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

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. 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...

RNA Interference Strategies for Future Management of Plant Pathogenic Fungi: Prospects and Challenges

“…Table 3, showed that exogenous application is effective in suppressing fungal growth. For example, a recent study by Werner and colleagues [38] showed that using spray-induced gene silencing The exogenous application of dsRNA can be very interesting also on horticultural produces at the postharvest stage [11] and against fungal pathogens, which are capable of producing mycotoxins very harmful to animal and human health [101,102]. Their control at the disposition stage is strictly limited to a few active ingredients due to residue concerns.…”

Advanced mycotoxin control and decontamination techniques in view of an increased aflatoxin risk in Europe due to climate change

“…Not surprisingly, a wide spectrum of RNA interference based technologies has been developed and tested in planta to control AF production by aflatoxigenic fungi ( McDonald et al, 2005 ; Majumdar et al, 2017 ; Wu, 2022 ). A frequently targeted gene is aflR in both peanut ( Arias et al, 2015 ; Faustinelli et al, 2018 ; Power et al, 2020 ) and maize ( Masanga et al, 2015 ). Other approaches targeted either the aflS regulatory gene ( Arias et al, 2015 ; Power et al, 2020 ) or AF biosynthetic genes including aflC (encoding polyketide synthase; Arias et al, 2015 ; Thakare et al, 2017 ; Power et al, 2020 ; Niño-Sánchez et al, 2021 ) and aflM (coding for versicolorin dehydrogenase; Raruang et al, 2020 ).…”

“…A frequently targeted gene is aflR in both peanut ( Arias et al, 2015 ; Faustinelli et al, 2018 ; Power et al, 2020 ) and maize ( Masanga et al, 2015 ). Other approaches targeted either the aflS regulatory gene ( Arias et al, 2015 ; Power et al, 2020 ) or AF biosynthetic genes including aflC (encoding polyketide synthase; Arias et al, 2015 ; Thakare et al, 2017 ; Power et al, 2020 ; Niño-Sánchez et al, 2021 ) and aflM (coding for versicolorin dehydrogenase; Raruang et al, 2020 ). It is noteworthy that a RNAi-based AF control system simultaneously silencing aflR , aflR , aflC , aflep (coding for AF efflux pump) and pes1 (encoding a non-ribosomal peptide synthase with hypothesized function in cyclopiazonic acid biosynthesis) was also constructed and tested in peanut using both transgenic and non-transgenic delivery tools ( Arias et al, 2015 ; Power et al, 2020 ).…”

“…Other approaches targeted either the aflS regulatory gene ( Arias et al, 2015 ; Power et al, 2020 ) or AF biosynthetic genes including aflC (encoding polyketide synthase; Arias et al, 2015 ; Thakare et al, 2017 ; Power et al, 2020 ; Niño-Sánchez et al, 2021 ) and aflM (coding for versicolorin dehydrogenase; Raruang et al, 2020 ). It is noteworthy that a RNAi-based AF control system simultaneously silencing aflR , aflR , aflC , aflep (coding for AF efflux pump) and pes1 (encoding a non-ribosomal peptide synthase with hypothesized function in cyclopiazonic acid biosynthesis) was also constructed and tested in peanut using both transgenic and non-transgenic delivery tools ( Arias et al, 2015 ; Power et al, 2020 ). In addition to host induced gene silencing strategies ( Majumdar et al, 2017 ; Wu, 2022 ), exogenous RNAi delivery-based, non-transgenic approaches are gaining ground including the application of RNAi-triggering dsDNA and dsRNA by gene gun ( Power et al, 2020 ), DsiRNA (Dicer-substrate siRNA) after wounding ( Faustinelli et al, 2018 ) and dsRNA by genetically engineered bacteria (RNAseIII-null mutant Escherichia coli , both living cells and crude whole-cell autolysates; Niño-Sánchez et al, 2021 ).…”

RNA Interference Strategies for Future Management of Plant Pathogenic Fungi: Prospects and Challenges

“…The exogenous application of dsRNA can be very interesting also on horticultural produces at the postharvest stage [ 11 ] and against fungal pathogens, which are capable of producing mycotoxins very harmful to animal and human health [ 44 , 160 ]. Their control at the disposition stage is strictly limited to a few active ingredients due to residue concerns.…”