Lecture 10 - Insect resistance - Casson

Question 1

How much of crops worldwide are lost to insects?

Answer 1

13%. Costs $5bil per annum.

Question 2

How have plants evolved to deter insect attack?

Answer 2

Physical barriers - spines, hairs, tough or sticky surfaces
Toxic secondary metabolites
Primary gene products e.g. digestive enzyme inhibitors (often rapidly induced following an insect attack)

Question 3

Do crops have natural resistance?

Answer 3

No, often it is reduced. This is because we are selecting for things like fruit size, not resistance. Also, selecting for flavour means reduction in production of toxic secondary metabolites.

Question 4

What do insects do in response?

Answer 4

Evolve resistance to insecticides

Question 5

What is an example of a gene that gives plants insect resistance?

Answer 5

Cowpea Trypsin Inibitor (CpTI)
It affects a broad range of insect species (butterflies, beetles)
Protein encoded by a single gene, so easy to engineer.

Question 6

How does CpTI work?

Answer 6

Stops digestion inside the insect, the insect starves, the population remains low.

Question 7

How did they first engineer CpTI?

Answer 7

Had a large cDNA library. Introduced CpTI gene to tobacco (using agro) with a CaMV35S promoter. Showed enhanced insect resistance (corn earworm etc).

Question 8

What are the potential problems with CpTI?

Answer 8

Might affect our digestion? Probably not. Insect gut is much simpler than ours. Intact CpTI would not reach the site of action (duodenum) in humans still intact. We already eat a lot of plants containing protease inhibitors naturally!
Also, the insect is still eating the leaf, so this is not an ideal solution.

Question 9

What are some other potential problems with use of CpTI?

Answer 9

Insects may evolve resistance - CpTI binds protease active site, so mutations around the active site would affect quality
Will it harm insects that pollinate? - Only affects insects that EAT the plant. Promoter should be used that directs expression in tissues likely to be targeted by pest.
The crop is still preyed upon - this doesn’t kill the insect, it just slows down its digestion. Also, eg Cowpea Weevil can feed upon Cowpea, as it doesn’t use a serine protease.
Also Pusztai affair has slowed commercialisation of GM crops.

Question 10

What is another example of a gene that confers insect resistance?

Answer 10

Bt-toxin. Bacillus thuringiensis strains produce crystalline proteins upon sporulation which are toxic to insect larvae.
Produced from Cry genes (Cry is precursor, cleaved to form Bt-toxin)

Question 11

What gene codes for Bt-toxin

Answer 11

Cry genes. Subgroups with differing insect specificities eg cryIA(a), cryIA(b) etc. Over 40 families of plasmid encoded proteins (Cryoproteins). Single gene for each one.

Question 12

How does Bt-toxin work?

Answer 12

Binds receptors in mid-gut. Opens cation channels, influx of water, burst epithelial cells.
Very effective insecticides.

Question 13

History of Bt-toxin?

Answer 13

Used since 1920 as an ‘organic’ insecticide. Low toxicity to animals as we do not have the receptors.

Question 14

Problems with Bt-toxin?

Answer 14

Difficult to produce large quantity - supply/demand issues.
Effect is short lived, it often runs off plants, it does not act systemically.
Also, many of the targets are sap-sucking insects, which bypass the surface entirely.

Question 15

What are the potential complications with engineering this gene into plants?

Answer 15

It is a bacterial gene. Plants/prokaryotes very different in gene organisation, transcription, translation.
Also potential problems with codon usage (how often a particular codon is used for a particular amino acid) - this is reflected in abundance of relative tRNA.

Question 16

Describe the first attempt to express Bt-toxin

Answer 16

First attempts used Cry1A and Cry3A. Used CaMV35S promoter, but expression was very low.
Monsanto then modified the gene seq of Cry1A - increased GC content - this would make it more ‘plant-like’ - also added a stronger promoter (2xCaMV35S). Translated 100x more efficiently.

Question 17

What do the three domains on the Cry protein (Bt-toxin) do?

Answer 17

I - alpha helix believed to create a pore in the gut
II - beta sheet domain involved in receptor recognition
III - variety of roles

Question 18

Did this work?

Answer 18

YES - gave good degree of protection against variety of insects

Question 19

Which crops used Bt-toxin first?

Answer 19

Variety of crops, but has been discontinued since.
Monsanto’s NewLeaf potato.
Commercialised in cotton and maize, with different Bt proteins to give resistance to specific pests.
Monsanto Bollgard - cotton expressing synthetic Cry1AC protects against budworm and bollworm pests
Bt brinjal - modified aubergine in India

Question 20

Which species frequently become resistant to pesticides?

Answer 20

Cotton bollworms! So, spraying is becoming ineffective.

Question 21

What is Monsanto Yieldgard?

Answer 21

European corn borer larvae feed on young maize leaves, bore into them and break the stems. Cause 55% yield loss in average year.
Maize expressing modified cry1Ab for resistance to ECB. Now, 85% of US crop is GM.

Question 22

What is the problem with Bt-toxin?

Answer 22

Insects evolve resistance to it. Early Bt-plants were single gene lines. As with any single gene-gene interaction, pests can achieve resistance through mutation of the relevant receptor.
This is increased if the protein level ingested was insufficient to kill the pest, and is couple with repeatedly growing in same area.

Question 23

What can we do to avoid bt resistance developing?

Answer 23

Stack traits. Introduce two or more resistance genes to elite line, but with different modes of action. eg different receptors. In the case of Bt-toxins, Bollgard II and Yieldgard plus contain two cry genes.
Also, stack with HT.

Question 24

What are other techniques to avoid resistance developing?

Answer 24

Non-Bt refuges.
This is an example of ‘integrated pest management.’ These utilise refuge areas planted with non-transgenic plants.
Farmers required to have 20% of total crop area refuge for a mono-Bt trait, but 5% for stacked trait.

Question 25

How do non-Bt refuges work?

Answer 25

The non-Bt area means that Bt-resistance will not develop in insects feeding there. These will breed with the insects possibly developing resistance (Bt area) and thus produce heterozygotes offspring, thus diluting the resistance allele down.

Question 26

Do natural refuges exist?

Answer 26

Yes, regions of weeds, wild hosts, or other planted crops that can serve as alternate plant hosts for tobacco budworm and cotton bollworm.

Question 27

Does Bt work?

Answer 27

Yes. 1996-2008 saving of 8.4% in pesticides. Reduction in 365mil kg active ingredient. When coupled with integrated pest management, Bt works.
90% of cotton in China is Bt.
Also in rice, but fear it will impact on China’s exporting ability due to different countries’ GM rules.

Question 28

Benefits of Bt-toxin?

Answer 28

Increased yields (10% for Bt cotton in China)
Increased opportunity for beneficial insects (eg honeybees) - Bt will not affect all insects - it is very selective
Reduced env impact due to less pesticide spraying. Up to 50% less pesticide for Bt cotton in China.
Reduced pesticide exposure to farmers and non-target organisms - Bt is essentially nontoxic to people, pets and wildlife.
Also less spread of viruses and fungi, due to reduced bore holes

Question 29

What is mycotoxin?

Answer 29

Toxins produced by fungi, often on cereals. Contamination by mycotoxin is big problem.
Middle ages in Europe, ergot poisoning from fungi on rye caused hallucination, manic depression, abortion etc.
Believed to help limit population until wheat and potatoes introduced 1700s.
Reduction in bore holes etc help to control the spread of this

Question 30

What are the potential knockon env effects of Bt toxin?

Answer 30

Aphid pop kept low
This means reduced food for birds, ladybirds. Will aphids become toxic to ladybirds or pollen to bees?
Bt crops may be less harmful to some species than some pesticides, therefore increasing the number of beneficial insects (honeybees) but also increased emergence of some pests, eg mealy worms.