Abstract
Summary RNA silencing is a universal mechanism involved in development, epigenetic modifications and responses to biotic and abiotic stresses. The major components of this mechanism are Dicer‐like (DCL), Argonaute (AGO) and RNA‐dependent RNA polymerase (RDR) proteins. Understanding the role of each component is of great scientific and agronomic importance. Plants, including Nicotiana benthamiana, an important plant model, usually possess four DCL proteins, each of which has a specific role, namely being responsible for the production of an exclusive small RNA population. Here, we used RNA interference (RNAi) technology to target DCL proteins and produced single and combinatorial mutants for DCL. We analysed the phenotype for each DCL knockdown plant, together with the small RNA profile, by next‐generation sequencing (NGS). We also investigated transgene expression, as well as viral infections, and were able to show that DCL suppression results in distinct developmental defects, changes in small RNA populations, increases in transgene expression and, finally, higher susceptibility in certain RNA viruses. Therefore, these plants are excellent tools for the following: (i) to study the role of DCL enzymes; (ii) to overexpress proteins of interest; and (iii) to understand the complex relationship between the plant silencing mechanism and biotic or abiotic stresses.
Highlights
Plants have developed sophisticated pathways to respond to environmental stimuli
DCL1 is involved in the micro-RNA biogenesis pathway producing 21-nucleotide (21-nt) small RNAs from miRNA precursors which are transcribed by a complex of proteins, including RNA polymerase II
We show that these plants can be used in multiple experimental set-ups, such as for the study of various aspects of DCL functions, transient expression, viral infection studies, as well as their interplay with RNA interference (RNAi) pathways
Summary
Plants have developed sophisticated pathways to respond to environmental stimuli. One such mechanism is RNA silencing, which isEach DCL carries out a specific function. Plants have developed sophisticated pathways to respond to environmental stimuli. One such mechanism is RNA silencing, which is. DCL1 is involved in the micro-RNA (miRNA) biogenesis pathway producing 21-nucleotide (21-nt) small RNAs from miRNA precursors which are transcribed by a complex of proteins, including RNA polymerase II. Primary miRNAs are initially trimmed by DCL1 to precursor miRNAs from which the protein further excises the miRNA/miRNA* duplexes (Achkar et al, 2016). Additional roles of DCL1 have been described in DNA methylation and transposon silencing (Laubinger et al, 2010). DCL1 is preferentially transcribed from the maternally inherited allele and is essential for the development of the embryo; homozygous mutations in this gene are embryo lethal (Bozorov et al, 2012; Castle et al, 1993; Ray et al, 1996)
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