15 - Evolution & BT Toxins

Does refugia work?

*You can use H-W equations to see what the frequency of the resistance gene, and it would be very very low. -However, this theory depends on resistance gene being truly recessive. If heterozygotes do at all better than homozygotes for non-resistance, the numbers change and resistance spreads much faster (but still slower than if there was no non-Bt field). The greatest reason this might not work is farmers cheating. So Monsanto sells seeds in premixed (and indistinguishable) mixes. It works quite well, because of the Halo effect. Having some Bt corn present helps all the corn do better. It doesn't matter if it is Bt and non-Bt corn mixed together, or non-Bt corn in a neighbor's field. Moths develop on corn, and then fly to other corn to deposit eggs. If there is some Bt corn present, it will kill the larvae that are born on it, but it will also kill the babies of moths that were living well on nearby non-Bt corn. So it reduces the effect of the pest on a landscape level, not only for the Bt plants themselves. *profits of Bt use are spread pretty well between Bt and non-Bt corn, so there is actually some incentive to buy and plant non-Bt corn because it is cheaper and still does well if it is near Bt corn (promoting refuge and polyculture). *So far, this strategy of refuge seems to be pretty self-sustaining. BUT, we need to start introducing stacked pesticide genes to prepare for the future resistance which will emerge

Benefits of GM Bt toxin plants

-Reduced environmental impacts from pesticides - When the plants are producing the toxins in their tissues there is no need to spray synthetic pesticides or apply Bt mixtures topically. -Increased opportunity for beneficial insects - Bt proteins will not kill beneficial insects. -Reduced pesticide exposure to farm workers and non-target organisms. ***Use of GM Bt toxin plants leads to fewer cotton crop losses and greatly reduced use of dangerous pesticides (in terms of how dangerous they are to ppl).

Benefits and dangers of Round UP

-benefits: great herbicide (kills all other plants) -simple, only one herbicide and one gene of resistance. before you had to use a cocktail of herbicides. -you don' have to apply it repeatedly -it doesn't have a lot of cross-toxicity (doesn't poison anything but its targets) -doesn't persist in environment very much, and has moved herbicide use away from dangerous ones that end up in drinking water that are toxic to non-plants. -crop rotation is good farming practice (helps keep pests and weeds down). but if you plant herbicide resistant soybeans this year, and the herbicide persists into next year, you must plant a resistant crop again next year. So if you use monsanto soybeans AND corn, you can use their herbicide and still rotate your crop between soybeans and corn -Glyphosate resistance has already emerged in weeds. First appeared in Australia, now is in major parts of the US. Now 63 weed species are resistant. We are a bit screwed when they spread.

Concerns/potential dangers of GM Bt plants

1)Does it effect on non-taget species? (pollinators! herbivores, decomposers, all other species it interacts with, above and below soil surface). Monarch butterflies: if you coat milkweed in Bt corn pollen, the monarch larvae die. Will Bt plants threaten monarchs and other butterflies? *No, b/c corn pollen is heavy and doesn't fly in the air, it falls straight down, so very little corn pollen spreads beyond a meter from the edge of a corn field, and very little milkweed is growing within a meter of cornfields. 2) Is it an allergen to some people? Key feature of an allergen is failure to break down in the digestive tract, allowing the protein to present itself to the intestinal epithelium and induce the allergic response. *So we must ask: is the new protein similar in structure to known allergens? does the gene come from a known allergen? ***If you have an allergic reaction, you have antibodies to that thing in your blood stream. if those antibodies are not present, there is no allergic reaction. ***There has been no evidence of allergic reactions to Bt proteins.

Adding nutritional value to food

1)fortifying food after harvest: adding iodine to salt, adding vitamin D to milk, adding vitamin C to juice 2)fortifying food before harvest: instead of enriching flour after, can we change the genes of the wheat plant so the flour has more vitamins/iron?

Why rice?

1/2 the world's population eats rice daily. White rice is just the seed; is a great source of calories (is just starch) but a poor source of everything else: almost no micronutrients and vitamins. Brown rice is the seed plus some hull; is a great source of nutrients, but also arsenic. Brown rice bran (the hull that makes it brown colored) goes rancid much faster than white rice, which is why most of the world eats white not brown. So ppl make brown rice syrup out of it as a "healthy" alternative to high fructose corn syrup, but that is where all the arsenic gets concentrated.

European Corn Borer

A pest worm thing that kills corn and cotton in US. One species grows in the stem, the other in the roots. If you put pesticide on outside of plant, it will not kill the borer. So you can alter the plant to express a toxin in every cell of the plant. Before bt corn the corn borer was killing more than 1 billion dollars of corn a year.

What is Bt?

Bacillus thuringiensis is a bacteria that kills worms. It was first used as a pesticide post-harvest, during storage. Meal worms would eat flour and other stored food, so bt toxins were used to protect flour. Bacillus thruingiensis forms spores, so it can live for years through bad conditions while dormant. it forms a crystal protein right next to the spore. It is a soil organism, as are many of the larvae it kills. The larva will be cruising around digesting soil, and when they eat the spore they will also eat the crystal protein. The larva's guts have a very high pH (alkaline, basic) which activates the crystal protein, which becomes toxic and kills the larva. The decaying larva is a perfect environment for the spore to grow in.

More on Bt toxins

Bt toxins are the favorite pesticide of organic growers. Counts as organic b/c it is a naturally occurring substance. Bt toxins (aka crystal proteins) are incredibly specific. Specific strains of Bt will kill only a specific species of pest. Different strains of Bt are specific to different receptors in insect gut wall. Each insect species possesses different types of receptors that will match only certain toxin proteins, like a lock to a key. It is because of this that farmers have to be careful to match the target pest species with a particular Bt toxin protein which is specific for that insect. This also helps the beneficial insects because they will usually not be harmed by that particular strain of Bt.

Cadherin

Cadherin is a protein expressed on the surface of the intestinal cells of grubs. The crystal protein comes in, the alkaline environment alters the crystal protein so it can bind with the cadherin. We don't know exactly how it happens but somehow pores are punched in the membranes of the intestinal cells so the insides rush out, killing the intestines and then the grub. 1st possibility: bonding to cadherin causes conformational change in crystal protein that makes it puncture cell wall. 2nd possibility: when crystal protein binds to cadherin (a membrane bound receptor protein), it causes a signaling cascade within the cell. the binding on surface leads to a series of misfired signals that causes pores in cell.

Cotton

Cotton grows in warm places where it never freezes, so the pests are never killed off. It is so vulnerable to pests that ppl use tons of pesticides and ruin soil.

From teosinte to corn

Example of traditional breeding: corn comes from teosinte. Corn is tall, single stalked, has a big head with MANY kernels. Teosinte is short with many stems and very small heads with one row of few kernels, which have an extremely hard outer coat that would break your teeth, and are not great to grind into four. So ancient farmers in Mexico would save the seeds with the softest outer coats over 1,000s of years. non-shattering pods are desirable on corn, and all similar plants: Evolution would encourage seeds to fall off/out of pod, so they can be dispersed. A farmer wants all the seeds to stay on the pod, so more food is gathered and so he can control where it grows.

Should it matter that we are switching genes from species to species?

Genes are simply polymers made of four letters of nucleotide. They speak the same language in every organism. The majority of our genome is the same as as plant genome and genes that are turned on in us can be turned on in plants and work exactly the same. genes from one species typically function normally in another. So if a plant is growing and reproducing healthily with a human gene in it, is it still a plant? *The human genome contains 50-100 genes that were transferred laterally from bacteria. Not only mitochondria but genes in our nuclear genome that we absorbed from environment some time after LUCA, and they are expressed in all humans, not even dormant. So genes move around between species and we have genes from a variety of sources. *And we talked about salamanders and sea slugs that pick up genes from algae.

What work is being done on plant genomes today?

Golden rice is being made as a humanitarian effort by governments, not big agribusiness. However, majority of work on plant genomes is done by agribusiness to introduce: -herbicide/pest/drought/salt/environmental stress resistance -more nutritional value (proteins, amino acids, micronutrients, etc.) -vaccines expressed in foods!

Example: pro-vitamin A in golden rice

Golden rice is genetically engineered to contain beta-carotene (a photosynthetic pigment and a precursor to vitamin A). People are terribly concerned about genetically modified organisms and the risks they might pose, so there are strict regulations, and it hasn't been adopted yet. It might be able to save a lot of lives.

Traditional Breeding vs. Genetic Engineering

Humans have been manipulated plant genomes for 10,000 years by breeding. But many people are very uncomfortable with genetic engineering. *In traditional breeding to solve the wheat rust problem: you would find a wild strain or weed of something resistant to rust and similar enough to our wheat; cross-pollinate, take only the resistant offspring and cross with wheat again, until you have enough plants that will pass on the resistant gene. So you are taking half the genome from another organism, you don't know what is in that other half. you are getting the resistance gene plus a bunch of other mystery genes. *In genetic engineering, you are taking exactly ONE gene or pathway, and moving that into wheat, so the rest of the genome is exactly the same. and you aren't limited to plants that are similar enough to reproduce with wheat.

Why Vitamin A?

In S. East. Asia, 70% of children under 5 have vitamin A defficiency. leads to blindness and a weak immune system (more death by disease). Improved vitamin A nutrition could save 1-2 million kids a year. (UNICEF)

The situation of our food:

In the last 50 years crop production increased 2.5 times, keeping up with population growth But, in the next 50 years we must do this again. We were choosing between all the choices that nature gave us, but now we have options that are not in nature: using genes from plants that would never cross pollinate, or from bacteria, fish, mammals, etc. Genetic Engineering can transcend the species barrier. ***The desired result: increased food production on less land with reduced environmental impact. we are loosing farmable land as it is paved over, and ruining more through bad farming practices. Irrigation brings water to a dry area, so after the land is watered the water evaporates, and leaves salt. this happened to the aztecs and mayans: over 1,000s of years of irrigation, the soil became saltier and saltier and they had less and less food. ***All the choices have risks and bad consequences, as well as good.

The best strategy to reduce resistance: stacking multiple pesticide genes.

In transgenic plants, "stack" multiple resistance genes. Occurrence of resistance to one Bt is low, simultaneous occurrence of resistance to two (or more) Bt toxins is much lower (multiplicative—product rule). ***This is exactly the same concept as multi drug cocktails in treatment of TB and AIDS. there are 30 types of crystal proteins, but we can get an infinite set of potential pesticides. MicroRNAs are very short RNA strands that aren't translated. they correspond with a specific part of an mRNA, making a short double-stranded portion on the mRNA. In plants, this cleaves it, so it is destroyed. In animals, it messes with translation so that it is not expressed. Insects have lots of genes that do not appear in anything else, so if you made a microRNA that targeted an essential insect-specific gene it could not possibly affect any other type of organism and would kill the insect. So monsanto's next big idea is plants with many insect resistance genes for microRNAs stacked in it.

Beta-carotene

Mammals make vitamin A from beta-carotene. If you take straight vitamin A pills, it is easy to poison yourself (it has toxicity). But if you eat tons of beta-carotene, your body can make exactly as much vitamin A as it wants, no limit except what is healthy. excess beta-carotene has no negative side effects. Rice makes beta-carotene in leaves, not the endosperm (the white part of rice grain). The PSY pathway uses two enzymes, phytoene synthase and phytoene desaturase, to make beta-carotene. Rice has the entire pathway (the PSY gene sequence) in its genome, but it is expressed in the leaves not the seed. So we just need to take a promoter/regulatory sequences of a gene that IS expressed in the endosperm, and use recombinant DNA to move them to in front of the PSY pathway genes. *So we must take an active endosperm promoter and replace the PSY promoter with it, so the endosperm promoter is now in front of the PSY coding sequence.

The Green Revolution

Norman Borlaug started the Green Revolution: he used conventional breeding to make semi-dwarf wheat, which didn't fall over and so was much more productive. It allowed countries like India to become self-sufficient and produce enough food to feed themselves. One problem he solved was wheat rust, a fungal pathogen that destroys crops. He used conventional breeding to introduce genes into the commercial cultivars that made them resistant to wheat rust. The problem was thought to be solved until a new strain of rust emerged that was resistant to the gene. He had done such a good job with his wheat rust defense that essentially every wheat plant grown in the world had it, so now every wheat plant is susceptible to the new rust. The rust spores are airborne, so eventually it will go around the world. Now breeders are racing to create wheat plants that are resistant to the new rust. 25% of calories on the planet are from wheat. The world needs more food because 1) there are increasing numbers of people 2) people are starting to eat better diets. As you go up trophic levels (up the food chain), each step up involves a 10% efficiency of biomass transfer. So it uses much less biomass/is more efficient, to eat from lower trophic levels. If instead of eating the plants, you eat the cow that eats those plants, you are getting only 10% of the food value of the plants. But India and China are moving towards meat-heavy diets.

Another strategy to reduce resistance: Refugia

Refugia: The approach used most widely to delay insect resistance to Bt crops is the refuge strategy, which requires refuges of host plants without Bt toxins near Bt crops to promote survival of susceptible pests. The theory = most of the rare resistant pests surviving on Bt crops will mate with abundant susceptible pests from refuges of host plants without Bt toxins (because there are many fewer survivors of the Bt field). Virtually all of the pests in the non-Bt field will be homozygous for non-resistant cadherin. If inheritance of resistance is recessive (which, because it must be a mutation, it probably will be and always has been so far), the hybrid offspring produced by such matings will be heterozygote for resistance and non-resistance, and be killed by Bt crops, markedly slowing the evolution of resistance. EPA policy: 20-50% of total on-farm maize must be non-Bt, planted within 0.8 km of Bt fields ***monoculture is the great danger to agriculture, so if each plant had variety in the genome (polyculture) the entire field would be less vulnerable to pests.

Herbicide Resistance: Round Up

Round Up is an herbicide produced by Monsanto. They also produce "Round Up Ready" crops (corn and soybeans) that are resistant. So you have to use both together. Round Up = glyphosate. Glyphosate inhibits an enzyme called EPSPS. EPSPS is needed for the synthesis of the three aromatic amino acids, so plants that don't have it can't make proteins and die. (We don't synthesize those but rather get them through food, so we don't need EPSPS, so it is not toxic to us.) *Glyphosate is absorbed through leaves and translocated to growing points in plant, so only effective as post-emergence (already growing), not on seeds. and works on broadleaf and cereal weeds. -To make round up, they found a bacterial (an agrobacterium) version of EPSPS that resists glyphosphate. So you need to put the bacterial gene in, and move a plant promoter to turn it on. ALSO, the genes for EPSPS are in nucleus, but aromatic amino acids are made in chloroplasts, so you also need a chloroplast targeting sequence to make sure the new version of enzyme is taken into cholorplasts after translation. So it is a combination of 3 parts (modularity).

How do we get a new gene into plants?

There is completely natural way of moving genes into a host organism: Agrobacterium bacteria infects wound sites on trees and transfers some of its DNA into the plant with its plasmid. Through recombinant DNA techniques we can introduce virtually any gene or combination of genes into Agrobacterium and then into many (but not all) plant species. But Agrobacterium is a plant pathogen, and so is subject to tons of regulations. *Biolistic transformation. There is another way to introduce DNA into plants: shoot it in. Micro-tungsten beads are coated with the desired DNA, and shot at high velocity into plant cells. This method of gene transfer has no regulation. This is crazy!

Risks

To humans: -possible long-term human health effects -eating food with genes that confer resistance to antibiotics, gene transfers to you, gene transfers to your gut flora and hurt your body!\ To environment: -pest resistance (very likely (no difference from resistance to chemical pesticides)). counter strategies = refuges, stacking Bt genes. **Monoculture is the real villain. It is convenient for the farmer to plant plants that are all the same. BUT polyculture is much better. Example: Several rice varieties, planted in mixtures across thousands of farms in Yunnan Province,China, showed enhanced resistance to rice blast, a fungal disease, than monocultures did. Darwin had seen this effect in wheat (that diversity was beneficial to survival of population), but had no explanation.

Monoculture

We are in this race to beat wheat rust because of our practice of monoculture. If all the wheat is identical, the one rare pest or bacterial or fungal pathogen that can evade the wheat's defenses will dominate and spread. So breeders are on a treadmill running to introduce the next line of defense for corn/wheat/etc. But they are still doing this one gene at a time: they put out one really strong gene into every plant that defends them for a while, until the next pathogen/pest learns how to evade it and now there is no defense, so they do one more really strong gene. **Borlaug was a conventional breeder, but embraced recombinant genes/transgenic plants, because it is a faster method and he believed we were in a reach to stop people from starving.

More info on gold rice

We need the two enzymes, phytoene synthase and phytoene desaturase, to be activated in the endosperm. Originally scientists took them from daffodils. This upset some groups like green peace, because daffodils are poisonous. So they switched to using genes from maize (safe, edible). It also worked much better (x20 more carotenoids in endosperm) **It just started being grown for human trials. Activists do not want the tests to happen. **The golden rice is being engineered by scientists using government money, and will be released for free to small farms.

How can we move the promoter?

We use the standard cloning tools. We have to construct a vector and put the desired genes (endosperm promoter) into the vector. We need a selectable marker (like a color change or glow), so that after the first generation we can select for the transgenic ones, because the success rate of transfer is low. *In plants scientist use a antibiotic resistant or herbicide resistant genes, b/c they will kill the untransformed plants and let the transgenic ones grow. *We use plasmids from agrobacteria to get the vector into the plants.

Will pests become resistant to Bt toxins?

Yes, definitely. If you use plants with the same defense all over the world, you are selecting for pests that can resist it, and eventually resistance will spread rapidly in pests. *How will it happen? The cry protein (crystal protein) interacts with cadherin. Different species have specific cadherin that can only interact with certain types of Bt toxin (each is highly specialized). So all that needs to happen is for a few amino acids in the pest's cadherin protein to change for it to become immune to one variety of Bt toxin.

Start of Bt toxins (crystal proteins) as pesticide

gene for making Bt crystal protein was cloned into corn using pBR322 plasmid. The advantage of plants that make their own Bt toxins is HUGE: it can kill pests in roots and inside plant, and you don't need to spray. Today many GM crops w/ Bt toxins (mainly corn and potatoes) are used around the world, to protect from corn borers, boll worms, and potato beetles. **You can also buy Bt toxins as spray or crystal powder, and put it on outside of plants.

How can we delay or evade resistance?

with "cocktails". provide multiple pesticides simultaneously. The likelihood of resistance to one pesticide is rare, the likelihood of resistance to a second is also rare; multiply them and it gets even smaller. Strategies to reduce resistance: -alternate Bt spray with another pesticide (but this would create pests that are resistant to all of them, step by step) -crop rotation (but this has the same problem as alternating the sprays) -the best way is to stack multiple resistance genes, so one organism would have to be resistant to several things at once: less likely