Genetic Modification of Plants to Produce Biopesticides: Chitinase

In a recent post, I mentioned the importance of identifying  genes and proteins besides Bacillus thuringiensis (Bt) that can be used in the fight against crop pests. One such protein is chitinase.

Chitinase (pronounced ′kītən′ās) is an enzyme that breaks down the protein chitin. Chitin is an important component of insect exoskeletons and fungal cell walls. Functionally, it helps the insect or fungus retain its structure. Destroy the chitin, and the insect or fungus dies.

Scientists from the Rao laboratory at the University of Napoli hypothesized that a plant that made its own chitinase could protect itself against pests. They generated genetically engineered (GE) plants that produced chitinase, then tested the effect of the chitinase-producing GE plants on fungi and tobacco budworm larvae.

Tobacco budworm (Heliothis virescens)

Tobacco budworm (Heliothis virescens)

The results, published in a 2008 article in Transgenic Research, showed reduced fungal growth and abnormally slow weight gain in the insect larvae after they ingested the plants. This suggests that chitinase-producing GE plants protect themselves against damage from fungi and insect larvae better than non-GE plants.

In a future article, I will report on research which suggests that combining TMOF and chitinase improves crop protection against insect pests.

Corrado, G, Arciello, A, Fanti, P, Fiandra, L, Garonna, A, Diglio, MC, Lorito, M, Giordana, B, Pennacchio, F, Rao, R. 2008. The chitinase A from the baculovirus AcMNPV enhances resistance to both fungi and herbivorous pests in tobacco. Transgenic Research 17: 557 – 571, DOI: 10.1007/s11248-007-9129-4.

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TMOF: A Protein That Could Prevent Crop Loss

One reason for genetically engineered (GE) cotton’s dramatic and rapid acceptance by farmers is its improved control of three major insect pests: tobacco budworm, cotton bollworm and pink bollworm.

Cotton and other crop plants that are genetically engineered to make Bacillus

Budworm Moth

Budworm Moth

thuringensis (Bt) proteins become resistant to damaging crop pests (see Promising Results in the Fight Against Rice Stem Borer Moths). Unfortunately, as with any pesticide, insects develop tolerance to Bt over time, so it is important to investigate other proteins that might have efficacy as biopesticides. One such protein is Trypsin Modulating Oostatic Factor (TMOF).

Tobacco Budworm Larvae

Tobacco Budworm Larvae

TMOF is a protein that prevents insects from synthesizing the digestive enzyme trypsin, which is critical for digestion. TMOF was first developed for use on mosquitoes. Mosquito larvae that eat TMOF die because they cannot digest their food.

TMOF can have a similar effect on crop pests. In articles published in 2002 and 2003, a research group from the University of Napoli in Italy generated GE plants that produce TMOF. The researchers then tested the effect of the TMOF-producing GE plants on insect pests. Tobacco budworm larvae that ate leaves from these plants developed at an abnormally slow rate. Later studies, which I will cover in a future post, also showed that TMOF reduced the number of larvae that survive to adulthood. Taken together, these reports show that TMOF-producing GE plants protect themselves against damage from tobacco budworm.

References:

Tortiglione C, Fanti P, Pennacchio F, Malva C, Breuer M, De Loof A, Monti L, Tremblay E, Rao R. The expression in tobacco plants of Aedes aegypti trypsin modulating oostatic factor (Aea-TMOF) alters growth and development of the tobacco budworm, Heliothis virescens. Molecular Breeding 2002; 9: 159 – 169. DOI: 10.1023/A:1019785914424.

Tortiglione C, Fogliano V, Ferracane R, Fanti P, Pennacchio F, Maria Monti L, Rao R. An insect peptide engineered into the tomato prosystemin gene is released in transgenic tabacco plants and exerts biological activity. Plant Molecular Biology 2003; 53: 891 – 902. DOI: 10.1023/B:PLAN.0000023667.62501.ef.

Disclaimer:

I also have researched the use of TMOF as a biopesticide, although not via GE plants. See one of my articles at Thompson DM, Young HP, Edens FW, Olmstead AW, LeBlanc GA, Hodgson E, Roe RM. Non-target toxicology of a new mosquito larvicide, trypsin modulating oostatic factor. Pesticide Biochemistry and Physiology 2004; 80: 131-142. DOI: 10.1016/j.pestbp.2004.06.009.


Genetic Modification without Genetic Engineering

Genetic engineering promises solutions to problems of agricultural importance including plant tolerance to stress caused by drought and salinity, resistance to insect and fungal pests, and weed prevention. However, consumer concerns about genetic engineering have slowed the release of biotech crops. A genetic modification technique called oligonucleotide-directed repair (ODR) can introduce subtle improvements to plants while avoiding some of those concerns.

ODR changes a single nucleotide in a gene that is already in a plant, animal, bacteria or fungus. Remember that a gene is a piece of DNA that provides instructions to the cell. If all the DNA in the cell represents the blueprint for a house, a gene represents instructions for making a portion of the house, the front door, for instance. The instructions are written using four nucleotides, shorthanded as “A”, “C”, “G” and “T”. Change one nucleotide and you can dramatically change the instructions.

ODR is a technique for changing one nucleotide in a plant, animal, bacterium or fungus. As an example, cystic fibrosis (CF) is an inherited disease. People with CF have an error of only one nucleotide in a critical gene. The single nucleotide change leads to a build up of mucus in the lungs, often leading to patient death in their 30’s. One potential application of this technology is for gene therapy in CF sufferers.

As the name suggests, ODR is based on the ability of an oligonucleotide to cause a change in DNA. An oligonucleotide is several nucleotides put together, but shorter than a gene. The oligonucleotide differs by only one nucleotide, or one letter of the DNA code, from a gene that has always been in the cell. The organism sees the oligonucleotide and changes its gene to match it. The result is identical to the original, with the exception of one change to the instructions (gene).

C. Dong and colleagues set out to prove that ODR would work in wheat. First they introduced a defective gene into the wheat plant, using traditional genetic engineering methods. Then they tried to correct the defective gene using ODR. They used an electric pulse to introduce an oligonucleotide, into the cell. ODR made the defective gene functional, proving the utility of the technique in wheat. This technology could be used to create healthier oils in food crops or generate new industrial oils for biofuels. In wheat, it could lead to production of gluten-free flour that would be a boon to individuals with Celiac Disease.

Dong, C, P Beetham, K Vincent, P Sharp. Oligonucleotide-directed gene repair in wheat using a transient plasmid gene repair assay system. Plant Cell Reports (2006) 25: 457-465.


Promising Results in the Fight Against Rice Stem Borer Moths

According to the USDA, over 430 million metric tons of rice were consumed worldwide in 2008, making it one of the world’s staple crops. But rice production has historically been threatened by disease and insect pests. Rice is Egypt’s second largest export crop. In Egypt, larvae of the rice stem borer moth (Chilo agamemnon) are major insect pests, which cause yield loss as high as 10 – 30%. Unfortunately, classical rice breeding has not improved resistance to this insect. Additionally, because the moth larva enters the rice stem, it is protected from most applied pesticides.

Since it’s difficult to externally apply the pesticide to the stem borer larvae, Egyptian genetic researcher Reda Moghaieb investigated the possibility of modifying the plant to make its own pesticide. Moghaieb chose a gene from a naturally occurring bacterium (Bacillus thuringiensis). This gene contains the instructions for producing a protein that is toxic to stem borer larvae. Moghaieb reasoned that when the gene was introduced into rice, the plant would produce the protein in cells of the stem, killing stem borer larvae that eat it. The gene was added to rice plants. When borer larvae were fed stems of these plants, the larvae died within four days; some died in only 24 hours. These results suggest that when the Bacillus thuringiensis gene is introduced into rice plants it acts as an effective pesticide against stem borer larvae.

REA Moghaieb. 2010. Transgenic rice plants expressing cry1Ia5 gene are resistant to stem borer (Chilo agamemnon). GM Crops 1:5, 1-6.

International Rice Research Institute has information about rice, its history, and its socio-economic relevance.


Good news for Celiac disease sufferers

Celiac disease (CD) is a digestive disorder that affects as many as 1 in 133 individuals. When CD sufferers eat foods containing gluten, which is present in wheat, rye and barley products, they exhibit a range of painful symptoms due to damage to the lining of the small intestine. Gluten is not a single protein, but rather comprises two families of proteins: glutenins and gliadins. It’s the gliadin proteins that cause the most trouble for CD sufferers. Currently the only treatment for CD is to avoid eating gluten entirely. This is challenging, given the amount of wheat, rye and barley products used as additives in prepared food. Also, gluten and yeast work together to make bread rise, and sometimes you just want a nice, chewy piece of bread.

Researchers thought that if they could remove just the gliadins from the gluten, CD sufferers would be able to safely eat gluten-containing products. Notice the difference between this and other biotech crops that we’ve seen in the news: instead of adding herbicide-tolerance or insect resistance to the plant, these researchers are removing proteins that cause disease in some people. Using a technique called “RNA interference”*, Gil-Humanes and colleagues showed that gliadin proteins could be reduced in wheat. Preliminary evidence reported in this paper suggests that CD sufferers won’t fall ill from the altered gluten proteins in the reduced-gliadin wheat.

You may be wondering: does reducing the gluten in wheat affect the texture of the bread? Apparently not, according to one bread-making quality test.

J Gil-Humanes, F Pistón, S Tollefsen, LM Sollid, F Barro. 2010. Effective shutdown in the expression of celiac disease-related wheat gliadin T-cell epitopes by RNA interference. PNAS 107: 17023 – 17028.

*RNA interference (RNAi) is a technique for preventing a gene from making protein. For more information, see this explanation on MedicineNet.

For more information about Celiac disease.