Flashcards in Lecture 10 - Insect resistance - Casson Deck (30)
How much of crops worldwide are lost to insects?
13%. Costs $5bil per annum.
How have plants evolved to deter insect attack?
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)
Do crops have natural resistance?
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.
What do insects do in response?
Evolve resistance to insecticides
What is an example of a gene that gives plants insect resistance?
Cowpea Trypsin Inibitor (CpTI)
It affects a broad range of insect species (butterflies, beetles)
Protein encoded by a single gene, so easy to engineer.
How does CpTI work?
Stops digestion inside the insect, the insect starves, the population remains low.
How did they first engineer CpTI?
Had a large cDNA library. Introduced CpTI gene to tobacco (using agro) with a CaMV35S promoter. Showed enhanced insect resistance (corn earworm etc).
What are the potential problems with CpTI?
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.
What are some other potential problems with use of CpTI?
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.
What is another example of a gene that confers insect resistance?
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)
What gene codes for Bt-toxin
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.
How does Bt-toxin work?
Binds receptors in mid-gut. Opens cation channels, influx of water, burst epithelial cells.
Very effective insecticides.
History of Bt-toxin?
Used since 1920 as an 'organic' insecticide. Low toxicity to animals as we do not have the receptors.
Problems with Bt-toxin?
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.
What are the potential complications with engineering this gene into plants?
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.
Describe the first attempt to express Bt-toxin
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.
What do the three domains on the Cry protein (Bt-toxin) do?
I - alpha helix believed to create a pore in the gut
II - beta sheet domain involved in receptor recognition
III - variety of roles
Did this work?
YES - gave good degree of protection against variety of insects
Which crops used Bt-toxin first?
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
Which species frequently become resistant to pesticides?
Cotton bollworms! So, spraying is becoming ineffective.
What is Monsanto Yieldgard?
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.
What is the problem with Bt-toxin?
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.
What can we do to avoid bt resistance developing?
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.
What are other techniques to avoid resistance developing?
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.
How do non-Bt refuges work?
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.
Do natural refuges exist?
Yes, regions of weeds, wild hosts, or other planted crops that can serve as alternate plant hosts for tobacco budworm and cotton bollworm.
Does Bt work?
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.
Benefits of Bt-toxin?
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
What is mycotoxin?
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