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2-Plants: New strategy to combat insect resistance to Bt-plants



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TITLE:  Overcoming insect resistance to Bt
SOURCE: ISB News Report, by Jennifer A. Thomson
DATE:   May 1999

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Dear GENET-news readers,

for more informations on the new Bt-resistance plans read
http://www.ncga.com/archives/news990419.html

Yours,
Hartmut Meyer

*****

OVERCOMING INSECT RESISTANCE TO Bt

A report describing what could be a significant breakthrough in the efforts to combat insect resistance to transgenic Bacillus thuringiensis (Bt)-expressing crops, has just been published. Kota et al. (1999) show that a combination of very high transgene expression due to insertion in the chloroplast genome, coupled with protein stability can result in mortality of even Bt-resistant insects.

In the U.S., millions of acres have been planted with Bt crops, mainly corn and cotton; however, permission to do so in European countries has not yet been granted. One of the main obstacles is the potential for insects to become resistant to the Bt toxin. This issue is also of concern in the U.S. and is being addressed by a number of organizations including the Environmental Protection Agency (EPA) and the National Corn Growers Association (NCGA). (See related article, pg. 8). U.S. producers of Bt crops strongly encourage farmers to grow non-engineered plants in plots alongside Bt-expressing varieties, hoping that creation of this Bt-free refuge community will postpone the evolution of Bt resistant insects. Seed companies, embroiled in a no-holds-barred marketing battle, have agreed on the importance of planting Bt-free refuges, which shows the importance they place on this issue. And rightly so, as evolving insect resistance could make or break Bt technology.

A general consensus has been reached on the need to maintain non-Bt refuges to thwart the emergence of insect resistance; however, some aspects of this insect resistance management (IRM) approach continue to be challenged. The rationale behind leaving Bt-free refuge communities is to provide a source of susceptible mates for any resistant insects that survive exposure to the Bt toxin. The strategy, though, is based on the assumption that resistance is a recessive trait, therefore the offspring of such a mating will be susceptible. This assumption has been questioned in some quarters but as yet there is little evidence that resistance is dominant.

A second unresolved issue is how to handle IRM when insects have access to more than one Bt crop. A prime example is corn earworm (Helicoverpa zea) which feeds on corn in the spring and early summer, then migrates to cotton where it is called cotton budworm. Also currently in contention is the size of the refuge area required to discourage evolution of resistant pests. The NCGA is currently recommending a 20% refuge in primary corn-growing regions and 50% in primary cotton-growing areas. These allotments may have to be increased if farmers find they need to use additional chemical pesticides to protect crops in times of unusually heavy insect predation, since sprays increase the risk of developing Bt resistance. There is also some concern that if farmers determine they need to spray a large percentage of their acreage, they may elect to spray the entire crop. Eventually they may find it more economical to spray than to employ Bt technology.

After considering some of the difficulties inherent in maintaining a Bt-free refuge, it becomes clear that continued advancements in Bt technology would be welcomed. One recommended strategy is to genetically alter crops to express multiple protein toxins, including non-Bt toxins. The availability of such "stacked" products could eventually permit a reduction in refuge size. Other suggestions include increasing the level of Bt expression, and targeting expression to tissues particularly sensitive to damage.

Kota et al. have outlined an approach for overcoming Bt resistance in insects that combines high levels of Bt gene expression with tissue specificity. Most commercial transgenic plants that target lepidopteran pests contain either the cry1Ab or cry1Ac genes. However, the proteins expressed by these genes share more than 90% homology, which increases the risk of cross-resistance. The authors chose Cry2Aa2 because it has limited homology to 1Ab and 1Ac and because its protoxin is only 65 kDa, compared with the 130-135 kDa proteins of 1Ab and 1Ac. As gene size can be a limiting factor for optimal expression in plants, this small size enabled them to introduce a gene encoding the entire protoxin, which is considered to be more environmentally stable.

They targeted the gene to the tobacco chloroplast which, due to its prokaryotic origin, could express the native DNA sequence without the necessity of codon optimization. The cry2Aa2 gene was cloned downstream of the spectinomycin-streptomycin resistance gene and driven by the chloroplast constitutive promoter Prrn. The chimeric gene was then integrated between the rbcL and accD genes, a region that is highly conserved among plants and can even be used to integrate genes into monocot chloroplasts.

Sixteen putative transformants were obtained of which two were shown to have a single insert in all the 5,000 to 10,000 chloroplasts. This high level of integration resulted in Cry2Aa2 representing 2% to 3% of total leaf protein, some 20- to 30-fold higher than current commercial nuclear transgenic plants. The importance of this high level of expression was clear when they tested the mortality of Bt-susceptible, Cry1Ac-resistant and Cry2A-resistant tobacco budworm (Heliothis virescens) by feeding them Bt-transgenic leaf material. They achieved 100% mortality, even though tobacco budworm is less sensitive to Cry2A than to Cry1Ac. They also obtained 100% mortality when leaves were fed to corn earworm and beet armyworm (Spodoptera exigua), despite the latter having a high tolerance to Cry2Aa2.

The authors state that the high levels of expression did not affect tobacco plant growth rates, photosynthesis, chlorophyll content, flowering, or seed setting in the laboratory. However, long-term tests under field conditions are needed before the full potential of this new technology can be determined. It should also be noted that a significant additional benefit of the introduction of the Bt gene into the chloroplast, rather than into the nuclear genome, is that the chloroplast genome is maternally inherited. This should help alleviate the fears of transgene spread via pollen to non-target plants.

This paper will be greeted enthusiastically by researchers involved in the development of Bt-expressing plants. It could well prove to be a watershed in the fight against insect resistance to Bt in transgenic crops.

Source:
Kota M et al. 1999. Overexpression of the Bacillus thuringiensis (Bt) CryA2Aa2 protein in chloroplasts confers resistance to plants against susceptible and Bt-resistant insects. Proceedings of the National Academy of Science USA 96:1840-1845.

Jennifer A Thomson
Department of Microbiology
University of Cape Town
jat@molbiol.uct.ac.za 



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