Transgenic Crops and Traits

So, the spouse has often complained that I don't have a post with an overview of what transgenesis means and the transgenic (GMO) crops themselves. They're scattered throughout the history of this blog, but not in a single place.

Transgenesis means taking a gene (or genes) from one species and sticking it into another. Unlike another process known as cisgenesis, transgenesis involves adding genes from a species that is sexually incompatible with the organism in question. Transgenesis is like taking a gene from a pomegranate and adding it to a Granny Smith apple. In contrast, cisgenesis is like taking a gene from a Red Delicious apple and adding it to a Granny Smith apple. For transgenesis, the species doesn't even have to be a plant: you can take a gene from an animal or bacteria and add it to a crop/plant or viceversa.

What does this mean? To explain, I have to go to the beginning: the working units within any cell are proteins. Proteins are made up by linking together amino acids in a given sequence. The exact amino acid sequence is defined in the cell's DNA; the DNA blueprint for a specific protein is known as a gene for that protein. In general, one gene encodes for one protein (of course, there are exceptions). Since there are thousands of proteins, there are thousands of genes. We're still figuring out what different genes/proteins accomplish.

Spouse: I think that you've been surprised by the fact that I can't just "make up" a protein. I wish!!! No, biotech still isn't at the point where I can say "I'm going to invent a DNA sequence that's a blueprint for a protein that will make the plants absorb more water". That would be AWESOME. The best we can do right now is to look in nature at the plants/animals/bacteria that have the trait that we want, find out what protein accomplishes that task, and then use it in transgenesis. The reason why this is important in discussions about transgenesis is that the proteins that have been added to GMOs are already in nature.

In transgenic crops, they've taken one or more genes from different species and added them to the plant's DNA so that you have new genes/proteins in the plant. That brings us to the main point of this article: what are some of the more popular genes/proteins that have been added to commercial transgenic crops or GMOs.

Transgenic proteins currently used in US agriculture can be split into 3 broad categories: herbicide tolerance, insect resistance and disease resistance. Here are some of the traits used in each category (NOTE: this is not a full list. You can find all traits in this database):

Herbicide tolerance

• EPSP synthase. A wonderfully short abbreviation for the painfully long "5-enolpyruvylshikimate-3-phosphate (EPSP) synthase". EPSP synthase is a protein that naturally exists in bacteria, plants, and fungi. The protein is part of a system that makes several crucial amino acids in these organisms. The active ingredient in weed killers such as Round-Up is "glyphosate", a synthetic compound that blocks EPSP synthase. The plant can't make the amino acids that it needs to survive so it dies. In order to make plants resistant to glyphosate, the EPSP synthase enzyme from a bacteria was added to the plants. This bacterial enzyme does the same thing (ie. it synthesizes the amino acids) but it's just different enough that glyphosate doesn't block it.
  
  It's important to note that EPSP synthase doesn't exist in mammals, which is why glyphosate has low toxicity. My previous post on glyphosate is here.
  
  In the US, the transgenic crops cultivated with the EPSP synthase gene are: alfalfa, canola, cotton, corn, soy, and sugar beet.


• AAD Enzyme. Another mercifully short abbreviation for "aryloxyalkanoate dioxygenase enzyme" and is from the bacterial species Sphingobium herbicidovorans. The protein breaks down 2,4-dichlorophenoxyacetic acid (2,4-D), a pesticide that's been used for many decades because it kills broadleaf weeds. 2,4-D mimics a natural plant hormone in these weeds, causing their leaves to grow uncontrollably, wither, and the plant eventually dies. The AAD-1 protein allows the plant to break down 2,4-D, so nothing happens to it (for a diagram of the biochemical reaction, please see here).
  
  In the US, there's only one transgenic crop with the AAD-1 gene approved for cultivation: corn made by Dow Agro was just granted approval this year. However, there are several others in the works. 

Insect resistance

It seems odd that no one is demanding for labeling of GM cotton
Image from Wikimedia commons

• Bt trait/Cry protein. There are several proteins from the bacteria Bacillus thuringiensis (Bt) that have been used in various crops and they're known as Cry proteins. Apparently, there are over 200 different Cry proteins from the Bt bacteria and they're toxic to specific orders of insects and nematodes. The insects that Cry proteins target are not all the same, which is why different proteins are used. Additionally, since the protein is toxic to insects, you may also see it referred to as "Bt-toxin". This website from UCSD offers a really simple explanation on how the Bt-toxin works: the protein dissolves in the high pH environment in the insect's gut. Then, it binds to receptors in the bug's gut causing the wall in gut to dissolve, which eventually kills the insect.
  
  Cry proteins are also used in organic farming (if you weren't aware that organic food production uses pesticides, please see bullet #2 here). The pesticide is considered to be benign to humans because the protein's mechanism of action doesn't work on mammals: our guts have a low pH and we don't have the receptors that the Cry protein binds to.
  
  Bt-corn and Bt-cotton have been commercialized. There's exciting work being done with Bt-eggplant in Bangladesh.

Disease Resistance

We had a papaya tree in our backyard in Venezuela.
I love the stuff, but the spouse can't even stand the smell.
Image from Wikimedia Commons. 

• Proteins from plant virus coats. In the United States, there are two commercial crops that have disease resistant traits: summer squash and papaya. Hawaii's Rainbow papaya is one of the great success stories of transgenesis: the papaya ringspot virus was threatening to wipe out this crop, which is a $17 million industry for Hawaiian farmers. In 1997, farmers started planting Rainbow papayas which have a protein from the virus itself. Likewise, transgenic summer squash carries proteins from several viruses which can harm this crop. I previously read up and shared my learning about how these proteins confer disease resistance to transgenic crops. Briefly, the transgene encodes for a protein from the virus (coat-protein) and this "blocks" the infection process from starting (interferes with the virus' disassembly). This is known as "coat-protein mediated resistance" or CP-MR. 

As you can see, there are no blue-strawberries or fish-tomatoes in the list. Such crops have never even made it far enough to start the regulatory approval process. I had written a conclusion for this article, with something along the lines of "See?? There's nothing scary about transgenesis! All you're doing is taking a protein that we know a lot about and moving it into a plant." But then I realized that to a lot of people, that can be scary, so I think I need to explain just a tad further.

You may have read arguments from GMO advocates stating that "we've been genetically modifying food for thousands of years. There's nothing different here." To a large extent, that's true. When you cross breed two compatible species, it's generally because there are specific qualities from species A and species B that you want to blend into a single species. For example, you may want to cross a rice strain that is naturally insect resistant with a second strain that grows very quickly. When you perform such a cross, you're blending all the genes from the two rice strains and then trying to find the hybrid that has all the traits that you're looking for.

Now, imagine instead that you know EXACTLY what gene/protein(s) caused the insect resistance in the first rice strain. Instead of crossing the two strains and blending together thousands of proteins, you specifically add this one protein to the second strain. How would you feel about that? My guess is that the vast majority of individuals would be OK with it. Now how would you feel if that gene/protein came from barley and you're adding it to rice? Again, I think many would be fine with it.

But what if it came from a bacteria?

I think that THIS is where the fear creeps in: the addition of a gene from a species that "doesn't belong". To be clear, I have no evidence to suggest this and have never polled anyone on this topic: it's just from conversations that I've had. And I think the reason why the majority of scientists don't have this fear is because we see things as proteins, and genes, and units, and no gene "belongs" to a species. We see genes/proteins as building blocks that came into existence in viruses and bacteria, and have changed, morphed, been copied, and erased throughout evolution. I work with enzymes (proteins) that have been mutated and morphed by companies so that they do what scientists need them to do in the lab. Back in grad-school, we added and removed genes in mice to figure out what they did in human disease. It was so common, that it had it's own term: "making a mouse". So the concept of adding a gene that we know a lot about into another species doesn't scare me nearly as much as it freaks out the spouse. In reviewing this piece, he agreed with my assessment adding that he views a species as a whole, whereas I view a species as bits and pieces that make a whole.

Feel free to comment below!

GMOs resistant to viruses. How does that work?

During a nerdy web-surfing episode, I stumbled upon a database that listed commercial GMOs, the regions where they've been approved, and a description of the genetic modification. Pretty handy. While scrolling through the list, I observed that one of the more common modifications is to confer viral resistance or immunity to the plant. The transgene that is added to the plant is a protein from the virus itself. And then I wondered, "how does that work? How does a protein from the virus give the plant immunity?" I knew that plants didn't have antibodies, but that was about it. During my bachelor's degree, I took quite a few plant courses ("Plants as Human Resources" was really popular), including plant biochem courses in later years to meet the requirements for my biochemistry major. But thinking back on everything I learned, I don't think that the topic of the plant immune system ever came up.

As usual, the interwebs didn't let me down. I found a very confusing 2006 review in Nature entitled "The plant immune system". Plants don't have "mobile defender cells". Despite the fact that Google will try to convince you that these are accessories for your cell phones, the role of these defender cells (which include our white blood cells) is to swoop in and "zap" foreign cells. Instead of this highly effective mobile defense system, plants have an "innate immune system" where proteins on the surface of cells and within them recognize signals from pathogens and microbes. One study found that the Arabidopsis plant turns on over 1100 genes in less than 1 hour when it recognizes bacterial pathogens. Successful pathogens either manage to suppress the plant's immune response or dodge it altogether.

The next paper I read was a fantastic review written in 1999 by one of the scientists who made the first transgenic plant with a virus protein. Before I continue, the spouse-like voice in my head is telling me that I'll need to explain the structure of a virus.

From Wikimedia Commons

Viruses are weird because it's hard to classify them as "living". They need a host cell so that they can replicate and spread, which is why they infect other organisms. They have several components and I'll focus on two of these in this article: the genetic material which is generally RNA, and a protein coat that protects the genetic material. In order to replicate and spread, the virus uses the host cell's machinery to make more of its protein components. Pretty sneaky, eh? Sort of like a thief breaking their way into your house and taking advantage of your lovely plumbing by taking a dump in your bathroom, on top of stealing all your stuff. That last analogy was inspired by my kid, who is currently pointing at his diaper and saying "Poop". I wonder what he needs?

Getting back to plant viruses. And yes... I did change his diaper.

So the review outlines that the first proposal to create a transgenic plant with a virus coat protein was made in 1981, when the author suggested creating plants resistant to the tobacco mosaic virus. The tobacco mosaic virus was the first virus ever discovered, is one of the most thoroughly studied viruses, and infects species other than tobacco (including tomatoes). The study was a joint collaboration between Monsanto and WashU in St Louis. It took several years until a tobacco plant was successfully made that was resistant to the virus, and where the next generation of plants were also resistant to infection by the virus.  Some plants were not resistant altogether, but it took longer for these plants to develop symptoms when compared to controls. The data were published in the journal Science in 1986 and the process was dubbed "coat-protein-mediated resistance" or CP-MR.

To understand how CP-MR works, several experiments were conducted. In the first, they infected the tobacco plants with the genetic material from the virus, meaning that the virus was missing the protective protein coat. They found that the genetically modified plants were more susceptible to infection by the genetic material when compared to control plants. That suggested that there was something in the coat-protein that had been added to the plant that interfered with the infection process early on. As an analogy, think of the virus as the pulp of an orange, as infection as the mess you leave on your counter when you squeeze out the juice, and the protein coat as the rind. In order to squirt the orange juice all over your kitchen counter, you have to remove the rind or at least cut through it. If there's something preventing you from cutting open the orange or removing the rind, then you'll never make a mess on your counter (i.e. infection will never happen). In this experiment, they handed over a pre-peeled orange for your squeezing pleasure, and found that a mess was easily made. So the results suggest that CP-MR doesn't have to do with the "squeezing" or infection process, rather, something prevents the removal of the rind. In the late 80's, several different studies were performed whose results backed up the hypothesis that the coat-protein transgene interferes with viral disassembly.

A few other interesting studies have been performed over the years, in attempts to better understand CP-MR. Some pretty cool experiments were the infection of the transgenic plants with different viruses that were similar to the tobacco virus, but not identical. They found that it's important for the coat proteins on the virus be similar to the coat protein genes in the transgenic plant, but that the sequence of the RNA in the virus doesn't really matter. In thinking about this, I thought it was an important finding because it would imply that the virus could mutate, but that CP-MR would continue working, although it may be to a lesser extent.

So, I like this whole story about CP-MR for many reasons. I think it's a cool, quirky oddity from nature. But I also think that it's a nice success story for the joint funding of research by public and private funds. Of course, the patent for CP-MR belongs to Monsanto and WashU, but apparently they've been working with different global institutions to create disease resistant crops. I have no doubt that Monsanto is making kajillions of dollars off of this, but it has led to the understanding of plant viruses, as well as the creation of some important crops. Some crops that use CP-MR are squash, papaya, and potato, and there are a few others approved in different countries.

Feel free to suggest future topics below.