Genetic engineering for biotic stress in plants.

1. Genetic engineering for resistance to
biotic stress.
Sreshti Bagati.
PhD. Biotechnology
.
J-12-D-02-BIOT

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. 
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. 
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.

5. HYBRID DEVELOPMENT
FOR HIGHER YIELD
NUTRITIONAL
QUALITY
BIOTIC STRESS
TOLERANCE
TRANSGENIC PLANTS
ENHANCED
SHELF LIFE
ABIOTIC STRESS
TOLERANCE
INDUSTRIAL
PRODUCTS
PHARMACEUTICALS
& EDIBLE VACCINE

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. The big five successful traits
 Insect Resistance
 Delayed Fruit Ripening
 Nutritional Enhancing
 Herbicide Resistance
 Virus Resistance

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. 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. 
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. Ti plasmid:

14. 
Direct DNA Transfer Method:

15. 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).

16. Other direct DNA Transfer Methods:
Electroporation:

17. 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.

18. 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.

20. 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)

21. 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.

22. 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.

23. 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.

24. 


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

25. 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.

26. 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.

27. 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

28. 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

29. 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.

30. 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.

31. 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.

32. 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.

33. 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.

34. 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).

35. 
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.

36. 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.

37. 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.

38. 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.

39. 
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 .

40. 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.

41. 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

42. 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.