Weeds Unwanted plants growing along with crops Compete with crops for water and nutrients and decrease yield Herbicide applied to eliminate weeds

1. MBB – 602 – Advances in crop biotechnology
Topic - Genetic engineering strategies for biotic stress resistance in plants
Department of Agricultural Biotechnology
Presented By -
Jyoti Prakash Sahoo
01ABT/PHD/17
12/22/2017 1
Course Instructor -
Prof. (Dr.) G. R. Rout
Professor and Head

2. 12/22/2017 2
Genetic engineering for herbicide resistance
Weeds
• Unwanted plants growing along with crops
• Compete with crops for water and nutrients and decrease yield
• Herbicide applied to eliminate weeds
Need to develop herbicides resistance crops
• Commercially available herbicides can’t discriminate weeds from crops; so difficult to
use in fields
• Hence need to develop herbicide resistance crops enabling use of herbicides to kill
weeds selectively

3. 12/22/2017 3
Herbicide resistance in plants
Several genes identified in bacterial and plants confers resistance to herbicides.
Herbicides Mode of development of herbicide resistance
Triazines Chloroplast D1 protein Mutated target (A mutation in PS-II
gene (psb A) prevents Triazine from binding with D1
Protein and confers resistance)
Substituted urea Chloroplast D1 protein Mutated target
Sulfonylureas Acetolactase synthase Mutated target
Imidazolinones Acetolactase synthase Mutated target
Pyrimidyl thiobenzoates Acetolactase synthase Mutated target
Aryloxyphenoxpropanoates Acetyl coenzyme A carboxylase Mutated target
Cyclohexanediones Acetyl coenzyme A carboxylase Mutated target
Glyphosate EPSPS Mutated target
EPSPS Over expression
Glyphosate oxidoreductase Detoxification
2,4-D Monooxygenase Detoxification
Glufosinate N-acetyl transferase Detoxification

4. Glyphosate herbicide effect
 Resistance to glyphosate can be generated by expressing transgenes that –
1. Encode a target enzyme that is tolerant to the herbicide
2. Lead to over production of the target enzyme
3. Produce an enzyme that inactivates glyphosate
12/22/2017 4
• Glyphosate – a broad spectrum herbicide inhibits the enzyme 5-enolpyruvyl shikimate-
3-phosphate (EPSP) synthase, which is important for synthesis of aromatic amino acids,
plants die due to unavailability of these amino acids.
Substrate

5. 12/22/2017
1. Encode a target enzyme that is tolerant to the herbicide
 Gene fusion between the transit peptide sequence of the petunia cDNA clone
and an E. coli gene which encodes glyphosate tolerant EPSP synthase yielded
transgenic tobacco plants which showed higher tolerance to glyphosate.
Express a mutant EPSPS from the CP4 strain of
Agrobacterium tumefaciens, using a 35S CaMV
promotor, a chloroplast transit peptide coding sequence
and a nopaline synthase (nos 3’) transcriptional
terminator from Agrobacterium tumefaciens.
Roundup Ready Soybeans
5
Padgette et al. 1995

6. 12/22/2017 6
The commercial soybean variety A5403 (Asgrow Seed Co.) transformed by gold
particle bombardment, with the PV-GMGT04 plasmid vector harvested from
Escherichia coli.
The PV-GMGT04 plasmid contained –
CP4 EPSPS gene coding for glyphosate tolerance
 gus gene for production of ß-glucuronidase as a selectable marker
 nptII gene for antibiotic (kanamycin) resistance
Development method
Plasmid map including genetic
elements of vector PV-GMGT04
used in the transformation of RR
soybean event 40-3-2 (taken from
Monsanto, 2000)

7. 2. Lead to over production of the target enzyme
• Glyphosate - tolerant cell cultures of Petunia hybrida was obtained after selection
on the herbicide.
• These petunia cell lines were found to overproduce EPSP synthase as a result of
a 20 - fold gene amplification.
• EPSPS gene encoding wild type EPSP synthase from petunia under the control
of cauliflower mosaic virus (CaMV) 35S promoter has been transformed into
other crop plants and over expressed lead to glyphosate tolerance.
7
12/22/2017

8. 12/22/2017 8
3. Produce an enzyme that inactivates glyphosate
• Glyphosate is metabolized by the enzyme glyphosate oxidoreductase (GOX).
• Two glyphosate metabolizing pathways have been identified in microorganisms.
1. Cleavage of the carbon-phosphorus bond yielding sarcosine and phosphate
2. Oxidative Cleavage of N-C bond to yield aminomethyl phosphonic acid (AMPA) and
glyoxylate
1
2
Inorganic
phosphate
Non-toxic Components
(GOX)

9. 12/22/2017 9
Representation of the cryIA(b) construct from
plasmid PVZMBK07 used in the transformation of
MON810, including the enhanced CaMV
35Spromoter, the maize hsp70 intron 1 and the
synthetic δ-endotoxin cryIA(b) gene followed by
the nos terminator.
Schematic representation of the
plasmid PV-ZMBK07 used in
engineering MON810
Resistance to the European Corn Borer (ECB)
modified from BATS, 2003

10. 12/22/2017 10
Genetic engineering for Insect resistance
• Approaches for insect resistance:
 Incorporating delta endotoxin sequence from Bacillus thuringiensis
 Incorporating plant derived genes like lectins, proteinase inhibitors etc.

11. 12/22/2017 11
Bacillus thuringiensis – Crystal proteins
• When Cry proteins are ingested by the insects,
they are solubilized in the alkaline environment
of insect midgut. The gut proteases process
them hydrolytically to yield a 60 KDa toxic
core fragments.
• The active toxins bind to receptors on midgut
epithelial cells, become inserted into the plasma
membrane and form pores leading to the cell
death through osmotic lysis.
Toxic action of cry proteins

12. 12/22/2017 12
Vegetative insecticidal protein (VIP)
 These proteins are produced during vegetative growth of cells and are secreted into the
growth medium.
 More than 50 Vip proteins have been identified so far. The ingestion of Vip proteins
causes swelling and disruption of the midgut epithelial cells by osmotic lysis in the
target insects.
Other genes for insect resistance
Bt transgenic plants currently dominate the market but there are many alternative
insecticidal proteins.

13. 12/22/2017 13
Proteinase inhibitors
• Interfere with the digestive enzymes of the insect, results in the nutrient
deprivation causing death of the insects.
• Most active inhibitor identified is cowpea trypsin inhibitor (CpTI)
• CpTI-transformed tobacco, field-tested and caused significant larval
mortality of cotton bollworm.

14. 12/22/2017 14
Lectins
• Lectins are carbohydrate-binding proteins
found in many plant tissues.
• Abundant in the seeds and storage tissues of
some plant species.
• Involves specific binding of lectin to
glyco-conjugates located in the
midgut of the insect.
• Tobacco plants expressing a pea lectin
were shown to be toxic to the Lepidoptera
insects.

15. 12/22/2017 15
alpha-Amylase inhibitors
• Insect larvae secrete a gut enzyme alpha- amylase to digest starch.
• Blocking the activity of this enzyme by α-amylase inhibitor, the larvae can
be starved and killed.
• α-Amylase inhibitor gene isolated from bean has been successfully transferred and
expressed in tobacco and provides resistance against Coleoptera insects.

16. 12/22/2017 16
Insect chitinases
• Chitin is an insoluble structural polysaccharide that occurs in
the exoskeletal and gut lining of insects.
• It is believed to protect the insect against water loss.
• Dissolution of chitin by chitinase is known to perforate
peritrophic matrix and exoskeleton and make insects
vulnerable to attack by different pathogens.

17. 12/22/2017 17
Risk associated with transgenic plants showing resistance against insect pest

18. 12/22/2017 18
Genetic engineering for fugal pathogen resistance
 The Food and Agriculture Organization (FAO) has estimated that each year,
25% of the world’s crops are affected by mycotoxins with an annual loss of
about 1 billion metric tons of food and food products.
- (The American Phytopathological Society, 2014)
 So it is an important aspect of farming community of the world to mitigate
fungal diseases in order to reduce yield losses in field and also during post-
harvest.

19. 19
• Pathogen virulent – Host plant suceptible – Compatible interaction (Disease)
• Pathogen avirulent – Host plant resistance – Incompatible interaction (No disease)
12/22/2017
Strategies of pathogen
to infect plants
Host pathogen interaction
 Innate immunity against pathogens
1. Anatomical defense (Structural barriers – bark, cuticle and waxes)
2. Pre – existing chemical and protein protection (Defensins)
 Inducible immunity against pathogens
1. Race-specific response (R-Avr interaction)
2. General Response (Hypersensitive response)
 Systemic immunity against pathogens
1. Systemic acquired resistance (SAR)
2. Induced systemic resistance (ISR)
Mechanisms of disease
resistance

20. 12/22/2017 20
R-Avrinteraction
 Small secreted proteins contain leucine-rich repeat (LRR) domain and most of them
have a nucleotide-binding site (NBS) or a toll or interleukine 1 receptor (TIR) site.
 Resistance to the disease requires two complementary gene products, one
from the pathogen called Avr protein other from the plant called R-protein, and
whenever there is an absence of either of the gene product it can lead to disease
development.
Recognition of Avr
(Avirulence) legand
Regulatory Domain
(CC)
Unaffected Domain

21. 12/22/2017 21
Systemic responses – Prevents spread of disease
 Systemic acquired resistance (SAR) – whole plant resistance response that
occurs following an earlier localized exposure to a pathogen
- Mediated by Salicyclic acid
 Induced systemic resistance (ISR) – resistance response of plant to pathogens,
benificial microbes, wounding etc.
- Mediated by Jasmonic acid and ethylene
Hypersensitive response
 Leads to localized cell death at the site of
pathogen invasion
 Prevents pathogen spread in the living
plant tissue hence associated with the
expression of resistance by the host plant.
1. R-Avr
2. Proteases
3. ROS , NO, SA
4. Autophagy
In response to
biotrophic pathogen
In response to
necrotrophic pathogen

22. 12/22/2017 22
Expression of non-host resistance
PR (Pathogen Related) proteins – Express in
response to pathogen attack
Pathogen-associated molecular patterns
(Abundant protein)
(Small peptide like proteins)

23. 12/22/2017 23
Ribosome inactivating proteins (RIP)
• Proteins having N-glycosidase activity
which remove adenine residue from 28S RNA
which prevents binding of 60s ribosomal
subunit to elongation factor and inhibit
protein elongation.
Small cysteine rich proteins
• Thionins form pores in fungal cell membranes
and cause death of fungal pathogen
• Defensin inhibits fungal growth in vitro. Some
of them are alpha amylase inhibitors.

24. 12/22/2017 24
Genetic engineering for fugal pathogen resistance – Case study

25. 12/22/2017 25
Genetic engineering for viral disease resistance
Virus resistance in plants can be achieved by transfering virus derived genes including -

26. 12/22/2017
26
Coat-protein mediated virus resistance (CP-MR)
 Virus coat protein has multiple functions
like – Encapsidation, viral RNA
translation, systemic movements and
vector transmission
 CP gene mediated resistance may arise
due to –
 Interaction between transgene CP and
virus CP
 Binding of transgenic CP to host factors
required for disassembly of virus
 Interaction of CP with nuclear inclusion
protein b (Specific to Poty viruses).
Examples –
• Potato mosaic virus – CP genes
expressing in transgenic potato
• TMV-CP genes expressing in
transgenic tobacco
• Papaya ring-spot virus –CP genes –
expressing in transgenic papaya

27. 12/22/2017 27
Replicase (Rep) mediated virus resistance
 Interactions between transgenic replicase proteins and other virus-encoded
proteins may affect the processes of replication and cell-to-cell movement.
 Interactions between non-functional (truncated) transgeic replicase may arise
due to RNA mediated silencing.
 Interactions between RNA of transgenic replicase and virus-encoded RNA
may lead to RNA-mediated silencing of virus replicase.
(RdRPs)

28. 12/22/2017 28
Movement protein (MP) mediated resistance
 Plant viruses included MPs facilitate the spread of virus locally and
systemically by modifying plasmodesmata.
 MPs interact with plasmodesmata or form tubules to allow intercellular
trafficking of virions.
 Transgenic plants expressing mutant MPs show resistance through
competition for plasmodesmatal binding sites.

29. 12/22/2017 29
ds RNA – mediated resistance
 Either cleavage of viral RNA or by bringing DNA methylation in ds DNA viral genome
 Virus resistance by gene silencing is known to spread systemically by movement of
siRNA through the vascular system of plant.

30. 12/22/2017 30
Plant R(Resistance)-gene mediated resistance
 Encodes proteins that participate in identifying specific pathogen

31. 12/22/2017 31
Plant ribosomal inactivating protein (RIP) mediated resistance
 RIPs catalyze depurination of ribosomes and show antiviral activity.
 Pokeweed antiviral protein (PAP) is stored in the cell wall matrix of leaf
mesophyll cells and enters cytoplasm only in response to injury and
inactivate ribosomes.
Example –
 RNA N-glycosidase specifically remove
purine base at 28s r RNA resulting in
seperation of 3’ end of substrate RNA
 RNA becomes incapable of participation in
translation.

32. 12/22/2017 32