Mina Tsagris

Generation of transgenic potato plants highly resistant to potato virus Y (PVY) through RNA silencing

In this study we applied RNA silencing to engineer potato plants that are resistant to potato virus Y (PVY). We expressed double-stranded (ds) RNA derived from the 3′ terminal part of the coat protein gene of PVY, which is highly conserved in sequence amongst different PVY isolates, in transgenic potatoes of the commercial variety ‘Spunta’. Transgenic plants were analyzed for generation of transgene-derived short interfering RNAs (siRNAs) prior to virus inoculation. Twelve of fifteen transgenic lines produced siRNAs and were highly resistant to three strains of PVY, each belonging to three different subtypes of the virus (PVYN, PVYO and PVYNTN). Infection of transgenic plants with Potato virus X (PVX) simultaneously or prior to the challenge with PVY did not interfere with PVY-resistance.

A Bromodomain-Containing Protein from Tomato Specifically Binds Potato Spindle Tuber Viroid RNA In Vitro and In Vivo

ABSTRACT 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.

Virp1 Is a Host Protein with a Major Role in Potato Spindle Tuber Viroid Infection in Nicotiana Plants

ABSTRACT Viroids are small, circular, single-stranded RNA molecules that, while not coding for any protein, cause several plant diseases. Viroids rely for their infectious cycle on host proteins, most of which are likely to be involved in endogenous RNA-mediated phenomena. Therefore, characterization of host factors interacting with the viroid may contribute to the elucidation of RNA-related pathways of the hosts. Potato spindle tuber viroid (PSTVd) infects several members of the Solanaceae family. In an RNA ligand screening we have previously isolated the tomato protein Virp1 by its ability to specifically interact with PSTVd positive-strand RNA. Virp1 is a bromodomain-containing protein with an atypical RNA binding domain and a nuclear localization signal. Here we investigate the role of Virp1 in the viroid infection cycle by the use of transgenic lines of Nicotiana tabacum and Nicotiana benthamiana that either overexpress the tomato Virp1 RNA or suppress the orthologous Nicotiana genes through RNA silencing. Plants of the Virp1-suppressed lines were not infected by PSTVd or Citrus exocortis viroid through mechanical inoculation, indicating a major role of Virp1 in viroid infection. On the other hand, overexpression of tomato Virp1 in N. tabacum and N. benthamiana plants did not affect PSTVd KF 440-2 infectivity or symptomatology in these species. Transfection experiments with isolated protoplasts revealed that Virp1-suppressed cells were unable to sustain viroid replication, suggesting that resistance to viroid infection in Virp1-suppressed plants is likely the result of cell-autonomous events.

Linear oligomeric potato spindle tuber viroid (PSTV) RNAs are accurately processed in vitro to the monomeric circular viroid proper when incubated with a nuclear extract from healthy potato cells

A nuclear extract for the processing of oligomeric viroid RNA in vitro has been prepared from nuclei isolated from healthy potato cells grown in suspension culture. Linear RNA molecules containing concatameric units of (+) or (−) strands, respectively, of the potato spindle tuber viroid (PSTV) were synthesized in vitro with the aid of the SP6 RNA polymerase and used as substrates for processing. When oligomeric linear PSTV (+)RNAs are incubated with the nuclear extract, monomeric linear molecules are accurately excised from them, and ligated to monomeric PSTV (+)RNA circles representing the viroid proper. Oligomeric PSTV (−)RNAs are likewise processed but with a much lower efficiency. Viroid‐processing operates although other nucleolytic activities are still present in the extract. These results substantiate our previous finding that oligomeric PSTV does not process autocatalytically under in vitro conditions where certain introns and other RNAs do. This is the first report of an in vitro RNA processing system derived from higher plants.

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

BackgroundExpression 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.ResultsHere 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.ConclusionsLight 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.

Processing of linear longer-than-unit-length potato spindle tuber viroid RNAs into infectious monomeric circular molecules by a G-specific endoribonuclease

Different cDNA constructs were used for the in vitro synthesis of RNA transcripts that contain a complete monomeric unit of the potato spindle tuber viroid (PSTVd) plus an additional repeat of a part of the circular RNA genome. These permutated linear longer-than-unit-length PSTVd RNAs were incubated with the G-specific endoribonuclease RNase T1 which generated monomeric circular PSTVd RNA molecules that were infectious when mechanically inoculated to tomato plants. Besides the correct monomeric PSTVd RNA, smaller and larger circular RNAs were also formed during the reaction. The comparison of different transcripts revealed that correct in vitro processing of PSTVd RNA can proceed at alternative sites indicating that circularization is driven by RNA structure and not governed by a particular sequence. Based on these data, we propose a novel model for the processing of multimeric replicative viroid RNA intermediates through RNA cleavage and ligation catalyzed by a host endoribonuclease.

Oligomeric potato spindle tuber viroid (PSTV) RNA does not process autocatalytically under conditions where other RNAs do.

In vitro-synthesized oligomeric linear RNAs representing the replicative intermediates of potato spindle tuber viroid (PSTV) were subjected to a large variety of in vitro conditions where self-splicing of group I introns occurs, and where self-cleavage and self-circularization of the satellite RNA from tobacco ringspot virus particles and self-cleavage of oligomeric forms of avocado sunblotch viroid RNA has been observed. No evidence whatsoever could be obtained that the PSTV RNA oligomers monomerize and subsequently circularize autocatalytically in vitro under these conditions. These results are at variance with the low level of self-cleavage of dimeric PSTV RNA reported by H. D. Robertson et al. Virology 142, 441-447, 1985). A comparable and seemingly autocatalytic in vitro cleavage we have initially observed under certain conditions could be related to an unknown heat-stable RNase contaminating a batch of autoclaved ultrapure ammonium sulfate used in the corresponding incubating mixtures as additive.

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) (hpRNACMV) produced CMV resistant Nicotiana tabacum plants. However, only 17% of the hpRNACMV-expressing plants generated substantial amounts of siRNAs that mediated CMV resistance (siRNAsCMV). Here, we demonstrate that the transcription of a hpRNACMV 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 hpRNACMV transcript was processed into 21-nt and 24-nt siRNAsCMV and lines containing this locus were resistant. At a second locus, where the transgene was shown to be transcribed, no siRNAsCMV 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.

Trafficking of the potato spindle tuber viroid between tomato and Orobanche ramosa.

Viroids, small RNA pathogens capable of infecting flowering plants, coexist in the field with parasitic plants that infest many crops. The ability of viroids to be exchanged between host and parasitic plants and spread in the latter has not yet been investigated. We studied the interaction between the Potato spindle tuber viroid (PSTVd) and Branched bromrape (Orobanche ramosa) using the tomato, Solanum lycopersicon, as a common host. We report the long distance trafficking of PSTVd RNA via the phloem from tomato to O. ramosa, but not vice versa. Furthermore, we identify O. ramosa as a novel host with the ability to facilitate the replication and processing of PSTVd. Finally, molecular variants of PSTVd with single nucleotide substitutions that replicate with different efficiencies in tomato were isolated from O. ramosa.

Viroid Replication Mechanisms

Viroids are pathogens which are the causal agents of some diseases of higher plants. At the molecular level, viroids consist only of a single-stranded, circular RNA molecule of 246–375 nucleotides. The autonomous replication of viroid RNA in the infected cell requires a close interaction with host factors. At least two replication mechanisms for different viroids can be envisaged. This chapter gives a brief review of possible mechanisms of viroid replication, summarizing our present knowledge of the synthesis of viroid RNA, the processing reactions which form the mature circular RNA molecule, and the relationships between viroid structure and replicability.