Resistance ppt


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Genetic engineering for biotic stress in plants.

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Resistance ppt

  1. 1. Genetic engineering for resistance to biotic stress. Sreshti Bagati. PhD. Biotechnology . J-12-D-02-BIOT
  2. 2. 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).
  3. 3.  Biotic and A biotic stresses reduce the average plant productivity by 65% to 87%. BIOTIC STRESS:  Occurs as a result of the damage done to the plants by other living organisms (bacteria, viruses, fungi, parasites).  Effects the economic growth as well as the practical development.  Arabidopsis thaliana was used as a model plant to study the responses of plants to different sources of stress.
  4. 4.  Genetic transformation has led to the possibility of transforming crops for enhanced resistance to insects and pathogens . Development of transgenics:  Since 1970’s rapid progress has been done in developing tools for the manipulation of genes in plants using recombinant DNA technology.
  6. 6. 1985 1988 1992 1st transgenic plants produced Particle bombardment developed 1994 Flavr-Savr tomato is released 1996 Herbicide- and insect-resistant crops approved for cultivation GM crops considered substantially equivalent to hybrid varieties 4.3 million acres of GM crops planted 1998 1999 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 2000 Golden rice with ß-carotene developed McDonald’s rejects GM potatoes
  7. 7. The big five successful traits  Insect Resistance  Delayed Fruit Ripening  Nutritional Enhancing  Herbicide Resistance  Virus Resistance
  8. 8.  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.
  9. 9. Vector based gene plant transformation: Characteristics of an ideal vector:  Should be of small size ( low molecular weight).  Confer a selectable phenotype on the host cells so that transformed cells can be selected.  Contain single sites for a large number of restriction enzymes to enable the efficient production of recombinant vectors.
  10. 10.  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)
  11. 11. Ti plasmid:
  12. 12.  Direct DNA Transfer Method:
  13. 13. CASE 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).
  14. 14. Other direct DNA Transfer Methods: Electroporation:
  15. 15. Genetic manipulation for herbicide resistance:  Over 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.
  16. 16. 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.
  17. 17. ROUNDUP (Glyphosate) RESISTANCE ( HERBICIDE TOLERANCE IN CROPS) Glucose ROUNDUP (Glyphosate ) 3 phosphoglycerate Glycolysis Phosphoenol pyruvate Tryptophan EPSP SYNTHASE EPSP SYNTHASE Tyrosine Phenylalaline ROUNDUP (Glyphosate ) In transgenic plant, herbicide cannot bind the mutant of EPSPS (Roundup resistant cotton and soybean)
  18. 18. Strategy1 for Glyphosate resistance : over expression of a plant EPSPS gene:  Isolation of petunia cDNA from Glyphosate resistant tissue cultures.  Stepwise selection of petunia cells capable of growing in presence of the increased amounts of Glyphosate led to the isolation of cultures in which the levels of EPSPS enzyme was much higher than normal.  The resistance was due to higher amounts of the enzyme produced.
  19. 19. Strategy 2 for Glyphosate resistance: mutant EPSPS genes:  Mutated EPSPS genes have been isolated from a number of Glyphosate resistant bacteria.  A mutated aroA gene from Salmonella typhimurium was inserted between the promoter and the terminator sequences of the ocs gene of the Agro bacterium tumifaceins Ti plasmid.  Only a moderate increase in the herbicide tolerance was obtained.
  20. 20. Strategy 3 for Glyphosate resistance: detoxification by heterologous genes  In soil micro organisms, Glyphosate can be degraded by cleavage of the C-N bond, catalyzed by an oxido reductase, to form amino methyl phosphonic acid (AMPA) and glyoxylate.  Gene encoding the enzyme Glyphosate oxidase (GOX) has been isolated from a soil organism, Ochrobactrum anthropi strain LBAA.  Transgenic crops such as oilseed rape transformed with this gene show very good Glyphosate resistance in the field.
  21. 21.    Glufosinate Resistance i. Glufosinate (the active ingredient being phosphinothricin) mimics the structure of the amino acid glutamine, which blocks the enzyme glutamate synthase. ii. Plants receive a gene from the bacterium Streptomyces (bar gene) that produce a protein that inactivates the 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
  22. 22. 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.
  23. 23. Insect resistance Anti-Insect Strategy - Insecticides a) Toxic crystal protein from Bacillus thuringensis Toxic crystals found during sporulation  Alkaline protein degrades gut wall of lepidopteron larvae • Corn borer caterpillars • Cotton bollworm caterpillars • Tobacco hornworm caterpillars • Gypsy moth larvae  Sprayed onto plants – but will wash off  The Bt toxin isolated from Bacillus thuringensis has been used in plants. The gene has been placed in corn, cotton, and potato, and has been marketed.
  24. 24. Insect Resistance 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. Corn hybrid with a Bt gene Corn hybrid susceptible to European corn borer
  25. 25. δ -endotoxin gene (Cry gene) of Bacillus thuriengenesis GENE FOR Bt TOXIN WAS TRANSFERRED TO OBTAIN BT TRANSGENIC PLANTS PLANT SYNTHESIZES INACTIVE PROTOXIN INSECT FEEDS ON TRANSGENIC PLANT PROTEINASE DIGESTION IN INSECT GUT MAKES THE ACTIVE TOXIN Toxin binds a receptor on the gut epithelial cells, forms a channel on the membrane. This causes electrolyte leakage and insect death
  26. 26. Case study :Resistance of Bt-maize to the European corn borer and other pests:  European corn borer ( Ostrinia nubilalis or ECB) a major pest of maize.  Larvae damages the crops by tunneling into the central pith of stalks .  The rate of adoption of Bt-corn has been rapid in the USA, growing from <5% of the crop acreage in 1996 to 25% in 2000.  three different transformation events with the cry 1Ab gene (176, Bt11 and Mon 810) have been developed by different companies and successfully marketed.
  27. 27. Problems of insect resistance to Bt:  Equally rapid appearance of resistant pests. This problem initially attracted widespread attention during the first commercial season of the Bt cotton crop.  A separate issue was brought to the lime light by a report that appeared in 1999 indicating that the pollen from Bt maize might be toxic to the larvae of the Monarch butterfly.
  28. 28. Virus resistance  a. b.  a. b. c. Pathogen Derived Resistance (PDR). Interactions involving viral proteins. Involving viral RNA. RNA Effects: Satellite sequences. Antisense and Ribozymes. Gene silencing /Co repression.
  29. 29. Pathogen Derived Resistance:  is the first and the main antiviral transgenic approach used; originally known as parasite-derived resistance.  Pathogen sequences are deliberately engineered into the host plants genome.  Cross-protection forms the basis of PDR i.e., the presence of the pathogen sequence may directly interfere with the replication of the pathogen or may induce some host defense mechanism.
  30. 30. Interactions involving viral proteins:  Most successful transgenic approach; involves the expression of the coat protein (CP) coding sequence.  CP mediated resistance was first reported with a TMV-tobacco model system in 1986.  Some degree of resistance has been found in many cases.  Variations in the levels of expression are due to transcriptional gene silencing, transgene position effects and the relationship between coding sequence and target virus.
  31. 31. RNA Effects:  I. I. I. II. Satellite sequences: Plant viral satellites RNAs are small RNA molecules that are unable to multiply in host cells without the presence of a specific helper virus. Satellite RNA is not used for viral replication but affects disease symptoms. It was noted that cucumber mosaic cucumovirus (CMV) symptoms were reduced when the virus was carrying a satellite. Transgenic Tobacco and tomato plants expressing CMV satellite RNA were tested in field in China (1990-1992).
  32. 32.  Although some reduction was seen but it was not strong enough to protect the plants.  To overcome this a strategy was developed in which satellite RNA PDR was developed in combination with CMV CPMR.  The resistance obtained was stronger than that of either CPMR or satellite –PDR alone.
  33. 33. Antisense and Ribozymes:  Constructs have been designed that express a negative sense RNA molecule that will hybridize with the infecting virus sequence.  Ribozymes are catalytic RNA molecules capable of catalyzing the cleavage of the target sense RNA sequence.  The aim is to both block replication by the formation of a double stranded RNA : RNA hybrid and to cut a key region of the virus genome before it is able to replicate.
  34. 34. Gene silencing/Co-repression  Involves post transcriptional Gene silencing (PTGS).  The transgenic petunia plants expressing introduced chalcone synthase genes, under the control of strong promoters (designed to produce deep purple color).  It was found that the pigment production in certain regions was turned off and white variegated flowers were produced.
  35. 35. Bacterial and Fungal Resistance:  For Fungal pathogens the genes that code for chitinase and glucanase enzymes have been isolated.  These enzymes degrade the cell walls of many fungi without affecting mammals.  Genes for the enzymes have been isolated from a number of sources like plants (rice , barley); bacteria (Serratia marcescens) and fungi ( Trichoderma harzianum).  Glucanases (PR proteins) have been used against fungal infection.
  36. 36.  When β-1,3 – glucanase (from barley) is expressed in transgenic tobacco plants under the control of 35S promoter, increased resistance was seen towards soil borne fungal pathogen Rhizoctonia solani.  Ribosome inhibiting proteins (RIP’s) are also used in the defense strategy. These enzymes remove an adenine residue from a specific site in the large rRNA of eukaryote and prokaryote ribosome's, thereby inhibiting protein synthesis.  Few antimicrobial proteins are used as well .
  37. 37. What has been commercialized in the West?  Yellow squash :  The As grow seeds company markets several varieties of squash under the names Independence II, Liberator III.  These lines are resistant to three important viral diseases: watermelon mosaic 2 poty virus (WMV -2) and cucumber mosaic virus (CMV).  The constructs used contain three separate CP coding sequences for the virus.
  38. 38. Papaya:  Was brought in a rapid time scale.  To overcome the damage caused by papaya ring spot poty virus (PRSV), workers at Cornell university and in Hawaii produced two genetically engineered lines.  Both these lines (Sun Up and Rainbow) have been available to farmers since 1998
  39. 39. Potato:  Monsanto marketed new leaf potato lines that had both Bt resistance and resistance to several virus lines.  Potato leafroll polerovirus, potato Y potyvirus are the viral lines.  Due to low take up of these lines they have been withdrawn from sale.