Power, Imana L.; Dang, Phat M.; Sobolev, Victor S.; Orner, Valerie; Powell, Joseph L.; Lamb, Marshall C.; and Arias, Renée S., "Characterization of small RNA populations in non-transgenic andaflatoxin-reducing-transformed peanut" (2017 t r a c tAflatoxin contamination is a major constraint in food production worldwide. In peanut (Arachis hypogaea L.), these toxic and carcinogenic aflatoxins are mainly produced by Aspergillus flavus Link and A. parasiticus Speare. The use of RNA interference (RNAi) is a promising method to reduce or prevent the accumulation of aflatoxin in peanut seed. In this study, we performed high-throughput sequencing of small RNA populations in a control line and in two transformed peanut lines that expressed an inverted repeat targeting five genes involved in the aflatoxin-biosynthesis pathway and that showed up to 100% less aflatoxin B 1 than the controls. The objective was to determine the putative involvement of the small RNA populations in aflatoxin reduction. In total, 41 known microRNA (miRNA) families and many novel miRNAs were identified. Among those, 89 known and 10 novel miRNAs were differentially expressed in the transformed lines. We furthermore found two small interfering RNAs derived from the inverted repeat, and 39 sRNAs that mapped without mismatches to the genome of A. flavus and were present only in the transformed lines. This information will increase our understanding of the effectiveness of RNAi and enable the possible improvement of the RNAi technology for the control of aflatoxins.

New technologies are needed to eliminate mycotoxins and/or fungal pathogens from agricultural products. RNA interference (RNAi) has shown potential to control fungi associated with crops. In RNAi, double-stranded RNA (dsRNA) targets homologous mRNA for cleavage, and can reach the mRNA of pathogens in contact with the plant. The key element in this process is the movement of RNA signals cell-to-cell and over long distances within the plant, and between host plants and parasites. In this study, we selected a regulatory gene in the aflatoxin biosynthesis pathway, aflS/aflR, necessary for the production of aflatoxins in Aspergillus spp. We designed a Dicer-substrate RNA (DsiRNA) to study the movement and stability of the duplex over time in in vitro peanut plants using stem-loop primers and RT-PCR for DsiRNA detection. The preliminary results demonstrated that DsiRNA was absorbed and moved away from the point of application, spread systemically and was transported rapidly, most likely through the phloem of the shoot, to the sink tissues, such as new auxiliary shoots, flowers and newly formed pegs. The DsiRNA remained detectable for at least 30 days after treatment. This is the first time that movement of exogenous DsiRNA in in vitro peanut plants has been described. Since DsiRNA was detectable in the pegs 15 days after treatment, aflatoxin reduction may be possible if the duplexes containing part of the aflatoxin biosynthesis pathogen gene induce silencing in the peanut seeds colonised by Aspergillus spp. The application of small RNAs could be a non-transformative option for mycotoxin contamination control.

Previously, we have shown that RNA interference (RNAi) can prevent aflatoxin accumulation in transformed peanuts. To explore aflatoxin control by exogenous delivery of double-strand RNA (dsRNA) it is necessary to understand the generation of small RNA (sRNA) populations. We sequenced 12 duplicate sRNA libraries of in-vitro-grown peanut plants, 24 and 48 h after exogenous application of five gene fragments (RNAi-5x) related to aflatoxin biosynthesis in Aspergillus flavus. RNAi-5x was applied either as double-stranded RNA (dsRNA) or RNAi plasmid DNA (dsDNA). Small interfering RNAs (siRNAs) derived from RNAi-5x were significantly more abundant at 48 h than at 24 h, and the majority mapped to the fragment of aflatoxin efflux-pump gene. RNAi-5x-specific siRNAs were significantly, three to fivefold, more abundant in dsDNA than dsRNA treatments. Further examination of known micro RNAs related to disease-resistance, showed significant down-regulation of miR399 and up-regulation of miR482 in leaves treated with dsDNA compared to the control. These results show that sRNA sequencing is useful to compare exogenous RNAi delivery methods on peanut plants, and to analyze the efficacy of molecular constructs to generate siRNAs against specific gene targets. This work lays the foundation for non-transgenic delivery of RNAi in controlling aflatoxins in peanut. One of the major constraints in peanut production is aflatoxin contamination, resulting in yearly losses that can add up to millions of dollars 1. Aflatoxins produced by Aspergillus flavus Link and A. parasiticus Speare are detrimental to human health, as they can lead to stunting in children 2 , immunosuppression, and acute hepatotoxicity 3. Aspergillus flavus and A. parasiticus can infect and produce aflatoxins in other important agricultural crops as well 4. Germplasm improvement, biological control, and crop management practices have so far been the main management strategies in peanut to combat aflatoxin contamination 5-7 though the problem still persists. There are several reports of the successful implementation of gene silencing or RNA interference (RNAi) against plant pathogenic fungi 8-12 ; Cooper and Campbell 8 found lower pustule development in bean (Phaseolus vulgaris) plants that expressed RNA targeting rust (Uromyces appendiculatus) effector genes; and Ghag et al. 9 developed transgenic banana (Musa spp.) lines expressing essential genes of Fusarium oxysporum f. sp. cubense and these transgenic lines were still disease-free eight months after inoculation. Options to reduce aflatoxin production using RNAi in crops have had varying levels of success 13-16. Control of plant diseases through RNAi is aimed at degradation of target-specific pathogenic mRNA, resulting in silencing of that gene in the pathogen. RNA interference (RNAi) is a biological process in which a double-stranded RNA (dsRNA) signal leads to silencing of a target gene 17. The process involves several enzymes and starts with the recognition of a dsRNA signal that binds to a protein complex called D...