Inhibition of Aspergillus flavus growth and aflatoxin production in transgenic maize expresing the a-amylase inhibitor from Lablab purpureus L

Authors: Rajasekaran, Kanniah - Rajah; SAYLER, RONALD - University Of Arkansas; Majumdar, Raj; Sickler, Christine; Cary, Jeffrey

Submitted to: Journal of Visualized Experiments
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/14/2018
Publication Date: 2/15/2019
Citation: Rajasekaran, K., Sayler, R.J., Majumdar, R., Sickler, C.M., Cary, J.W. 2019. Inhibition of Aspergillus flavus growth and aflatoxin production in transgenic maize expresing the a-amylase inhibitor from Lablab purpureus L. Journal of Visualized Experiments. 144:e59169. https://doi.org/10.3791/59169.
DOI: https://doi.org/10.3791/59169

Interpretive Summary: Aflatoxin contamination of maize kernels by the fungus Aspergillus flavus poses a serious health hazard to humans and animals worldwide. Control of preharvest aflatoxin contamination is possible through cultural practices, breeding of resistant varieties, development of transgenic maize expressing potent antifungal proteins or peptides and by use of biological control of toxin producing strains by non-toxin producing strains. However, total control of this fungal species is nearly impossible because of the non-specific nature of infection and spread in fatty acid-rich seed such as maize kernels. Development of transgenic crops, using naturally available antifungal proteins or synthetic ones, offers a quick solution to increase the host plant resistance to aflatoxin contamination. One such natural plant protein is from the hyacinth beans (AILP) which blocks the action of a key fungal enzyme called alpha-amylase that is essential for fungal growth and infection. We generated transgenic maize lines expressing the AILP and they demonstrated significant control of fungal growth infection in maize kernels and reduced aflatoxin contamination by 62-88%. The results will be useful to other scientists to develop resistant maize lines with reduced aflatoxin contamination upon harvest, thereby increasing the food and feed safety to humans and animals.

Technical Abstract: Aflatoxin contamination in food and feed crops is a major challenge worldwide. Aflatoxins, produced by the fungus Aspergillus flavus (A. flavus) are potent carcinogens that substantially reduce crop value in maize and other oil rich crops like peanut besides posing serious threat to human and animal health. Different approaches including traditional breeding and advanced functional genomics techniques such as transgenic expression of resistance associated proteins, RNAi based approach are being evaluated in addition to finding new A. flavus potential gene targets to increase aflatoxin resistance in susceptible crops. A few studies in the past have shown an important role of a-amylase in A. flavus pathogenesis and aflatoxin production, suggesting this gene/enzyme is a potential target to reduce both A. flavus growth and aflatoxin production. In this regard, the current study was undertaken to evaluate heterologous expression (under the constitutive 35S promoter) of a Lablab purpureus a-amylase inhibitor-like protein (AILP) in maize against A. flavus. AILP is a 36-kDa protein, which is competitive inhibitor of A. flavus a-amylase enzyme and belongs to the lectin–arcelin–a-amylase inhibitor family protein in common bean. In vitro studies prior to the current work had demonstrated the role of AILP in inhibition of A. flavus a-amylase activity and fungal growth. The goal of the current work was to implement this previous knowledge in a commercially important crop like maize to increase aflatoxin resistance. Our results show a 35-72% reduction in A. flavus growth in AILP expressing transgenic maize kernels which translated into a 62-88% reduction in aflatoxin levels.