2. A transgenic approach for controlling
Lygus in cotton
Authors: Anilkumar Gowda, Timothy J. Rydel, Andrew M. Wollacott,w, Robert S.
Brown, Waseem Akbar,
Thomas L. Clark, Stanislaw Flasinski, Jeffrey R. Nageotte, Andrew C. Read,w,
Xiaohong Shi, Brent J. Werner,
Michael J. Pleau & James A. Baum
Affiliation: Monsanto Company, Chesterfield, Missouri 63017, USA. wPresent addresses: Visterra Inc, Cambridge,
Massachusetts 02139, USA (A.M.W.); Plant
Pathology and Plant-Microbe Biology, School of Integrative Plant Science, Cornell University, 334 Plant Science, Ithaca,
New York 14853, USA (A.C.R.).
Correspondence and requests for materials should be addressed to A.G. (email: anilkumar.gowda@monsanto.com)
Nature Communications 7,
Article number: 12213
doi:10.1038/ncomms12213
Received 27 Nov 2015 | Accepted 13 Jun 2016 | Published 18 Jul 2016

3. A transgenic approach for controlling
Lygus in cotton
Course: Plant Physiology
Course code: GEB301
Course Instructor: Md. Tofazzal Islam, Ph D
Professor, Department of Biotechnology
Bangabandhu Sheikh Mujibur Rahman Agricultural University
Gazipur-1706
E-mail: tofazzalislam@yahoo.com
Presented By: [Group-2]
1. Monowara islam; 2015-1-77-002
2. Shadia Rahman; 2015-1-77-009
3. Md. Mohsin; 2015-1-77-007
4. Mohtashim Mohammad Zahin; 2015-1-77-019
5. Md. Shabab Mehebub; 2015-1-77-004
6. Bitali Islam; 2015-1-77-017

5. Keywords
• Lygus- Lygus bugs are known for their destructive feeding habits - they puncture
plant tissues with their piercing mouthparts, and feed by sucking sap. The
term lygus bug is used for any member of genus Lygus. The genus Lygus includes
over 40 species of plant-feeding insects in the family Miridae.
• Transgenic- denoting an organism that contains genetic material into
which DNA from a different organism has been artificially introduced.
Lygus spp.

6. Keywords
• Bt Cotton- Bt [Bacillus thuringiensis] cotton is a genetically modified
organism (GMO) cotton variety, which produces an inhibitory
substrate to bollworm.
Bollworm on Cotton Bollworm on Bt cotton Boll Bt cotton

7. Background of the Research
• Lygus species of plant-feeding insects have emerged as economically important
pests of cotton in the United States
• Lygus bug attack Cotton, Strawberries, and seed crops such as Alfalfa.
• These species are not controlled by commercial Bacillus thuringiensis (Bt) cotton
varieties.
• Resulting in economic losses and increased application of insecticide.
• Previously, a Bt crystal protein (Cry51Aa2) was reported with insecticidal activity
against Lygus spp.
• However, transgenic cotton plants expressing this protein did not exhibit
effective protection from Lygus feeding damage.
Fig: Lygus bug damage the cotton boll

8. Introduction
History of Bt Cotton
1901 - the bacterium Bacillus thuringiensis (Bt) was first isolated as the cause of sotto
disease.
1911 - a bacteria was isolated that had killed a Mediterranean flour moth and rediscovered Bt.
1915 - the existence of a crystal within Bt was reported, but the activity of this crystal was
not discovered until much later.
1920- Farmers started to use Bt as a pesticide.
1956 - Researchers found that the main insecticidal activity against lepidoteran (moth) insects
was due to the parasporal crystal. With this discovery came increased interest in the crystal
structure, biochemistry, and general mode of action of Bt.
Starting in 1958 - In the US, Bt was used commercially.
1961 - Bt was registered as a pesticide to the EPA [Environmental Protection Agency].

9. Introduction
History of Bt Cotton
1980- Use of Bt increased when insects became increasingly resistant to the synthetic insecticides and
chemicals were harming the environment. Bt is organic and it affects specific insects and does not persist in
the environment. Because of this, governments and private industries started to fund research on Bt.
1995 - With the advancement in molecular biology, it soon became feasible to move the gene that encodes
the toxic crystals into a plant. The first genetically engineered plant, corn, was registered with the EPA.
1996:Bt cotton was introduced into US agriculture. Introduced insect-protected in-seed insect protection
against the cotton bollworm, tobacco budworm and pink bollworm.
1997: Monsanto introduced a stacked trait cotton offering with Bollgard and Roundup Ready®
technologies.
2002: Bollgard cotton became the first biotech crop technology approved for commercialization in India.
More than 6 million farmers adopted the technology on their farms.
2003: Monsanto introduced a second-generation trait product in cotton with insect-protected cotton in
2003. The product provides farmers with the same benefits as the original Bollgard product, as well as
expanded protection against other cotton pests.
2015- In Bangladesh Bt Cotton is going on field trial inside the greenhouse.

10. Introduction
• The development and cultivation of transgenic crops has
revolutionized agriculture worldwide.
• In 2014, the global cultivation of insect-protected crops was estimated
at 78.8 M ha.
• In the United States, the plant bugs Lygus hesperus and L. lineolaris
have attained status as the two most economically damaging pests of
cotton.

11. Introduction
• On cotton, Lygus feeding on small- to medium-sized squares
cause abscission resulting in yield loss, while feeding on large
squares results in damage to anthers leading to abnormal flowers.
• The traditional Bt cotton technology does not show any toxicity in
lygus bug, if they change the structure of cry51Aa2 then it may
work on lygus.

12. Objective
• Their objective of this research was to modify the Bt crystal
protein (Cry51Aa2) for further development of cotton varieties
that could potentially reduce or eliminate insecticide
application for control of Lygus and the associated
environmental impacts.
• Transgenic cotton may advanced for further development as
a Lygus control product.

13. Method and Materials
Structure determination of Cry51Aa2
X-ray crystal structure of Cry51Aa2 was obtained by crystallizing
Mutagenesis strategies
Structure design approaches
were used to improve the
activity of Cry51Aa2 .

14. Method and Materials
Cry51Aa2 variant construction and protein production
Plasmid pMON106128 served as the base vector
Variant clones generated by
Transformed into
B. thuringiensis using
electroporation for producing
protiens.

15. Method and Materials
Protein accumulation in plants
Field insect bioassays
Insect sources and diet bioassays
L. hesperus and L. lineolaris eggs
were obtained from the laboratory
culture at National Biological Control
Laboratory, USDA-ARS (Stoneville,
MS).
Cry51Aa2.834_16 was designed to
enable efficient cotton expression
Cotton transformation
Transgenic events and DP393 were
planted in eight 30-ft rows and grown
as per the
cotton growing recommendations.
Statistical analyses

16. Result and Discussion
Fig: The Cry51Aa2 crystal structure.
HYS repeat is missing in Cry51Aa1 but present in
Cry51Aa12
Key finding: HYS is present and the structure
of Cry51Aa2

17. Result and Discussion
Fig: Cry51Aa1 and Cry51Aa2 display a high level of structural similarity
Key finding: Cry51Aa1 and Cry51Aa2 is not
same.

18. Result and Discussion
Key Findings: Sequence difference between these protein in 196 –
201 amino acid.
Fig: Alignment of TIC853 ,Cry51Aa2 and Cry51Aa1 protein.

19. Result and Discussion
Cry51Aa2 is not work on L.
lineolaris.
Cry51Aa2.834_5 is marginally
more active on both species of
Lygus.
Cry51Aa2.834_5 Mutagenesis
and variant improved protein
activity is tested.
Key finding: Cry51Aa2.834_16 shows highest activity against L. lineolaris
and L. hesperus

20. Result and Discussion
Fig: Cry51Aa2.834_16 accumulation in various tissues of transgenic cotton plant
Key finding:
In every transgenic event square tissue accumulate highest amount of
Cry51Aa2.834_16 protein.
But after 90 days of planting transgenic event GH_A710504 exhibited reduced
Cry51Aa2.834_16 accumulation in square tissue.

21. Result and Discussion
• Concentration-response assay demonstrating improved activity of select
Cry51Aa2 variants towards L. hesperus.
Key finding: The most active variants showed higher activity compared with the native Cry51Aa2
protein in a concentration response assay with an estimated LC50 of B12–20 p.p.m. These variants
included E125A, F147A and Q149A, and the double variants T145A-F147A, F147A-Q149A and
Q149A-S151A. Subsequent combinatorial mutagenesis identified several other variants with
significant improvements in activity compared with the native Cry51Aa2 protein .
Fig: Concentration-response assay towards L. hesperus.

22. Result and Discussion
Activity of the cryAa2.834 alanine substitution variants towards l.
hespersus.
Key finding: T93A shows higher mortality rate.

23. Result and Discussion
Fig: Efficiency of transgenic cotton plants on L. lineolaris.Key Finding: in average GHQ710504
Transgenic event shows better activity
All four Cry51Aa2.834_16 events
were efficacious in reducing
numbers of L. ineolaris (a) and L.
hesperus (b) when compared with
the negative control .

24. Result and Discussion
Fig: Efficacy of transgenic cotton plants on L hesperus
Key Finding: in average GH_A772050 Transgenic event shows better activity

25. Result and Discussion
The expression cassette in pMON139006 contains the 35S promoter from the
figwort mosaic virus, untranslated leader sequence from Hsp81 of Arabidopsis
thaliana, Cry51Aa2.834_16 coding region and the cauliflower mosaic virus 35S
terminator for transformation in the plant.
Fig 6: coding region and expression cassette of cry51Aa2.834_16

26. Result and Discussion
Key Finding: HYS deletion in Cry51Aa2.807_5 protein does not impact
activity towards L. lineolaris.
Fig: impact on activity toward L. lineolaris for HYS deletion in Cry51Aa2.807_5

27. Conclusion
Modification of Cry51Aa2 protein by deletion of Alanine Cry51Aa2.834_16 protein created.
This protein increase its specific activity towards Lygus spp.
The level of efficacy observed with Cry51Aa2.834_16 should provide economic control of Lygus
bugs in cotton.
Transgenic cotton event GH_A710504 is advanced for further development as a Lygus control
product and designated as MON88702.
A transgenic option for Lygus management using MON88702 could reduce or eliminate
sprays required for Lygus control and lessen the environmental impact of chemical
insecticide use on cotton for desire yield.
Bangladesh is going to introduced the Bt cotton by upcoming years. So the research is
very important for introducing new developed variety MON88702 towards the farmers
rather than the old one.

28. Acknowledgement
We are grateful to Anilkumar Gowda et al.
and also grateful to Md. Tofazzal Islam.
We are thankful to : Effi Haque
Some pictures are available in www.google.com

29. Thank You!