Detection of exogenous double‐stranded RNA movement in in vitro peanut plants

Abstract

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.

Exogenous RNAs for Gene Regulation and Plant Resistance

“…Koch et al [13] reported on the fluorescent signal detection of labeled dsRNA in barley vascular tissue 24 h after spraying. Faustinelli et al [74] demonstrated that exogenously applied synthetic 27 nt siRNAs spread systemically and were stable for at least 30 days in in vitro peanut plants. In addition, there are several studies reporting on the stability of dsRNA applied onto plant leaf surfaces and showing that dried naked dsRNA or dsRNA loaded into LDH nanosheets were stable on plant surfaces for up to 30 days [10,19].…”

Analysis of small RNA populations generated in peanut leaves after exogenous application of dsRNA and dsDNA targeting aflatoxin synthesis genes

“…Our results also show that siRNAs were generated from the introduced dsRNA insert, and that the majority of the siRNAs mapped to afl ep, followed by pes 1, while fewer mapped to afl R. Since the siRNAs that mapped the RNAi-5x insert showed high abundance on specific areas of the insert, both for dsRNA and for dsDNA treatments, the sRNAs were interpreted as being generated de novo; a uniform coverage would be expected if they had originated from dsRNA degradation. In our previous study, we had observed a quantitative increase in the amount of RNA duplexes over time 25 .…”

“…204243 (Qiagen, Germantown, MD, USA). The stemloop primers were designed as described previously 25 . Stemloop, forward and reverse primers, and synthetic sequences of the small RNAs that were validated (the DNA oligonucleotides), were obtained from Integrated DNA Technologies (IDT, Coralville, Iowa, USA) ( Table 2).…”

“…In both treatments, dsRNA and dsDNA, more and higher abundance of siRNA types were generated from the dsRNA insert at 48 h than at 24 h; in most cases twice as many siRNAs at 48 h, and up to twice as many siRNA types at 48 h. In this study, a Gene Gun was used to ensure delivery of the RNAi signal to the peanut plant, and thus avoiding uncertainties of loss or degradation of sRNAs when using different application methods such as soil drench or spraying the foliage. In a different experiment of our RNAi research 25 , Dicer-substrate siRNAs (dsiRNAs) were applied to the top of the peanut plant, after wounding. We were able to demonstrate that the dsiRNA moved systemically from the application point through the vascular system to new auxiliary shoots, flowers, and newly formed pegs, within 15 days after treatment.…”

Double-Stranded RNAs in Plant Protection Against Pathogenic Organisms and Viruses in Agriculture

“…Treatment of leaves with artificially synthesized dsRNAs targeted at the genes involved in insect development significantly increases mortality and inhibits insect growth [88][89][90][91][92]. This effect can be achieved by irrigating the roots of plants by dsRNA, which leads to effective suppression of the target gene and abnormal development of insect pests [74,76,93]. RNA interference using exogenous dsRNAs can be used for a very wide range of insect genes.…”