IDENTIFICATION AND ENHANCEMENT OF SEED-BASED BIOCHEMICAL RESISTANCE IN CROPS TO AFLATOXIN PRODUCING PATHOGENS
Location: Food and Feed Safety Research
Title: Antifungal traits of a 14 kDa maize kernel trypsin inhibitor protein in transgenic cotton
Submitted to: Journal of Crop Improvement
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 9, 2008
Publication Date: May 1, 2008
Citation: Rajasekaran, K., Cary, J.W., Chen, Z.-Y., Brown, R.L., Cleveland, T.E. 2008. Antifungal traits of a 14 kDa maize kernel trypsin inhibitor protein in transgenic cotton. Journal of Crop Improvement. 22(1):1-16.
Interpretive Summary: Contamination of food and feed crops such as corn, cotton, peanut, and tree nuts with aflatoxins not only causes severe financial loss to growers, but also poses a serious health hazard to animals and humans. Availability of disease resistance genes in germplasm is important in developing new varieties with resistance to aflatoxin-producing fungal pathogens. This approach is especially useful in crops like cotton where there is no known resistance to Aspergillus mold. However, resistance genes from other sources could be genetically engineered into cotton to develop resistance. A corn kernel protein, called trypsin inhibitor, was determined to be present at higher levels in lines naturally resistant to Aspergillus mold. This protein was found in laboratory tests to inhibit Aspergillus growth and germination. The gene coding for this protein was genetically engineered into cotton and transgenic cotton lines expressing the corn protein showed resistance to one of the fungal pathogens that causes Verticillium wilt, but not to Aspergillus mold. The corn protein was not produced in sufficient levels in transgenic cotton plants to confer resistance to Aspergillus mold, but the levels were adequate to control Verticillium. These results confirm that this protein plays an important role in corn resistance against Aspergillus growth and aflatoxin contamination. Enhancing the expression of this protein in commercially-useful corn lines and other crops, such as cotton and peanuts, could result in the development of lines that resist aflatoxin contamination. This would, in turn, enhance the safety of food and feed for humans and animals, and increase financial savings to growers.
Transgenic cotton plants expressing the maize kernel trypsin inhibitor (TI) protein were produced and evaluated for antifungal traits. This 14 kD trypsin inhibitor protein has been previously associated with resistance to aflatoxin-producing fungus Aspergillus flavus. Successful transformation of cotton and expression of trypsin inhibitor was demonstrated by PCR and Northern analysis respectively. Proteins extracted from cottonseed and leaf tissues of transgenic plants were separated using SDS-PAGE and it indicated the presence of a 15-16 kDa protein in transgenic tissues as compared to control. Only transgenic cottonseed tissue reacted with the TI antibody indicating the expression in cottonseed. No cross-reaction to the TI antibody was detected from leaf extracts, indicating the TI was either not expressed or expressed at a level too low to be detected by Western blot. Crude leaf extracts from transgenic cotton plants did not show significant control of colonies from pre-germinated spores of Aspergillus flavus or Verticillium dahliae; however, extracts from transgenic cottonseed tissue showed about 60% reduction of V. dahliae colonies indicating the antifungal nature of the maize TI by itself. Cotton bolls inoculated with a green fluorescent protein (GFP)-expressing A. flavus strain showed no difference among controls or transgenic cotton plants indicating that the expression of TI cottonseed is not high enough to prevent A. flavus colonization.