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

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.

RNA silencing machinery contributes to inability of BSBV to establish infection in Nicotiana benthamiana: evidence from characterization of agroinfectious clones of Beet soil-borne virus

“…3c), confirming efficient viral replication. This result is in agreement with studies that showed that down regulation of those two DCL in N. benthamiana was associated with increased viral titres for other (+)ss RNA viruses, while individual down regulation of DCL2 or four did not lead to such a result [43].…”

“…Seeds of this cultivar were first uncoated using 10 % (v/v) EtOH. Transgenic N. benthamiana lines down regulated for DCL (DCL1.13i, DCL2.11i, DCL3.10i, DCL4.9i, DCL2/4.5i) have been described before [42, 43].…”

Genetic evidence for the involvement of Dicer-like 2 and 4 as well as Argonaute 2 in the Nicotiana benthamiana response against Pelargonium line pattern virus

“…These results suggested a main involvement of DCL4 and DCL2 in anti-PLPV silencing as reported in other plant–virus combinations [11, 38, 39]. In order to confirm, or refute, this possibility, we used a genetic approach by taking advantage of the previous creation of a collection of N. benthamiana transgenic lines in which the different DCL genes were downregulated, either individually or in combinations, through an RNAi approach [33, 34, 40]. This collection included either single DCL RNAi lines (henceforth DCL1i, DCL2i, DCL3i or DCL4i) or double (DCL2/4i) and triple (DCL2/3/4i) DCL RNAi combinations.…”

Viral Detection: Past, Present, and Future

“…[44] The benefit of this method is the detection of different strains of the same virus with the use of only one probe. [48,49] In 1977, a modification of this technique, named northern blot, was created, specialized in the detection of RNA molecules, soon used also for viral and viroidal detection.…”