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

Imana L Power,Imana L Power,Imana L Power,Imana L Power,Valerie A Orner,Valerie A Orner,Valerie A Orner,Paola C Faustinelli,Paola C Faustinelli,Paola C Faustinelli,Renee S Arias,Renee S Arias,Renee S Arias,Victor S Sobolev

doi:10.1038/s41598-020-70618-6

Abstract

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.

Highlights

We have shown that RNA interference (RNAi) can prevent aflatoxin accumulation in transformed peanuts
Small interfering RNAs are derived from double-stranded RNA (dsRNA) and cleaved into 20–24 nucleotides by DCL2, DCL3 or DCL4, and micro RNAs (miRNAs) are derived from single-stranded RNA and cleaved by DCL1 into 19–24 nucleotides[18,19,20]
First we examined the efficiency of two methods of introduction of RNAi signals for silencing aflatoxin biosynthesis genes into peanut plants, by measuring the production of construct-specific small RNA (sRNA) in plant tissue

Summary

Introduction

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. 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. Since there was a range in ability to reduce the aflatoxin accumulation in the transformed peanut lines, we previously performed high-throughput sequencing of sRNAs to identify transgene-derived sRNAs, and possible differences in abundance of sRNA populations in the control and transformed peanut lines[15]. These results had raised several questions, e.g.: were construct-specific sRNAs generated? If so, what was the timeframe for their production, and for how long were these sRNAs detectable? In addition, it was important to know whether sRNA were generated by all five gene fragments in the RNAi construct or only some of them

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Conclusion