Lecture 8: Bacillus thuringiensis (Bt) toxin Flashcards
– why is it such a good insecticide for biotechnological applications?
The requirement for biotechnological solutions for control of insect pests in agriculture
*Chemical solutions to pest problems are still viable in many instances, but major problems have accumulated where pests have become resistant to pesticides (viral, bacterial, fungal or insect)
*Previous use of pesticides with toxicity to higher animals and humans has resulted (rightly!) in a regulatory regime that requires rigorous safety testing of existing and new compounds
*Pace of introduction of new pesticide classes has declined drastically – largely due to commercial reasons
Insects are generalists they land on a plant and begin feeding – there is very little time for a response e.g. locust plague in Kenya cleared crop fields in weeks – these situations are rare but devestating
> Chemical pesticides in common use are divided into a series of classes based on chemical structure. Most of these compounds are toxic to higher animals.
Californian farms import 3million honey bees yearly to pollinate fruit and almonds due to a lack of natural pollinators as a result of pesticide use
Note many “natural” compounds are equally toxic!
One response to pests/disease:
‘natural’ pesticides
”Naturally” occurring compounds from plants and other sources are widely suggested as a replacement for “synthetic” pesticides
Problems
*Often very limited efficacy, and thus may not be usable in commercial agriculture
*Compounds can be more toxic than the synthetic pesticides they are meant to replace
Natural does not mean non-toxic!!
Another response to pests/disease: endogenous resistance
If “natural” endogenous resistance to insect pests is available in the crop then pesticide application can be minimised
Problems:
*Often, no suitable naturally occurring resistance genes are available in the crop.
*Insects may already contain genes which enable them to adapt to resistance
Not a fast enough response for chewing insects (locusts etc.)
Can we use biotechnology to produce endogenous resistance to insect pests that is not limited by the genetic resources available in a crop species?
Syngenta testing site at Bracknell is a chemical testing warehouse where weather conditions are controlled and plants are infected with pests then sprayed to test for kill effectiveness
^the chemicals are then tested to avoid harm to mammals
^ each chemical developed costs £80,000 to make market ready
*The availability of technology to introduce genes into the plant genome opens up the possibility of engineering plants to produce novel insecticidal compounds
*Introducing novel biosynthetic pathways to produce molecules similar to existing “synthetic” pesticides is technically difficult (although not impossible) – and accumulation of “synthetic” pesticides in plant tissues may be significantly deleterious to the plant.
*Exploiting insecticidal compounds that can be produced from a single introduced gene is technically much more straightforward
– but what are these compounds?
*Insecticidal proteins are the major types of pesticides produced in engineered plants. One type of protein, the toxin from the bacterium Bacillus thuringiensis, accounts for almost all the commercially deployed insect resistant engineered crops.
*Insecticidal nucleic acids (RNAs) are a recent introduction in commercial insect-resistant crops.
Toxins from Bacillus thuringiensis (Bt toxins): naturally occurring insecticidal proteins
*Bacillus thuringiensis (Bt) is a bacterium found in soil and in the general environment
*A common feature of this species is the formation of bacterial spores containing crystalline deposits
*The crystalline deposits are made up of one or more proteins, which are toxic to insects (and other invertebrates in some cases). These are called Cry (for Crystal) proteins
Variability in Bt toxins between strains
*There is a high level of variability in toxins and their accumulation between different naturally occurring isolates of Bacillus thuringiensis – or, put more simply, different Bt strains contain different toxins
*Bt strains which produce similar proteins with other biological activities are known; the bacterium is closely related to other soil Bacillus spp.
*Bt toxins show a high level of specificity in toxic action - I.e. a given toxin is generally only highly toxic to a limited range of target species. Toxicity is often described as a specificity towards insect orders (e.g. lepidoptera, coleoptera), but this is misleading in that significant levels of toxicity are only shown towards a restricted range of insects, often corresponding to subfamily level
see: Toxicity of different Bt toxins towards different phyla, orders, families and species
From Baranek et al. (2020) Nature Scientific Reports, 1976
^ 97.9% of characterised Bt toxins are effective against Arthropods, of which 67.3% are effective against Lepidoptera, but only 0.5% are effective against (for example) Plutella xylostella.
(^ Note: data are incomplete, and biased towards toxicity to crop pests)
Bacillus thuringiensis interaction with pest lifecycle
see figure:
Bacteria accumulate on the surfaces of plants, especially young plants, and insect feeding may increase their accumulation – however, evidence for insecticidal levels of Bt on plants in field situations is limited the spores germinate within the insect that consumes it and it grows on the dead insect ready to infect more insects
Within the insect:
Toxin binds to brush border
membrane of microvilli of midgut
epithelial cells; insertion of toxin
into membrane forms an open pore
leading to collapse of ionic
gradients across cell membrane and
leakage of components
Bacillus thuringiensis toxins (Bt toxins)
genetic composition
*Genes encoding Bt toxins are carried on plasmids; this allows exchange between different strains, and allows the population in the field to carry a wide range of variants of the plasmid-encoded toxins
*Bacillus thuringiensis contains plasmids 4,000 – 150,000 bp in size; a single strain can carry multiple plasmids (up to 17). Genes encoding insecticidal Cry proteins are found on “megaplasmids” , which are >50,000 bp in size
*Megaplasmids main function is as a reservoir for genes encoding Cry proteins
*The “typical” Bt toxin is a large protein, up to 200,000 Da, which contains a conserved structure of approx. 600 amino acids arranged in 3 distinct domains
*Most Bt toxins belong to this three-domain toxin family, designated Cry proteins - however, other types of toxin have been identified in some strains - two-component toxins (also designated Cry) and single-domain cytolytic proteins (Cyt)
*Bt also produces other insecticidal proteins during the normal growing phase - these are termed vegetative insecticidal proteins (Vip)
The “typical” Bt toxin is synthesised as an inactive precursor.
Activation requires proteolysis in the insect gut
The “three domain” structure is found in the N-terminal active toxin part of the molecule (red in the diagram below); the remainder of the inactive protoxin precursor (green in the diagram below) does not have a clearly defined structure.
The term “three domain toxin” strictly only refers to the active toxin, but is often used more loosely to refer to the whole protoxin.
3 domains: pro-toxin (cleaved when infecting a host) , N terminal , C terminal
Sub-families of Bt toxins are designated by a unified nomenclamature scheme based on sequence similarity; subdivisions are based on degree of similarity. Specificity of toxicity generally correlates well with sequence similarity (I.e. all toxins in a family have similar specificities)
“Cry” and “Cyt” are used as designations for all proteins found in crystalline deposits; not all Cry proteins are three-domain toxins!
See figures in notes:
^ Left: Conserved sequences in a range of different Bt toxin families; conserved sequence “blocks” are colour-coded. “Short” toxins do not fit 3-domain structure model.
From Trends in Genetics, 17, 193-199 (2001)
^Right: Clustal “tree” of sequence similarities in Bt toxins.
From http://www.lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/tree.gif
The three domains in a ”typical” Bt toxin have distinct structures and play different roles in the mechanism of action.
All three domains are globular, and the overall protein is globular and soluble
3 domains: pro-toxin (cleaved when infecting a host) , N terminal , C terminal
activated form depicted in notes:
Domain 1: Pore forming
Domain 2: Carbohydrate binding
Domain 3: Protein-protein interaction
The mechanism of action of three-domain Bt toxins involves multiple steps
(see figures in notes) :
1) Protein dissolves, and the protoxin is activated by proteases in insect gut, involving removal of variable C-terminal region to generate the three-domain active toxin. This process may be involved in determining overall specificity between insect orders.
2) Binding of activated toxin to an abundant membrane-anchored protein in cells of the gut epithelium (typically aminopeptidase N or alkaline phosphatase) in a low-affinity interaction involving domains 2 and 3.
3) Binding of the toxin to an integral membrane protein (typically cadherin-like) in a high affinity interaction involving domain 2 - normally determines fine specificity between insect species.
4) Proteolytic cleavage of the N-terminal pro-region on the active toxin (domain 1), followed by oligomerisation. Removal of the pro-region on domain 1 is necessary for oligomerisation, and pore formation (step 6) to occur.
5) Binding of the oligomerised toxin to the abundant membrane anchored protein in a high-affinity interaction involving domain 2, allowing the oligomerised toxin to interact with the membrane of the gut epithelial cell.
6) The oligomerised toxin forms an open pore by insertion of domain 1 of Bt toxin into the membrane of the gut epithelial cell. Free passage of ions through the channel leads to cell lysis and death - not normally involved in determining specificity.
Evidence for the 2-receptor/oligomerisation model
*Insects carrying mutations which make them insensitive to specific Bt toxins can be found
*Some insects resistant to specific Bt toxins have mutations in Aminopeptidase N or alkaline phosphatase genes (loss of function)
*Transfer of aminopeptidase N genes between insect species can transfer sensitivity to specific Bt toxins (gain of function) – specific example in transfer of lepidopteran aminopeptidase N to Drosophila (fly)
*Some insects resistant to specific Bt toxins have mutations in cadherin genes – this technique has been used to map the binding sites for Bt toxins on cadherin proteins
*Bt toxins that have been modified by removal of the N-terminal pro-region show enhanced toxicity, and do not require binding to cadherin to be toxic, due to oligomerisation occurring spontaneously
*Bt toxins can be shown to bind to aminopeptidase N, cadherin and other membrane proteins in vitro
Data are consistent with binding to both the membrane anchored receptor (aminopeptidase N or alkaline phosphatase) and the integral membrane receptor (cadherin) being necessary for toxicity
Binary toxins from Bacillus thuringiensis contain a protein which interacts with membranes, and a pore-forming protein
Both components of the binary toxin are necessary for insecticidal effects. The mechanism of action appears to involve binding to the surface of gut cells by Cry35, and formation of pores by Cry34, but details remain to be fully elucidated.
The diagram right (from Palma et al. Toxins 2014, 6(12), 3296-3325) shows the presence of motifs found in carbohydrate-binding proteins in Cry35; Cry34 has a structure resembling the bacterial pore-forming toxin aerolysin.
Cry binary toxins, such as Cry34/35 show toxicity towards coleopteran insects (beetles) – they have proved useful for control of pests such as corn rootworm
Proposed model for the action of Cry toxin. Cry toxin binds to BT-R and stimulates G protein and AC, which promotes production of intracellular cAMP. In turn, PKA activation destabilizes the cytoskeleton and ion channels, leading to cell death.
Bt toxins can be used as a “conventional” exogenously-applied insecticide
Easy to bottle in crystalline form and spray apply – but it is indiscriminate and large quantities needed – better to engineer into plant
*Preparations of Bt spores (produced by growing the bacterium in vitro) have been in use since the 1920s as a conventional, exogenously applied (spray) insecticide
*Approved as an “organic” insecticide (“natural” product)
*See http://www.ext.colostate.edu/pubs/insect/05556.html
Exogenous application not very effective due to:
*Run-off; most compound lost from plant surfaces
*Wash-off; rain removes compound from plant surfaces
*Inactivation; compound susceptible to degradation in the environment, inactivated by uv in sunlight
*Poor targeting to pest species; spraying distributes compound over both crop and surroundings
*Comparatively high dose required for toxicity (compared to organophosphate or pyrethroid insecticides)
*Not widely used as a commercial insecticide prior to increase in “organic” agriculture
- Fairly expensive to produce
Why is Bt toxin a good candidate for engineering endogenous resistance to insect pests in plants?
*Bt toxin is a protein insecticide and is thus suitable for expression in transgenic plants
–The insecticide is a direct gene product, thus only a single gene needs to be transferred to confer insecticidal properties
*Plants contain no potential targets for Bt toxin; bacterium is part of the normal plant microbiome
*Endogenous synthesis of this protein in crops would overcome many of the problems associated with its use as a conventional exogenously applied treatment
–Avoids run-off and potential exposure of non-target organisms
–Avoids inactivation
*The compound has a history of use as a conventional insecticide, and has been proven to have minimal deleterious effects on non-target organisms, and to be safe to humans
+ Pesticide run-off has become increasingly unacceptable due to pollution of watercourse
Expressing 3 domain toxins in transgenic plants
See figures in notes
The Bt toxin gene is a bacterial gene and will not function if it is transferred to a plant cell. In order to make this gene suitable for expression in plant cells, extensive changes to its structure must be carried out.
The Bt toxin gene is modified for expression in plant cells by replacement of the promoter, truncation and modification of the coding sequence, and replacement of the terminator.
Initial attempts to express Bt toxins in transgenic plants showed that
extensive modification of the bacterial gene was necessary to achieve
protein levels sufficient to cause insect mortality
^ modern promotors are able to create much higher levels of protein than original forms
Recent testing has proven transgenic Bt toxin expressing plants to be effective in pest management:
yellow corn stem borer stunted growth observed and eventually death results
(see figures in notes)
Transgenic plants expressing Bt toxins are a highly successful product - main commercial uses
Cotton expressing Cry1 toxins for resistance to lepidopteran pests - widely employed throughout the world
Corn (maize) expressing Cry1 toxins for resistance to lepidopteran pests - widely used in US, some use in Europe
Corn (maize) expressing Cry3 or Cry34/35 binary toxin for resistance to corn rootworm (coleopteran pest) - widely used in US
Rice expressing Cry1 toxins for resistance to lepidopteran pests - used on a significant scale in China
Major Bt expressing crops
Cotton: cry1Ac, cry1F, and vip3A(a). Resistance to lepidopteran pests including, cotton bollworm, pink bollworm, tobacco budworm.
Maize: cry1Ab. cry9C, cry1F cry1Ac, cry1Ab European corn borer (Ostrinia nubilalis)
cry3Bb1, cry3A resistance to corn root worm (Coleoptera, Diabrotica spp.)
Stacked: cry34Ab1 and cry35Ab1, resistance to corn root worm (Coleoptera, Diabrotica spp.)
And a series of minor crops, including:
Eggplant (brinjal): cry1Ac. Shoot borer.
Potato: cry3A. Resistance to Colorado potato beetle (Leptinotarsa decemlineata, Say). Not continued for commercial agriculture
http://www.agbios.com/dbase.php
Deploying insect-resistant transgenic crops expressing Bt toxins in the field
Problem: Insect resistant Bt crops in agriculture
*Bt-expressing transgenic plants have become a major success in modern commercial agriculture
*This success has been dependent on control over deployment, particularly on resistance management in target pests
*Unfortunately, Bt crops have not been as much of a success as they could have been in benefitting smaller farmers and growers
*Commercial self-interest, and regulatory and “environmental” concerns, have reinforced each
other in preventing this technology from getting into the hands of those who might benefit most from it
Terminator gene can be introduced to Bt seeds resulting in dependency on bioengineering companies
This could wipe out the ‘pest’ problem – in other words decimating insect populations globally
An overall success: insect resistant cotton
Host Organism / Variety Gossypium hirsutum L. (Cotton) Bollgard®, Bollgard II
Trait Resistance to lepidopteran pests including, but not limited to, cotton bollworm, pink bollworm, tobacco budworm.
Expresses Cry1Ac (Bollgard); Cry1Ac + Cry2Ab2 (Bollgard II)
Trait Introduction Method Agrobacterium tumefaciens-mediated plant transformation.
Proposed Use Production of cotton for fibre, cottonseed and cottonseed meal for livestock feed, and cottonseed oil for human consumption.
Company Information Monsanto Company
How Bollgard cotton functions
Pink Bollworm (Pectinophora gossypiella)
- is a pest susceptible to Bt Cry1Ac which is controlled by Bollgard cotton
Female moths lay their eggs in the cotton bolls, and larvae emerge to feed on the cotton seed, chewing through the cotton lint and damaging the boll.
The ‘Cotton boll’ is the harvested part of the cotton plant and includes the cotton itself (white fibres; lint) which encase the cotton seed. Cotton seed contains high levels of protein and oil and is used as animal feed.
Economic Benefits to the Farmer: Bt Technology
see photo examples for cotton in notes
e.g. Cotton field showing mixed planting where Bt cotton has set more bolls. The conventional cotton has suffered insect damage to developing bolls, and has failed to set as much harvestable product, compensating by producing extra vegetative growth.
e.g. Cotton bolls. Upper panels show insect damage (left) to developing boll on conventional cotton, resulting in aborted development; transgenic cotton (right) is undamaged and develops normally.
Lower panels show damaged (left) and undamaged bolls (transgenic and conventional plants respectively); insect damage has reduced cotton fibre production significantly even where bolls do develop.
But then pink Bollworm developed resistance to Bollgard cotton
The initial transgenic Bt cotton varieties broke down in the field in India
NEW DELHI—Monsanto has revealed that a common insect pest has developed resistance to its flagship genetically modified (GM) product in India. The agricultural biotechnology leader says it “detected unusual survival” of pink bollworms that fed on cotton containing the Cry1Ac gene from the bacterium Bacillus thuringiensis (Bt), which codes for a protein that’s toxic to many insect pests. In a statement to Science, Monsanto claims that the finding from western India “is the first case of field-relevant resistance to Cry1Ac products, anywhere in the world.”
Science 327, 1439 (2010)
“Breaking” the resistance of Bollgard cotton to pink bollworm emerged
^ how and why the problem was solved by the introduction of Bollgard II cotton is covered later
Transgenic crops can have major environmental benefits: Pesticide usage on corn – a case study
European Corn Borer
&Corn Rootworm
^ Both of these species are difficult to control with conventional insecticides; corn borer tunnels inside stems, whereas corn rootworm is in the soil.
Other major pests include corn earworm and armyworm (lepidopteran), grasshoppers (orthopteran) and wireworms (coleopteran)
Insect pests also carry viruses, fungi (mycotoxins) and bacteria therefore reducing their impact on crops has multiple benefits – reducing toxin load and disease
Reduced insecticide use
- Data shows that increasing adoption of Bt corn in USA correlated with
decreased insecticide usage
see figure: http://www.vox.com/cards/genetically-modified-foods/are-gmo-
crops-good-or-bad-for-the-environment
Bt corn shows vigour and yields comparable to conventional varieties
- No evidence for yield penalty associated with transgene expression
Bt corn offers full-season protection against corn borer, while retaining high yields and vigour of non-transgenic varieties. Yield increases averaging 80% compared to non-treated conventional corn are routinely observed.
Conclusion: Yield increase and reduced need for toxic chemical spray
