A beneficial fungal root endophyte triggers systemic RNA silencing and DNA methylation of a host reporter gene.

A growing body of evidence suggests that RNA interference (RNAi) plays a pivotal role in the communication between plants and pathogenic fungi, where a bi-directional trans-kingdom RNAi is established to the advantage of either the host or the pathogen. Similar mechanisms acting during plant association with non-pathogenic symbiotic microorganisms have been elusive to this date. To determine whether root endophytes can induce systemic RNAi responses to their host plants, we designed an experimental reporter-based system consisting of the root-restricted, beneficial fungal endophyte, strain K (FsK) and its host .

Genetically Modified Organism-Free RNA Interference: Exogenous Application of RNA Molecules in Plants.

The latest advances in the field exogenous application of RNA molecules in plants help to protect and modify them through RNA interference (RNAi).

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

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.

Induction of Silencing in Plants by High-Pressure Spraying of In vitro-Synthesized Small RNAs.

In this report, we describe a method for the delivery of small interfering RNAs (siRNAs) into plant cells. In vitro synthesized siRNAs that were designed to target the coding region of a GREEN FLUORESCENT PROTEIN (GFP) transgene were applied by various methods onto GFP-expressing transgenic Nicotiana benthamiana plants to trigger RNA silencing. In contrast to mere siRNA applications, including spraying, syringe injection, and infiltration of siRNAs that all failed to induce RNA silencing, high pressure spraying of siRNAs resulted in efficient local and systemic silencing of the GFP transgene, with comparable efficiency as was achieved with biolistic siRNA introduction.

RNA-directed DNA methylation efficiency depends on trigger and target sequence identity.

RNA-directed DNA methylation (RdDM) in plants has been extensively studied, but the RNA molecules guiding the RdDM machinery to their targets are still to be characterized. It is unclear whether these molecules require full complementarity with their target. In this study, we have generated Nicotiana tabacum (Nt) plants carrying an infectious tomato apical stunt viroid (TASVd) transgene (Nt-TASVd) and a non-infectious potato spindle tuber viroid (PSTVd) transgene (Nt-SB2).

Replicating Potato spindle tuber viroid mediates de novo methylation of an intronic viroid sequence but no cleavage of the corresponding pre-mRNA.

In plants, Potato spindle tuber viroid (PSTVd) replication triggers post-transcriptional gene silencing (PTGS) and RNA-directed DNA methylation (RdDM) of homologous RNA and DNA sequences, respectively. PTGS predominantly occurs in the cytoplasm, but nuclear PTGS has been also reported. In this study, we investigated whether the nuclear replicating PSTVd is able to trigger nuclear PTGS.

Engineering viroid resistance.

Viroids are non-encapsidated, non-coding, circular, single-stranded RNAs (ssRNAs). They are classified into the families Pospiviroidae and Avsunviroidae, whose members replicate in the nucleus and chloroplast of plant cells, respectively. Viroids have a wide host range, including crop and ornamental plants, and can cause devastating diseases with significant economic losses.

Viroid-induced DNA methylation in plants.

In eukaryotes, DNA methylation refers to the addition of a methyl group to the fifth atom in the six-atom ring of cytosine residues. At least in plants, DNA regions that become de novo methylated can be defined by homologous RNA molecules in a process termed RNA-directed DNA methylation (RdDM). RdDM was first discovered in viroid-infected plants.

An endogene-resembling transgene is resistant to DNA methylation and systemic silencing.

In plants, endogenes are less prone to RNA silencing than transgenes. While both can be efficiently targeted by small RNAs for post-transcriptional gene silencing (PTGS), generally only transgene PTGS is accompanied by transitivity, RNA-directed DNA methylation (RdDM) and systemic silencing. In order to investigate whether a transgene could mimick an endogene and thus be less susceptible to RNA silencing, we generated an intron-containing, endogene-resembling GREEN FLUORESCENT PROTEIN (GFP) transgene (GFP(endo)).

An endogene-resembling transgene delays the onset of silencing and limits siRNA accumulation.

In plants, transgenes are generally more sensitive against RNA silencing than endogenes are. In this study, we generated a transgene that structurally mimicks an endogene. It is composed of endogenous promoter, 5'-UTR, introns, 3'-UTR and terminator elements.

Transgenerational maintenance of transgene body CG but not CHG and CHH methylation.

In plants, RNA-directed DNA methylation (RdDM) can target both transgene promoters and coding regions/gene bodies. RdDM leads to methylation of cytosines in all sequence contexts: CG, CHG and CHH. Upon segregation of the RdDM trigger, at least CG methylation can be maintained at promoter regions in the progeny.

Local RNA silencing mediated by agroinfiltration.

Agroinfiltration is a very fast and powerful method to express in planta any sequences in a transient fashion. Agroinfiltration has proven very useful for the overexpression of proteins in the infiltrated zone when a short-term effect can be informative. However, it has been a real success story in the induction of local and eventually systemic silencing.