RNAi-mediated control of aflatoxins in peanut: Method to analyze mycotoxin production and transgene expression in the peanut/Aspergillus pathosystem

Authors: Arias De Ares, Renee; Dang, Phat; Sobolev, Victor

Submitted to: Journal of Visualized Experiments
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/28/2015
Publication Date: 12/22/2015
Publication URL: http://doi:10-3791/53398
Citation: Arias De Ares, R.S., Dang, P.M., Sobolev, V. 2015. RNAi-mediated control of aflatoxins in peanut: Method to analyze mycotoxin production and transgene expression in the peanut/Aspergillus pathosystem. Journal of Visualized Experiments. Issue 106:e53398.

Interpretive Summary: This method allows analyze aflatoxins and gene expression in peanut seeds that harbor RNA-interference (RNAi) signals for silencing aflatoxin-synthesis genes in the fungus Aspergillus flavus. Plant-RNAi-mediated control of mycotoxins has not been done before, thus, the physiology, chemistry, detection methods, and most importantly the sample size need to be considered.

Technical Abstract: The Food and Agriculture Organization of the United Nations estimates that 25% of the food crops in the world are contaminated with aflatoxins. That represents 100 million tons of food being destroyed or diverted to non-human consumption each year. Aflatoxins are powerful carcinogens normally accumulated by the fungi Aspergillus flavus and Aspergillus parasiticus in cereals, nuts, root crops and other agricultural products. We have used for the first time the silencing of aflatoxin-synthesis genes by RNA interference (RNAi) in peanut plants to control aflatoxin accumulation. Thus, no method existed to analyze the effectiveness of RNAi against aflatoxins. Peanut plants from individual transgenic events usually produce few seeds. Therefore, field testing hundreds of plants in the field under aflatoxin-conducive conditions was not an option, since the probability of finding naturally contaminated seeds can be from one in a hundred to one in a thousand, and these mycotoxins are not normally distributed. Given the limited seed supply from individual RNAi plants, we developed a method to minimize the number of tested seeds per transgenic event coupled with a high-sensitivity assay, allowing analysis of small seed pieces for gene expression by real-time PCR (RT-PCR), small RNA sequencing, and for aflatoxin accumulation by ultra-performance liquid chromatography (UPLC). Solving the aflatoxin problem in crops would be equivalent to increase food production by millions of tons that could be safely consumed instead of being discarded or diverted to other uses.