Maize Rayado Fino Virus, Maize Streak Virus, Maize
Stripe Virus, Maize Rough Dwarf Virus; Sorghum: Exserohilum
turcicum, Colletotrichum graminicola (Glomerella graminicola),
Cercospora sorghi, Gloeocercospora sorghi, Ascochyta sorghina,
Pseudomonas syringae p.v. syringae, Xanthomonas campestris p.v.
holcicola, Pseudomonas andropogonis, Puccinia purpurea,
Macrophomina phaseolina, Perconia circinata, Fusarium moniliforme,
Alternaria alternata, Bipolaris sorghicola, Helminthosporium
sorghicola, Curvularia lunata, Phoma insidiosa, Pseudomonas avenae
(Pseudomonas alboprecipitans), Ramulispora sorghi, Ramulispora
sorghicola, Phyllachara sacchari, Sporisorium reilianum
(Sphacelotheca reiliana), Sphacelotheca cruenta, Sporisorium
sorghi, Sugarcane mosaic H, Maize Dwarf Mosaic Virus A & B,
Claviceps sorghi, Rhizoctonia solani, Acremonium strictum,
Sclerophthona macrospora, Peronosclerospora sorghi,
Peronosclerospora philippinensis, Sclerospora graminicola, Fusarium
graminearum, Fusarium oxysporum, Pythium arrhenomanes, Pythium
graminicola, etc.
[0019] Nematodes include parasitic nematodes such as root knot,
cyst and lesion nematodes, etc.
[0020] Insect pests include insects selected from the orders
Coleoptera, Diptera, Hymenoptera, Lepidoptera, Mallophaga,
Homoptera, Hemiptera, Orthoptera, Thysanoptera, Dermaptera,
Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., particularly
Coleoptera and Lepidoptera. Insect pests of the invention for the
major crops include: Maize: Ostrinia nubilalis, European corn
borer; Agrotis ipsilon, black cutworm; Helicoverpa zea, corn
earworm; Spodoptera frugiperda, fall armyworm; Diatraea
grandiosella, southwestern corn borer; Elasmopalpus lignosellus,
lesser cornstalk borer; Diatraea saccharalis, surgarcane borer;
Diabrotica virgifera, western corn rootworm; Diabrotica longicornis
barberi, northern corn rootworm; Diabrotica undecimpunctata
howardi, southern corn rootworm; 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 maidiradicis, corn root aphid;
Blissus leucopterus leucopterus, chinch bug; Melanoplus
femurrubrum, redlegged grasshopper; Melanoplus sanguinipes,
migratory grasshopper; Hylemya platura, seedcorn maggot; Agromyza
parvicornis, corn blot leafininer; Anaphothrips 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, lesser 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; Blissus leucopterus
leucopterus, chinch bug; Contarinia sorghicola, sorghum midge;
Tetranychus cinnabarinus, carmine spider mite; Tetranychus urticae,
twospotted spider mite; Wheat: Pseudaletia unipunctata, army worm;
Spodoptera frugiperda, fall armyworm; Elasmopalpus lignosellus,
lesser cornstalk borer; Agrotis orthogonia, western cutworm;
Elasmopalpus lignosellus, lesser cornstalk borer; Oulema melanopus,
cereal leaf beetle; Hypera punctata, clover leaf weevil; Diabrotica
undecimpunctata howardi, southern corn rootworm; Russian wheat
aphid; Schizaphis graminum, greenbug; Macrosiphum avenae, English
grain aphid; Melanoplus femurrubrum, redlegged grasshopper;
Melanoplus differentialis, differential grasshopper; Melanoplus
sanguinipes, migratory grasshopper; Mayetiola destructor, Hessian
fly; Sitodiplosis mosellana, wheat midge; Meromyza americana, wheat
stem maggot; Hylemya coarctata, wheat bulb fly; Frankliniella
fusca, tobacco thrips; Cephus cinctus, wheat stem sawfly; Aceria
tulipae, wheat curl mite; Sunflower: 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, cotton budworm; Helicoverpa zea, cotton
bollworm; Spodoptera exigua, beet armyworm; Pectinophora
gossypiella, pink bollworm; Anthonomus grandis, boll weevil; 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,
twospotted spider mite; Rice: Diatraea saccharalis, sugarcane
borer; Spodoptera frugiperda, fall armyworm; Helicoverpa zea, corn
earworm; Colaspis brunnea, grape colaspis; Lissorhoptrus
oryzophilus, rice water weevil; Sitophilus oryzae, rice weevil;
Nephotettix nigropictus, rice leafhopper; Blissus leucopterus
leucopterus, chinch bug; 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, cotton budworm; Helicoverpa zea, cotton bollworm;
Epilachna varivestis, Mexican bean beetle; Myzus persicae, green
peach aphid; Empoasca fabae, potato leafhopper; 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; Tetranychus turkestani, strawberry
spider mite; Tetranychus urticae, twospotted spider mite; Barley:
Ostrinia nubilalis, European corn borer; Agrotis ipsilon, black
cutworm; Schizaphis graminum, greenbug; Blissus leucopterus
leucopterus, chinch bug; Acrosternum hilare, green stink bug;
Euschistus servus, brown stink bug; Delia platura, seedcorn maggot;
Mayetiola destructor, Hessian fly; Petrobia latens, brown wheat
mite; Oil Seed Rape: Brevicoryne brassicae, cabbage aphid;
Phyllotreta cruciferae, Flea beetle; Mamestra configurata, Bertha
armyworm; Plutella xylostella, Diamond-back moth; Delia ssp., Root
maggots.
[0021] The method of the present invention comprises genetically
transforming a plant to generate a hypersensitive-like response
that is nonspecific for the invading pathogen. By
"hypersensitive-like response" is intended a response whereby cells
in immediate contact with the pathogen rapidly collapse and dry in
a necrotic fleck, leading to a localized lesion mimicking that seen
for the native plant-pathogen hypersensitive response. In
accordance with the present invention, death of these cells, which
is mediated by oxygen free radicals, results in limiting pathogen
invasion to cells within the lesion area. By "nonspecific" is
intended the hypersensitive-like response is triggered by any plant
pathogen, as listed above, without the need for a gene-for-gene
relationship between a resistance gene in the plant and a
corresponding avirulence gene in the pathogen, as is required to
illicit the native hypersensitive response.
[0022] As disclosed in the present invention, the method of
generating a hypersensitive-like response to an invading pathogen
comprises genetic manipulation of porphyrin levels within the cells
that are in contact with the invading pathogen. In the native
state, cell porphyrin levels are regulated in the C-5 porphyrin
metabolic pathway. This pathway includes a series of enzymatic
reactions that convert porphobilinogen, the immediate monopyrrole
precursor to the porphyrins, to protoporphyrin IX, a cyclic
tetrapyrrole precursor of heme-containing proteins. The pathway is
important in both animals and plants for the eventual production of
cytochromes, peroxidases, catalases, vitamin B12 and other corrins,
and in the production of chlorophylls in plants. Important enzymes
in this pathway include porphobilinogen deaminase (EC 4.3.1.8) and
uroporphorinogen-Ill (co)synth(et)ase (EC 4.2.1.75), which enable
condensation of 4 molecules of porphobilinogen to uroporphorinogen
III; uroporphyrinogen decarboxylase (EC 4.1.1.37), which converts
uroporphorinogen III to coproporphyrinogen III; coproporphyrinogen
oxidase (EC 1.3.3.3), which converts coproporphyrinogen III to
protoporphyrinogen IX; and protoporphyrinogen oxidase (EC 1.3.3.4),
which oxidizes protoporphyrinogen IX to protoporphyrin IX.
[0023] Mutations of human genes encoding enzymes in the porphyrin
pathway result in a metabolic disorder that is generally called
porphyria. This disorder is characterized by elevated levels of
porphyrins in blood and urine. One consistent clinical
manifestation of this disorder is skin sensitivity to light. In the
case of porphyria cutanea tarda, intensely fluorescent, free
uroporphyrin(ogen) III is deposited under the surface of the skin.
On exposure to light, easily photoexcitable uroporphyrin III
molecules readily react with oxygen to produce singlet oxygen and
other reactive oxygen species that damage skin cells. This
particular porphyria disorder is a result of mutations in the gene
encoding uroporphyrinogen decarboxylase (urod gene). In humans,
urod mutations inherit as mendelian dominants.
[0024] The urod gene and other genes involved in the porphyrin
pathway have been highly conserved through evolution. Compositions
of the present invention provide for the nucleotide sequence of a
maize urod gene and a mutant phenotype, Les22, where one copy of
the urod gene has become nonfunctional, causing phytoporphyria to
develop. By "phytoporphyria" is intended a metabolic disorder in
plants that is manifested by a lesion mimic phenotype that exhibits
a dominant mode of inheritance similar to that found for mutations
of the human urod gene that result in human uroporphyria. The maize
urod nucleotide sequence, as well as nucleotide sequences for other
urod genes and any other genes encoding enzymes of the C-5
porphyrin pathway, are useful in the method of the present
invention.
[0025] In the method of the present invention, cell porphyrin
levels are manipulated by stably transforming a plant with an
antisense DNA nucleotide sequence for a targeted gene to inhibit
expression of the targeted gene, where the targeted gene comprises
the known DNA nucleotide sequence for one of the native genes
encoding an enzyme of the C-5 porphyrin pathway. By "antisense DNA
nucleotide sequence" is intended a sequence that is in inverse
orientation to the 5' to 3' normal orientation of that nucleotide
sequence. When delivered into a plant cell, the antisense DNA
sequence prevents normal expression of the DNA nucleotide sequence
for the native gene. The antisense nucleotide sequence encodes an
RNA transcript that is complementary to and capable of hybridizing
to the endogenous messenger RNA (mRNA) produced by transcription of
the DNA nucleotide sequence for the native gene. Once bound to the
endogenous mRNA, the antisense RNA product prevents production of
the native enzyme involved in the porphyrin pathway. Depending upon
the gene targeted for inhibition, specific porphyrin substrates can
be targeted for accumulation.
[0026] These porphyrin substrates are photoexcitable, or
photosensitive, and their accumulation in the presence of light
brings about oxidative damage to cellular structural components. It
is the accumulation of these substrates that will be manipulated in
the present invention to bring about a hypersensitive-like response
to pathogen invasion of a cell.
[0027] Use of antisense nucleotide sequences to inhibit or control
gene expression is well known in the art. See particularly Inouye
et al., U.S. Pat. Nos. 5,190,931 and 5,272,065; Albertsen et al.,
U.S. Pat. No. 5,478,369; Shewmaker et al., U.S. Pat. No. 5,453,566;
Weintrab et al. (1985) Trends Gen. 1:22-25; and Bourque and Folk
(1992) Plant Mol. Biol. 19:641-647. Antisense nucleotide sequences
are particularly effective in manipulating metabolic pathways to
alter the phenotype of an organism.
[0028] The antisense nucleotide sequence for any of the genes
encoding the enzymes of the C-5 metabolic pathway that are involved
in the production of protoporphyrin IX from delta-aminolevulinic
acid may be used in the method of the present invention. Nucleotide
sequences for a number of these genes are available in the art
which include, but are not limited to, the sequenced genes encoding
porphobilinogen deaminase (Arabidopsis thaliana, Accession No.
X73535; Euglena gracilus, Accession No. X15743),
uroporphyrinogen-III (co)synth(et)ase (yeast, Accession No.
X04694), uroporphyrinogen decarboxylase (yeast, Accession No.
X63721; barley, Accession No. X82832; tobacco, Accession No.
X82833; maize, as disclosed in the present invention),
coproporphyrinogen oxidase (soybean, Accession No. X71083;
Arabidopsis thaliana, Accession No. T20727 for partial sequence),
and protoporphyrinogen oxidase (yeast, Accession No. Z71381;
barley, Accession No. Y13466; tobacco, Accession No. Y13465).
[0029] The invention encompasses isolated or substantially purified
nucleic acid or protein compositions. More particularly,
compositions of the present invention include isolated nucleic acid
molecules comprising the nucleotide sequence for the naturally
occurring maize urod gene (set forth in SEQ ID NO: 1), and
fragments and variants thereof. Compositions of the present
invention also include the naturally occurring uroporphyrinogen
decarboxylase (UROD) protein (whose sequence is set forth in SEQ ID
NO: 2) encoded by this maize urod gene, as well as any
substantially homologous and functionally equivalent variants
thereof.
[0030] An "isolated" or "purified" nucleic acid molecule or
protein, or biologically active portion thereof, is substantially
free of other cellular material, or culture medium when produced by
recombinant techniques, or substantially free of chemical
precursors or other chemicals when chemically synthesized.
Preferably, an "isolated" nucleic acid molecule is free of
sequences (preferably protein encoding sequences) that naturally
flank the nucleic acid molecule (i.e., sequences located at the 5'
and 3' ends of the nucleic acid) in the genomic DNA of the organism
from which the nucleic acid is derived. For example, in various
embodiments, the isolated nucleic acid molecule can contain less
than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of
nucleotide sequences that naturally flank the nucleic acid molecule
in genomic DNA of the cell from which the nucleic acid molecule is
derived. A protein that is substantially free of cellular material
includes preparations of protein having less than about 30%, 20%,
10%, 5%, (by dry weight) of contaminating protein. When the protein
of the invention or biologically active portion thereof is
recombinantly produced, preferably, culture medium represents less
than about 30%, 20%, 10%, or 5% (by dry weight) of chemical
precursors or non-protein-of-interest chemicals.
[0031] Fragments and variants of the native nucleotide and amino
acid sequences are also encompassed by the present invention. By
"fragment" is intended a portion of a nucleotide or amino acid
sequence. Fragments of a nucleotide sequence may encode protein
fragments that retain the biological activity of the native UROD
protein, i.e., the sequence set forth in SEQ ID NO: 1, and hence
confer UROD activity, which results in conversion of
uroporphorinogen III to coproporphyrinogen III. Alternatively,
fragments of a coding nucleotide sequence that are useful as
hybridization probes generally do not encode fragment proteins
retaining biological activity. Thus, fragments of a nucleotide
sequence may range from at least about 20 nucleotides, about 50
nucleotides, about 100 nucleotides, and up to the entire nucleotide
sequence encoding the UROD protein of the invention.
[0032] A fragment of a urod nucleotide sequence that encodes a
biologically active portion of a UROD protein of the invention will
encode at least 15, 25, 30, 40, 50, 75, 100, 150, 200, 250, 300, or
350 contiguous amino acids, or up to the total number of amino
acids present in the full-length UROD protein of the invention
(i.e., 394 amino acids; SEQ ID NO: 2). Fragments of a urod
nucleotide sequence that are useful as hybridization probes for PCR
primers generally need not encode a biologically active portion of
a UROD protein.
[0033] A biologically active portion of a UROD protein can be
prepared by isolating a portion of the urod nucleotide sequence of
the invention, expressing the encoded portion of the UROD protein
(e.g., by recombinant expression in vitro), and assessing the
activity of the encoded portion of the UROD protein. Nucleic acid
molecules that are fragments of a urod nucleotide sequence comprise
at least 15, 20, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450,
500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1550,
or 1600 nucleotides, or up to the number of nucleotides present in
the full-length urod nucleotide sequence disclosed herein (i.e.,
1604 nucleotides; SEQ ID NO: 1).
[0034] By "variants" is intended sequences having substantial
similarity with a nucleotide sequence or protein disclosed herein.
For nucleotide sequences, conservative variants include those
sequences that, because of the degeneracy of the genetic code,
encode the amino acid sequence of the UROD protein of the
invention. Naturally occurring allelic variants such as these can
be identified with the use of well-known molecular biology
techniques, as, for example, with polymerase chain reaction (PCR)
and hybridization techniques as outlined below. Variant nucleotide
sequences also include synthetically derived nucleotide sequences,
such as those generated, for example, by using site-directed
mutagenesis but which still encode a UROD protein of the invention.
Generally, nucleotide sequence variants of the invention will have
at least 40%, 50%, 60%, 70%, generally, 80%, preferably 85%, 90% to
95%, even 98% or more sequence identity to the respective native
nucleotide sequence.
[0035] By "variant" protein is intended a protein derived from the
native protein by deletion (so-called truncation) or addition of
one or more amino acids to the N-terminal and/or C-terminal end of
the native protein; deletion or addition of one or more amino acids
at one or more sites in the native protein; or substitution of one
or more amino acids at one or more sites in the native protein.
Such variants may result from, for example, genetic polymorphism or
from human manipulation. Methods for such manipulations are
generally known in the art.
[0036] For example, amino acid sequence variants of the polypeptide
can be prepared by mutations in the cloned DNA sequence encoding
the native protein of interest. Methods for mutagenesis and
nucleotide sequence alterations are well known in the art. See, for
example, Walker and Gaastra, eds. (1983) Techniques in Molecular
Biology (MacMillan Publishing Company, New York); Kunkel (1985)
Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods
Enzymol. 154:367-382; Sambrook et al. (1989) Molecular Cloning: A
Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press,
Plainview, N.Y.); U.S. Pat. No. 4,873,192; and the references cited
therein; herein incorporated by reference. Guidance as to
appropriate amino acid substitutions that do not affect biological
activity of the protein of interest may be found in the model of
Dayhoff et al. (1978) in Atlas of Protein Sequence and Structure
(Natl. Biomed. Res. Found., Washington, D.C.), herein incorporated
by reference. Conservative substitutions, such as exchanging one
amino acid with another having similar properties, may be
preferred. In constructing variants of the UROD protein of
interest, modifications to the nucleotide sequences encoding the
variants will be made such that variants continue to possess the
desired activity. Obviously, any mutations made in the DNA encoding
the variant protein must not place the sequence out of reading
frame and preferably will not create complementary regions that
could produce secondary mRNA structure. See EP Patent Application
Publication No. 75,444.
[0037] Thus nucleotide sequences of the invention and the proteins
encoded thereby include the naturally occurring forms as well as
variants and fragments thereof. The variant nucleotide sequences
and variant proteins will be substantially homologous to their
naturally occurring sequence. A variant of a native nucleotide
sequence or protein is "substantially homologous" to the native
nucleotide sequence or protein when at least about 50%, 60%, to
70%, preferably at least about 80%, more preferably at least about
85%, 90%, and most preferably at least about 95%, to 98% of its
nucleotide or amino acid sequence is identical to the native
nucleotide or amino acid sequence. A variant protein will be
functionally equivalent to the native protein. By "functionally
equivalent" is intended that the sequence of the variant defines a
chain that produces a protein having substantially the same
biological effect as the native protein of interest. Thus, for
purposes of the present invention, a functionally equivalent
variant will confer UROD activity, which results in conversion of
uroporphorinogen III to coproporphyrinogen III. Such functionally
equivalent variants that comprise substantial sequence variations
are also encompassed by the invention.
[0038] The following terms are used to describe the sequence
relationships between two or more nucleic acids or polynucleotides:
(a) "reference sequence", (b) "comparison window", (c) "sequence
identity", (d) "percentage of sequence identity", and (e)
"substantial identity".
[0039] (a) As used herein, "reference sequence" is a defined
sequence used as a basis for sequence comparison. A reference
sequence may be a subset or the entirety of a specified sequence;
for example, as a segment of a full-length cDNA or gene sequence,
or the complete cDNA or gene sequence.
[0040] (b) As used herein, "comparison window" makes reference to a
contiguous and specified segment of a polynucleotide sequence,
wherein the polynucleotide sequence in the comparison window may
comprise additions or deletions (i.e., gaps) compared to the
reference sequence (which does not comprise additions or deletions)
for optimal alignment of the two sequences. Generally, the
comparison window is at least 20 contiguous nucleotides in length,
and optionally can be 30, 40, 50, 100, or longer. Those of skill in
the art understand that to avoid a high similarity to a reference
sequence due to inclusion of gaps in the polynucleotide sequence a
gap penalty is typically introduced and is subtracted from the
number of matches.
[0041] Methods of alignment of sequences for comparison are well
known in the art. Optimal alignment of sequences for comparison may
be conducted by the local homology algorithm of Smith et al. (1981)
Adv. Appl. Math. 2:482; by the homology alignment algorithm of
Needleman et al. (1970) J. Mol. Biol. 48:443; by the search for
similarity method of Pearson et al. (1988) Proc. Natl. Acad. Sci.
85:2444; by computerized implementations of these algorithms,
including, but not limited to: CLUSTAL in the PC/Gene program by
Intelligenetics, Mountain View, Calif.; GAP, BESTFIT, BLAST, FASTA,
and TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer Group (GCG), 575 Science Drive, Madison, Wis., USA; the
CLUSTAL program is well described by Higgins et al. (1988)Gene
73:237-244 (1988); Higgins et al. (1989) CABIOS 5:151-153; Corpet
et al. (1988) Nuc. Acids Res. 16:10881-90; Huang et al. (1992)
Computer Applications in the Biosciences 8:155-65, and Person et
al. (1994) Methods of Mol. Biol. 24:307-331; preferred computer
alignment methods also include the BLASTP, BLASTN, and BLASTX
algorithms (see Altschul et al. (1990) J. Mol. Biol. 215:403-410).
Alignment is also often performed by inspection and manual
alignment.
[0042] (c) As used herein, "sequence identity" or "identity" in the
context of two nucleic acid or polypeptide sequences makes
reference to the residues in the two sequences that are the same
when aligned for maximum correspondence over a specified comparison
window. When percentage of sequence identity is used in reference
to proteins it is recognized that residue positions which are not
identical often differ by conservative amino acid substitutions,
where amino acid residues are substituted for other amino acid
residues with similar chemical properties (e.g., charge or
hydrophobicity) and therefore do not change the functional
properties of the molecule. When sequences differ in conservative
substitutions, the percent sequence identity may be adjusted
upwards to correct for the conservative nature of the substitution.
Sequences that differ by such conservative substitutions are said
to have "sequence similarity" or "similarity". Means for making
this adjustment are well known to those of skill in the art.
Typically this involves scoring a conservative substitution as a
partial rather than a full mismatch, thereby increasing the
percentage sequence identity. Thus, for example, where an identical
amino acid is given a score of 1 and a non-conservative
substitution is given a score of zero, a conservative substitution
is given a score between zero and 1. The scoring of conservative
substitutions is calculated, e.g., as implemented in the program
PC/GENE (Intelligenetics, Mountain View, Calif.).
[0043] (d) As used herein, "percentage of sequence identity" means
the value determined by comparing two optimally aligned sequences
over a comparison window, wherein the portion of the polynucleotide
sequence in the comparison window may comprise additions or
deletions (i.e., gaps) as compared to the reference sequence (which
does not comprise additions or deletions) for optimal alignment of
the two sequences. The percentage is calculated by determining the
number of positions at which the identical nucleic acid base or
amino acid residue occurs in both sequences to yield the number of
matched positions, dividing the number of matched positions by the
total number of positions in the window of comparison, and
multiplying the result by 100 to yield the percentage of sequence
identity.
[0044] (e)(i) The term "substantial identity" of polynucleotide
sequences means that a polynucleotide comprises a sequence that has
at least 70% sequence identity, preferably at least 80%, more
preferably at least 90%, and most preferably at least 95%, compared
to a reference sequence using one of the alignment programs
described using standard parameters. One of skill in the art will
recognize that these values can be appropriately adjusted to
determine corresponding identity of proteins encoded by two
nucleotide sequences by taking into account codon degeneracy, amino
acid similarity, reading frame positioning, and the like.
Substantial identity of amino acid sequences for these purposes
normally means sequence identity of at least 60%, more preferably
at least 70%, 80%, 90%, and most preferably at least 95%.
[0045] Another indication that nucleotide sequences are
substantially identical is if two molecules hybridize to each other
under stringent conditions. Generally, stringent conditions are
selected to be about 5.degree. C. to about 20.degree. C. lower than
the thermal melting point (T.sub.m) for the specific sequence at a
defined ionic strength and pH. The T.sub.m is the temperature
(under defined ionic strength and pH) at which 50% of the target
sequence hybridizes to a perfectly matched probe. Typically,
stringent wash conditions are those in which the salt concentration
is about 0.02 molar at pH 7 and the temperature is at least about
50, 55, or 60.degree. C. However, nucleic acids that do not
hybridize to each other under stringent conditions are still
substantially identical if the polypeptides they encode are
substantially identical. This may occur, e.g., when a copy of a
nucleic acid is created using the maximum codon degeneracy
permitted by the genetic code. One indication that two nucleic acid
sequences are substantially identical is when the polypeptide
encoded by the first nucleic acid is immunologically cross reactive
with the polypeptide encoded by the second nucleic acid.
[0046] (e)(ii) The term "substantial identity" in the context of a
peptide indicates that a peptide comprises a sequence with at least
70% sequence identity to a reference sequence, preferably 80%, more
preferably 85%, most preferably at least 90% or 95% sequence
identity to the reference sequence over a specified comparison
window. Preferably, optimal alignment is conducted using the
homology alignment algorithm of Needleman et al. (1970) J. Mol.
Biol. 48:443. An indication that two peptide sequences are
substantially identical is that one peptide is immunologically
reactive with antibodies raised against the second peptide. Thus, a
peptide is substantially identical to a second peptide, for
example, where the two peptides differ only by a conservative
substitution. Peptides that are "substantially similar" share
sequences as noted above except that residue positions that are not
identical may differ by conservative amino acid changes.
[0047] The nucleotide sequences encoding the enzymes of the
porphyrin metabolic pathway can be the naturally occurring
sequences or they may be synthetically derived sequences.
Alternatively, the nucleotide sequence for the maize urod gene of
the present invention, as well as previously published nucleotide
sequences for other urod genes or other genes involved in the C-5
porphyrin metabolic pathway, can be utilized to isolate homologous
genes from other plants, including Arabidopsis, sorghum, Brassica,
wheat, tobacco, cotton, tomato, barley, sunflower, cucumber,
alfalfa, soybeans, sorghum, etc. Methods are readily available in
the art for the hybridization of nucleic acid sequences. Coding
sequences from other plants may be isolated according to well-known
techniques based on their sequence homology to the maize urod
coding sequence set forth herein or to other known coding sequences
for other urod genes or for other genes in the porphyrin pathway.
In these techniques, all or part of the known coding sequence is
used as a probe that selectively hybridizes to other coding
sequences for genes of the porphyrin pathway that are present in a
population of cloned genomic DNA fragments or cDNA fragments (i.e.,
genomic or cDNA libraries) from a chosen plant.
[0048] For example, the entire maize urod gene sequence disclosed
herein or portions thereof may be used as probes capable of
specifically hybridizing to corresponding coding sequences and
messenger RNAs. To achieve specific hybridization under a variety
of conditions, such probes include sequences that are unique among
urod coding sequences and are preferably at least about 10
nucleotides in length, and most preferably at least about 20
nucleotides in length. Such probes may be used to amplify urod
coding sequences from a chosen plant by the well-known process of
polymerase chain reaction (PCR).
[0049] Such techniques include hybridization screening of plated
DNA libraries (either plaques or colonies; see, for example,
Sambrook et al., eds. (1989) Molecular Cloning: A Laboratory Manual
(2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.) and
amplification by PCR using oligonucleotide primers corresponding to
sequence domains conserved among the amino acid sequences (see, for
example, Innis et al. (1990) PCR Protocols, a Guide to Methods and
Applications (Academic Press, New York).
[0050] For example, hybridization of such sequences may be carried
out under conditions of reduced stringency, medium stringency, or
even stringent conditions (e.g., conditions represented by a wash
stringency of 35-40% Formamide with 5x Denhardt's solution, 0.5%
SDS, and 1x SSPE at 37EC; conditions represented by a wash
stringency of 40-45% Formamide with 5x Denhardt's solution, 0.5%
SDS, and 1x SSPE at 42EC; and conditions represented by a wash
stringency of 50% Formamide with 5x Denhardt's solution, 0.5% SDS,
and 1x SSPE at 42EC, respectively) to DNA encoding the wild-type
maize urod gene disclosed herein in a standard hybridization assay.
See Sambrook et al (1989) Molecular Cloning: A Laboratory Manual
(2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.). In
general, sequences that code for a UROD protein and hybridize to
the wild-type maize urod gene disclosed herein will be at least 40%
to 50% homologous, about 60% to 70% homologous, and even 85%, 90%,
95% to 95% homologous or more with the maize sequence. That is, the
sequence similarity of sequences may range, sharing at least about
40% to 50%, about 60% to 70%, and even at least about 80%, 85%,
90%, 95% to 98% sequence similarity.
[0051] Generally, since leader peptides are not highly conserved
between monocots and dicots, sequences can be utilized from the
carboxyterminal end of the protein as probes for the isolation of
corresponding sequences from any plant. Nucleotide probes can be
constructed and utilized in hybridization experiments as discussed
above. In this manner, even gene sequences that are divergent in
the aminoterminal region can be identified and isolated for use in
the methods of the invention.
[0052] Thus known nucleotide sequences or portions thereof for any
urod gene, or any other gene encoding an enzyme in the C-5
porphyrin metabolic pathway, can be used as probes for identifying
nucleotide sequences for similar genes in a chosen plant or
organism. Once similar genes are identified, their respective
antisense nucleotide sequences can be utilized in the present
invention to inhibit or control expression of the genes encoding
UROD or other enzymes of the C-5 porphyrin metabolic pathway.
Although it is preferable to use the specific antisense nucleotide
sequence corresponding to the nucleotide sequence for a targeted
native urod gene, the antisense nucleotide sequence for the
nucleotide sequence for any urod gene can be used in the invention
to regulate the native urod gene. Likewise, the antisense
nucleotide sequence for any alad gene can be used to regulate the
targeted native alad gene of a plant; and so forth for all other
genes associated with the pathway. In this manner, the degree of
sequence homology between the gene serving as the template for the
antisense nucleotide sequence and the targeted native gene will
determine the degree of binding between the antisense nucleotide
sequence and the nucleotide sequence for the targeted native gene.
The greater the sequence homology, the greater the binding of the
antisense sequence, and hence the greater the inhibition of
expression of the targeted gene. In this manner, the degree of
inhibition of specific enzyme activity, and hence accumulation of
specific substrates to bring about the hypersensitive-like response
to pathogen invasion, can be regulated.
[0053] Degree of suppression or inhibition of expression of the
targeted gene may also be regulated by length of the antisense
nucleotide sequence. Hence, the antisense nucleotide sequence can
be designed to encode an RNA transcript that is complementary to
and thus hybridizes to any portion of the endogenous mRNA produced
by transcription of the DNA nucleotide sequence for the targeted
native gene. That is, the hybridizing site may be proximal to the
5'-terminus or capping site, downstream from the capping site,
between the capping site and the initiation codon, and may cover
all or only a portion of the noncoding region, may bridge the
noncoding and coding region, be complementary to all or part of the
coding region, complementary to the 3'-terminus of the coding
region, or complementary to the 3'-untranslated region of the mRNA.
See particularly Shewmaker et al., U.S. Pat. No. 5,453,566; Inouye,
U.S. Pat. No. 5,190,931; and Helene and Toulme, Biochemica et
Biophysica Acta (1990):99-125. For the purposes of disease
resistance, the antisense nucleotide sequence will encode an RNA
product that hybridizes to about 50% of, preferably to about 75%
of, more preferably to the entire endogenous mRNA, with the latter
enabling maximum suppression of gene expression, and hence maximum
hypersensitive-like response associated with accumulation of
photoexcitable substrate.
[0054] The method of the present invention relies upon expression
of the introduced antisense nucleotide sequence in response to
pathogen invasion of a cell. Expression of the antisense sequence
then effectively disrupts porphyrin metabolism such that
photosensitive porphyrins accumulate. The presence of these
porphyrins causes oxidative damage, leading to a
hypersensitive-like response within the invaded cell and
development of a localized lesion wherein the spread of the
pathogen is contained.
[0055] Because expression of the introduced antisense DNA sequence
in a plant cell causes cell death, an inducible promoter is used to
drive expression of this sequence. The inducible promoter must be
tightly regulated to prevent unnecessary cell death yet be
expressed in the presence of a pathogen to prevent spread of the
infection and disease symptoms. Generally, it will be beneficial to
express the gene from an inducible promoter, particularly from a
pathogen-inducible promoter. Such promoters include those from
pathogenesis-related proteins (PR proteins), which are induced
following infection by a pathogen; e.g., PR proteins, SAR proteins,
beta-1,3-glucanase, chitinase, etc. See, for example, Redolfi et
al. (1983) Neth. J Plant Pathol. 89:245-254; Uknes et al. (1992)
Plant Cell 4:645-656; and Van Loon (1985) Plant Mol. Virol.
4:111-116; and the copending applications both entitled "Maize
Inducible Promoters," U.S. Patent Application Ser. No. 60/076,100,
filed Feb. 26, 1998, and U.S. Patent Application Ser. No.
60/079,648, filed Mar. 27, 1998; herein incorporated by
reference.
[0056] Of particular interest are promoters that are expressed
locally at or near the site of pathogen infection. See, for
example, Marineau et al. (1987) Plant Mol. Biol. 9:335-342; Matton
et al. (1989) Molecular Plant-Microbe Interactions 2:325-331;
Somsisch et al. (1986) Proc. Natl. Acad. Sci. USA 83:2427-2430;
Somsisch et al. (1988) Molecular and General Genetics 2:93-98; and
Yang (1996) Proc. Natl. Acad. Sci. USA 93:14972-14977. See also
Chen et al. (1996) Plant J. 10:955-966; Zhang and Sing (1994) Proc.
Natl. Acad. Sci. USA 91:2507-2511; Warner et al. (1993) Plant J.
3:191-201; Siebertz et al. (1989) Plant Cell 1:961-968; 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.
(1992) Physiological and Molecular Plant Pathology 41:189-200).
[0057] The antisense nucleotide sequences for the native genes
encoding enzymes involved in the C-5 porphyrin pathway are useful
in the genetic manipulation of any plant when operably linked to an
inducible promoter, more preferably a pathogen-inducible promoter.
In this manner, the antisense sequences of the invention are
provided in expression cassettes for expression in the plant of
interest.
[0058] Such expression cassettes will comprise a transcriptional
initiation region linked to the antisense nucleotide sequence for
the native gene or genes targeted for inhibition. Such an
expression cassette is provided with a plurality of restriction
sites for insertion of the antisense sequence to be under the
transcriptional regulation of the regulatory regions. The
expression cassette may additionally contain selectable marker
genes.
[0059] The transcriptional initiation region, the inducible
promoter, may be native or analogous or foreign or heterologous to
the plant host. Additionally, the promoter may be the natural
sequence or alternatively a synthetic sequence. By "foreign" is
intended that the transcriptional initiation region is not found in
the native plant into which the transcriptional initiation region
is introduced. As used herein, a chimeric gene comprises a coding
sequence operably linked to transcription initiation region that is
heterologous to the coding sequence. The transcriptional cassette
will include in the 5'-3' direction of transcription, a
transcriptional and translational initiation region, an antisense
DNA sequence for the targeted gene of interest, and a
transcriptional and translational termination region functional in
plants. The termination region may be native with the
transcriptional initiation region, may be native with the DNA
sequence of interest, or may be derived from another source.
Convenient termination regions are available from the Ti-plasmid of
A. tumefaciens, such as the octopine synthase and nopaline synthase
termination regions. See also, Guerineau et al. (1991) Mol. Gen.
Genet. 262:141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et
al. (1991) Genes Dev. 5:141-149; Mogen et al. (1990) Plant Cell
2:1261-1272; Munroe et al. (1990) Gene 91:151-158; Ballas et al.
1989) Nuc. Acids Res. 17:7891-7903; Joshi et al. (1987) Nuc. Acid
Res. 15:9627-9639.
[0060] The antisense sequences of the invention are provided in
expression cassettes for expression in the plant of interest. The
cassette will include 5' and 3' regulatory sequences operably
linked to the gene of interest. The cassette may additionally
contain at least one additional gene to be cotransformed into the
organism. Alternatively, the additional gene(s) can be provided on
another expression cassette.
[0061] For example, flow of substrates into the porphyrin pathway
is regulated by feedback inhibition of 5-aminolevulinic acid
dehyratase (ALAD), which generates 2 molecules of porphobilinogen
from 2 molecules of 5-aminolevulinic acid. For the antisense
nucleotides of the present invention to be effective in generating
a hypersensitive-like response, ALAD activity must be high enough
to support accumulation of photoexcitable porphyrin substrates.
This is achieved naturally in developing tissues, where the demand
for protoporphyrin IX to support chlorophyll and heme synthesis is
high. In developmentally mature tissues, demand for protoporphyrin
IX is decreased, and ALAD activity is correspondingly decreased. To
enable continued elevated activity, the expression cassette can
also comprise a nucleotide sequence encoding the alad gene, which
is also operably linked to the inducible promoter.
[0062] Where appropriate, the antisense sequence and additional
gene(s) may be optimized for increased expression in the
transformed plant. That is, these nucleotide sequences can be
synthesized using plant-preferred codons for improved expression.
Methods are available in the art for synthesizing plant-preferred
genes. See, for example, U.S. Pat. Nos. 5,380,831, 5,436,391, and
Murray et al. (1989) Nuc. Acids Res. 17:477-498, herein
incorporated by reference.
[0063] Additional sequence modifications are known to enhance gene
expression in a cellular host. These include elimination of
sequences encoding spurious polyadenylation signals, exon-intron
splice site signals, transposon-like repeats, and other such
well-characterized sequences, which may be deleterious to gene
expression. The G-C content of the sequence may be adjusted to
levels average for a given cellular host, as calculated by
reference to known genes expressed in the host cell. When possible,
the sequence is modified to avoid predicted hairpin secondary mRNA
structures.
[0064] The expression cassettes may additionally contain 5' leader
sequences in the expression cassette construct. Such leader
sequences can act to enhance translation. Translation leaders are
known in the art and include: picornavirus leaders, for example,
EMCV leader (Encephalomyocarditis 5' noncoding region) (Elroy-Stein
et al. (1989) Proc. Nat. Acad. Sci. USA 86:6126-6130); potyvirus
leaders, for example, TEV leader (Tobacco Etch Virus) (Allison et
al. (1986)); MDMV leader (Maize Dwarf Mosaic Virus) (Virology
154:9-20); human immunoglobulin heavy-chain binding protein (BiP)
(Macejak et al. (1991) Nature 353:90-94); untranslated leader from
the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling
et al. (1987) Nature 325:622-625); tobacco mosaic virus leader
(TMV) (Gallie et al. (1989) Molecular Biology of RNA, pages
237-256); and maize chlorotic mottle virus leader (MCMV) (Lommel et
al. (1991) Virology 81:382-385). See also Della-Cioppa et al.
(1987) Plant Physiology 84:965-968. Other methods known to enhance
translation can also be utilized, for example, introns, and the
like.
[0065] In preparing the expression cassette, the various DNA
fragments may be manipulated, so as to provide for the DNA
sequences in the proper orientation and, as appropriate, in the
proper reading frame. Toward this end, adapters or linkers may be
employed to join the DNA fragments or other manipulations may be
involved to provide for convenient restriction sites, removal of
superfluous DNA, removal of restriction sites, or the like. For
this purpose, in vitro mutagenesis, primer repair, restriction,
annealing, resubstitutions, e.g. transitions and transversions, may
be involved.
[0066] The antisense nucleotide sequences of the present invention
can be used to transform any plant. In this manner, genetically
modified plants, plant cells, plant tissue, seed, and the like can
be obtained. Transformation protocols 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. (1986) Biotechniques
4:320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad.
Sci. USA 83:5602-5606, Agrobacterium-mediated transformation
(Townsend et al., U.S. Pat. No. 5,563,055); direct gene transfer
(Paszkowski et al. (1984) EMBO J 3:2717-2722), and ballistic
particle acceleration (see, for example, Sanford et al., U.S. Pat.
No. 4,945,050; Tomes et al. (1995) "Direct DNA Transfer into Intact
Plant Cells via Microprojectile Bombardment," in Plant Cell,
Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and
Phillips (Springer-Verlag, Berlin); and McCabe et al. (1988)
Biotechnology 6:923-926). Also see Weissinger et al. (1988) Annual
Rev. Genet. 22:421-477; Sanford et al. (1987) Particulate Science
and Technology 5:27-37 (onion); Christou et al. (1988) Plant
Physiol. 87:671-674 (soybean); McCabe et al. (1988) Bio/Technology
6:923-926 (soybean); Finer and McMullen (1991) In Vitro Cell Dev.
Biol. 27P:175-182 (soybean); Singh et al. (1998) Theor. Appl.
Genet. 96:319-324 (soybean); Datta et al. (1990) Biotechnology
8:736-740 (rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA
85:4305-4309 (maize); Klein et al. (1988) Biotechnology 6:559-563
(maize); Tomes, U.S. Pat. No. 5,240,855; Buising et al., U.S. Pat.
Nos. 5,322,783 and 5,324,646; Tomes et al. (1995) "Direct DNA
Transfer into Intact Plant Cells via Microprojectile Bombardment,"
in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed.
Gamborg (Springer-Verlag, Berlin) (maize); Klein et al. (1988)
Plant Physiol. 91:440-444 (maize); Fromm et al. (1990)
Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren et al.
(1984) Nature (London) 311:763-764; Bytebier et al. (1987) Proc.
Natl. Acad. Sci. USA 84:5345-5349 (Liliaceae); De Wet et al. (1985)
in The Experimental Manipulation of Ovule Tissues, ed. Chapman et
al. (Longman, New York), pp. 197-209 (pollen); Kaeppler et al.
(1990) Plant Cell Reports 9:415-418; and Kaeppler et al. (1992)
Theor. Appl. Genet. 84:560-566 (whisker-mediated transformation);
D'Halluin et al. (1992) Plant Cell 4:1495-1505 (electroporation);
Li et al. (1993) Plant Cell Reports 12:250-255 and Christou et al.
(1995) Annals of Botany 75:407-413 (rice); Osjoda et al. (1996)
Nature Biotech. 14:745-750 (maize via Agrobacterium tumefaciens);
all of which are herein incorporated by reference.
[0067] The cells that have been transformed may be grown into
plants in accordance with conventional ways. See, for example,
McCormick et al. (1986) Plant Cell Reports 5:81-84. These plants
may then be grown, and either pollinated with the same transformed
strain or different strains, and the resulting hybrid having the
desired phenotypic characteristic identified. Two or more
generations may be grown to ensure that the subject phenotypic
characteristic is stably maintained and inherited and then seeds
harvested to ensure the desired phenotype or other property has
been achieved.
[0068] The methods of the invention can be used with other methods
available in the art for enhancing disease resistance in
plants.
[0069] The antisense nucleotide sequences of the present invention
also find use in targeting specific tissues for cell death. In this
manner, a plant of choice can be stably transformed with an
expression cassette comprising a chimeric gene that comprises an
antisense nucleotide sequence for a gene encoding an enzyme in the
C-5 porphyrin metabolic pathway, wherein the antisense sequence is
operably linked to a stamen promoter to achieve male sterility. In
this manner, a promoter that normally enables stamen development
now drives expression of the antisense sequence, whose expression
ultimately leads to cell death in tissues that normally would have
become fertile stamens. Such promoters are available in the art
(see, for example, EPA0344029 and U.S. Pat. No. 5,470,359, herein
incorporated by reference). In another embodiment of the present
invention, a method for overcoming herbicide resistance during crop
rotation is provided. Following harvest of a first crop of the
season, herbicide treatment may routinely be used to eliminate
unwanted weeds during preparation of the field site for a
subsequent crop of the season. However, this herbicide application
is ineffective at removing volunteer plants of the first crop,
which may be overlooked during field preparation or which may
germinate from previously buried or dispersed seed. An abundance of
these volunteer plants effectively poses competition for
environmental resources similar to that seen with weeds, and hence
has the potential to decrease yield of the subsequent crop.
[0070] The antisense nucleotide sequences of the present invention
are useful in overcoming this problem. Herbicide resistant crop
plants can be stably transformed with an expression cassette
comprising a chimeric gene that comprises an antisense nucleotide
sequence for a gene encoding an enzyme in the C-5 porphyrin
metabolic pathway. For the purpose of overcoming herbicide
resistance, the antisense nucleotide sequence is operably linked to
a chemical-inducible promoter, such that contact of the plant with
a known chemical substance induces expression of the antisense
nucleotide sequence. As before, expression of this sequence results
in accumulation of photosensitive porphyrins, ultimately leading to
photooxidative damage to cell membranes and death of the plant
tissues.
[0071] 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-1a
promoter, which is activated by salicylic acid.
[0072] In this manner, seed of the transformed crop plants, and
transformed seedlings germinating therefrom, would effectively die
following application of the chemical substance whose inducible
promoter is part of the stably incorporated chimeric gene.
Following harvest of the desired crop product, the remaining plant
parts can be treated with an application of the chemical substance.
Furthermore, any volunteer seedlings germinating from seed can be
similarly treated to eliminate the undesired crop from the
field.
[0073] The invention further finds use in therapies for mammals,
particularly humans, for preventing growth of malignant cells. In
this embodiment of the present invention, a method for killing, and
thereby preventing the proliferation of, malignant or nonmalignant
abnormal cells in an affected tissue is provided. The antioxidant
defense capabilities of these abnormal cells, particularly
malignant cancer cells, are compromised relative to normal cells.
By "antioxidant" is intended the ability to keep photosensitive
compounds, such as tetrapyrrole-containing porphyrins, and other
reactive oxygen species, including hydrogen peroxide, at relatively
low cellular concentrations to prevent oxidative damage to cell
membranes. These normal defense capabilities include tight
regulation of the C-5 porphyrin pathway and the presence of
catalases and peroxidases, which are heme-containing enzymes. Thus,
manipulation of the C-5 porphyrin pathway to disrupt an already
compromised defense capability would be an effective means of
preventing further proliferation of these abnormal cells.
[0074] In accordance with this method, a pharmaceutical composition
comprising an antisense nucleotide sequence complementary to the
mRNA for any one of the human genes encoding the key enzymes
outlined in the C-5 porphyrin pathway can be administered to the
affected tissue in such a manner as to target the proliferating
abnormal cells. Human genes encoding these enzymes have been
identified and sequenced and are well known in the art. Once at the
targeted site, the antisense nucleotide sequence will hybridize to
mRNA of the targeted gene of the C-5 pathway, effectively blocking
transcription, leading to accumulation of photosensitive porphyrin
substrate. Subsequent exposure of the treated tissue to light of
photoactivating wavelengths in the visible region for an
experimentally determined length of time would lead to
photooxidative damage and death of the treated cells.
[0075] Antisense nucleotide sequences that are directly
complementary to the targeted mRNA transcripts include not only the
native polymers of the biologically active nucleotides, but also
sequences that are modified to improve stability and/or lipid
solubility. Modifications, such as substitution of methyl or sulfur
groups in the internucleotide phosphodiester linkage, can be used
to improve lipid solubility and prevent nuclease cleavage of the
antisense sequence, thereby effectively increasing availability of
the sequence for hybridization to mRNA.
[0076] Such antisense oligonucleotides may be oligonucleotides
wherein at least one, or all, of the intemucleotide bridging
phosphate residues are modified phosphates, such as methyl
phosphonates, methyl phosphonothioates, phosphoromorpholidates,
phosphoropiperazidates and phosphoramidates. For example, some, for
example, every other one, of the intemucleotide bridging phosphate
residues may be modified as described. In another example, such
antisense oligonucleotides are oligonucleotides wherein at least
one, or all, of the nucleotides contain a 2 loweralkyl moiety
(e.g., C1-C4, linear or branched, saturated or unsaturated alkyl,
such as methyl, ethyl, ethenyl, propyl, 1-propenyl, 2-propenyl, and
isopropyl). See also Furdon et al. (1989) Nucleic Acids Res.
17:9193-9204; Agrawal et al. (1990) Proc. Natl. Acad. Sci. USA
87:1401-1405; Baker et al. (1990) Nucl. Acids Res. 18:3537-3543;
Sproat et al. (1989) Nuc. Acids Res. 17:3373-3389; Walder et al.
(1988) Proc. Natl. Acad. Sci. USA 85:5011-5015.
[0077] Modification of the phosphodiester backbone has been shown
to impart stability and may allow for enhanced affinity and
increased cellular penetration of ODNs. Additionally, chemical
strategies may be employed to replace the entire phosphodiester
backbone with novel linkages. Phosphorothioate and
methylphosphonate modified ODNs may be made through automated ODN
synthesis.
[0078] A phosphorodithioate version of the phosphorothioate can be
synthesized. In the dithioate linkage, the nonbridging oxygens can
be substituted with sulfur. This linkage is highly nuclease
resistant.
[0079] Sugar modifications may also be used to enhance stability
and affinity of the molecules. The alpha-anomer of a 2'-deoxyribose
sugar has the base inverted with respect to the natural
beta-anomer. ODNs containing alpha-anomer sugars are resistant to
nuclease degradation.
[0080] This method of treatment depends upon successful delivery of
the pharmaceutical composition comprising the antisense nucleotide
sequence to the targeted cells, with limited accumulation in cells
of normal tissue. In the event that normal cells accumulate the
pharmaceutical composition, exposure to light following treatment
should be minimized. However, unlike other cancer treatment methods
that rely upon systemic doses of exogenous porphyrins, such as
hemotoporphyrin IX or hematoporphyrin derivatives, this method
relies on accumulation of naturally occurring porphyrins in the
targeted abnormal cells. The residence time of naturally occurring
porphyrins is greatly reduced (on the order of days) when compared
to residence time of exogenous porphyrins (on the order of weeks ),
so that photosensitive levels do not persist for long periods
following treatment. This greatly reduces the risk of
photosensitivity of treated tissues.
[0081] Additionally, this method relies upon the presence of
5-aminolevulinic acid (ALA) as a precursor for porphyrin
production. This substrate is regulated by tight feedback
inhibition of the C-5 porphyrin metabolic pathway. As an
alternative, additional amounts of ALA can be administered
separately or with the pharmaceutical composition comprising the
antisense nucleotide sequence.
[0082] The present invention also provides a maize lesion mimic
phenotype, designated Les22, that represents a dominant mutant
situation whose molecular basis resides in the disruption of the
maize urod gene disclosed herein. In Les22 individuals, one copy of
the urod gene comprises at least one Mutator (Mu) transposable
element inserted within its nucleotide sequence. This insertion
results in a null mutation within this copy of the gene. By "null
mutation" is intended a mutation that results in loss-of-function
of the gene. Thus Les22 individuals have one copy of the urod gene
that is nonfunctional. For example, in the maize lesion mimic
mutant designated Les22-7, the nonfunctional copy of the urod gene
has a Mu transposable element inserted between bp 102 and bp 103 of
the nucleotide sequence set forth in SEQ ID NO: 1, and in the maize
lesion mimic mutant designated Les22-3, the nonfunctional copy of
the urod gene has a Mu transposable element inserted between bp 196
and bp 197 of SEQ ID NO: 1. In Les22 lesion mimics, the single
functional copy of urod produces an insufficient amount of UROD
protein, leading to a partial block in the porphoryin metabolic
pathway that results in accumulation of this enzyme's substrate,
uroporphoryin III. Accumulation of this highly photoreactive
substrate leads to development of phytophoria in the presence of
photoactivating wavelengths in the visible region.
[0083] The maize lesion mimic mutant phenotype Les22 and its
molecular basis as disclosed in this invention are novel in the
plant kingdom. The implications of this novelty are significant for
purposes beyond the methods of the present invention. First, being
the first identified mutation of the porphyrin pathway in plants,
Les22 provides an excellent tool to understand how the production
of chlorophyll and heme is regulated.
[0084] Second, this apparently represents the first case of a
mutation of a conserved gene that has parallel phenotypic
manifestations in both humans and plants. The dominant nature of
this defect suggests that the porphyrin pathway, which although is
expected to operate in different subcellular locations in plant and
human cells, is regulated very similarly in both organisms. Since
mutations of most genes of the porphyrin pathway in humans result
in porphyria, one consistent clinical manifestation of which is
heightened skin sensitivity to light, mutations with a phenotype
like that of Les22 may also result from defects in other genes of
the porphyrin pathway in plants. In fact, genetic allelism tests
between various Les22-mutants support this hypothesis.
[0085] Third, the dominant nature of Les22 is caused not by a gain
of a new function, but rather is the result of a null,
loss-of-function mutation in one copy of the urod gene. This
represents a rare, if not the only, case of haplo-insufficiency
(gene dosage dependence) in plants. Haplo-insufficiency, which has
been well established in the case of human uroporphyria, is thought
not to exist in plants (Birchler (1993) Annu. Rev. Genet.
27:181).
[0086] Fourth, Les22, being cell autonomous, visually discernible,
and nonlethal, provides an elegant molecular tool to probe into the
phenomenon of Mu suppression in maize. This enigmatic phenomenon
seems to epitomize the mechanism(s) by which plants keep the
activity of transposons in check. The phenotypic effects of certain
mutations caused by Mutator (Mu) insertions sometimes become
dependent on the activity of the Mu system. For example, the mutant
phenotype of a mutation will express if the plant has Mu activity.
However, when Mu turns off, the mutant phenotype reverts back to
the normal wild-type phenotype, and this happens without the loss
of the Mu insertion. Such mutations, and the phenomenon they
exhibit, are called Mu suppressible. What causes a plant to lose Mu
activity remains enigmatic, but it often happens during vegetative
development of the plant as well as following inbreeding, even
though intact Mu elements remain in the plant. At the DNA level, Mu
elements of plants with the suppressed mutant phenotype show
hypermethylation.
[0087] This phenomenon of dominant negative regulation was first
uncovered with hcf-106 (Martienssen et al. (1990) Genes Dev.
4:331-343) and later shown to suppress coordinately the phenotypes
of both hcf-106 and Les28 (a lesion mimic mutant phenotypically
identical to Les22) (Martienssen and Baron (1994) Genetics
136:1157-1170). A few alleles of Les22, including Les22-7, are also
Mu-suppressible.
[0088] Finally, from a practical viewpoint, Les22 may provide a
simplified system for the development of effective sunscreens
needed to protect human skin from high-intensity light and UV
damage. Cell lines, or plants, that have been transformed with
expression cassettes comprising an antisense nucleotide sequence
for a urod gene or other gene encoding an enzyme of the C-5 pathway
can be used to test for effectiveness of sunscreens. In this case,
antisense sequences may be operably linked to an inducible
promoter, such as a chemical inducible promoter, as previously
described. These cell lines, or plants, can be administered the
chemical inducer in the presence of putative sunscreen substances,
and treated cell lines or plants can subsequently be exposed to
light of photoactivating wavelengths. Effectiveness of a putative
sunscreen can be measured in terms of its ability to prevent
photooxidative damage to the cells. By "photooxidative damage" is
intended loss of cellular functions, such as loss of membrane
integrity and normal function of organelles, including cell death.
This damage results from the interaction of intercellular
components with reactive oxygen species, which are the reaction
products of photoreactive substrates, such as the photosensitive
porphyrins, and oxygen in the presence of photoactivating
wavelengths. Thus, in the case of a test plant, an effective
putative sunscreen composition would, for example, prevent
development of necrotic spots and lesions on the treated leaf
tissue. Alternatively, a plant assay system with a Les22 phenotype
(e.g., maize Les22 seedlings) may be used to rapidly screen a large
number of potential sunscreen creams or compositions. Since the
Les22 phenotype is completely dependent on irradiation, the
effectiveness of various creams can be rapidly determined, where
application of an effective sunscreen composition to a leaf would
prevent the Les22 phenotype, i.e., lesions, from developing during
exposure to light of photoactivating wavelengths, such as
wavelengths of normal sunlight.
[0089] The following examples are offered by way of illustration
and not by way of limitation.
EXPERIMENTAL
[0090] To elucidate the molecular basis of a dominant lesion mimic
mutation of maize, Les22 (previously designated Les*-2552; see
Johal (1994) Maydica 89:69), which is characterized by the
formation of discrete, tiny whitish-gray bleached or necrotic spots
on leaf blades that partly resemble hypersensitive response lesions
in appearance, was selected. Like most lesion mimics of maize, the
expression of Les22 lesions is cell autonomous, developmentally
dictated, and light-dependent (Johal (1994) Maydica 39:69). Lesions
do not initiate on leaf regions that are protected from the light.
However, lesions form in the albino sectors of double mutants of
Les22 with ij1, a recessive mutation characterized by alternate
green and albino leaf stripes (Han et al. (1992) EMBO J. 11:4037),
suggesting that the expression of Les22 lesions is mediated
primarily by incident light. Two other mutations that exhibit a
phenotype identical to Les22 are Les2 (Neuffer et al. (1975) J.
Hered. 66:265) and Les28 (Martienssen et al. (1994) Genetics
136:1157). Interestingly, Les22, like Les2, maps to the short arm
of chromosome 1. The map location of Les28 has not been reported
yet, although some mutant alleles of Les22, including Les22-7 (see
below), exhibit a Mu-suppressible phenomenon previously described
for Les28.
Example 1: Plant Material
[0091] The first Les22 mutant that allowed us to define the Les22
locus was a gift from Dr. Don Robertson of Iowa State University.
It had appeared spontaneously in one of his Mutator nurseries. This
mutant has been designated as Les22-17.
[0092] All other Les22 mutants, Les22-1 through Les22-16, with the
exception of Les22-7, were recovered from various Mutator
populations at the Pioneer nurseries. These Mutator populations
were generated in the laboratory either to tag various genes,
including Hm1, Br2, Lls1, and Bk2, by directed mutagenesis or to
identify new mutant phenotypes of interest by developing random F2
populations.
[0093] The Les22-7 mutant, which allowed Les22 to be cloned, was
isolated at the University of Missouri in 1993. The progeny from
which it was isolated as a single event was developed at the
Pioneer Winter Nursery in Hawaii in 1989 as follows. A plant from
the row HW89-37-326#3, with the genotype Pr1xCSH89-9-8-1, was
pollinated with pollen from the inbred Pr1.
[0094] The source of all Mutator stocks that happened to generate
Les22 was a laboratory at Pioneer Hi-Bred. Original Mutator
material was received from Dr. Don Robertson, Iowa State
University.
Example 2: Determination of Les22 Homozygous Phenotype
[0095] An outcross progeny of Les22-9 with A632 was used to map a
number of RFLP markers from the short arm of chromosome 1 to
determine what would be the phenotype of a plant homozygous for
Les22. Two RFLP markers, UMC194 and UMC76, were identified that
mapped 2.6 cM distal and 9.8 cM proximal to Les22, respectively.
These markers were used to genotype an F2 population derived from a
Les22 mutant. Contrary to what was thought previously (Johal (1994)
Maydica 39:69), densely lesioned F2 plants were not homozygous for
Les22. Instead, a yellow seedling lethal (ysl) plant that scalded
easily in sunlight was found to segregate completely with both the
flanking RFLP markers, raising the possibility that this ysl may
very well be the phenotype of a Les22 homozygote.
Example 3: Cloning of the Mutant Les22 Gene
[0096] To clone Les22, a Mutator (Mu) transposon-based gene tagging
approach was used that relied on the random appearance of this
mutant phenotype in various Mu populations (Johal (1994) Maydica
39:69-76). Being a dominant mutation, Les22 was easy to spot even
in populations other than F2s, and as a result, 16 cases of
independent origin, designated Les22-1 through Les22-16, were
collected. To identify Mu elements that may have caused these
mutations, each mutant was backcrossed three times with either B73
or A632 (Johal (1994) Maydica 39:69-76), and the progeny from the
last cross was subjected to a gel-blot-based analysis that examined
the linkage of each of the nine Mu elements with each mutant allele
(Walbot (1992) Annu. Rev. Plant Physiol. Plant Mol. Biol. 43:49-82;
Bennetzen et al. (1993) Crit. Rev. Plant Sci. 12:57-95).
[0097] Genomic DNA from maize seedlings was extracted by the
CTAB-based method as previously described (Hulbert et al. (1991)
Mol. Gen. Genet. 226:377-382). Southern blot analysis to identify
RFLP markers and to perform cosegregation analysis was done as
previously described (Gardiner et al. (1993) Genetics 134:917-930).
Cosegregation analysis, to look for Mu elements linked to various
Les22 mutant alleles, was first performed with pooled (involving at
least 15 plants) DNAs from either the mutant or wild-type siblings
of each mutant. DNA samples were digested with seven restriction
enzymes, and the blots were hybridized with each of the nine Mu
elements as described earlier (Gray et al. (1997) Cell
89:25-31).
[0098] From the Les22-7 family, a Mul-hybridizing 6.5 kb Xho I
restriction fragment was identified that was present in the DNA of
all 39 mutants and absent in the DNA of all 27 wild-type sibs (data
not shown), suggesting that this restriction fragment either
carries at least a part of the Les22 gene or contains a Mu1 element
that is closely linked to it. This restriction fragment was cloned
in .lambda. ZapII vector (Stratagene), followed by rescuing of this
fragment as a phagemid using in vivo excision.
Example 4: PCR Verification of Cloned Les22 Gene
[0099] To verify the cloning of Les22, a PCR approach was used
(Gray et al. (1997) Cell 89:25). A 500 bp fragment flanking on the
left side of Mu1 insertion in this clone, designated LF7, was
amplified using a Mu-TIR primer (SEQ ID NO: 3) (Gray et al. (1997)
Cell 89:25) and the reverse primer from the 6.5 kb Xho I clone. LF7
was then subcloned in the TA cloning vector (Invitrogen) and then
sequenced. Two oppositely orienting PCR primers were designed from
the sequence of LF7 and each was used in combination with the
Mu-TIR primer in a PCR reaction in which the template DNA was
derived from each of the 16 Les22 mutants. The primer sequences
were LF7-A (see SEQ ID NO: 4) and LF7-B (see SEQ ID NO: 5).
Conditions for the PCR were as previously described (Gray et al.
(1997) Cell 89:25).
[0100] A 300 base-pair amplification product, which hybridized with
LF7, was obtained from the DNA of the Les22-3 mutant, demonstrating
that a Mu element was present in the vicinity of the LF7 region in
this mutant allele. Subsequent sequence analysis of this PCR
product revealed that a Mu element had inserted in the Les22-3
mutant allele 95 nucleotides away from the Mu1 insertion in
Les22-7. Multiple insertions of this sort in independent mutants
are considered a proof for the correct cloning of a gene (Gray et
al. (1997) Cell 89:25).
Example 5: DNA Polymorphism and Northern Analysis
[0101] Unequivocal evidence that Les22 had been cloned came from
two additional experiments. First, to detect polymorphism between
the Les22-7 mutant allele (which was found from a single plant in
the progeny of a cross between Pr1 (an inbred) and a Mu active
line) and its wild-type progenitor, DNA from 50 wild-type siblings
of the original Les22-7 mutant was compared with the DNA of the
Les22-7 mutant allele from one of the advanced generations of
Les22-7 with A632 mentioned in the text. Respective DNAs were
digested with Xba I, which does not cut within Mu1, and the blot
was hybridized with LF7. Examination of the DNA blot revealed a
restriction fragment length polymorphism, with the size difference
between the band for the wild-type progenitor allele and the upper
band for the mutant allele of Les22-7 being 1.4 kb (data not
shown). This DNA polymorphism is of the size expected from a Mu1
insertion (Bennetzen et al. (1993) Crit. Rev. Plant Sci.
12:57).
[0102] Second, RNA extraction and subsequent Northern analysis was
performed as described previously (Johal and Briggs (1992) Science
258:985), except that total RNA (30 .mu.g per lane) was used in
this study. The entire cDNA was used as a probe. Northern analysis
showed that the steady-state level of a 1.5 kb transcript, which
was found fairly abundantly in wild-type plants, was reduced to
about 50% of the wild-type level in the Les22 alleles of not only
Les22-3 and Les22-7 (both of which are caused by Mu insertions),
but also of Les22-15 (data not shown). Furthermore, this transcript
was completely missing in the ysl mutants that segregated
recessively in the self-pollinated populations of each of Les22-3,
Les22-7, and Les22-15, confirming that the ysl phenotype
constitutes the homozygous form of Les22. Additionally, these
results indicate that all three of the mutant alleles characterized
here by Northern analysis are the result of null mutations of
Les22.
Example 6: Determination of the Molecular Nature of Les22
[0103] To ascertain the molecular nature of Les22, a 1.5 kb cDNA
clone corresponding to the sequence of LF7 was recovered from the
maize EST collection at Pioneer Hi-Bred International, Inc., and
sequenced. DNA sequences were determined by automated sequencing on
an ABI377 sequencer (Perkin Elmer) situated at the DNA Core
Facility of the University of Missouri. DNA sequence analysis was
performed using ALIGN and MEGALIGN programs of the DNASTAR software
package (DNASTAR Inc., Madison, Wisconsin). Searches of the GenBank
database were performed using the National Center for Biotechnology
Information's BLAST WWW Server.
[0104] Blast analysis indicated that Les22 encodes uroporphyrinogen
decarboxylase (UROD), the fifth enzyme of the porphyrin pathway
that is required in plants to produce the tetrapyrrole rings of
both chlorophyll and heme. The cDNA sequence for the maize urod
gene is set forth in SEQ ID NO: 1. Consistent with this revelation
is the observation that plants homozygous recessive for Les22
exhibit a chlorophyll-less ysl phenotype. Les22 mutants also appear
to be deficient in heme. Protein extraction and catalase activity
assays were carried out as previously described (Anderson et al.
(1995) Plant Physiol. 109:1247) for wild-type (Wt), Les22 mutant
(M), and ysl (Y) plants. Protein concentration was quantified using
a protein assay kit (Bio-Rad) and 30 .mu.g total protein was loaded
per lane. Four units of catalase (Sigma) were loaded in the control
lane. Catalase activity, which depends on a heme prosthetic group,
is significantly reduced and eliminated in Les22 mutants and
homozygotes, respectively, as compared to the level detected in
wild-type siblings (data not shown).
[0105] The urod gene and the porphyrin pathway, in which UROD
catalyzes the sequential decarboxylation of uroporphyrinogen III to
coproporphyrinogen III (Elder and Roberts (1995) J. Bioener.
Biomem. 27:207-214; von Wettstein et al. (1995) Plant Cell
7:1039-1057), have been highly conserved through evolution (see,
for example, Jordan, ed. (1991) in Biosynthesis of Tetrapyrroles
(Elsevier Science Publishers), pages 1-66; Labbe-Bois et al. (1977)
Mol. Gen. Genet. 156:177; Chamnongpol et al. (1996) Plant J.
10:491; Zoladek et al. (1996) Photochem. Photobiol. 64:957). Not
unexpectedly therefore, the predicted protein of the maize urod
gene (set forth in SEQ ID NO: 2) exhibits a 97%, 93% and 54% amino
acid similarity to the corresponding proteins from barley, tobacco,
and humans, respectively (Romeo et al. (1986) J. Biol. Chem.
261:9825; Mock et al. (1995) Plant Mol. Biol. 28:245). Compared to
the 391 amino acid protein of tobacco, the maize urod gene
translates into a protein of 393 amino acids, the first 62 amino
acids of which, like the 60 amino acids of the tobacco UROD but
from which it has diverged significantly, may constitute the
transit peptide that is expected to localize the enzyme in the
chloroplast (Mock et al. (1995) Plant Mol. Biol. 28:245). In the
mutant alleles of Les22-7 and Les22-3, Mu elements had inserted
between bp 102 and bp 103 and between bp 196 and bp 197,
respectively, of the nucleotide sequence for the maize urod gene
set forth in SEQ ID NO: 1. Thus insertion of the Mu elements was 34
nucleotides upstream and 59 nucleotides downstream, respectively,
from the first nucleotide (bp 137 of SEQ ID NO: 1) of the ATG start
codon. The locations of both of these Mu insertions are critical
and are expected to cause null mutations in the Les22 gene, as has
been demonstrated by the transcript analysis. In addition, the
location of the Mu1 element in Les22-7, which appears to be between
the transcription and translation start sites of urod, is
consistent with what has been found previously with Mu-suppressible
mutants whose phenotypic manifestations are dependent on Mu
activity (Barkan et al. (1991) Proc. Natl. Acad. Sci. USA
88:3502-3506).
[0106] Accepting that Les22 results from a disruption of urod, how
does this deficiency lead to a lesion mimic phenotype that exhibits
a dominant mode of inheritance? A compelling explanation emerges
from the examination of urod mutations in humans which, like Les22,
inherit as mendelian dominants, are dependent on light for
phenotypic manifestations, and result from a loss-of-function of
the urod gene (Romeo (1977) Hum. Genet. 39:261-276; De Verneuil et
al. (1986) Science 234:732-734; Moore et al. (1987) Disorders of
Porphyrin Metabolism (Plenum Publishing Corp., New York). These
urod defects are responsible for a metabolic disorder called
porphyria cutanea tarda. As previously mentioned, the major
clinical manifestation of this defect is hypersensitivity of skin
to the damaging effects of sunlight, apparently caused by the
excessive accumulation of easily photoexcitable uroporphyrin III
(Moore et al. (1987) Disorders of Porphyrin Metabolism (Plenum
Publishing Corp., New York); Straka et al. (1990) Annu. Rev. Med.
41:457-469; Moore (1993) Int. J. Biochem. 25:1353-1368; McCarrol
(1995) Analytical Chem. 67:425R-428R). The reason for this
manifestation is that when an allele of the urod gene becomes
inactive as a result of a null mutation, the activity of UROD is
reduced to one half of its normal level, leading to a partial block
in the porphyrin metabolic pathway and resulting in uroporphyrin
accumulation. On exposure to light, excited uroporphyrin, like all
other porphyrin intermediates, readily reacts with oxygen to
produce singlet oxygen and other reactive oxygen species that
damage skin cells (Moore et al. (1987) Disorders of Porphyrin
Metabolism (Plenum Publishing Corp., New York); Straka et al.
(1990) Annu. Rev. Med. 41:457-469; Zoladek et al. (1996) Photochem.
Photobiol 64:957-962).
[0107] Several features of Les22 suggests that it has much in
common with human porphyria cutanea tarda and may therefore be
caused by the same mechanism. For instance, the phenotypic
manifestation of both Les22 and porphyria is conditioned by
sunlight. They both inherit as dominant mutations, and this
dominance is not the result of a gain of a new function, as is
usually the case with most dominant mutations (Hodkin (1993) Trends
Genet. 9:1-2), but is the consequence of a loss of function of one
copy of the urod gene.
[0108] To evaluate whether the pathologic basis of Les22 also has
its roots in porphyria, uroporphyrin(ogen) and its natural product,
coproporphyrin(ogen), were extracted from both Les22 heterozygotes
(with the lesion mimic phenotype) and homozygotes(ysl mutants) and
compared with those of their Wt siblings. Extractions were obtained
from 10 day-old maize seedlings of an F2 population of Les22-15.
The methods used to extract and HPLC analyze these porphyrin
intermediates were as previously described (Mock and Grimm (1997)
Plant Mol. Biol. 28:245-256; and Kruse et al. (1995) EMBO J.
14:3712-3720). The entire foliar tissue (pooled) was used for ysl
mutants. For Les22 mutants (heterozygotes), only the second leaf
(from the bottom), partitioned into lesion-containing (apical) and
lesion-lacking (bottom) parts and pooled from a number of plants,
as used. Pooled tissues from Wt siblings were equivalent to the
corresponding tissue from Les22 mutants. Compared to Wt controls,
uroporphyrin levels were found to be elevated in Les22 plants.
While Les22 mutants exhibited a 2- to 3-fold increase in
uroporphyrin levels (Table 1), as would be expected from their
heterozygous genotype with only one functional copy of the urod
gene, Les22 homozygotes had as much as 60 times the amount of
uroporphyrin as compared to Wt siblings (Table 1). In contrast, no
such increases in coproporphyrin were detected in either of the
Les22 genotypes (data not shown). These results are consistent with
the interpretation that the porphyrin pathway is partly blocked at
the step catalyzed by UROD in the Les22 lesion mimic mutants, and
that this disorder is responsible for the etiology of Les22.
Supporting this conclusion is the finding that tobacco transgenics
over-expressing antisense urod, besides showing stunted growth,
exhibited light-dependent induction of necrotic leaf lesions, the
intensity of which correlated with the reduction of UROD activity
(Mock and Grimm (1997) Plant Physiol. 113:1101).
[0109] Table 1. Uroporphyrin III levels in the leaf tissues
(apical, basal, or whole) of a Les22 mutant, its homozygote (ysl),
and a WT (wild-type) sibling. The data presented represent the mean
of four replications.
1 Uroporphyrin III Tissue (nmol/g fresh wt) WT apical 0.259 .+-.
0.008 WT basal 0.216 .+-. 0.005 Les22 apical 0.608 .+-. 0.018 Les22
basal 0.476 .+-. 0.014 ysl total leaf 14.042 .+-. 0.421
Example 7: Characterization of Disease Resistance
[0110] Seeds of Les22-7 were segregating for Les22 and wild-type
phenotype. They were planted in 8.89-cm pots in Strong-Lite
Universal Mix potting soil (Universal Mix, Pine Buff, Ariz.) and
grown in a greenhouse (16-h day, 20 to 35.degree. C., 50% relative
humidity, 0.56 to 0.62 mE s.sup.-1 m.sup.-2 of light from both the
sun and halogen lamps). Plants were grown to the V-9 stage (see
Simmons et al. (1998) Mol. Plant-Microbe Interact. 11:1110-1118).
At this stage, plants expressing the Les22 phenotype had leaf 10
and older leaves completely covered with lesions. Leaf 11 of these
plants had a basal portion that was lesion free; the middle of leaf
11 represented a zone where lesions were initiating; and the tip of
leaf 11 had fully formed lesions. The upper leaf 13 was completely
free of lesions.
[0111] A Texas isolate of C. heterostrophus (Drechs.) from a fungal
culture collection was used to assay for corn leaf blight. Ten
microliters of spore suspension (2.times.10.sup.4 conidia/mL) in
0.02% Tween 20 were placed on sterile, 6 mm-diameter filter paper
disks (Whatman #1). Using transparent, polyethylene adhesive tape
(3M), the disks were attached to the abaxial surface of the basal,
middle, or tip of the blade on both sides of the mid-vein of leaf
11 and the middle of leaf 13. Plants were covered with plastic bags
for the first 18 hours after inoculation, after which, both bags
and tape squares were removed. Control plants received the same
treatment but without spores. Plants received standard greenhouse
care and were evaluated for development of symptoms 10 days after
inoculation. Lesions were traced onto clear plastic film,
digitized, and total lesion area/inoculation site determined.
[0112] Conjugates of salicylic acid (SA) were extracted and
quantified after chemical (base followed by acid) hydrolysis
(Enyedi et al. (1992) Proc. Natl. Acad. Sci. USA 89:2480-2482).
Samples were analyzed with a liquid chromatography system (Waters
Corp., Milford, Mass.). Ten microliters of each extract were
injected at a flow rate of 1.5 mL/min into a Luna 3 .mu.m C-18
column (4.6 cm.times.100 mm; Phenomenex, Torrance, Calif.). The
column was maintained at 40.degree. C. and equilibrated in 22%
acetonitrile against 78% of 0.1% citrate buffer, pH 3.3. Salicylic
acid was eluted isocratically under these conditions (R.sub.1,3.1
min) and quantified using a scanning fluorescence detector (Model
474, Waters Corp.) using excitation and emission wavelengths of 300
and 405 nm, respectively. The identity of SA in maize extracts was
confirmed by its co-elution with authentic standard and by analysis
of its UV light absorption spectrum, as measured with a photodiode
array detector (Model 996, Waters Corp.).
[0113] When compared to wild-type siblings, plants expressing Les22
show enhanced resistance against infection by C. heterostrophus
(FIG. 1). Resistance is not only manifested in leaf tissue that at
the time of inoculation expresses a Les22 phenotype but also in
younger tissue that has not formed any lesions.
[0114] Levels of free plus conjugated forms of salicylic acid
(total SA) in leaves of Les22 do not seem to differ significantly
from those found in wild-type sibs (FIG. 2). Levels of free SA are
slightly higher in Les22 leaf tissue compared to equivalent tissue
of wild-type plants. However, it is not clear if this difference is
sufficient to account for the enhanced resistance of Les22 against
C. heterostrophus.
[0115] All publications and patent applications mentioned in the
specification are indicative of the level of those skilled in the
art to which this invention pertains. All publications and patent
applications are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference.
[0116] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be obvious that certain changes and
modifications may be practiced within the scope of the appended
claims.
Sequence CWU 1
1
5 1 1604 DNA Zea mays CDS (137)..(1318) 1 cccttccgat tctccgtcgc
ctgagcggct gagccaactt gccaagccca agcaagccaa 60 gtcgtcgcct
ccccgaccca acgccgcgac ccccttgccc gtccgcgacc gctgcagcac 120
ctcggatccc gcccca atg gca aca gcg tgt ccg ccg ctc tcg ctg ccg tcc
172 Met Ala Thr Ala Cys Pro Pro Leu Ser Leu Pro Ser 1 5 10 acc tcc
ctc ttc cgc ggc agg tcc gcc cgc gcc ggg ccc aac gca ggc 220 Thr Ser
Leu Phe Arg Gly Arg Ser Ala Arg Ala Gly Pro Asn Ala Gly 15 20 25
agc tca cgg ccg tcc gct gca gcg ccg tcg gag agg cgg tcg tgg agg 268
Ser Ser Arg Pro Ser Ala Ala Ala Pro Ser Glu Arg Arg Ser Trp Arg 30
35 40 agg cct cgc cca gac ggc gga aga gcc gct gct ggt gag cgc aat
cag 316 Arg Pro Arg Pro Asp Gly Gly Arg Ala Ala Ala Gly Glu Arg Asn
Gln 45 50 55 60 agg gag gaa gtc gag agg cca ccc gtc tgg ctc atg agg
cag gcc ggg 364 Arg Glu Glu Val Glu Arg Pro Pro Val Trp Leu Met Arg
Gln Ala Gly 65 70 75 agg tac atg aag agc tac caa ttg ctc tgc gag
cgg tat cct tcg ttc 412 Arg Tyr Met Lys Ser Tyr Gln Leu Leu Cys Glu
Arg Tyr Pro Ser Phe 80 85 90 cgt gaa aga tca gaa aat gtc gac cta
gtt gtt gag atc tct ttg caa 460 Arg Glu Arg Ser Glu Asn Val Asp Leu
Val Val Glu Ile Ser Leu Gln 95 100 105 cca tgg aag gtt ttc aag cct
gat gga gtc atc ttg ttc tcg gac atc 508 Pro Trp Lys Val Phe Lys Pro
Asp Gly Val Ile Leu Phe Ser Asp Ile 110 115 120 ctt act cca ctt cct
ggg atg aac ata cct ttt gac att gtg aag gga 556 Leu Thr Pro Leu Pro
Gly Met Asn Ile Pro Phe Asp Ile Val Lys Gly 125 130 135 140 aaa ggt
cca gtg atc tat gat cca ttg aga acg gca gca gct gtg aat 604 Lys Gly
Pro Val Ile Tyr Asp Pro Leu Arg Thr Ala Ala Ala Val Asn 145 150 155
gaa gtc aga gaa ttt gtt cct gag gag tgg gtc cct tat gtg ggg cag 652
Glu Val Arg Glu Phe Val Pro Glu Glu Trp Val Pro Tyr Val Gly Gln 160
165 170 gct ctg aat att ttg aga caa gag gtt aaa aat gaa gct gct gta
cta 700 Ala Leu Asn Ile Leu Arg Gln Glu Val Lys Asn Glu Ala Ala Val
Leu 175 180 185 ggt ttt gtt gga gct ccg ttt acc ttg gca tct tat tgt
gtg gaa gga 748 Gly Phe Val Gly Ala Pro Phe Thr Leu Ala Ser Tyr Cys
Val Glu Gly 190 195 200 ggt tca tca aag aac ttt aca ttg att aag aaa
atg gcc ttc tca gaa 796 Gly Ser Ser Lys Asn Phe Thr Leu Ile Lys Lys
Met Ala Phe Ser Glu 205 210 215 220 cca gcg att tta cac aat ttg cta
cag aag ttc aca aca tca atg gct 844 Pro Ala Ile Leu His Asn Leu Leu
Gln Lys Phe Thr Thr Ser Met Ala 225 230 235 aac tat att aaa tac caa
gcg gac aat ggg gcg cag gct gtc caa att 892 Asn Tyr Ile Lys Tyr Gln
Ala Asp Asn Gly Ala Gln Ala Val Gln Ile 240 245 250 ttc gat tca tgg
gct act gaa ctc agc ccg gct gat ttt gag gag ttt 940 Phe Asp Ser Trp
Ala Thr Glu Leu Ser Pro Ala Asp Phe Glu Glu Phe 255 260 265 agc ctg
cct tat cta aag cag ata gtg gat agt gtt agg gaa aca cat 988 Ser Leu
Pro Tyr Leu Lys Gln Ile Val Asp Ser Val Arg Glu Thr His 270 275 280
cct gac ttg cct ctg ata ctt tac gca agt gga tct ggg ggc ttg ctg
1036 Pro Asp Leu Pro Leu Ile Leu Tyr Ala Ser Gly Ser Gly Gly Leu
Leu 285 290 295 300 gag agg ctt cct ttg aca ggt gtt gat gtt gtc agc
ttg gac tgg acg 1084 Glu Arg Leu Pro Leu Thr Gly Val Asp Val Val
Ser Leu Asp Trp Thr 305 310 315 gtc gat atg gca gag ggc agg aaa aga
ttg gga tct aac aca gca gtc 1132 Val Asp Met Ala Glu Gly Arg Lys
Arg Leu Gly Ser Asn Thr Ala Val 320 325 330 caa ggg aac gtg gac cct
ggt gtt ctt ttt gga tcc aaa gag ttt ata 1180 Gln Gly Asn Val Asp
Pro Gly Val Leu Phe Gly Ser Lys Glu Phe Ile 335 340 345 acg agg cgg
att tac gac act gtg cag aag gct ggc aat gtt gga cat 1228 Thr Arg
Arg Ile Tyr Asp Thr Val Gln Lys Ala Gly Asn Val Gly His 350 355 360
gta ttg aac ctt ggc cat ggc atc aag gtt gga act ccg gag gaa aat
1276 Val Leu Asn Leu Gly His Gly Ile Lys Val Gly Thr Pro Glu Glu
Asn 365 370 375 380 gtt gct cac ttt ttt gag gtc gca aaa ggg atc aga
tat taa 1318 Val Ala His Phe Phe Glu Val Ala Lys Gly Ile Arg Tyr
385 390 agaacctggc atggtttttt cctttttcca aatcggcaga agttgtagag
tcggcggtcg 1378 aggatagatg cagaaagccc atgtgcagta tagagtgcct
gaaaaaattt ttgggactga 1438 ttttgtttgt tgcatttcaa gttccggttt
cagtgtaata ttgtaagcag atttgagtgg 1498 aggcgtaatg aagtgcctaa
ttgtttatag caatatagtt ttgtacaacc agtatccttg 1558 tttatgagag
tacgaagcag aaatactgat catgtgttga cagata 1604 2 393 PRT Zea mays 2
Met Ala Thr Ala Cys Pro Pro Leu Ser Leu Pro Ser Thr Ser Leu Phe 1 5
10 15 Arg Gly Arg Ser Ala Arg Ala Gly Pro Asn Ala Gly Ser Ser Arg
Pro 20 25 30 Ser Ala Ala Ala Pro Ser Glu Arg Arg Ser Trp Arg Arg
Pro Arg Pro 35 40 45 Asp Gly Gly Arg Ala Ala Ala Gly Glu Arg Asn
Gln Arg Glu Glu Val 50 55 60 Glu Arg Pro Pro Val Trp Leu Met Arg
Gln Ala Gly Arg Tyr Met Lys 65 70 75 80 Ser Tyr Gln Leu Leu Cys Glu
Arg Tyr Pro Ser Phe Arg Glu Arg Ser 85 90 95 Glu Asn Val Asp Leu
Val Val Glu Ile Ser Leu Gln Pro Trp Lys Val 100 105 110 Phe Lys Pro
Asp Gly Val Ile Leu Phe Ser Asp Ile Leu Thr Pro Leu 115 120 125 Pro
Gly Met Asn Ile Pro Phe Asp Ile Val Lys Gly Lys Gly Pro Val 130 135
140 Ile Tyr Asp Pro Leu Arg Thr Ala Ala Ala Val Asn Glu Val Arg Glu
145 150 155 160 Phe Val Pro Glu Glu Trp Val Pro Tyr Val Gly Gln Ala
Leu Asn Ile 165 170 175 Leu Arg Gln Glu Val Lys Asn Glu Ala Ala Val
Leu Gly Phe Val Gly 180 185 190 Ala Pro Phe Thr Leu Ala Ser Tyr Cys
Val Glu Gly Gly Ser Ser Lys 195 200 205 Asn Phe Thr Leu Ile Lys Lys
Met Ala Phe Ser Glu Pro Ala Ile Leu 210 215 220 His Asn Leu Leu Gln
Lys Phe Thr Thr Ser Met Ala Asn Tyr Ile Lys 225 230 235 240 Tyr Gln
Ala Asp Asn Gly Ala Gln Ala Val Gln Ile Phe Asp Ser Trp 245 250 255
Ala Thr Glu Leu Ser Pro Ala Asp Phe Glu Glu Phe Ser Leu Pro Tyr 260
265 270 Leu Lys Gln Ile Val Asp Ser Val Arg Glu Thr His Pro Asp Leu
Pro 275 280 285 Leu Ile Leu Tyr Ala Ser Gly Ser Gly Gly Leu Leu Glu
Arg Leu Pro 290 295 300 Leu Thr Gly Val Asp Val Val Ser Leu Asp Trp
Thr Val Asp Met Ala 305 310 315 320 Glu Gly Arg Lys Arg Leu Gly Ser
Asn Thr Ala Val Gln Gly Asn Val 325 330 335 Asp Pro Gly Val Leu Phe
Gly Ser Lys Glu Phe Ile Thr Arg Arg Ile 340 345 350 Tyr Asp Thr Val
Gln Lys Ala Gly Asn Val Gly His Val Leu Asn Leu 355 360 365 Gly His
Gly Ile Lys Val Gly Thr Pro Glu Glu Asn Val Ala His Phe 370 375 380
Phe Glu Val Ala Lys Gly Ile Arg Tyr 385 390 3 27 DNA Artificial
Sequence Description of Artificial SequenceSynthetic
oligonucleotide 3 cgccaacgcc tccatttcgt cgaatcc 27 4 21 DNA
Artificial Sequence Description of Artificial SequenceSynthetic
oligonucleotide 4 cttgccttca tgtacctccc g 21 5 21 DNA Artificial
Sequence Description of Artificial SequenceSynthetic
Oligonucleotide 5 cgggaggtac atgaaggcaa g 21
* * * * *