Abstract
Exogenous application of double-stranded RNAs (dsRNAs) and small-interfering RNAs (siRNAs) to plant surfaces has emerged as a promising method for regulation of essential genes in plant pathogens and for plant disease protection. Yet, regulation of plant endogenous genes via external RNA treatments has not been sufficiently investigated. In this study, we targeted the genes of chalcone synthase (CHS), the key enzyme in the flavonoid/anthocyanin biosynthesis pathway, and two transcriptional factors, MYBL2 and ANAC032, negatively regulating anthocyanin biosynthesis in Arabidopsis. Direct foliar application of AtCHS-specific dsRNAs and siRNAs resulted in an efficient downregulation of the AtCHS gene and suppressed anthocyanin accumulation in A. thaliana under anthocyanin biosynthesis-modulating conditions. Targeting the AtMYBL2 and AtANAC032 genes by foliar dsRNA treatments markedly reduced their mRNA levels and led to a pronounced upregulation of the AtCHS gene. The content of anthocyanins was increased after treatment with AtMYBL2-dsRNA. Laser scanning microscopy showed a passage of Cy3-labeled AtCHS-dsRNA into the A. thaliana leaf vessels, leaf parenchyma cells, and stomata, indicating the dsRNA uptake and spreading into leaf tissues and plant individual cells. Together, these data show that exogenous dsRNAs were capable of downregulating Arabidopsis genes and induced relevant biochemical changes, which may have applications in plant biotechnology and gene functional studies.
Highlights
The current RNA-based crop improvement studies employ the RNA interference (RNAi) phenomenon for downregulation of gene targets in plants for further plant disease control and crop management [1,2]
RNAi is a natural regulatory mechanism that involves sequence-specific degradation of target mRNAs or translation inhibition induced by short small-interfering RNAs or microRNAs originated from long double-stranded RNA precursors that may vary in length and origin [3,4]
NAs and small-interfering RNAs (siRNAs) have been shown to spread systemically into plant tissues and were double-stranded RNAs (dsRNAs) and siRNAs have been shown to spread systemically into plant tissues and were uptaken by the fungal cells inducing a RNAi-mediated silencing of the targeted genes of uptaken by the fungal cells inducing a RNAi-mediated silencing of the targeted genes of the the pathogens [16,17,21,27]. This strategy of plant disease control was termed as spraypathogens [16,17,21,27]. This strategy of plant disease control was termed as spray-induced induced gene silencing (SIGS)
Summary
The current RNA-based crop improvement studies employ the RNA interference (RNAi) phenomenon for downregulation of gene targets in plants for further plant disease control and crop management [1,2]. The principles of the RNAi phenomenon are being actively exploited in plant gene functional studies. RNAi is a natural regulatory mechanism that involves sequence-specific degradation of target mRNAs or translation inhibition induced by short small-interfering RNAs (siRNAs) or microRNAs (miRNAs) originated from long double-stranded RNA (dsRNA) precursors that may vary in length and origin [3,4]. In the course of RNAi, long dsRNAs precursors are recognized and processed by a ribonuclease DICER into small RNA duplexes of 20–24-nucleotide (nt)-long, i.e., siRNAs and miRNAs [3]. These siRNAs are incorporated into the RNA-induced silencing complex (RISC) that drives silencing of the target mRNAs via their cleavage, destabilization, or hindering translation
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