Gustavo Gómez

A Long-Distance Translocatable Phloem Protein from Cucumber Forms a Ribonucleoprotein Complex In Vivo with Hop Stunt Viroid RNA

ABSTRACT 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.

Identification of an In Vitro Ribonucleoprotein Complex Between a Viroid RNA and a Phloem Protein from Cucumber Plants

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.

High-Throughput Sequencing, Characterization and Detection of New and Conserved Cucumber miRNAs

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.

Viroid-Induced Symptoms in Nicotiana benthamiana Plants Are Dependent on RDR6 Activity

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.

High-throughput sequencing of Hop stunt viroid-derived small RNAs from cucumber leaves and phloem.

Small RNA (sRNA)-guided processes, referred to as RNA silencing, regulate endogenous and exogenous gene expression. In plants and some animals, these processes are noncell autonomous and can operate beyond the site of initiation. Viroids, the smallest self-replicating plant pathogens known, are inducers, targets and evaders of this regulatory mechanism and, consequently, the presence of viroid-derived sRNAs (vd-sRNAs) is usually associated with viroid infection. However, the pathways involved in the biogenesis of vd-sRNAs are largely unknown. Here, we analyse, by high-throughput pyrosequencing, the profiling of the Hop stunt viroid (HSVd) vd-sRNAs recovered from the leaves and phloem of infected cucumber (Cucumis sativus) plants. HSVd vd-sRNAs are mostly 21 and 22 nucleotides in length and derived equally from plus and minus HSVd RNA strands. The widespread distribution of vd-sRNAs across the genome reveals that the totality of the HSVd RNA genome contributes to the formation of vd-sRNAs. Our sequence data suggest that viroid-derived double-stranded RNA functions as one of the main precursors of vd-sRNAs. Remarkably, phloem vd-sRNAs accumulated preferentially as 22-nucleotide species with a consensus sequence over-represented. This bias in size and sequence in the HSVd vd-sRNA population recovered from phloem exudate suggests the existence of a selective trafficking of vd-sRNAs to the phloem tissue of infected cucumber plants.

A pathogenic non-coding RNA induces changes in dynamic DNA methylation of ribosomal RNA genes in host plants

Viroids are plant-pathogenic non-coding RNAs able to interfere with as yet poorly known host-regulatory pathways and to cause alterations recognized as diseases. The way in which these RNAs coerce the host to express symptoms remains to be totally deciphered. In recent years, diverse studies have proposed a close interplay between viroid-induced pathogenesis and RNA silencing, supporting the belief that viroid-derived small RNAs mediate the post-transcriptional cleavage of endogenous mRNAs by acting as elicitors of symptoms expression. Although the evidence supporting the role of viroid-derived small RNAs in pathogenesis is robust, the possibility that this phenomenon can be a more complex process, also involving viroid-induced alterations in plant gene expression at transcriptional levels, has been considered. Here we show that plants infected with the ‘Hop stunt viroid’ accumulate high levels of sRNAs derived from ribosomal transcripts. This effect was correlated with an increase in the transcription of ribosomal RNA (rRNA) precursors during infection. We observed that the transcriptional reactivation of rRNA genes correlates with a modification of DNA methylation in their promoter region and revealed that some rRNA genes are demethylated and transcriptionally reactivated during infection. This study reports a previously unknown mechanism associated with viroid (or any other pathogenic RNA) infection in plants providing new insights into aspects of host alterations induced by the viroid infectious cycle.

Noncoding RNA mediated traffic of foreign mRNA into chloroplasts reveals a novel signaling mechanism in plants.

The import of diverse nucleus-encoded proteins into chloroplasts is crucial for plant life. Although this crosstalk is mainly dependent on specific transit peptides, it has been recently reported that a non protein-coding RNA (ncRNA) based on a viroid-derived sequence (vdRNA) and acting as a 5´UTR-end mediates the functional import of GFP-mRNA into chloroplasts. This observation unearths a novel plant cell signaling pathway able to control the accumulation of the nuclear-encoded proteins in this organelle. The mechanisms regulating this chloroplast-specific localization remain yet unclear. To unravel the functional nature of this chloroplastic signal, here we dissect the 5´UTR-end responsible for the chloroplast targeting. A confocal microscopy analysis in Nicotiana benthamiana leaves of the transcripts expression carrying partial deletions of the 5`UTR-end indicate that an internal 110 nucleotides-length fragment is sufficient to mediate the traffic of functional GFP-mRNA into chloroplasts. However, the capability of this motif to act as a chloroplastic localization signal was enhanced when fused to either the 5` or the 3`region of the vd-5´UTR sequence. These findings suggest that the chloroplast-specific RNA targeting is dependent on a structural motif rather than on the RNA sequence.Communication between chloroplasts and the nucleus is one of the milestones of the evolution of plants on earth. Proteins encoded by ancestral chloroplast-endogenous genes were transferred to the nucleus during the endosymbiotic evolution and originated this communication, which is mainly dependent on specific transit-peptides. However, the identification of nuclear-encoded proteins targeted to the chloroplast lacking these canonical signals suggests the existence of an alternative cellular pathway tuning this metabolic crosstalk. Non-coding RNAS (NcRNAs) are increasingly recognized as regulators of gene expression as they play roles previously believed to correspond to proteins. Avsunviroidae family viroids are the only noncoding functional RNAs that have been reported to traffic inside the chloroplasts. Elucidating mechanisms used by these pathogens to enter this organelle will unearth novel transport pathways in plant cells. Here we show that a viroid-derived NcRNA acting as a 5′UTR-end mediates the functional import of Green Fluorescent Protein (GFP) mRNA into chloroplast. This claim is supported by the observation at confocal microscopy of a selective accumulation of GFP in the chloroplast of the leaves expressing the chimeric vd-5′UTR/GFP and by the detection of the GFP mRNA in chloroplasts isolated from cells expressing this construct. These results support the existence of an alternative signaling mechanism in plants between the host cell and chloroplasts, where an ncRNA functions as a key regulatory molecule to control the accumulation of nuclear-encoded proteins in this organelle. In addition, our findings provide a conceptual framework to develop new biotechnological tools in systems using plant chloroplast as bioreactors. Finally, viroids of the family Avsunviroidae have probably evolved to subvert this signaling mechanism to regulate their differential traffic into the chloroplast of infected cells.

Phloem RNA-binding proteins as potential components of the long-distance RNA transport system

RNA-binding proteins (RBPs) govern a myriad of different essential processes in eukaryotic cells. Recent evidence reveals that apart from playing critical roles in RNA metabolism and RNA transport, RBPs perform a key function in plant adaptation to various environmental conditions. Long-distance RNA transport occurs in land plants through the phloem, a conducting tissue that integrates the wide range of signaling pathways required to regulate plant development and response to stress processes. The macromolecules in the phloem pathway vary greatly and include defense proteins, transcription factors, chaperones acting in long-distance trafficking, and RNAs (mRNAs, siRNAs, and miRNAs). How these RNA molecules translocate through the phloem is not well understood, but recent evidence indicates the presence of translocatable RBPs in the phloem, which act as potential components of long-distance RNA transport system. This review updates our knowledge on the characteristics and functions of RBPs present in the phloem.

Studies on Subcellular Compartmentalization of Plant Pathogenic Noncoding RNAs Give New Insights into the Intracellular RNA-Traffic Mechanisms

Intracellular RNA-trafficking and specific subcellular compartmentalization are key regulatory processes controlling gene expression. Viroids are plant-pathogenic non-coding RNAs that specifically replicate in the nucleus (family Pospiviroidae) or chloroplasts (Avsunviroidae) of infected cells. Subcellular compartmentalization is a critical step in guaranteeing their access to the machineries involved in their RNA transcription and processing. However, how the members of the Avsunviroidae are imported into chloroplasts is a poorly understood process. Elucidating the mechanism used by viroids to enter chloroplasts will enhance our understanding of viroid infection and reveal significant aspects of intracellular RNA trafficking. Here, we report the use of a combined approach involving the cytoplasmic and nuclear expression of monomeric viroid-RNAs (vd-RNA) to study in vivo aspects of the intracellular movement of the Avsunviroidae. We observed that, in leaves infected with a Potato virus X-(PVX)/Green fluorescence protein-(GFP) construct interrupted by an intron carrying a full-length Eggplant latent viroid-(ELVd) sequence, the vd-RNA mediates in cis the nuclear import of the chimeric GFP-mRNA expressed in the cytoplasm, indicating that the vd-RNA has a nuclear import signal. Subsequently, it was demonstrated that a linear monomeric form of the vd-RNA mediates the selective and functional trafficking of GFP-mRNA from the nucleus to chloroplasts. These findings support the idea that during the initial phase of their life-cycle the Avsunviroidae are first transported from the cytoplasm into the nucleus and are then specifically delivered from the nucleus to the chloroplasts, where they can replicate, revealing a novel and unexpected route that could explains their selective subcellular compartmentalization.

Viroids: a light in the darkness of the lncRNA‐directed regulatory networks in plants

The authors would like to thank J. A. Daros and S. F. Elena for the critical reading of the manuscript. This work was supported by grant BIO2011-25018 from the Spanish granting agency Direccion General de Investigacion Cientifica and from the Prometeo program 2011/003 from the Generalitat Valenciana. The authors thank the anonymous reviewers for their valuable comments and suggestions.