L15-16: Development of pest and disease

resistant plants

Engineering plants resistant to microbial pathogens (bacteria)

Engineering virus-resistant plants (viral coat protein, RNAi)

Engineering insect-resistant plants

Production of herbicide-tolerant transgenic plants

 Engineering virus-resistant plants
1. Viral entry: aphids and other insects tap into the phloem to feed
These vectors can also pick up virus particles and spread to new hosts
2. Other viruses infect plant cells through a wound site created by a
leaf-munching insect such as a beetle
3. Following entry, virus uncoats releasing viral RNA
(most plant viruses are RNA viruses)

Viral
infection

2
https://www.the-scientist.com/infographics/how-viruses-attack-plants-30340
Coat-protein (viral-encoded)-
mediated resistance
No uncoating, no transcription
Transgenic plant genome
No replication, no propagation
engineered to express viral
coat protein (CP) gene
Coat protein interferes
with viral uncoating CP mRNA

Viral coat
protein

Virus entered the cell Transgenic plant

Virus starts uncoating resistant to
viral infection
3
Coat protein (CP)-mediated resistance
• Express viral coat protein in transgenic plants
• Copies of viral DNA or virus-related nucleic acid
sequences were introduced into plants to develop
resistance
• 1st : Transgenic tobacco plants expressing coat
protein of tobacco mosaic virus (TMV) develop
resistance to TMV infection
• Successful and most widely applicable

4
http://www.ualberta.ca/~mingchen/pics/m-tmv.jpg
 CP-mediated resistance
Similar results obtained with other viruses
e.g. alfafa mosaic virus, cucumovirus, potexvirus,
potyvirus, tobamovirus, tobravirus and luteovirus
groups potexvirus
cucumovirus potyvirus

alfafa mosaic
tobamovirus
http://sdb.im.ac.cn/vide/images/a2.jpg

http://sdb.im.ac.cn/vide/images/d6.jpg
http://www.dpvweb.net/dpvfigs/a1.jpg

luteovirus tobravirus

https://microbewiki.kenyon.edu/ima
ges/thumb/8/82/Tobamo1.jpg/300p
https://www.sciencephoto.co x-Tobamo1.jpg
m/image/249632/800wm

https://s10.lite.msu.edu/res/msu/bo
http://sdb.im.ac.cn/vide/images/c2.jpg tonl/b_online/library/multimedia-
virology/Tobravirus.GIF 5
 Properties of CP-mediated resistance

Coat protein must be expressed in

• epidermis (viral entry point) and
• vascular tissue (through which virus spreads systemically) to
achieve optimal levels of resistance

Resistance manifested at different levels:

• Fewer infection sites on inoculated leaves of CP-expressing
plants
• Systemic disease development reduced
• Accumulation of virus much lower than control

6
 Properties of CP-mediated resistance

• In most cases, CP-mediated protection effective

against virus from which CP was derived or against
closely-related strains

• Some cross-protection against heterologous viruses

• e.g. tobacco expressing soybean mosaic potyvirus (SMV)
CP also immune to
• 2 other potyviruses - tobacco etch virus and potato virus Y
which show ~ 60% homology in CP a.a. seq with SMV
• e.g. tobacco expressing TMV CP
• resistant to tobamoviruses that share 60-82% CP with TMV

7
 Examples: CP-mediated resistance

• Field trials with tomato expressing TMV CP

• Highly resistant to TMV
• Somewhat less to tomato mosaic virus strains

• Transgenic potato expressing CP genes of

potato virus X (PVX) and PVY
• Highly resistant to infection by PVX and PVY
under field conditions

8
 Tomato plants expressing TMV CP
Effect of TMV

Wildtype Trangenic
Wildtype Tomato plant Field test (1988) Control & transgenic tomato
expressing seedlings were grown in greenhouse &
TMV CP transplanted 6 weeks later to field test site.
TMV (10 µg/ml) strain PV230 was applied 2
weeks after planting.
The photo was taken 4 weeks later. 9
Gasser & Fraley 1989 Science 244, 1293-1299.
Example: Virus-resistant rice

10
 CP-mediated resistance
Selfed progeny of transgenic rice plants
4 months after virus inoculation.
Plants showing disease symptoms (%)

Seedlings at the four-leaf stage were used

15 days after infection
49 days after infection

WT Transgenic KVII5071 KVII5072

No CP
expression expressing CP
sample 17 20 17 14
size
Resistant progeny Susceptible progeny

11
Hayakawa et al. 1992 PNAS 89, 9865-9869.
 GM Papaya takes on Ringspot
Virus & Wins
Papaya ringspot virus (PRSV) is a non-enveloped,
flexous rod-shaped particle that is between 760–
800 nm long and 12 nm in diameter

Transmitted between plants by mechanical

activities like pruning and by numerous aphid
species

The virus interferes with the plant’s ability

to photosynthesize
Symptoms of PRSV on
Papaya tree (a) and fruit (b)
Hawaiian papaya production severely
affected by PRSV in 1990s

12
https://upload.wikimedia.org/wikipedia/commons/4/49/Papaya_Ringspot_Virus_Symptoms.jpg
 GM Papaya takes on Ringspot
Virus & Wins
Scientists at Cornell and the
University of Hawaii
inserted a viral coat protein
gene into papaya genome
with a gene gun

Immature zygotic embryo

cultures were used

CP mRNA expression in
transgenic papaya confirmed by
Northern blot analysis
13
Plant Cell Reports (1990) 9:189-194
 GM Papaya takes on Ringspot
Virus & Wins
Transgenic papaya at the center of field (darker patch of green)
surrounded by non-transgenic plants

Transgenic papaya varieties that are resistant to PRSV entered production in 1998
Science 320, 472 (Apr. 25, 2008) 14
Photo courtesy of Stephen A. Ferreira (https://www.eurekalert.org/multimedia/pub/web/7757_web.jpg)
 GM Papaya takes on Ringspot
Virus & Wins
World's first GM fruit successfully commercialized (over 20-
year safety record)

Virus threatens all papaya trees but environmental groups

against adoption elsewhere

No evidence GM papaya are allergenic

No transgenes in seeds from an orchard 400 m downwind

Acceptance rests on politics and economics

• Brazil: exports conventional papayas to Europe or Japan, which
prohibit GM papaya
• Mexico: permits planting because major market US allows GM fruit
15
Science 320, 472 (Apr. 25, 2008)
Engineering virus-resistant plants
(viral coat protein, RNAi)

RNAi
Refer to RNAi Lectures 9-10

16
Example Exam Qs:

Discuss how scientists have produced transgenic rice lines that are resistant to
infection by the following pathogens:
a) bacteria;
b) viruses.

Why are coat protein genes from viruses expressed in transgenic plants? Give
2 specific examples on the successful application of this strategy in
agriculture.

Describe “coat protein-mediated resistance” and explain how it can be applied

in plant genetic engineering.
Include a figure in your answer to help explain a possible mechanism of
its action.
What are the advantages and limitations in using this method?
L15-16: Development of pest and disease
resistant plants

Engineering plants resistant to microbial pathogens (bacteria)

Engineering virus-resistant plants (viral coat protein, RNAi)

Engineering insect-resistant plants

Production of herbicide-tolerant transgenic plants

Engineering plants resistant to insects
Use of Bacillus thuringiensis (Bt) toxin
to combat insect pests
Colorado beetle

potato
Tobacco budworm bug

attacks alfalfa, clover, cotton, flax, attacks genus Solanum

soybean, tobacco, cabbage, cantaloupe, (potato, eggplant, tomato, larva
lettuce, pea, pepper, pigeon pea, squash, pepper)
tomato …

https://upload.wikimedia.org/wikipedia/commons/thumb/2/21/Colorado_potato_beetle.jpg/250px-Colorado_potato_beetle.jpg
http://entnemdept.ufl.edu/creatures/field/tobacco_budworm02.jpg 19
https://static1.squarespace.com/static/5761e7869de4bbe3608910c4/594e4a7486e6c02442824ed7/594e4a74be6594fef2605ec6/1498303205692/Colorado-Potato-Beetle-Larva-picture-from-Purdue-Extension.jpg
 Why protection against insect pests?
Need from data on worldwide expenditure on insecticide &
crop losses
Expenditure on insecticides Crop losses caused by insect pests

20
Maagd, Bosch & Stiekema, January 1999. Bacillus thuringiensis toxin-mediated insect resistance in plants. Trends in Plant Science 4(1), 9-13.
 Bacillus thuringiensis (Bt)
• Gram positive soil bacterium
• Produce proteinaceous crystalline inclusions
with insecticidal activity during sporulation
• Insecticidal crystal proteins (ICP) non-toxic to
organisms other than insects
• Four major classes
• Cry I (Lepidoptera-specific): moths & butterflies
• Cry II (Lepidoptera- and Diptera-specific)
• Cry III (Coleoptera-specific): beetles
• Cry IV (Diptera-specific): flies
https://upload.wikimedia.org/wikipedia/commons/6/61/Halteres-Tipule.jpg
http://myrmecos.net/wp-content/uploads/2008/10/chrysochusauratus2.jpg https://pbs.twimg.com/profile_images/603951470074064896/ZDAci4a8.jpg 21
https://upload.wikimedia.org/wikipedia/commons/7/72/ComputerHotline_-_Lepidoptera_sp._(by)_(24).jpg http://www.troplep.org/1.jpg
Bt bacteria produce insecticidal proteins

Bacillus
thuringiensis
expressing Bt
toxin

Bacillus thuringiensis expressing insecticidal

Bt toxin
which can be sprayed onto plants
Used as insecticide by farmers before
transgenic plants were generated
 Spore preparations of Bt have been used
for many years as a biological insecticide
Electron micrograph:
spore round spores and angular Bt toxin crystals
ICP produced when B. thuringiensis
sporulate

Electron micrograph:
Pseudomonas transformed with Bt toxin
gene,
cultivated, harvested, heat-treated and
used as biopesticide

23
Chrispeels, M.J. and D.E. Sadava. Plants, genes, and agriculture.
 ICPs
In the midgut of insect larvae
• Protoxins; proteolytically converted to smaller toxic
polypeptides
• Toxins bind to midgut epithelial cells
• Disruption of cell membrane: paralysis
• Insect stops feeding & die
• Highly specific, bind to specific receptors

24
Transgenic plant producing Bt toxin
Intestine

Processing

Receptor
Plant cell binding
expressing Intestinal
Bt toxin lumen Leakage of cellular
content and gut
paralysis

Bt toxin affects only some insects because to be effective it

has to be processed and bind to a specific receptor protein
 Why Bt toxin expression in plants?

• Proteinaceous nature
• Highly specific: proven safety
• ICPs sensitive to sunlight/UV
Regular spraying not cost-effective
• Ideal for heterologous expression in plants

ICP: Insecticidal crystal proteins 26

 Effectiveness of Bt toxins
Transgenic rice Wild-type
containing Bt gene control

https://www.researchgate.net/profile/Bao-Rong_Lu/publication/303459169/figure/fig1/AS:365201493905408@1464082229390/GM-
27
versus-non-GM-crops-An-insect-resistant-GM-rice-line-green-plants-containing-the.png
Insect resistance through introduction of
the Bt toxin gene
Transgenic peanut plant
Wild-type peanut plant expressing the Bt gene

Photo by Herb Pilcher USDA

 Genes encoding Bt toxin
• cloned from several strains of Bacillus thuringiensis and
sequenced
• Deletion analysis: region essential for toxicity resides in
N-terminus

29
Maagd et al (1999) Trends in Plant Science 4: 9-13
 Transgenic plants expressing ICP

• First successful application of plant transformation for

crop improvement

• CaMV 35S promoter-modified ICP genes (e.g.

truncated chimera encoding functional toxin) -
introduced into tobacco and tomato plants by
Agrobacterium-mediated transformation

30
 Tobacco plants infested with Manduca sexta
Transgenic plants
contain chimeric
gene expressing the
B. thuringiensis var.
kurstaki endotoxin

Plants infested with

larvae of Manduca sexta
(tobacco hornworm)

Transgenic Wild type Transgenic

Infested with larvae Transgenic expression
of Bt toxin protects
31
http://www.educationalscience.com/media/catalog/category/canstockphoto7272055.jpg
against insect larvae
 Post harvest protection of seeds
• Post harvest storage of seeds support growth
of seed-feeding beetles
• Family Bruchidae
• Cowpea weevil: Callosobruchus maculatus
• Azuki bean weevil: C. chinensis
• Substantial economic & nutritional losses
• Fumigants undesirable: environmental & food
safety concerns

https://upload.wikimedia.org/wikipedia/commons/thumb/8/88/Callosobruchus_chinensis_%28Linn%C3%A9%2C_1758%29_male.jpg/220px-
Callosobruchus_chinensis_%28Linn%C3%A9%2C_1758%29_male.jpg http://i149.photobucket.com/albums/s75/41714049/IMG_8016.jpg 32
http://entnemdept.ufl.edu/insectid/images/Cowpea-weevil-Callosobruchus-maculatus-Buss.jpg
 What can be done?
• Seeds of common bean (Phaseolus vulgaris) resistant
to these weevils contain a protein, αAI-Pv, toxic to
larvae

• Larvae secrete gut enzyme (α-amylase) to digest the

starch in pulses

• αAI-Pv inhibits α-amylase, preventing digestion

• Starving weevil to death

Amylase Inhibitor
https://upload.wikimedia.org/wikipedia/com 33
mons/6/6a/Phaseolus_vulgaris_003.JPG
Tested on pea, Pisum sativum
Bio/Technology 12, 793-796 (1994)

34
 Expression of αAI in pea
PHA: phytohemagglutinin OCS: octopine synthase
: Basta resistance
αAI: α-Amylase Inhibitor

35S: CaMV 35S

: β-glucuronidase

kanamycin resistance : : chloramphenicol

acetyltransferase
NOS: nopaline synthase (non-functional)

BR, BL: Right & left borders of T-DNA

35
Shade et al. 1994 Bio/Technology 12, 793-796
 Expression of αAI in pea
Use of strong seed-specific promoter dlec2:
dlec2 encodes PhytoHemAgglutinin-L

Pea seeds
1.0-1.2 % of αAI-Pv

Transgenic Wild type

Transgenic seeds resistant to both cowpea & Azuki
bean weevils Shade et al. 1994 Bio/Technology 12, 793-796 Schmidt 1994. Science 265, 739
36
Example Exam Qs:

What is coat protein mediated resistance? Illustrate with specific examples its applications in plant
biotechnology.

What are insecticidal crystal proteins? Describe their use as pest control agents.

Discuss how insect-resistant transgenic crops can be produced by

a) nuclear transformation;
b) chloroplast transformation.

Discuss how plants in the field and post-harvest plant products can be protected against insect pests.
Using a specifically-named protein, describe the mechanism used to achieve protection, and
the advantages and limitations (if any) of each approach?
L15-16: Development of pest and disease
resistant plants

Engineering plants resistant to microbial pathogens (bacteria)

Engineering virus-resistant plants (viral coat protein, RNAi)

Engineering insect-resistant plants

Production of herbicide-tolerant transgenic plants

 Herbicide resistance
Herbicides - weed control
Herbicides affect processes unique to plants:
# photosynthesis
* biosynthetic pathways for essential amino acids

*
*
*
*
#
#

39
 Herbicide resistance
Since crops = plants
competing weeds = plants

Pathways are same and

herbicides are non-selective!

WHY CROP PROTECTION IS NEEDED?

“Between 26-40% of world’s potential crop production lost annually from weeds, pests
and diseases and losses could double without use of crop protection practices”
FAO (UN Food & Agri Org)

40
 GM Example: Herbicide resistance
Plants compete with other plants
eg weeds for sunlight and
nutrients.

Many farmers use herbicides to

eliminate weeds (undesired
plants) from their fields.

Left – corn rows sprayed with herbicide to eliminate competing plants

Right – corn being choked by giant foxtail (Setaria faberi)

Photo credit: Doug Buhler, Bugwood.org

41
 Herbicide resistance

Concerns - environment, toxicity to animals, residues in soil & water

- new herbicides that are improved to decrease concerns

42
2 strategies in engineering herbicide resistance in plants:

I Modification of target:
Glyphosate

II Detoxification/degradation:
Phosphinotricin (PPT)

43
2 strategies in engineering herbicide resistance in plants:
I Modification of the target of herbicide action
Sensitivity of plant to herbicide decreased by
a) Overproduction of sensitive target enzyme
b) Expression of modified target that is insensitive to herbicide but retains enzymatic
activity
II Detoxification/ degradation of herbicide
Plant is provided with detoxifying pathway

44
Specific E.g.
I Modification of the target of herbicide action e.g. glyphosate

Sensitivity of plant to herbicide decreased by

a) Overproduction of sensitive target enzyme
E.g. overproduction of plant 5-enolpyruvyl-shikimate-3-phosphate synthase (EPSPS) in
glyphosate resistant transgenic petunia

b) Expression of modified target that is insensitive to herbicide but retains enzymatic activity
E.g. expression of mutant bacterial aroA in glyphosate resistance

Plant and bacterial genes encoding herbicide-sensitive or -insensitive targets identified

II Detoxification/ degradation of herbicide e.g. PPT

Plant is provided with detoxifying pathway

E.g. expression of bar in phosphinothricin (PPT)-resistant transgenic tobacco

45
 GLYPHOSATE affects a.a. biosynthesis

Glyphosate - most extensively used nonselective herbicide

Active ingredient in “Roundup”

Broad spectrum - controls annual and biennial weeds

Rapidly absorbed by foliar tissue; quickly translocated to various organs; rapidly

metabolized in soil (no residual activity)

Inhibits EPSPS, key enzyme in aromatic a.a. biosynthesis in bacteria and plants

46
 47 Snustad,
E.D. Gardner, Siimmons,
Principles of genetics
2 strategies in engineering herbicide resistance in plants:

I Modification of the target of herbicide action

II Detoxification/ degradation of herbicide

Plant is provided with detoxifying pathway

48
II Detoxification/ degradation of herbicide

Plant is provided with detoxifying pathway

E.g. expression of bar in phosphinothricin-resistant transgenic tobacco

Such detoxification/ degradation pathways exist in plants or microbes that

naturally are tolerant

Identify detoxifying enzymes in plants/ microorganisms

Transfer such genes to crops to inactivate herbicide

49
 L-Phosphinothricin (PPT/Basta)

PPT- irreversible inhibitor (analogue) of glutamine synthetase (GS) in bacteria and plants

GS adds glutamate to ammonia (by-product of photosynthesis) forming harmless glutamine

PPT resembles glutamate, binds GS, blocks action

Ammonia accumulation in chloroplasts destroys them, kills plant

50
 Phosphinothricin (PPT)

PPT-degrading enzyme in biaolaphos biosynthesis pathway

Herbicide biaolophos (tripeptide) produced by Streptomyces viridochromogenes

and Streptomyces hydroscopicus

Consists of PPT and 2 L-alanine residues

Removal Ala releases herbicide

Bar from S. hydroscopicus encodes acetyl transferase (PAT), acetylates free amino
group of PPT & shows substrate specificity for PPT

PAT converts PPT to acetylated non-herbicide

51
 L-Phosphinothricin (PPT)

• Basta contains PPT;

used just before sprouting
(before leaves produced)

• Wild-type sugar beet

requires 2-6 kg herbicide
per hectare

• Using Basta on transgenic

beet cuts down to 0.6-1.5
kg per hectare

(New Scientist 139, 16 11 Sept 1993)

52
 L-Phosphinothricin (PPT)

• Bar gene under CaMV 35S pro for Agrobacterium-mediated transformation of

tobacco, tomato, canola, alfafa, sugar-beet, aspen, poplar

• Greenhouse plants resistant

• Field-tested transgenic potato, tobacco and alfafa resistant even at low

intracellular PAT levels

• Growth of transgenic same as wild-type

• By particle bombardment, CaMV 35S-bar transferred to maize and wheat

Bar also used as selectable marker

• Herbicide resistant rice from protoplasts using CaMV 35S-bar

53
 L-Phosphinothricin (PPT)

• Biaolophos is toxic to fungal pathogens like Rhizoctania solani that infects rice

• Biaolophos treatment of transgenic rice expressing bar prevented R. solani

infection (sheath blight)

• Simultaneous control of weeds and fungal pathogen using bar

54
PPT-resistant plant

55
Herbicide resistance - BACTERIAL detoxifying enzymes

PPT-
resistant
rye

56
Herbicide resistance - BACTERIAL detoxifying enzymes

PPT-
resistant
rye

57
 PPT-resistant turfgrass

58
Herbicide Resistant Turfgrass (Agrostis palustris Huds.) by Biolistic Transformation. BIO/TECHNOLOGY 12, 919-923 SEPTEMBER 1994
 Herbicide tolerant plants are
environmentally friendly
Farmers that plant herbicide-
tolerant crop plants use less Soybean
herbicide, herbicides that are less
toxic, and till (plow) less, saving *
soil and fuel.

Cerdeira, A.L. and Duke, S.O. (2006). The Current Status and Environmental Impacts of Glyphosate-Resistant Crops. J.
Environ. Qual. 35: 1633-1658. Photo credit Hunt Sanders, University of Georgia, bugwood.org.

59
 Biotech (GM) Crops Good for Farmers and Environment Science (2010) 328: 295

In 2010, 14 y after GM crops in US, benefits to farmers are clear (Nat Research Council)

Going green - farmers control weeds by spraying crops with herbicides (lessens soil
erosion and reduces fuel costs in tillage)

• Bt corn - farmers spray less of dangerous insecticides (good for wildlife)

• Glyphosate-resistant soybean reduces tilling fields to control weeds (less soil erosion)

Overall amount of glyphosate increased but glyphosate replaces >toxic herbicides

• Reduced tillage "largest single environmental benefit of GM crops" (less sediment,

fertilizer, pesticides wash into streams)

• Data from biotech (GM) corn, soy, cotton in US and other engineered crops will
inform policymakers in countries trying to weigh environmental benefits and risks of
biotech (GM) crops 60
 Herbicide-resistant Soybean
(BIOL3402 video)

PSI - Are my soybeans wearing different genes?,

Author: Burkhard Schulz
https://www.youtube.com/watch?v=xMKN9Q5FJh4 (6 min)

Soybean plants can survive herbicide treatments and grow on fields

without weed competition is investigated using DNA extraction,
polymerase chain reaction (PCR), gene analysis etc.

Transgenes are found that make the soybean plants herbicide resistant.

These genes can be detected in soybean plants as well as in food products

such as tofu that are found on the supermarket shelf.

This video addresses questions about genes and transgenes in food – we all
eat DNA and genes every day - and the use of transgenic crops for food
production.
61
http://www.chlorofilms.org/index.php/crpVideo/display/videoid/29
Example Exam Qs:

Describe how a bacterial-derived gene can be used for the generation of

herbicide-resistant crops.

Write short notes on the following: (a) AoPR1 promoter; (b) bar gene.

Comment on the statement "Herbicide-tolerant plants are environmentally

friendly".
Discuss how crops can be engineered to acquire resistance to the herbicide L-
phosphinothricin.

62