Location: National Peanut Research LaboratoryTitle: Analysis of small RNA populations generated in peanut leaves after exogenous application of dsRNA and dsDNA targeting aflatoxin synthesis genes
Submitted to: Scientific Reports
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/27/2020
Publication Date: 8/14/2020
Citation: Power, I.L., Faustinelli, P.C., Orner, V.A., Sobolev, V., Arias De Ares, R.S. 2020. Analysis of small RNA populations generated in peanut leaves after exogenous application of dsRNA and dsDNA targeting aflatoxin synthesis genes. PLoS One. 10:13820. https://doi.org/10.1038/s41598-020-70618-6.
Interpretive Summary: We have shown that RNA interference can prevent aflatoxin accumulation upon fungal pathogen infection. The effectiveness of this technology depends on multiple molecular factors: such as type and quantity of small RNAs [small interferring RNA (siRNA) and micro RNAs (miRNAs)] being produced, the form of application of nucleic acids either DNA or double strand RNA (dsRNA), orientation of the siRNAs and microRNAs being produced, their base-matching with the messenger (mRNA) of the gene target in the fungal pathogen, and side effects such as altered expression of small RNAs towards non-target genes in the plant. In this work, we analyzed in duplicate three treatments at two sampling dates for all the molecular aspects mentioned above.
Technical Abstract: Aflatoxins produced by Aspergillus flavus and A. parasiticus can lead to millions of dollars in yearly losses in peanut production and processing. The use of RNA interference (RNAi) to control aflatoxins by genetically transforming the plant host has in general been successful. Here, we examined for the first time the populations of small RNA (sRNA) generated in 12 libraries of in-vitro-grown peanut plants, at 24 and 48 h after exogenous delivery of fragments of five genes involved in aflatoxin biosynthesis (RNAi-5x insert) and the control, all in duplicate. The RNAi-5x insert was applied by particle bombardment, either as double-stranded RNA (dsRNA) or as plasmid DNA with inverted repeats (dsDNA). Examination of known micro RNAs (miRNAs) related to disease resistance, showed on the treated-leaf areas significant down-regulation of miR399 and up-regulation of miR482 (both, p = 0.05) on leaves treated with RNAi-5x insert as dsDNA compared to the control. Small interfering RNAs (siRNAs) derived from the RNAi-5x insert were compared among libraries, and showed that the majority of siRNAs mapped to the aflatoxin efflux pump (AFL2G_0527, aflep). The number of RNAi-5x-specific siRNAs significantly increased between 24 and 48 (p = 0.01) for applications as dsDNA; also, dsDNA generated significantly (p = 0.05) more RNAi-5x-specific siRNAs than the dsRNA treatment, both at 24 and 48 h, respectively. These results show that sequencing sRNAs after exogenous application of dsRNA or plasmid DNA with inverted repeats, can be used to determine the effectiveness of molecular constructs to generate siRNAs inside the peanut plant against target genes in the fungal pathogen. This work, describes the early production of small RNAs after dsRNA and dsDNA applications, and lays the foundation for the non-transgenic delivery of RNAi in the control of aflatoxins in peanut.