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Overexpression of
a Novel Bt Protein in Chloroplasts
Provides Resistance to Plants
Against Insects

Madhuri Kota, Henry Daniell, Sam Varma,
Steven F. Garczynski, Fred Gould, and William J. Moar

ElementaryT.gif (1015 bytes)HE USE OF COMMERCIAL CROPS expressing Bacillus thuringiensis (Bt) toxins has increased in recent years due to their advantages over crops that require traditional chemical insecticides. In Alabama alone, between 300,000 and 400,000 acres of Bt cotton have been grown annually since 1996. However, in crops such as cotton that are plagued by several pests with varying degrees of susceptibility to Bt, there is concern that the toxins will not be strong enough to kill all pests. The result would be reduced efficacy of the Bt and increased risk of pests developing Bt resistance. Additionally, reliance on a single (or similar) Bt protein(s) for insect control increases the likelihood of Bt resistance development. Plant-specific recommendations to reduce Bt resistance development include increasing Bt expression levels (high dose strategy), expressing multiple toxins (gene pyramiding), or expressing the protein only in tissues highly sensitive to damage (tissue specific expression).

   In the past, the Bt gene has been incorporated into the nucleus of plant cells. More recently AAES scientists have incorporated genes that provide insect and herbicide (glyphosate or Round-Up) resistance into the chloroplast of plants. Incorporating these genes in the chloroplast offers several advantages, including the ability to place foreign genes at a specific location in the plant cell and to increase the levels of toxic proteins in the plants. Furthermore, because chloroplast genes are inherited through the mother (ovary) instead of through the father (pollen), the risk for outcrossing (foreign genes escaping to other species) to other plants, such as weeds, is reduced.

   Most current commercial transgenic plants in the United States that target caterpillar pests contain either the Bt CrylAb (corn) or CrylAc (cotton) proteins. Bt corn is targeted primarily against the European corn borer, although other pests such as the corn earworm (cotton bollworm) may also be affected. Bt cotton is targeted primarily against the tobacco budworm and cotton bollworm in the southeastern United States. Especially with cotton, other pests, such as armyworms, can also be economically damaging, but have only limited susceptibility to CrylAc. Use of single Bt proteins to control insects such as tobacco budworm and cotton bollworm could lead to relatively rapid Bt resistance development.

   Additionally, because CrylAb and CrylAc are very similar in their structure and function, resistance to one CrylA protein would most likely impart resistance to another CrylA protein, as has already been observed with the tobacco budworrn. Nowhere is this more of a concern than with cotton bollworm/corn earworm that usually feeds on corn in the spring and early summer, then migrates to cotton to complete several more generations. Clearly, different Bt proteins are needed to decrease the development of resistance.

   Another class of Bt proteins that are toxic to many caterpillars, such as the European corn borer and tobacco budworm, but are quite different in structure and function (resulting in less cross resistance) than the CrylA proteins are the Cry2A proteins. Cry2A proteins are about half the size of CrylA proteins, and therefore should be expressed at higher levels. Research was conducted at Auburn University to overexpress the Cry2Aa2 protein in tobacco chloroplasts as a model system, and as a possible solution to the evolution of Bt resistance observed in the field.

   Transgenic tobacco plants were made by inserting the Bt cry2Aa2 gene into the plants' chloroplasts using a device called a Gene-Gun, which literally shoot foreign DNA into plant cells. After bombardment with the Gene-Gun, the leaves were cut into small pieces and placed on agar medium containing antibiotics that select for those leaf pieces that contain foreign DNA. Green tissue and shoot formation were observed after about five to eight weeks of antibiotic selection. Leaf segments were allowed to grow and produce additional leaflets on the agar medium for another four to five weeks. Shoots were then selected and transferred to bottles or test-tubes with rooting medium. Shoots with sufficient leaves and roots were transferred to potting-soil and grown in growth chambers.

   Further tests were run to distinguish cry2Aa2-chloroplast transgenic plants from mutants and to determine the level of Bt toxins expressed in the plant cells. Results showed high levels of toxin in the chloroplasts of the transgenic plants. Researchers then tested the response of insects exposed to these plants. Insects used in the test included susceptible and resistant strains (at both neonate and older ages) of tobacco budworm, cotton bollworm, and beet armyworm. The caterpillars were placed in petri dishes containing both the transgenic and nontransgenic plant materials (which were used as a control) and checked for mortality daily for five days. Treatments were replicated at least twice, but four to five times in most cases.

   There was 100% mortality of the neonate susceptible budworm caterpillars feeding on the Cry2Aa2 leaves and the leaf pieces were essentially intact, while the control leaf pieces were completely devoured (Fig.1). Similar results were obtained with resistant budworms. Even older insects, which are typically more tolerant than the neonates, showed 100% mortality (data not shown).

   When leaves from Cry2Aa2 leaves were fed to cotton bollworm and beet armyworm, 100% mortality was observed, whereas no mortality was observed in the control group (data not shown). Although there was no detectable feeding damage on cry2Aa2 leaves with cotton bollworm, there was some leaf damage observed with beet armyworm, indicative of the high tolerance of this insect species to Cry2Aa2.

   In summary, this study showed that there is a high level of expression of Cry2Aa2 in tobacco chloroplasts compared to Bt genes inserted into plant nuclei. This is likely because chloroplasts can read the DNA better than plant nuclei and because there are many chloroplasts within a plant cell, but only one nucleus. DNA placed in the chloroplasts will be copied 5,000 to 10,000 times in a cell, while typically there are only one to four copies of the gene per cell when it is contained within the nucleus. Also, plant cells can express smaller genes (cry2Aa2) better than larger (crylAc) genes.

   Another advantage of expressing insecticidal proteins in chloroplasts is that it puts the toxins in the part of a plant where they are most likely to be consumed. Most caterpillars feed on green tissues that are rich in chloroplasts; therefore, they consume the highest level of insecticidal toxins if the toxins are placed in cholorplasts. Results also showed that high levels of expression of cry2Aa2 in transgenic tobacco did not affect growth rates, photosynthesis, chlorophyll content, flowering, or seed setting in a laboratory setting.

   With the successful introduction of cry2Aa2 into the chloroplast genome, the high-dose strategy should be attainable for insects such as tobacco budworm, cotton bollworm, and beet armyworm. In this context, plants expressingcry2Aa2 through the chloroplast, either singly or as part of a gene-pyramid with other insect proteins (preferably non-Bt proteins with different modes of action), could become an invaluable tool for resistance management.

   Long-term tests using agronomically important crops grown under field conditions are needed before validation of this potential new methodology can be obtained.

Moar is Associate Professor, Garczynski is Research Associate, and Kota is Graduate Research Assistant of Entomology, Auburn University; Daniell is former Professor at Auburn (now Professor, Department of Molecular Biology and Microbiology, University of Central Florida) and Varma is Graduate Research Assistant of Botany and Microbiology, Auburn University; Gould is Professor Department of Entomology, North Carolina State University.



For additional information wmoar@acesag.auburn.edu



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