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Texas A&M's Keerti Rathore on Using RNAi to Build an Edible Cottonseed

Name: Keerti Rathore
Position: Associate professor/director, Institute for Plant Genomics & Biotechnology, Texas A&M University
Background:Assistant professor, Institute for Plant Genomics & Biotechnology, Texas A&M — 1997-2003
Research scientist, Crop Biotechnology Center, Texas A&M — 1995-1997
Research scientist, botony/plant pathology, Purdue University — 1991-1995
Postdoc, Purdue University — 1985-1990
Postdoc, Imperial College, London — 1982-1984
PhD, plant physiology, Inperial College, London — 1981
MSc, plant biochemistry/physiology/cell biology/ecology/taxonomy/anatomy — Gujarat University

In this week’s issue of the Proceedings of the National Academy of Sciences, Keerti Rathore and colleagues from Texas A&M published data showing that RNAi can eliminate a toxin in cotton plants that has prevented the plant’s seeds from being used as a food source.
According to the paper, cotton has been cultivated for more than 7,000 years for its fiber, but its seeds, which contain significant amounts of high-quality protein, have remained an inaccessible source of nutrition to humans and animals because of the presence of the toxin gossypol, which protects the plants from pests.
While traditional breeding techniques have been used to successfully eliminate gossypol from cotton, the plants were commercially unviable because of their increased susceptibility to insects. Using RNAi, however, Rathore and his colleagues have been able to silence the production of gossypol only in the cottonseed.
This week, RNAi News spoke with Rathore about his research.
How did this work come about? Were you working with cottonseed or RNAi?
This was my very first project when I came to Texas A&M about 10 years ago. Cotton is a big crop in Texas, so obviously one of the things that I thought would be good to do is work on some of the problems associated with cotton.
Cottonseed toxicity due to gossypol is a long-standing problem, and people have tried to fix it but haven’t been able to through traditional plant breeding. My area of research is plant transgenics, so I thought about using some molecular approaches to address this problem.
When I started this work, we [had already identified] the gene that we targeted in this [most recent] research. At the time, I started using antisense technology to do this job and we had some success but not a lot. We were able to bring down gossypol in the seed by about 80 percent using antisense, but that was only in one line; we screen over a hundred lines and only found one that had this much reduction in the seed. That was okay, but not enough to really make it safe for human consumption, so about four or five years ago we [turned] to RNAi, [which appeared] to be a much more powerful gene-suppression technology.
One of the things that we had to be careful about was to do the suppression of gossypol in a highly seed-specific manner [because] gossypol and some related terpenoids … are present throughout [cotton] plants, including the seed, and protect the plants from insect attacks as well as diseases. So you don’t want to knock down gossypol throughout the plant.
We started using RNAi and at the same time identified a seed-specific promoter from cotton. We utilized these two bits and made some hairpin RNA constructs to transform cotton plants. We screened more than 70 or 80 lines and found some lines where the levels of gossypol in the seed were substantially reduced. In fact, in some of the best lines we have the gossypol has been reduced by about 98 percent. When we looked at these plants at the level of the leaves and other floral organs and roots, we found that the level of gossypol and other terpenoids are what they should be in the wild-type plant. So [the RNAi-based reduction of gossypol] seems to be working in a very seed-specific manner.
In the paper we only discuss about two generations [of the transformed cotton plants], but we have gone to one more generation and the results still look pretty good: The trait is stable generation after generation. The levels of gossypol in the seeds now are below the level considered safe by the [US Food and Drug Administration].
So in theory these seeds are ready for consumption?
Technically they should be, but this is genetic engineering, and one has to jump through all the hoops. And those are probably much more challenging than the science ever was, although the science was not easy.
That’s a topic I want to get to. But before that, how is it that the silencing didn’t spread to the rest of the plant?
That was our concern also when we started this project, although when you use a seed-specific promoter you should limit [silencing] to the seed. We have characterized this promoter and it is only active during embryo development, and most of the gossypol is in that embryo, which is what you call the kernel. We know that the promoter is highly specific — it gets turned on before the gossypol synthesis begins in the embryo and is there all through embryo development.
At the same time, there is a danger that an RNAi signal can spread from its point of origin to other parts of the plant. That was certainly a concern, but when we checked the leaves and the other parts of the plant … we found that [they] were just as normal as wild-type plants … [which] tells you that the RNAi is not spreading.
But we also thought that it might be possible in cotton that RNAi spread does not happen [at all]. To address this, we utilized some materials that we already had in the lab when we created GFP-expressing cotton plants. We took [these] and did RNAi on them, so we had both lines — lines [in which RNAi suppressed GFP expression], as well as GFP-expressing lines. We did some grafting studies and tried to directly address the question, “Does the RNAi silencing signal spread from one part of the plant to the other?” When we checked these grafts after two months, when we knew the graft had taken nicely and the scion was growing, we sliced open the part of the stem where the graft junction should be. We did not see the silencing signal move beyond the graft junction.
One other reason why we think our silencing remains confined to the embryo is that … for most plant embryos the embryos develop in isolation — there is not a direct connection to the maternal tissue. So it’s possible that as the embryos are developing in isolation, the signal remains confined and does not go to the maternal tissue and affect other parts of the plant.
Specifically, what RNAi approach did you use [in this work]?
Unlike what they do in animal studies, we — and this is what most plant people do — made a hairpin RNAi construct using a 600-base pair [sequence], and are stably expressing this construct in the plant using … an agrobacterium method. Once [the agrobacterium] has done the job of transferring this bit of gene into the plant cells, we killed it off [and] the RNAi construct becomes part of the genome.
To get back to the point you raised earlier, you talked about dealing with political issues regarding genetically modified foods. This is a big issue, and certainly golden rice has never taken off because of those concerns. What is the next step for you guys in getting this work to become a reality?
Golden rice is a good example. It has faced many hurdles, although because it has received a lot of publicity things are going through. Several field studies are being done in various countries, and I think one day it will be out there in the hands of farmers.
[For our cotton], we still have a few more studies to do — we want to make sure the trait is stable, so we’ll go for a couple more generations. We also are going to do some field studies.

At the same time, we are trying to find some partners and will probably be looking at charitable foundations to help us out in terms of doing all kinds of testing that is required before a genetically engineered plant is approved for food or feed. We are in the very early stages and have a lot of ideas in mind, but we need to pursue those. Hopefully, we can find some sort of partnership that will allow us to do them.


This is the short term. I want to see this [plant available] in my lifetime, but at the same time, one has to take a long-term view. We’ve been dealing with this problem [of cottonseed toxicity] for hundreds and thousands of years, and right now there are many hurdles when you are dealing with a genetically modified plant. But I think in the next 15 or 20 years a lot of these regulations that we have to satisfy will be eliminated or reduced substantially.