Resistance ppt

Genetic engineering for resistance to Genetic engineering for resistance to biotic stress.biotic stress.

Sreshti Bagati.Sreshti Bagati.

PhD. BiotechnologyPhD. Biotechnology

J-12-D-02-BIOTJ-12-D-02-BIOT..

STRESS:

Any external conditions that adversely affects the

growth , development or productivity of a plant.

These stresses can trigger a wide range of plant

responses like, 1. Altered Gene expression.

2. Cellular Metabolism.

3. Changes in the growth rates and crop

yields.

Stresses are classified as :-

a. Biotic stress ( by other organisms).

b. A biotic stress (excess or deficit in the

environment).

Biotic and A biotic stresses reduce the average plant Biotic and A biotic stresses reduce the average plant

productivity by 65% to 87%.productivity by 65% to 87%.

BIOTIC STRESS:BIOTIC STRESS:

Occurs as a result of the damage done to the plants by Occurs as a result of the damage done to the plants by

other living organisms (bacteria, viruses, fungi, other living organisms (bacteria, viruses, fungi,

parasites).parasites).

Effects the economic growth as well as the practical Effects the economic growth as well as the practical

development.development.

Arabidopsis thaliana Arabidopsis thaliana was used as a model plant to study was used as a model plant to study

the responses of plants to different sources of stress.the responses of plants to different sources of stress.

Genetic transformation has led to the possibility of Genetic transformation has led to the possibility of

transforming crops for enhanced resistance to transforming crops for enhanced resistance to

insects and pathogens .insects and pathogens .

Development of transgenics: Development of transgenics:

Since 1970’s rapid progress has been done in Since 1970’s rapid progress has been done in

developing tools for the manipulation of genes in developing tools for the manipulation of genes in

plants using recombinant DNA technology.plants using recombinant DNA technology.

TRANSGENIC PLANTS

NUTRITIONALQUALITY

BIOTIC STRESSTOLERANCE

ABIOTIC STRESSTOLERANCE

PHARMACEUTICALS & EDIBLE VACCINE

HYBRID DEVELOPMENTFOR HIGHER YIELD

ENHANCED SHELF LIFE

INDUSTRIAL PRODUCTS

1985

19921988

1994

1998

1996

1999

2000

1st transgenic plants producedParticle bombardment developed

GM crops considered substantially equivalent to hybrid varieties

Flavr-Savr tomato is released

Herbicide- and insect-resistant crops approved for cultivation

4.3 million acres of GM crops planted

GM food is dangerous (UK TV)Monarch butterfly paper causes uproar

GM corn is excluded from its baby food

Greenpeace starts anti-GM campaign

75 million acres of GM crops planted

Golden rice with ß-carotene developedMcDonald’s rejects GM potatoes

Insect ResistanceInsect Resistance Delayed Fruit Ripening Delayed Fruit Ripening Nutritional Enhancing Nutritional Enhancing Herbicide ResistanceHerbicide Resistance

Virus ResistanceVirus Resistance

The big five successful traitsThe big five successful traits

Techniques For Plant Transformation:Techniques For Plant Transformation:

Are widely used as methods to understand how

plants work and to improve crop plant

characteristics.

Depends upon the stable introduction of transgene into the genome of the plant.

Most commonly used methods are : vector based

(Dicotyledons) as well as the direct DNA transfer

methods (biolistics) for monocots.

Vector based gene plant Vector based gene plant transformation:transformation:

Characteristics of an ideal vector:Characteristics of an ideal vector: Should be of small size ( low molecular weight).Should be of small size ( low molecular weight).

Confer a selectable phenotype on the host cells Confer a selectable phenotype on the host cells so that transformed cells can be selected.so that transformed cells can be selected.

Contain single sites for a large number of Contain single sites for a large number of restriction enzymes to enable the efficient restriction enzymes to enable the efficient production of recombinant vectors.production of recombinant vectors.

Agro bacterium mediated gene transfer: (vector based) :

Agro bacterium tumifaceins is a soil borne , gram- negative bacterium, rod shaped motile bacterium found in the rhizosphere region.

When the plants ( like grapes, walnuts, apples and roses)are wounded or damaged, causes “crown gall” disease.

Crown gall formation in plants depends on the presence of Ti plasmid (Tumour- inducing plasmid)

Ti plasmid:Ti plasmid:

Direct DNA Transfer Method:Direct DNA Transfer Method:

CASE STUDY:Biolistic transformation of riceCASE STUDY:Biolistic transformation of rice

In this strategy two plasmids are introduced into the plant cell together.

a. One plasmid (pOZ) carries the transgene of interest.

b. Other (pHAG) carries a selectable marker (hyg) ; confers resistance to hygromysin and gus A reporter gene ( can be assayed histochemically ).

The plant tissues are screened for dual transformation by selecting them on a selective medium ( with hygromysin).

Other direct DNA Transfer MethodsOther direct DNA Transfer Methods: : Electroporation:Electroporation:

Genetic manipulation for herbicide Genetic manipulation for herbicide resistance:resistance:

Over expression of the target proteinOver expression of the target protein: : involves the titrating out of herbicide by overproduction of the target protein.

Mutation of the target protein: the logic behind this is to find a modified target protein that substitutes functionally for the native protein.

Detoxification of the herbicide using a single gene from a foreign source: means converting the herbicide to a less toxic form and removing it from the system.

Glyphosate resistance: is a broad spectrum herbicide that is effective

against 76 of the world’s worst 78 weeds.

Marketed as “ round up” by the American chemical company Monsanto.

Is a simple glycine derivative , acts as a competitive inhibitor of the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS).

EPSPS is a key enzyme in the biosynthetic pathways of the aromatic amino acids phenylalanine, tyrosine and tryptophan.

EPSP SYNTHASE

Phosphoenol pyruvate

Tryptophan

Tyrosine

Phenylalaline

ROUNDUP(Glyphosate )

Glucose

3 phosphoglycerate

GlycolysisEPSP SYNTHASE

ROUNDUP(Glyphosate )

In transgenic plant, herbicide cannot bind the mutant of EPSPS (Roundup resistant cotton and soybean)

ROUNDUP (Glyphosate) RESISTANCEROUNDUP (Glyphosate) RESISTANCE(( HERBICIDE TOLERANCE IN CROPS) HERBICIDE TOLERANCE IN CROPS)

Strategy1 for Glyphosate resistance : over Strategy1 for Glyphosate resistance : over expression of a plant EPSPS gene:expression of a plant EPSPS gene:

Isolation of petunia cDNA from Glyphosate Isolation of petunia cDNA from Glyphosate resistant tissue cultures.resistant tissue cultures.

Stepwise selection of petunia cells capable of Stepwise selection of petunia cells capable of growing in presence of the increased amounts of growing in presence of the increased amounts of Glyphosate led to the isolation of cultures in Glyphosate led to the isolation of cultures in which the levels of EPSPS enzyme was much which the levels of EPSPS enzyme was much higher than normal.higher than normal.

The resistance was due to higher amounts of the The resistance was due to higher amounts of the enzyme produced.enzyme produced.

Strategy 2 for Glyphosate resistance: mutant Strategy 2 for Glyphosate resistance: mutant EPSPS genes:EPSPS genes:

Mutated EPSPS genes have been isolated from a Mutated EPSPS genes have been isolated from a number of Glyphosate resistant bacteria.number of Glyphosate resistant bacteria.

A mutated aroA gene from A mutated aroA gene from Salmonella Salmonella typhimurium typhimurium was inserted between the promoter was inserted between the promoter and the terminator sequences of the ocs gene of and the terminator sequences of the ocs gene of the the Agro bacterium tumifaceins Agro bacterium tumifaceins Ti plasmid.Ti plasmid.

Only a moderate increase in the herbicide Only a moderate increase in the herbicide tolerance was obtained.tolerance was obtained.

Strategy 3 for Glyphosate resistance: detoxification by Strategy 3 for Glyphosate resistance: detoxification by heterologous genesheterologous genes

In soil micro organisms, Glyphosate can be In soil micro organisms, Glyphosate can be degraded by cleavage of the C-N bond, catalyzed degraded by cleavage of the C-N bond, catalyzed by an oxido reductase, to form amino methyl by an oxido reductase, to form amino methyl phosphonic acid (AMPA) and glyoxylate.phosphonic acid (AMPA) and glyoxylate.

Gene encoding the enzyme Glyphosate oxidase Gene encoding the enzyme Glyphosate oxidase (GOX) has been isolated from a soil organism, (GOX) has been isolated from a soil organism, Ochrobactrum anthropi Ochrobactrum anthropi strain LBAA.strain LBAA.

Transgenic crops such as oilseed rape Transgenic crops such as oilseed rape transformed with this gene show very good transformed with this gene show very good Glyphosate resistance in the field.Glyphosate resistance in the field.

Glufosinate Resistance Glufosinate Resistance i.i. Glufosinate (the active ingredient being Glufosinate (the active ingredient being

phosphinothricin) mimics the structure of the amino phosphinothricin) mimics the structure of the amino acid glutamine, which blocks the enzyme glutamate acid glutamine, which blocks the enzyme glutamate synthase.synthase.

ii.ii. Plants receive a gene from the bacterium Streptomyces Plants receive a gene from the bacterium Streptomyces (bar gene) that produce a protein that inactivates the (bar gene) that produce a protein that inactivates the herbicide.herbicide.

Bromoxynil Resistance i. A gene encoding the enzyme bromoxynil nitrilase (BXN)

is transferred from Klebsiella pneumoniae bacteria to plants.

ii. Nitrilase inactivates the Bromoxynil before it kills the plant.

Sulfonylurea. i. Kills plants by blocking an enzyme needed for synthesis

of the amino acids valine, leucine, and isoleucine. ii. Resistance generated by mutating a gene in tobacco

plants (acetolactate synthase), and transferring the mutated gene into crop plants

Roundup Ready™ Soybeans

A problem in agriculture is the reduced growth of crops imposed by the presence of unwanted weeds.

Herbicides such as RoundupTM and Liberty LinkTM are able to kill a wide range of weeds and have the

advantage of breaking down easily. Development of herbicide resistant crops allows the elimination of

surrounding weeds without harm to the crops.

Insect resistanceInsect resistance

Anti-Insect Strategy - InsecticidesAnti-Insect Strategy - Insecticidesa) Toxic crystal protein from a) Toxic crystal protein from Bacillus Bacillus

thuringensisthuringensis Toxic crystals found during sporulationToxic crystals found during sporulation Alkaline protein degrades gut wall of Alkaline protein degrades gut wall of

lepidopteron larvae lepidopteron larvae • Corn borer caterpillarsCorn borer caterpillars• Cotton bollworm caterpillarsCotton bollworm caterpillars• Tobacco hornworm caterpillarsTobacco hornworm caterpillars• Gypsy moth larvaeGypsy moth larvae

Sprayed onto plants – but will wash offSprayed onto plants – but will wash off

The Bt toxin isolated from The Bt toxin isolated from Bacillus thuringensisBacillus thuringensis has has been used in plants. The gene has been placed in been used in plants. The gene has been placed in corn, cotton, and potato, and has been marketed.corn, cotton, and potato, and has been marketed.

Insect Resistance

Corn hybrid with a Bt gene Corn hybrid susceptible to European corn borer

Various insect resistant crops have been produced. Most of these make use of the Cry

gene in the bacteria Bacillus thuringiensis (Bt); this gene directs the production of a protein that causes paralysis and death to

many insects.

δ -endotoxin gene (Cry gene) of Bacillus thuriengenesis

GENE FOR Bt TOXIN WAS TRANSFERREDTO OBTAIN BT TRANSGENIC PLANTS

PLANT SYNTHESIZES INACTIVE PROTOXIN

PROTEINASEDIGESTION ININSECT GUT MAKES THEACTIVE TOXIN

Toxin binds a receptor on the gut epithelial cells, forms a channel on the membrane. This causes

electrolyte leakage and insect death

INSECT FEEDS ONTRANSGENIC PLANT

Case study :Resistance of Bt-maize to the Case study :Resistance of Bt-maize to the

European corn borer and other pestsEuropean corn borer and other pests::

European corn borer ( European corn borer ( Ostrinia nubilalis Ostrinia nubilalis or ECB) a or ECB) a major pest of maize.major pest of maize.

Larvae damages the crops by tunneling into the central Larvae damages the crops by tunneling into the central pith of stalks .pith of stalks .

The rate of adoption of Bt-corn has been rapid in the The rate of adoption of Bt-corn has been rapid in the USA, growing from <5% of the crop acreage in 1996 to USA, growing from <5% of the crop acreage in 1996 to 25% in 2000.25% in 2000.

three different transformation events with the cry 1Ab three different transformation events with the cry 1Ab gene (176, Bt11 and Mon 810) have been developed by gene (176, Bt11 and Mon 810) have been developed by different companies and successfully marketed.different companies and successfully marketed.

Problems of insect resistance to Problems of insect resistance to Bt:Bt:

Equally rapid appearance of resistant pests. This Equally rapid appearance of resistant pests. This problem initially attracted widespread attention problem initially attracted widespread attention during the first commercial season of the Bt during the first commercial season of the Bt cotton crop.cotton crop.

A separate issue was brought to the lime light by A separate issue was brought to the lime light by a report that appeared in 1999 indicating that a report that appeared in 1999 indicating that the pollen from Bt maize might be toxic to the the pollen from Bt maize might be toxic to the larvae of the Monarch butterfly.larvae of the Monarch butterfly.

Pathogen Derived Resistance (PDR).Pathogen Derived Resistance (PDR).

a.a. Interactions involving viral proteins. Interactions involving viral proteins.

b.b. Involving viral RNA. Involving viral RNA.

RNA Effects:RNA Effects:

a.a. Satellite sequences.Satellite sequences.

b.b. Antisense and RibozymesAntisense and Ribozymes..

c.c. Gene silencing /Co repression.Gene silencing /Co repression.

Virus resistanceVirus resistance

Pathogen Derived Resistance:Pathogen Derived Resistance:

is the first and the main antiviral transgenic is the first and the main antiviral transgenic approach used; originally known as parasite-approach used; originally known as parasite-derived resistance.derived resistance.

Pathogen sequences are deliberately engineered Pathogen sequences are deliberately engineered into the host plants genome.into the host plants genome.

Cross-protection forms the basis of PDR i.e., the Cross-protection forms the basis of PDR i.e., the presence of the pathogen sequence may directly presence of the pathogen sequence may directly interfere with the replication of the pathogen or interfere with the replication of the pathogen or may induce some host defense mechanism.may induce some host defense mechanism.

Interactions involving viral proteins:Interactions involving viral proteins:

Most successful transgenic approach; involves the Most successful transgenic approach; involves the expression of the coat protein (CP) coding sequence.expression of the coat protein (CP) coding sequence.

CP mediated resistance was first reported with a CP mediated resistance was first reported with a TMV-tobacco model system in 1986.TMV-tobacco model system in 1986.

Some degree of resistance has been found in many Some degree of resistance has been found in many cases.cases.

Variations in the levels of expression are due to Variations in the levels of expression are due to transcriptional gene silencing, transgene position transcriptional gene silencing, transgene position effects and the relationship between coding effects and the relationship between coding sequence and target virus.sequence and target virus.

RNA Effects:RNA Effects: Satellite sequences:Satellite sequences:

I.I. Plant viral satellites RNAs are small RNA molecules Plant viral satellites RNAs are small RNA molecules that are unable to multiply in host cells without the that are unable to multiply in host cells without the presence of a specific helper virus.presence of a specific helper virus.

I.I. Satellite RNA is not used for viral replication but Satellite RNA is not used for viral replication but affects disease symptoms.affects disease symptoms.

I.I. It was noted that cucumber mosaic cucumovirus It was noted that cucumber mosaic cucumovirus (CMV) symptoms were reduced when the virus was (CMV) symptoms were reduced when the virus was carrying a satellite.carrying a satellite.

II.II. Transgenic Tobacco and tomato plants expressing Transgenic Tobacco and tomato plants expressing CMV satellite RNA were tested in field in China (1990-CMV satellite RNA were tested in field in China (1990-1992).1992).

Although some reduction was seen but it was not Although some reduction was seen but it was not strong enough to protect the plants.strong enough to protect the plants.

To overcome this a strategy was developed in To overcome this a strategy was developed in which satellite RNA PDR was developed in which satellite RNA PDR was developed in combination with CMV CPMR.combination with CMV CPMR.

The resistance obtained was stronger than that The resistance obtained was stronger than that of either CPMR or satellite –PDR alone.of either CPMR or satellite –PDR alone.

Antisense and Ribozymes:Antisense and Ribozymes: Constructs have been designed that express a Constructs have been designed that express a

negative sense RNA molecule that will hybridize negative sense RNA molecule that will hybridize with the infecting virus sequence.with the infecting virus sequence.

Ribozymes are catalytic RNA molecules capable of Ribozymes are catalytic RNA molecules capable of catalyzing the cleavage of the target sense RNA catalyzing the cleavage of the target sense RNA sequence.sequence.

The aim is to both block replication by the The aim is to both block replication by the formation of a double stranded RNA : RNA hybrid formation of a double stranded RNA : RNA hybrid and to cut a key region of the virus genome before and to cut a key region of the virus genome before it is able to replicate.it is able to replicate.

Gene silencing/Co-repressionGene silencing/Co-repression

Involves post transcriptional Gene silencing Involves post transcriptional Gene silencing (PTGS).(PTGS).

The transgenic petunia plants expressing The transgenic petunia plants expressing introduced chalcone synthase genes, under the introduced chalcone synthase genes, under the control of strong promoters (designed to produce control of strong promoters (designed to produce deep purple color). deep purple color).

It was found that the pigment production in It was found that the pigment production in certain regions was turned off and white certain regions was turned off and white variegated flowers were produced.variegated flowers were produced.

Bacterial and Fungal Resistance:Bacterial and Fungal Resistance: For Fungal pathogens the genes that code for For Fungal pathogens the genes that code for

chitinase and glucanase enzymes have been chitinase and glucanase enzymes have been isolated.isolated.

These enzymes degrade the cell walls of many fungi These enzymes degrade the cell walls of many fungi without affecting mammals.without affecting mammals.

Genes for the enzymes have been isolated from a Genes for the enzymes have been isolated from a number of sources like plants (rice , barley); number of sources like plants (rice , barley); bacteria (bacteria (Serratia marcescensSerratia marcescens) and fungi ) and fungi ( ( Trichoderma harzianumTrichoderma harzianum).).

Glucanases (PR proteins) have been used against Glucanases (PR proteins) have been used against fungal infection.fungal infection.

When When ββ-1,3 – glucanase (from barley) is expressed -1,3 – glucanase (from barley) is expressed in transgenic tobacco plants under the control of in transgenic tobacco plants under the control of 35S promoter, increased resistance was seen 35S promoter, increased resistance was seen towards soil borne fungal pathogen towards soil borne fungal pathogen Rhizoctonia Rhizoctonia solani.solani.

Ribosome inhibiting proteins (RIP’s) are also used Ribosome inhibiting proteins (RIP’s) are also used in the defense strategy. These enzymes remove an in the defense strategy. These enzymes remove an adenine residue from a specific site in the large adenine residue from a specific site in the large rRNA of eukaryote and prokaryote ribosome's, rRNA of eukaryote and prokaryote ribosome's, thereby inhibiting protein synthesis.thereby inhibiting protein synthesis.

Few antimicrobial proteins are used as well .Few antimicrobial proteins are used as well .

What has been commercialized in the West?What has been commercialized in the West?

Yellow squash :Yellow squash :

The As grow seeds company markets several The As grow seeds company markets several varieties of squash under the names varieties of squash under the names Independence II, Liberator III.Independence II, Liberator III.

These lines are resistant to three important viral These lines are resistant to three important viral diseases: watermelon mosaic 2 poty virus (WMV diseases: watermelon mosaic 2 poty virus (WMV -2) and cucumber mosaic virus (CMV).-2) and cucumber mosaic virus (CMV).

The constructs used contain three separate CP The constructs used contain three separate CP coding sequences for the virus.coding sequences for the virus.

Papaya:Papaya:

Was brought in a rapid time scale.Was brought in a rapid time scale.

To overcome the damage caused by papaya ring To overcome the damage caused by papaya ring spot poty virus (PRSV), workers at Cornell spot poty virus (PRSV), workers at Cornell university and in Hawaii produced two university and in Hawaii produced two genetically engineered lines.genetically engineered lines.

Both these lines (Sun Up and Rainbow) have Both these lines (Sun Up and Rainbow) have been available to farmers since 1998been available to farmers since 1998

Potato:Potato:

Monsanto marketed new leaf potato lines that Monsanto marketed new leaf potato lines that had both Bt resistance and resistance to several had both Bt resistance and resistance to several virus lines.virus lines.

Potato leafroll polerovirus, potato Y potyvirus are Potato leafroll polerovirus, potato Y potyvirus are the viral lines.the viral lines.

Due to low take up of these lines they have been Due to low take up of these lines they have been withdrawn from sale.withdrawn from sale.