DCL‐suppressed Nicotiana benthamiana plants: valuable tools in research and biotechnology

Plants have developed sophisticated pathways to respond to environmental stimuli. One such mechanism is RNA silencing, which is capable of regulating gene expression in response to various biotic and abiotic stresses (Khraiwesh et al., 2012; Pumplin and Voinnet, 2013). Key proteins involved in this complex mechanism are ribonucleases named Dicer‐like proteins (DCL) (Martinez de Alba et al., 2013), with most plants, including Nicotiana benthamiana, encoding four distinct DCL proteins (Nakasugi et al., 2013). Each DCL carries out a specific function. 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). After excision, miRNAs are bound to a protein complex, involving at least one Argonaute (AGO) protein (RNA‐induced silencing complex, RISC), and are directed to complementary RNA sequences to be silenced (Martinez de Alba et al., 2013). Additional roles of DCL1 have also 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; thus, homozygous mutations in this gene are embryo lethal (Bozorov et al., 2012; Castle et al., 1993; Ray et al., 1996). The DCL2 protein processes double‐stranded RNA (dsRNA) molecules of exogenous origin and natural antisense small interfering RNAs (siRNAs) into 22‐nt primary siRNAs (Borsani et al., 2005; Xie et al., 2004). DCL2 is mainly involved in antiviral defence; however, there is mounting evidence that its role is masked by DCL4 activity, as it has been clearly demonstrated that DCL2 is also involved in transitivity and the RNA decoy mechanism (Mlotshwa et al., 2008; Parent et al., 2015; Qin et al., 2017; Taochy et al., 2017; Zhang et al., 2015). DCL3 is involved in the production of 24‐nt small RNAs which operate in the RNA‐dependent DNA methylation (RdDM) pathway (Blevins et al., 2015; Qi et al., 2005; Xie et al., 2004; Xie and Yu, 2015). DCL3 has also been reported to produce long miRNAs of 24 nt in Arabidopsis thaliana, rice, tomato and N. benthamiana (Kangquan et al., 2015; Kravchik et al., 2014a; Vazquez et al., 2008; Wu et al., 2010), and also phased siRNAs in rice (Song et al., 2012). DCL4 is the primary DCL enzyme involved in antiviral defence, as DCL4 A. thaliana mutants are more susceptible to different viruses (Bouche et al., 2006; Deleris et al., 2006; Henderson et al., 2006). DCL4 is responsible for the production of 21‐nt‐long siRNAs and is further involved in multiple endogenous processes, such as the production of 21‐nt trans‐acting siRNAs (tasiRNAs), which regulate major developmental processes (D'Ario et al., 2017; Pulido and Laufs, 2010), the production of miR822, miR839 and miR869 in A. thaliana (Ben Amor et al., 2009; Rajagopalan et al., 2006) and, finally, in transcription termination (Duc et al., 2013; Liu et al., 2012). Although the role of each DCL protein is, to some extent distinct. It is to note that important functional redundancies have also been reported, especially in A. thaliana (Blevins et al., 2006; Bouche et al., 2006; Deleris et al., 2006; Gasciolli et al., 2005; Henderson et al., 2006; Kasschau et al., 2007; Xie et al., 2005). At present, the majority of studies addressing the role of DCL proteins have been performed with A. thaliana knockout plants. This approach has proven to be very fruitful indeed, as it has shed light onto key aspects of the RNA silencing pathways. Nevertheless, there are certain limitations to this methodology. (i) Although different mutants of the same DCL are often treated as equally informative, the plants used are usually either T‐DNA or point mutants with different phenotypes, depending on the efficiency of the mutation. To the point at hand, the analysis of three A. thaliana dcl4 mutants (dcl4‐6, dcl4‐70b1 and dcl4‐8) showed differences in the processing of mir822, TAS3 and TAS1, respectively (Montavon et al., 2018). (ii) Arabidopsis thaliana is a major plant model, but not necessarily the most representative species to address all plant phenomena. Arabidopsis thaliana cannot be infected by certain viruses and viroids, is not ideal for the study of systemic processes and is not used for fast transient methods, such as agroinfiltration or virus‐induced gene silencing (VIGS) experiments (Daros and Flores, 2004; Goodin et al., 2008; Wroblewski et al., 2005). In order to overcome these limitations and to address the function of DCL proteins in other plant models, we aimed to generate N. benthamiana DCLi knockdown plants using RNA interference (RNAi). Nicotiana benthamiana is a plant species of the Solanaceae family. Nicotiana benthamiana is susceptible to many plant viruses, allows for relatively easy experiments on intercellular macromolecular movement, as well as VIGS experiments, and its genome has been sequenced recently and made publically available (Bally et al., 2015; Bombarely et al., 2012; Goodin et al., 2008; Nakasugi et al., 2013). In this study, we present the molecular and phenotypic characterization of plants suppressed for each and every DCL, as well as their combinations. 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 RNAi pathways.