Title of Invention	USE OF NUCLEOTIDE SEQUENCE ENCODING A MUTATION INDUCED RECESSIVE ALLELE FOR CONFERRING DISEASE RESISTANCE
Abstract	The present invention relates to the isolation of nucleotide sequence that can stimulate defence responses in plants against plant pathogens. The present invention also encompasses within its frame work, the expression product of any of the nucleic acid sequences of the gene and the methods of making the expression product under suitable conditions in suitable host cells, i.e., prokaryotic or eukaryotic.

Title of Invention

USE OF NUCLEOTIDE SEQUENCE ENCODING A MUTATION INDUCED RECESSIVE ALLELE FOR CONFERRING DISEASE RESISTANCE

Abstract

The present invention relates to the isolation of nucleotide sequence that can stimulate defence responses in plants against plant pathogens. The present invention also encompasses within its frame work, the expression product of any of the nucleic acid sequences of the gene and the methods of making the expression product under suitable conditions in suitable host cells, i.e., prokaryotic or eukaryotic.

Full Text	pathogen defence response is modulated, particularly stimulated. Various approaches enable diagnostic determination of the presence of susceptibility or resistance alleles in plants. W09947S52 patent application relates to isolating DNA molecules encoding Mlo proteins, wherein such Mlo proteins confer resistance of plants to fungal pathogens. Description Diseases in plants are caused by biotic and abiotic causes. Biotic causes include fungi, viruses, bacteria, and nematodes. Of these, fungi are the most frequent causative agent of disease on plants. Abiotic causes of disease in plants include extremes of temperature, water, oxygen, soil pH, plus nutrient-element deficiencies and imbalances, excess heavy metals, and air pollution. A host of cellular processes enables 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. This limitation of the pathogen intruder is frequently accomplished by localized containment of the intruder following a coordinated complex defense response. 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 neighboring 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. The hypersensitive response in many plant-pathogen interactions is specified by and dependent on the presence of two complementary genes, one from the host and one from the pathogen. These complementary genes are the resistance (R) gene in the plant and a corresponding avinilence (avr) gene in the pathogen. The interaction of the genes is associated with the rapid, localized cell death of the hypersensitive response. R genes that respond to specific bacterial, fungal, or viral pathogens, have been isolated from a variety of plant species and several appear to encode cytoplasmic proteins. The resistance gene in the plant and the avinilence 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. Because avr-R gene-for-gene relationships are observed in many plant-pathogen systems and are accompanied by a characteristic set of defense responses, a common molecular mechanism underlying avr-R gene mediated resistance has been postulated. A simple model which has been proposed is that pathogen avr genes directly or indirecdy generate a specific molecular signal (ligand) that is recognized by cognate receptors encoded by plant R genes. Both plant resistance genes and corresponding pathogen avirulence genes have been cloned. The plant kingdom contains thousands of R genes with specific specificities for viral, bacterial, fungal, or nematode pathogens. Although there are differences in the defense responses induced during different plant-pathogen interactions, some common themes are apparent among R gene-mediated defenses. The function of a given R gene is dependent on the genotype of the pathogen. Plant pathogens produce a diversity of potential signals, and in a fashion analogous to the production of antigens by mammalian pathogens, some of these signals are detectable by some plants. The avirulence gene causes the pathogen to produce a signal that triggers a strong defense response in a plant with the appropriate R gene. However, expressing an "avirulence gene does not stop the pathogen from being virulent on hosts that lack the corresponding R gene, A single plant can have many R genes, and a pathogen can have many avr genes. Monogenic resistance mediated by recessive (mlo) alleles of the Mlo locus is different. It differs from race-specific incompatibility to single pathogen strains in that it is believed to confer a broad spectrum resistance to almost all known isolates of the fungal pathogen, and the resistance is apparently durable in the field despite extensive cultivation. Further, mlo resistance alleles have been obtained by mutagen treatment of susceptible wild-type Mlo varieties. These mlo plants exhibit a spontaneous leaf cell death phenotype under pathogen-free or even axenic conditions. Mutations have been described in several plants in which defence responses to pathogens appear to be constitutively expressed. Mutation-induced recessive alleles (mlo) of the barley Mlo locus exhibit a leaf lesion phenotype and confer an apparently durable, broad spectrum resistance to the powdery mildew pathogen, Erysiphe graminis f sp hordei. Resistance responses to the powdery mildew pathogen have been genetically well characterized (Wiberg, 1974; Sgaard and Jrgensen, 1988; Jrgensen, 1994). In most analyzed cases resistance is specified by race-specific resistance genes following the rules of Flor's gene-for-gene hypothesis (Flor, 1971). In this type of plant/pathogen interaction, resistance is specified by and dependent on the presence of two complementary genes, one from the host and one from the fungal pathogen. The complementary genes have been termed operationally (pathogen) resistance ("R") gene and avirulence gene, respectively. Most of the powdery mildew resistance genes (Mix) act as dominant or semidominant traits (Jergensen, 1994). This cDNA clone is of potential value in plant biotechnology programmes, and as a transdominant mutant gene, can confer, resistance to plant pathogens. The resultant product of the nucleic acid sequence may be used in producing antibodies and polypeptides comprising of antigen binding regions of the antibodies and also used in identifying homologues in other species. Procedure 1. Total RNA was extracted from ten day old coleoptile tissue. 2. Subsequently, mRNA was isolated from the total RNA. 3. A cDNA library was constructed by making use of the GIBCOBRL Superscript Plasmid System with Gateway Technology for cDNA Synthesis and Cloning Kit. 4. The clones were screened and selected for sequencing and subjected to a data base search in order to ascertain their identity. 5. Functional details of the clones were collected and one cDNA clone was designated as cDGsCo01B03, which was predicted to be a homologue of barley Mlo gene. The present invention provides new and effective strategies to control fungal diseases in economically important crops, potentially reducing amounts of chemicals applied to crops and reducing the risk of appearance of pathogens resistant to control agents. Claims 1. A method for cloning cDNAs encoding disease-resistance proteins, mutation induced recessive allele nucleotide sequences comprising (a) Providing tissue induced to systemic acquired resistance or localized acquired resistance using biological inducers, and (b) Isolating cDNA clones encoding disease-resistance proteins. 2. A method of producing a plant with an enhanced hypersensitive response to invading pathogens, characterized by transforming a plant with the genetic sequence of mutation induced recessive allele. 3. A claim as in claim 2, wherein the transgenic plants could apply to all varieties of plants. 4. A claim as in claim 2 & 3, wherein the transgenic plants confer increased tolerance to pathogenic infections, thereby providing novel methods for improving plant quality and yield in the presence of pathogen. 5. A chimeric DNA constructs encoding the mutation induced recessive allele useful for producing transgenic disease-resistant plants and to genetic engineering of plants to produce the phenotype of disease resistance.

Full Text

pathogen defence response is modulated, particularly stimulated. Various approaches enable diagnostic determination of the presence of susceptibility or resistance alleles in plants.
W09947S52 patent application relates to isolating DNA molecules encoding Mlo proteins, wherein such Mlo proteins confer resistance of plants to fungal pathogens.
Description
Diseases in plants are caused by biotic and abiotic causes. Biotic causes include fungi, viruses, bacteria, and nematodes. Of these, fungi are the most frequent causative agent of disease on plants. Abiotic causes of disease in plants include extremes of temperature, water, oxygen, soil pH, plus nutrient-element deficiencies and imbalances, excess heavy metals, and air pollution.
A host of cellular processes enables 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. This limitation of the pathogen intruder is frequently accomplished by localized containment of the intruder following a coordinated complex defense response.
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 neighboring 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.
The hypersensitive response in many plant-pathogen interactions is specified by and dependent on the presence of two complementary genes, one from the host and one from the pathogen. These complementary genes are the resistance (R) gene in the plant and a corresponding avinilence (avr) gene in the pathogen. The interaction of the genes is associated with the rapid, localized cell death of the hypersensitive response. R genes that respond to specific bacterial, fungal, or viral pathogens, have been isolated from a variety of plant species and several appear to encode cytoplasmic proteins.
The resistance gene in the plant and the avinilence 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. Because avr-R gene-for-gene relationships are observed in many plant-pathogen systems and are accompanied by a characteristic set of defense responses, a common molecular mechanism underlying avr-R gene mediated resistance has been postulated. A simple model which has been

proposed is that pathogen avr genes directly or indirecdy generate a specific molecular signal (ligand) that is recognized by cognate receptors encoded by plant R
genes.
Both plant resistance genes and corresponding pathogen avirulence genes have been cloned. The plant kingdom contains thousands of R genes with specific specificities for viral, bacterial, fungal, or nematode pathogens. Although there are differences in the defense responses induced during different plant-pathogen interactions, some common themes are apparent among R gene-mediated defenses. The function of a given R gene is dependent on the genotype of the pathogen. Plant pathogens produce a diversity of potential signals, and in a fashion analogous to the production of antigens by mammalian pathogens, some of these signals are detectable by some plants.
The avirulence gene causes the pathogen to produce a signal that triggers a strong
defense response in a plant with the appropriate R gene. However, expressing an "avirulence gene does not stop the pathogen from being virulent on hosts that lack the corresponding R gene, A single plant can have many R genes, and a pathogen can have many avr genes.
Monogenic resistance mediated by recessive (mlo) alleles of the Mlo locus is different. It differs from race-specific incompatibility to single pathogen strains in that it is believed to confer a broad spectrum resistance to almost all known isolates of the fungal pathogen, and the resistance is apparently durable in the field despite extensive cultivation. Further, mlo resistance alleles have been obtained by mutagen treatment of susceptible wild-type Mlo varieties. These mlo plants exhibit a spontaneous leaf cell death phenotype under pathogen-free or even axenic conditions.
Mutations have been described in several plants in which defence responses to pathogens appear to be constitutively expressed. Mutation-induced recessive alleles (mlo) of the barley Mlo locus exhibit a leaf lesion phenotype and confer an apparently durable, broad spectrum resistance to the powdery mildew pathogen, Erysiphe graminis f sp hordei.
Resistance responses to the powdery mildew pathogen have been genetically well
characterized (Wiberg, 1974; Sgaard and
Jrgensen, 1988; Jrgensen, 1994). In most analyzed cases resistance is specified by race-specific resistance genes following the rules of Flor's gene-for-gene hypothesis (Flor, 1971). In this type of plant/pathogen interaction, resistance is specified by and dependent on the presence of two complementary genes, one from the host and one from the fungal pathogen. The complementary genes have been termed operationally (pathogen) resistance ("R") gene and avirulence gene, respectively. Most of the powdery mildew resistance genes (Mix) act as dominant or semidominant traits (Jergensen, 1994).
This cDNA clone is of potential value in plant biotechnology programmes, and as a transdominant mutant gene, can confer, resistance to plant pathogens. The resultant product of the nucleic acid sequence may be used in producing antibodies and polypeptides comprising of antigen binding regions of the antibodies and also used in identifying homologues in other species.

Procedure
1. Total RNA was extracted from ten day old coleoptile tissue.
2. Subsequently, mRNA was isolated from the total RNA.
3. A cDNA library was constructed by making use of the GIBCOBRL Superscript Plasmid System with Gateway Technology for cDNA Synthesis and Cloning Kit.
4. The clones were screened and selected for sequencing and subjected to a data base search in order to ascertain their identity.
5. Functional details of the clones were collected and one cDNA clone was designated as cDGsCo01B03, which was predicted to be a homologue of barley Mlo gene.
The present invention provides new and effective strategies to control fungal diseases in economically important crops, potentially reducing amounts of chemicals applied to crops and reducing the risk of appearance of pathogens resistant to control agents.

Claims
1. A method for cloning cDNAs encoding disease-resistance proteins, mutation induced recessive allele nucleotide sequences comprising
(a) Providing tissue induced to systemic acquired resistance or localized acquired resistance using biological inducers, and
(b) Isolating cDNA clones encoding disease-resistance proteins.
2. A method of producing a plant with an enhanced hypersensitive response to
invading pathogens, characterized by transforming a plant with the genetic
sequence of mutation induced recessive allele.
3. A claim as in claim 2, wherein the transgenic plants could apply to all
varieties of plants.
4. A claim as in claim 2 & 3, wherein the transgenic plants confer increased
tolerance to pathogenic infections, thereby providing novel methods for
improving plant quality and yield in the presence of pathogen.
5. A chimeric DNA constructs encoding the mutation induced recessive allele
useful for producing transgenic disease-resistant plants and to genetic
engineering of plants to produce the phenotype of disease resistance.

Documents:

773-che-2003 abstract-17-07-2009.pdf

773-che-2003 claims-17-07-2009.pdf

773-che-2003 correspondence others-17-07-2009.pdf

773-che-2003 description(complete)-17-07-2009.pdf

773-che-2003 drawings-17-07-2009.pdf

773-che-2003 form-1-17-07-2009.pdf

773-che-2003-abstract.pdf

773-che-2003-claims.pdf

773-che-2003-correspondnece-po.pdf

773-che-2003-description(complete).pdf

773-che-2003-form 1.pdf

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Patent Number

241635

Indian Patent Application Number

773/CHE/2003

PG Journal Number

30/2010

Publication Date

23-Jul-2010

Grant Date

16-Jul-2010

Date of Filing

24-Sep-2003

Name of Patentee

Avestha Gengraine Technologies Pvt Ltd

Applicant Address

'DISCOVERER' 9TH FLOOR, UNIT 3, INTERNATIONAL TECH PARK WHITEFIELD ROAD BANGALORE 560 066

Inventors:

#	Inventor's Name	Inventor's Address
1	DR. VILLOO MORAWALA PATELL	'DISCOVERER' 9TH FLOOR, UNIT 3, INTERNATIONAL TECH PARK WHITEFIELD ROAD BANGALORE 560 066

PCT International Classification Number

C12N15/9

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA