Hairpin transcription does not necessarily lead to efficient triggering of the RNAi pathway.

Previously, we had shown that stable expression of a hairpin RNA sharing homology with the coat protein (CP) of the Cucumber mosaic virus (CMV) (hpRNA(CMV)) produced CMV resistant Nicotiana tabacum plants. However, only 17% of the hpRNA(CMV)-expressing plants generated substantial amounts of siRNAs that mediated CMV resistance (siRNAs(CMV)). Here, we demonstrate that the transcription of a hpRNA(CMV) per se is not sufficient to trigger cytoplasmic and nuclear RNAi. A multiple-transgene copy line showed a strong resistance phenotype. Segregation of individual copies revealed that in one locus, the transgene-produced hpRNA(CMV) transcript was processed into 21-nt and 24-nt siRNAs(CMV) and lines containing this locus were resistant. At a second locus, where the transgene was shown to be transcribed, no siRNAs(CMV) were produced and lines harbouring only this locus were susceptible. In addition, the second locus failed to trigger de novo RNA-directed DNA methylation (RdDM) in cis, of its cognate sequence. However, after being induced in trans, methylation in the transcribed region of the transgene was maintained in both CG and CHG residues. Sequence-specific maintenance of methylation in transcribed regions, as well as diverse RNA degradation pathways in plants are discussed in view of our observations.

Light intensity affects RNA silencing of a transgene in Nicotiana benthamiana plants.

BACKGROUND: Expression of exogenous sequences in plants is often suppressed through one of the earliest described RNA silencing pathways, sense post-transcriptional gene silencing (S-PTGS). This type of suppression has made significant contributions to our knowledge of the biology of RNA silencing pathways and has important consequences in plant transgenesis applications. Although significant progress has been made in recent years, factors affecting the stability of transgene expression are still not well understood. It has been shown before that the efficiency of RNA silencing in plants is influenced by various environmental factors. RESULTS: Here we report that a major environmental factor, light intensity, significantly affects the induction and systemic spread of S-PTGS. Moreover, we show that photoadaptation to high or low light intensity conditions differentially affects mRNA levels of major components of the RNA silencing machinery. CONCLUSIONS: Light intensity is one of the previously unknown factors that affect transgene stability at the post-transcriptional level. Our findings demonstrate an example of how environmental conditions could affect RNA silencing.

Native electrophoresis and western blot analysis: method and applications.

Native electrophoresis and western blot analysis (NEWeB) has been developed for the study of plant virus characteristics, among others, virus particle-protein interactions, electrophorotype formation, and strain separation. The method is based on the property of electrophoretic mobility of virus particles (VP) and proteins and combines the analytical capacity of electrophoresis with the specificity of western blot. One of its advantages is that it deals with entire VP that can be studied in cause and effect or in time-interval experiments. Some of the most interesting approaches include VP structural studies, VP interaction with host or viral proteins, and also the characterization of VP-protein complexes. In this protocol, NEWeB is used to demonstrate the interaction of Plum pox virus particles with the helper component, a virus encoded protein. It is expected that the method could be used in analogous studies of other viruses or large protein complexes, where similar principles apply.

Characterization of the RNA motif responsible for the specific interaction of potato spindle tuber viroid RNA (PSTVd) and the tomato protein Virp1.

Viroids are small non-coding parasitic RNAs that are able to infect their host plants systemically. This circular naked RNA makes use of host proteins to accomplish its proliferation. Here we analyze the specific binding of the tomato protein Virp1 to the terminal right domain of potato spindle tuber viroid RNA (PSTVd). We find that two asymmetric internal loops within the PSTVd (+) RNA, each composed of the sequence elements 5'-ACAGG and CUCUUCC-5', are responsible for the specific RNA-protein interaction. In view of the nucleotide composition we call this structural element an 'RY motif'. The RY motif located close to the terminal right hairpin loop of the PSTVd secondary structure has an approximately 5-fold stronger binding affinity than the more centrally located RY motif. Simultaneous sequence alterations in both RY motifs abolished the specific binding to Virp1. Mutations in any of the two RY motifs resulted in non-infectious viroid RNA, with the exception of one case, where reversion to sequence wild type took place. In contrast, the simultaneous exchange of two nucleotides within the terminal right hairpin loop of PSTVd had only moderate influence on the binding to Virp1. This variant was infectious and sequence changes were maintained in the progeny. The relevance of the phylogenetic conservation of the RY motif, and sequence elements therein, amongst various genera of the family Pospiviroidae is discussed.

Induction of RNA interference in Caenorhabditis elegans by RNAs derived from plants exhibiting post-transcriptional gene silencing.

The term 'gene silencing' refers to transcriptional and post-transcriptional control of gene expression. Related processes are found across kingdoms in plants and animals. We intended to test whether particular RNA constituents of a silenced plant can induce silencing in an animal. We generated Nicotiana benthamiana lines that expressed green fluorescent protein (GFP) from a transgene. Plants in which GFP expression was spontaneously silenced showed siRNAs characteristic of post-transcriptional gene silencing (PTGS). RNA extracts prepared from silenced plants were injected into a GFP-expressing strain of Caenorhabditis elegans, where they induced RNA interference (RNAi). Extracts from non-silenced plants were inactive. This directly demonstrates a relationship and a mechanistic link between PTGS and RNAi. Controls confirmed that the silencing agent was an RNA. Size fractionation on denaturing gels revealed that an RNA of approximately 85 nt was most active in inducing silencing in the worm. Northern blot analysis of the region in question did not detect a prominent GFP-specific RNA of sense or antisense polarity, indicating that the RNA species which induced silencing was present only in low concentration or did not hybridize due to formation of an intramolecular double strand. In view of its high activity, it is possible that this agent is responsible for the systemic spread of silencing in plants and it might represent the aberrant RNA, a previously postulated inducer of silencing.

Viroids: petite RNA pathogens with distinguished talents.

Viroids are small, circular, single-stranded RNA molecules that cause several infectious plant diseases. Viroids do not encode any pathogen-specific peptides but nonetheless, the subviral pathogens replicate autonomously and spread in the plant by recruiting host proteins via functional motifs encoded in their RNA genome. During the past couple of years, considerable progress has been made towards comprehending how viroids interact with their hosts. Here, we summarize recent findings on the structure-function relationships of viroids, their strategies and mechanisms of replication and trafficking, and the identification and characterization of interacting host proteins. We also describe the impact of the RNA silencing machinery of plants on viroid RNAs and how this has started to influence our models of viroid replication and pathogenicity.

Native Electrophoresis and Western Blot Analysis (NEWeB): Methods and Applications.

Native Electrophoresis and Western Blot Analysis (NEWeB) has been developed for the study of plant virus characteristics, among others, virus particle-protein interactions, electrophorotype formation, and strain separation. The method is based on the property of electrophoretic mobility of virus particles (VP) and proteins and combines the analytical capacity of electrophoresis with the specificity of western blot. One of its advantages is that it deals with entire VP that can be studied in cause and effect or in time-interval experiments. Some of the most interesting approaches include VP structural studies, VP interaction with host or viral proteins, and also the characterization of VP-protein complexes. In this protocol, NEWeB is used to demonstrate the interaction of Plum pox virus particles with the helper component, a virus encoded protein. It is expected that the method could be used in analogous studies of other viruses or large protein complexes, where similar principles apply.

Infectious long non-coding RNAs.

Long non protein coding RNAs (lncRNAs) constitute a large category of the RNA world, able to regulate different biological processes. In this review we are focusing on infectious lncRNAs, their classification, pathogenesis and impact on the infected organisms. Here they are presented in two separate groups: 'dependent lncRNAs' (comprising satellites RNA, Hepatitis D virus and lncRNAs of viral origin) which need a helper virus and 'independent lncRNAs' (viroids) that can self-replicate. Even though these lncRNA do not encode any protein, their structure and/or sequence comprise all the necessary information to drive specific interactions with host factors and regulate several cellular functions. These new data that have emerged during the last few years concerning lncRNAs modify the way we understand molecular biology's 'central dogma' and give new perspectives for applications and potential therapeutic strategies.

The occurrence of CMV-specific short Rnas in transgenic tobacco expressing virus-derived double-stranded RNA is indicative of resistance to the virus.

Expression or introduction of double-stranded (ds)RNA in eukaryotic cells can trigger sequence-specific gene silencing of transgenes, endogenes, and viruses. Transgenic plants producing dsRNAs with homology to viral sequences are likely to exhibit pathogen-derived resistance to the virus. Cucumber mosaic virus (CMV), a very widespread virus with over 1,000 host species, has the natural ability to suppress silencing in order to establish infection. Here, we report the generation of transgenic tobacco lines, where a DNA transgene containing an inverted repeat of CMV cDNA had been introduced. Expression of this DNA construct delivered an RNA transcript that is able to form an intramolecular double strand. Transgenic plants were challenged with CMV. Three categories of plants could be discriminated: susceptible plants, which typically reacted with milder symptoms than the wild-type control; a "recovery" phenotype, in which newly emerging leaves were free of symptoms; and plants that showed complete resistance. Northern analysis showed that the expression of CMV dsRNA caused, in some transgenic lines, the generation of short RNAs characteristic of posttranscriptional gene silencing. Those lines were CMV resistant. The correlation between the detection of short RNAs and virus resistance provides a molecular marker that makes it possible to predict success in attempts to engineer virus resistance by dsRNA.

Spontaneous short-range silencing of a GFP transgene in Nicotiana benthamiana is possibly mediated by small quantities of siRNA that do not trigger systemic silencing.

A green fluorescent protein (GFP) transgene under the control of the 35S cauliflower mosaic virus (CaMV) promoter was introduced by Agrobacterium-mediated transformation into Nicotiana benthamiana to generate fourteen transgenic lines. Homozygous lines that contained one or two copies of the transgene showed great variation of GFP expression under ultraviolet (UV) light, which allowed classification into three types of transgenic plants. Plants from more than half of the transgenic lines underwent systemic RNA silencing and produced short interfering RNA (siRNA) as young seedlings, while plants of the remaining lines developed, in a spontaneous manner, defined GFP-silenced zones on their leaves, mostly in the form of circular spots that expanded to about 4-7 mm in size. In some of the latter lines, the GFP-silenced spots remained stable, but no systemic silencing occurred. Here we characterize this phenomenon, which we term spontaneous short-range silencing (SSRS). Biochemical analysis of silenced spot tissue did not reveal detectable levels of siRNA. However, agro-infiltration with the suppressor proteins P19 of cymbidium ring spot virus (CymRSV), HC-Pro of tobacco etch virus (TEV), and crosses to a P19 transgenic line, nevertheless suggests that low concentrations of siRNA may have a functional role in the locally silenced zone. We propose that small alterations in the steady-state concentration of siRNAs and their cognate mRNA are decisive with regard to whether silencing remains local or spreads in a systemic manner.

Short interfering RNAs specific for potato spindle tuber viroid are found in the cytoplasm but not in the nucleus.

Short interfering (si) and micro (mi) RNAs influence gene expression at post-transcriptional level. In plants, different classes of DICER-LIKE (DCL) enzymes are responsible for the generation of these small regulatory RNAs from different precursors. To characterize the cellular site of their generation and accumulation, we purified nuclei from tomato plants infected with potato spindle tuber viroid (PSTVd) RNA, which is known to replicate in the nucleus via double-stranded (ds) RNA intermediates. We could detect PSTVd-specific siRNAs in the cytoplasmic fraction, but not in the nuclear fraction. To correlate the localization of the PSTVd-specific siRNAs with that of similarly sized small RNAs, we studied the compartmentalization of a naturally occurring miRNA. We could detect the precursor of miR167 in the nucleus, but the mature miRNA was found only in the cytoplasmic fraction. We discuss the consequences of this finding for the model of viroid replication and heterochromatin formation.

A bromodomain-containing protein from tomato specifically binds potato spindle tuber viroid RNA in vitro and in vivo.

For the identification of RNA-binding proteins that specifically interact with potato spindle tuber viroid (PSTVd), we subjected a tomato cDNA expression library prepared from viroid-infected leaves to an RNA ligand screening procedure. We repeatedly identified cDNA clones that expressed a protein of 602 amino acids. The protein contains a bromodomain and was termed viroid RNA-binding protein 1 (VIRP1). The specificity of interaction of VIRP1 with viroid RNA was studied by different methodologies, which included Northwestern blotting, plaque lift, and electrophoretic mobility shift assays. VIRP1 interacted strongly and specifically with monomeric and oligomeric PSTVd positive-strand RNA transcripts. Other RNAs, for example, U1 RNA, did not bind to VIRP1. Further, we could immunoprecipitate complexes from infected tomato leaves that contained VIRP1 and viroid RNA in vivo. Analysis of the protein sequence revealed that VIRP1 is a member of a newly identified family of transcriptional regulators associated with chromatin remodeling. VIRP1 is the first member of this family of proteins, for which a specific RNA-binding activity is shown. A possible role of VIRP1 in viroid replication and in RNA mediated chromatin remodeling is discussed.

Viroid RNA systemic spread may depend on the interaction of a 71-nucleotide bulged hairpin with the host protein VirP1.

Viroids are noncoding circular single-stranded RNAs that are propagated systemically in plants. VirP1 is a protein from tomato, which is an excellent host for potato spindle tuber viroid (PSTVd), and it has been isolated by virtue of its specific in vitro binding to PSTVd RNA. We report on the specific in vivo interaction of VirP1 with full-length viroid RNA as well as with subfragments in the three-hybrid system. The terminal right domain (TR) of PSTVd was identified as a strong interacting partner for VirP1. A weaker partner is provided by a right-hand subfragment of hop stunt viroid (HSVd), a viroid that infects tomato poorly. We present a sequence and structural motif of the VirP1-interacting subfragments. The motif is disturbed in the replicative but nonspreading R+ mutant of the TR. According to our in vivo and in vitro binding assays, the interaction of this mutant with VirP1 is compromised. We propose that the AGG/CCUUC motif bolsters recognition of the TR by VirP1 to achieve access of the viroid to pathways that propagate endogenous RNA systemic signals in plants. Systemic trafficking has been suggested for miRNA precursors, of which the TR, as a stable bulged hairpin 71 nt long, is quite reminiscent.