Viroids are highly structured plant pathogenic RNAs that do not code for any protein, and thus, their long-distance movement within the plant must be mediated by direct interaction with cellular factors, the nature of which is presently unknown. In addition to this type of RNAs, recent evidence indicates that endogenous RNAs move through the phloem acting as macromolecular signals involved in plant defense and development. The form in which these RNA molecules are transported to distal parts of the plant is unclear. Viroids can be a good model system to try to identify translocatable proteins that could assist the vascular movement of RNA molecules. Here, we demonstrate by use of immunoprecipitation experiments, that the phloem protein 2 from cucumber (CsPP2) is able to interact in vivo with a viroid RNA. Intergeneric graft assays revealed that both the CsPP2 and the Hop stunt viroid RNA were translocated to the scion. The translocated viroid is symptomatic in the nonhost scion, indicating that the translocated RNA is functional. The CsPP2 gene was cloned and sequenced. The analysis of its primary structure revealed the existence of a potential double-spaced-RNA-binding motif, previously identified in a set of proteins that bind to highly structured RNAs, which could explain its RNA-binding properties. The possible involvement of this phloem protein in assisting the long-distance movement of the viroid RNA within the plant is discussed.

We used the interaction of Hop stunt viroid (HSVd) and cucumber plants to investigate the involvement of phloem proteins in the systemic transport of RNA molecules. A ribonucleoprotein complex, stable even at high salt and temperature conditions, was detected in vitro between HSVd-RNA and the phloem exudate obtained from sectioned internodes from cucumber plants. The phloem protein 2 was recovered from this ribonucleoprotein complex and its RNA-binding properties as demonstrated by gel retardation analysis. The involvement of this protein in the movement of RNAs in cucumber is discussed.

Viroids are small self-replicating RNAs that infect plants. How these noncoding pathogenic RNAs interact with hosts to induce disease symptoms is a long-standing unanswered question. Recent experimental data have led to the suggestive proposal of a pathogenic model based on the RNA silencing mechanism. However, evidence of a direct relation between key components of the RNA silencing pathway and symptom expression in infected plants remains elusive. To address this issue, we used a symptomatic transgenic line of Nicotiana benthamiana that expresses and processes dimeric forms of Hop stunt viroid (HSVd). These plants were analyzed under different growing temperature conditions and were used as stocks in grafting assays with the rdr6i-Nb line, in which the RNA-dependent RNA polymerase 6 (RDR6) is constitutively silenced. Here, we show that the symptom expression in N. benthamiana plants is independent of HSVd accumulation levels but dependent on an active state of the viroid-specific RNA silencing pathway. The scion of rdr6i-Nb plants remained asymptomatic when grafted onto symptomatic plants, despite an accumulation of a high level of mature forms of HSVd, indicating the requirement of RDR6 for viroid-induced symptom production. In addition, the RDR6 requirement for symptom expression was also observed in wild-type N. benthamiana plants mechanically infected with HSVd. These results provide biological evidence of the involvement of the viroid-specific RNA silencing pathway in the symptom expression associated with viroid pathogenesis.

Micro RNAS (miRNAs) are a class of endogenous small non coding RNAs involved in the post-transcriptional regulation of gene expression. In plants, a great number of conserved and specific miRNAs, mainly arising from model species, have been identified to date. However less is known about the diversity of these regulatory RNAs in vegetal species with agricultural and/or horticultural importance. Here we report a combined approach of bioinformatics prediction, high-throughput sequencing data and molecular methods to analyze miRNAs populations in cucumber (Cucumis sativus) plants. A set of 19 conserved and 6 known but non-conserved miRNA families were found in our cucumber small RNA dataset. We also identified 7 (3 with their miRNA* strand) not previously described miRNAs, candidates to be cucumber-specific. To validate their description these new C. sativus miRNAs were detected by northern blot hybridization. Additionally, potential targets for most conserved and new miRNAs were identified in cucumber genome. In summary, in this study we have identified, by first time, conserved, known non-conserved and new miRNAs arising from an agronomically important species such as C. sativus. The detection of this complex population of regulatory small RNAs suggests that similarly to that observe in other plant species, cucumber miRNAs may possibly play an important role in diverse biological and metabolic processes.