RNAi-mediated Control of Aflatoxins in Peanut: Method to Analyze Mycotoxin Production and Transgene Expression in the Peanut/<em>Aspergillus</em> Pathosystem

Abstract

Citation: Arias, R.S., Dang, P.M., Sobolev, V.S. RNAi-mediated Control of Aflatoxins in Peanut: Method to Analyze Mycotoxin Production and Transgene Expression in the Peanut/Aspergillus Pathosystem. J. Vis. Exp. (106), e53398, doi:10.3791/53398 (2015). AbstractThe 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 A. parasiticus in cereals, nuts, root crops and other agricultural products. Silencing of five aflatoxin-synthesis genes by RNA interference (RNAi) in peanut plants was used to control aflatoxin accumulation following inoculation with A. flavus. Previously, no method existed to analyze the effectiveness of RNAi in individual peanut transgenic events, as these usually produce few seeds, and traditional methods of large field experiments under aflatoxin-conducive conditions were not an option. In the field, the probability of finding naturally contaminated seeds is often 1/100 to 1/1,000. In addition, aflatoxin contamination is not uniformly distributed. Our method uses few seeds per transgenic event, with small pieces processed for real-time PCR (RT-PCR) or small RNA sequencing, and for analysis of aflatoxin accumulation by ultra-performance liquid chromatography (UPLC). RNAi-expressing peanut lines 288-72 and 288-74, showed up to 100% reduction (p≤0.01) in aflatoxin B 1 and B 2 compared to the control that accumulated up to 14,000 ng . This protocol describes the application of RNAi-mediated control of aflatoxins in transgenic peanut seeds and methods for its evaluation. We believe that its application in breeding of peanut and other crops will bring rapid advancement in this important area of science, medicine and human nutrition, and will significantly contribute to the international effort to control aflatoxins, and potentially other mycotoxins in major food crops.

Characterization of small RNA populations in non-transgenic and aflatoxin-reducing-transformed peanut

“…Transformed peanut lines (288-10 and 288-11) were obtained through Agrobacterium-mediated transformation, by integrating dsRNA targeting five genes involved in aflatoxin biosynthesis (RNAi-5x) as previously reported [22]. The five genes were AFL2G 07223 (aflS or aflJ), AFL2G 07224 (aflR), AFL2G 07228 (aflC/pksA/pksL1), AFL2G 07731 (pes1), and AFL2G 05027 (aflatoxin efflux pump, aflep); numbers correspond to accessions in A. flavus genome annotation (BROAD Institute, Cambridge MA), other names are in parentheses [28].…”

“…Complementary DNAs were synthesized using 1 g of RNA per sample, combining random hexamers and oligo-dT in Superscript III First Strand Synthesis Super Mix (Invitrogen, MA). Detection of the selectable marker NPTII was carried out using single-tube nested PCRs (STN-PCR), as described by Gomes et al [30] with modifications by Arias et al [22], using external primers PCAPD 5714F: 5 -AGGCTATTCGGCTATGACTG-3 and PCAPD 6446R: 5 -CGTCAAGAAGGCGATAGAAG-3 , and internal primers PCAPD 5730F: 5 -ACTGGGCACAACAGACAATC-3 and PCAPD 6249R: 5 -ATATTCGGCAAGCAGGCATC-3 . Briefly, the STN-PCR involves two PCR reactions, performed in one tube, in 20 l total volume, containing 4 l of 5x Phire reaction buffer (Carlsbad, CA), 1.25 mM dNTPs, 0.4 l Phire Hot start II DNA polymerase (Carlsbad, CA), 10 pmol of each forward and reverse external primers, 1.0 l DNA template, and 13.4 l RNase-DNase free water.…”

“…First-generation immature and mature seeds harvested from transformed and control plants, were challenged with A. flavus isolate NRRL 3357 (provided by Dr. Bruce Horn), as described by Arias et al [22]. Briefly, seeds were surface sterilized and imbibed in sterile deionized water, after which the seed coats and embryos were removed.…”

“…Cleanup procedure and Ultra-Performance Liquid Chromatography (UPLC) of aflatoxins in experimental and control samples were performed as described by Sobolev and Dorner [33] with modifications as described by Arias et al [22]. Briefly, seeds were extracted with methanol followed by a cleanup procedure on a mini column packed with basic aluminum oxide.…”

“…Recently, Arias et al [22] transformed peanut plants to generate dsRNA simultaneously targeting five genes in the aflatoxin biosynthesis pathway of A. flavus. In several of the transformed peanut lines obtained there was an 84-100% reduction in aflatoxin accumulation compared to the control.…”