gene 411 quiz 3 Genetic engineering for insect resistance

Lepidoptera

butterflies and moths

plants have their own systems for defense against insects

constitutive vs. Induced 3 major classes of secondary metabolites that can help confer insect resistance A) Terpenes - act in many ways B) Tennis - make plants unpalatable and indigestible C) Alkaloids- affect the central nervous system

diptera

flies and mosquitoes

Domestic crops

has provided conditions beneficial to insects. There is an abundant food source that is carefully cultivated allowing the insects to multiply rapidly. Has resulted in loss of insect resistance.

Bt corn slide 1

lines have been marketed by several companies. first( primary) target - European corn borer (1996) Annual Us damage from corn borer was nearly a billion dollars Protection against added in Corn rootworms (2003) Corn earworms (2010)

Strategies for pest control (human)1b

many of the pesticides used today in the US and Europe are much less poisonous than the ones used 50 years ago. Most farmers in the developing world cant afford the expensive new pesticides and are more likely to turn to older, more toxic versions that are still quite dangerous to health and the environment.

Protease (proteinase) inhibitors slide 2

- At least 14 different plant PI genes have been used to produce transgenic plants and several have increased insect resistance. Example: CpTI - cowpea trypsin inhibitor - Most active inhibitor identified - has been transferred into at least 10 species - is effective against a range of field and storage pests.

Application of Bacteria

- died and sprayed or dusted on plants - Bt protoxinsis not very stable when applied this way. Mycogen introduced a Bt gene into Pseudomonas. -can be cultured on large scale and then killed -the killed bacteria are then sprayed on the target plants. -protxin crystals remain in bacterial cells until consumed by an insect and are therefore more stable - effective against insects that live on the surface of leaves -less effective with larvae that eat roots or live inside the plant

Bt slide 1

- product of the Bt genes are protoxins -larval digestive enzymes cleave the protoxin producing an active toxin - toxin causes holes in the gut and eventual starvation - specificity in the interaction between the Bt toxin and membranes of gut cells

Protease (Proteinase) Inhibitors slide 1

- proteases found in insects are responsible for digestion of proteins nutrients for growth and development - 4 classes: serine, cysteine, aspartic, and metalo - Protease inhibitors (PI) are part of a plant's natural defense system.

Bt slide 2

- some Bt toxins kill lepidoptera (butterfly and moth larvae) , thers kill coleoptera (beetles and weevils), dipetera (mosquitoes) or nematodes -a single Bt toxin may kill some Lepidopetera but not others - Bt is not toxic to mammanls

Protease (Proteinase) inhibitors- limitations

- some insect species can compensate for protease inhibition by suing an alternative proteolytic activity or by overproducing the protease. - In some cases, resistance to an insect is greater in one engineered species than another - Within a species, a PI may confer resistance to one insect species but not another -Unlike Bt endotoxins which are acutely toxic, protease inhibitors need to be expressed at very high levels to be effective. They do not give complete insect control but have in some cases, caused a 50% or more reduction in damage over controls. - Sometimes, the introduction of 2 different insecticidal proteins can decrease damage even furter.

For the genes to be expressed at high levels in a plant, they needed to be altered extensively including:

- using regulatory elements recognized by plants -altering the codon usage -increasing the G/c content -removing any sequences that might be recognized as splice sites within a plant - remove polyadenylation signals within the coding region and other features which reduce transcript stability - in Plants expressing such synthetic genes, the level of expression is 0.02 to 1 % of leaf soluble proteins

alpha-Amylase Inhibitors

-Amylase: enzymes that break down starch -Amylase inhibitors: a class of plant proteins that can interfere with food digestion by insects. -some but not all amylases are inhibited by each inhibitor. -alpha- amylase inhibitors can be effective against seed weevils because they are dependent on starch. Example: -An alpha-AI common bean forms a complex with insect -Amylase Inhibitors but not with plant -Amylase Inhibitors -The alpha-AL confers resistance to coleopteran stored- product pests.

Vip3: susceptibe Insect Species

-Fall armyworm - Black cutworms - Corn earworm - Mediterranean corn borer - Beet armyworm - Yellow striped armyworm - Cabbage Looper - Soybean looper - Tobacco budworm - Cotton budworm - Native budworm - Southwestern corn borer - Sugarcane borer - Asian cor borer - Tobacco hornworm - Diamondback moth

Lectins slide 1

-Sugar- binding proteins - found in plants, viruses, microorganisms, and animals - little is known of their function -The roles are likely to vary in different organisms. - In plants, they are found in many plant tissues and are abundant in the storage organs. - Mode of action- some plant lectins bind to the gut wall in insects and are toxic. Some plant lectins also possess chitinase activity which may damage some insect membranes. - Some lectins are toxic to mammals. Lectins from snowdrop and garlic are toxic to insects but not mammals

Strategies to reduce the chances resistant population will develop

1) the EPA requires the planting of reuges of nonBt cotton to produce susceptible insects to mate with resistant ones from the Bt coton- thereby diluting the resistance genes - 20% refuge that can be sprayed with noonBt insecticides -4% refuge that cant be sprayed 2) plants expressing the Bt toxins at very high levels may be needed to cause greater mortality and less opportunity for resistance to develop 3) Use of multiple- toxin genes. The gene or genes controlling resistance to one toxin must not confer resistance to any of the other toxins

Strategies for pest control (human)1a

1. Chemicals - Increase yield but at a cost to the environment. - only a small amount of the chemicals applied are delivered to the plant part where they are needed. - insects can evolve resistance. -annual global cost ~10 billion

Strategies for pest control (human) list

1. chemicals 2. biological control by other insects or organisms 3. cultivation practices 4. Breeding 5. Genetic engineering

Bt cotton

1990 Bt genes were used to produce transgenic cotton with resistance to cotton bollworm. This was accomplished by increasing the level of insecticidal protein 100-fold by using a strong promoter and sequence modifications 1996 1st year of commercial production (potatoes were first in 1995) > 1.8 million acres in the US (approx. 13% of US acres) this resulted in a reduction of 250,000 gallons of formulated insecticide products used 1999 14 million acres of GM cotton (insect and/or herbicide resistance) in the US ~55% of US acres 2006 165,600 km^2 (40.9 million acre) of Bt corn and 115,900 (28.4 million acres) of Bt cotton currently, 85 percent of US cotton areas are planted with genetically engineered, insect-resistant seeds.

Strategies for pest control (human)2

2) Biological control by other insects or organisms. - bacteria (ex. Bacillus thuringiensis-BT) - Other insects -nematodes and other animals -viruses -fungi - male sterile insects There is potential for using genetic engineering of these organisms to make them more powerful.

Do insect resistant crops reduce the use of pesticides?

2003- in the US Bt varieties with resistance to tabacco bud-worn and pink bollworm increased production by more than 362 million pounds, improved farm income by nearly $191 million and reduced pesticide use by more than 3.2 million pounds

Strategies for pest control (human) 3, 4

3. Cultivation practices (crop rotations, crop health, etc.) 4. Breeding -can take a very long time (12-15 years) - insects can evolve resistance -genes for resistance dont always exist in populations of the desired species.

Strategies for pest control (human) 5

5. Genetic engineering Concerns/ disadvantages -limited number of available genes -difficult and expensive -insects evolve resistance -are there dangers to other organisms? - social factors Advantages/ positives - faster than breeding -can introduce genes from other organisms (plant, animal, microbe) -can transfer several new genes

Their are also proteins that can help confer resistance

A) Toxic proteins B) lectins - bind to proteins in the gut and interfere with the absorption of nutrients C) inhibitors of digestive enzymes -amylase inhibitors (amylases digest starch) -protease inhibitors (proteases digest protiens) Amylase and protease inhibitors may cause impairment of nurittion

Crop damage by insects

Adult insects: -eat leaves and decrease the ability of the plant to photosynthesis -suck sap from the phloem and starve plant organs Larvae: -eat roots, leaves, cambium seeds, etc. Can completely serve a stem

Bt Transgenic Plants

Allows the entire plant to be protected, especially against insects that infect plant parts that sprays cannot reach 1987- first attempts to transfer cry genes to tobacco and tomato - cry1Aa, cry1Ab, cry1Ac from a lepidopteran active BT - The genes were expressed but at levels too low to provide adequate protection (<0.001% of leaf soluble proteins)

Bt

Baccillus thuringiensis

Several species of bacteria produce proteins that kill insect larvae

Baccillus thuringiensis (Bt) -most studied - large number of subspecies and strains -most strains produce several Cry proteins, each of which shows a rather narrow host range - great diversity of endotoxin encoding genes (> 200 + human created mutations)

Bt corn slide 2

Before Bt corn was commercially introduced, the European corn Borer was only partially controlled using chemical insecticides. Chemical use was not always profitable, and timely application was difficult. After Bt corn, adopters lowered their pesticide costs and increased yields.

The bean -amylase provided protection from weevils in field grown peas.

Pea weevil emergence in nontransgenic and transgenic peas. AI-1 is a more effective inhibitor of B. pisorum -Amylase (80%) than AI-2 and is active over a greater pH range.

molecular characterization of a Novel Vegetative insecticidal Protein from Bacillus thuringiensis Effective Against Sap- Sucking Insect pest

Cotton aphid id one of the dangerous pests of various crop plants including cotton. A VIP2A is toxic to the aphids. There is a receptor in the brush border membrane vesicles of the cotton aphids only, but not in the lepidopteran insects.

Background and criticisms Cry9C

Cry9C - different from the other Bt toxins. - heat-stable -resistant to degradation in gastric juices (generally considered the most important indicators of potential allergenicity) No known history of human dietary exposure to the Cry9C protein. People have been exposed to other Cry proteins due to Bt spraying StarLink corn produces high levels of Cry9C in kernels, roots, and leaves The Cry9C [ ] is 10 to 400 times as high as that of the other Cry proteins in Bt corn

Lectins slide 2

Effective against sap sucking insects. Examples- A lectin isolated from snowdrop (GNA) can cause 80% mortality of rice brown planthopper when added to their liquid diet. - The GNA gene has been cloned and introduced into many different crop species. - Some increase in resistance to sap sucking insects such as aphids and to a Lepidopteran larvae has been observed but in general, levels of expressions have been too low to give good levels of resistance.

PNAS

Fall 2001, 6 other studies were published in PNAS Bt176, kind of Bt corn, produces pollen that is highly toxic to Monarch larvae and some other butterfly species. Bt176 contains a promoter that is very active in pollen. less than 2% of the corn acreage contained Bt176 at that time and that has decreased. More commonly grown varieties don't produce much toxin in pollen and larvae can eat large amount without showing ill effects

How did StarlInk corn get into human foods?

Farmers clam they weren't told they couldn't sell this corn for human consumption or that it would be approved for human consumption by harvest time. The taco shells were manufactured in Mexico using corn processed in Plano, TX but StarLink corn was sent to many plants StarLink seeds were planted on 350,000 of America's 80 million acres of corn

A comparison between maize and B. thuringiensis

GC content good Leucine codon UUA - Maize 6.42% -B. Thuringiensis 47.72 B. Thuringiensis will need to be changed

GNA was exressed in rice phloem and epidermal layers (tissue specific RSs promoter) or constitutively

GNA was exressed in rice phloem and epidermal layers (tissue specific RSs promoter) or constitutively

Bt or not Bt... Transgenic Corn vs. Monarch Butterflies

In 1999, John Losey showed Bt toxic proteins makes corn pollen poisonous to Monarch butterflies. Monarch caterpillars eat on milkweed plants growing near corn fields Losey simulated Bt corn field conditions and showedonly 56% of the larvae survived. Surviving caterpillars weighed on average less than half the controls

Why are plants eaten if they have all these defense compounds?

Insects evolve mechanisms to detoxify, sequester, or otherwise neutralize harmful chemicals.

Concerns: insects may become resistant to Bt toxins

Probably the greatest concern with plants containing BT genes is that the insects will evolve a resistance to the toxin. Insects become resistant to sprayed Bt by transgenic plants may speed up this process. Bt is one of the few forms of insect control available to organic gardeners/ producers and they are concerned that if insects evolve resistance, they will lose the use of Bt the risk of rapid pest adaptation to an insecticide is highly dependent on the initial frequency of resistance alleles in the population.

Resistance genes from higher plants

Proteins produced by plants have the potential for use in genetic engineering for insect resistance. Genes encoding proteins are being used more than those encoding micromolecules such as terpenes, tannins, and alkaloids. Why? -Proteinase inhibitors (also from animals) -Amylase inhibitors -lectins -Chitinases (not effective) -Anionic peroxidase -Tryptophan decarboxylase (TDC) because these genes are isolated from plants, they don't need to be as extensively modified as the Bt genes

StarLink (Cry9C) Corn

Taco Bell (and other) taco shells were recalled in 2000 when it was discovered they contained corn transformed with the Cry9C gene. Starlink corn was allowed in feed bit was not certified for human consumption. 24 people claimed to have had severe allergic reactions. 17 had blood test that showed they had an allergic reaction but not to the StarLink corn.

Coleoptera

beetles and weevils

VIP3 corn - fall armyworm

VIPS activity is similar to -endotoxins - induce gut paralysis - complete lysis of the gut epithelium cells - larval mortality

Other insect resistance genes from Bacillus

VIPS- Vegative insecticidal proteins -Clarified culture supernatant fluids collected during vegetative growth of Bacillus species are a rich source of insecticidal activies besides the delta endotoxins - VIPs are effective against some species such as rootworms and cutworms that are fairly resistant to other known insecticidal proteins - in 2005, Syngenta petioned for non-regulated status of a cotton variety resistant to Lepidopteran pests because if was modified with a synthetic VIP3A(a) gene.

Corn Pollen

most of the pollen produced in a corn field falls within the field or nearby [ ] of pollen fall rapidly as you move away from the corn fields caterpillars feeding on milkweed plants at a short distance from corn fields are unlikely to encounter any pollen

Bt cotton problems in China

other insects have become a problem on Bt cotton 2nd country after the US to plant Bt cotton by 3rd year, farmers with Bt cotton cut pesticide use by more than 70% and had 36% higher earnings than nonBt farmers By 2004 Bt farmers had to use just as much spray and Bt seed tripled in cost of conventional seeds. This was not caused by bollworms but by secondary insects that used to be controlled while spraying for bollworms. Benefical insects and arthopods have also increased

Insect resistance

~15 - 25% of the plant food grown worldwide is consumed by insects. 3% of the US wheat crop is lost to insects. This is enough wheat to feed 2 million people. Farmers in the tropics lose up to 50% of their crops to insects and diseases. In order to feed the projected 9 billion people who will inhabit the earth by 2050, farmers must increase their cereal yields by 40% The development of insect-resistant crops would increase our ability to feed the world.