Viroids:Small Probes for Exploring the Vast Universe of RNA Trafficking in Plants

Cell-to-cell and long-distance trafficking of RNA is a rapidly evolving frontier of integrative plant biology that broadly impacts studies on plant growth and development, spread of infectious agents and plant defense responses. The fundamental questions being pursued at the forefronts revolve around function, mechanism and evolution. In the present review, we will first use specific examples to illustrate the biological importance of cell-to-cell and long-distance trafficking of RNA. We then focus our discussion on research findings obtained using viroids that have advanced our understanding of the underlying mechanisms involved in RNA trafficking. We further use viroid examples to illustrate the great diversity of trafficking machinery evolved by plants, as well as the promise for new insights in the years ahead. Finally, we discuss the prospect of integrating findings from different experimental systems to achieve a systems-based understanding of RNA trafficking function, mechanism and evolution.     

Plasmodesmata as a supracellular control network in plants

The evolution of intercellular communication had an important role in the increasing complexity of both multicellular and supracellular organisms. Plasmodesmata, the intercellular organelles of the plant kingdom, establish an effective pathway for local and long-distance signalling. In higher plants, this pathway involves the trafficking of proteins and various forms of RNA that function non-cell-autonomously to affect developmental programmes.     

A systemic small RNA signaling system in plants

Systemic translocation of RNA exerts non-cell-autonomous control over plant development and defense. Long-distance delivery of mRNA has been proven, but transport of small interfering RNA and microRNA remains to be demonstrated. Analyses performed on phloem sap collected from a range of plants identified populations of small RNA species. The dynamic nature of this population was reflected in its response to growth conditions and viral infection. The authenticity of these phloem small RNA molecules was confirmed by bioinformatic analysis; potential targets for a set of phloem small RNA species were identified. Heterografting studies, using spontaneously silencing coat protein (CP) plant lines, also established that transgene-derived siRNA move in the long-distance phloem and initiate CP gene silencing in the scion. Biochemical analysis of pumpkin (Cucurbita maxima) phloem sap led to the characterization of C. maxima Phloem SMALL RNA BINDING PROTEIN1 (CmPSRP1), a unique component of the protein machinery probably involved in small RNA trafficking. Equivalently sized small RNA binding proteins were detected in phloem sap from cucumber (Cucumis sativus) and lupin (Lupinus albus). PSRP1 binds selectively to 25-nucleotide single-stranded RNA species. Microinjection studies provided direct evidence that PSRP1 could mediate the cell-to-cell trafficking of 25-nucleotide single-stranded, but not double-stranded, RNA molecules. The potential role played by PSRP1 in long-distance transmission of silencing signals is discussed with respect to the pathways and mechanisms used by plants to exert systemic control over developmental and physiological processes.     

Destination-selective long-distance movement of phloem proteins

The phloem macromolecular transport system plays a pivotal role in plant growth and development. However, little information is available regarding whether the long-distance trafficking of macromolecules is a controlled process or passive movement. Here, we demonstrate the destination-selective long-distance trafficking of phloem proteins. Direct introduction, into rice (Oryza sativa), of phloem proteins from pumpkin (Cucurbita maxima) was used to screen for the capacity of specific proteins to move long distance in rice sieve tubes. In our system, shoot-ward translocation appeared to be passively carried by bulk flow. By contrast, root-ward movement of the phloem RNA binding proteins 16-kD C. maxima phloem protein 1 (CmPP16-1) and CmPP16-2 was selectively controlled. When CmPP16 proteins were purified, the root-ward movement of CmPP16-1 became inefficient, suggesting the presence of pumpkin phloem factors that are responsible for determining protein destination. Gel-filtration chromatography and immunoprecipitation showed that CmPP16-1 formed a complex with other phloem sap proteins. These interacting proteins positively regulated the root-ward movement of CmPP16-1. The same proteins interacted with CmPP16-2 as well and did not positively regulate its root-ward movement. Our data demonstrate that, in addition to passive bulk flow transport, a destination-selective process is involved in long-distance movement control, and the selective movement is regulated by protein-protein interaction in the phloem sap.     

Viroid trafficking: a small RNA makes a big move

RNA trafficking has broad implications in the systemic spread of infectious agents, plant defense, and the systemic regulation of gene expression. The mechanisms that regulate trafficking remain poorly understood. The non-coding, infectious viroid RNAs are emerging as highly tractable model systems for the investigation of the basic mechanisms of RNA trafficking. Recent studies on viroids have led to new insights into the direct role of RNAs in intracellular and systemic trafficking, and to the identification of cellular proteins that might play a role in RNA trafficking. Here, we discuss these areas of progress, emphasizing on the unifying principles that control the trafficking of viroid, viral and endogenous RNAs.     

Direct role of a viroid RNA motif in mediating directional RNA trafficking across a specific cellular boundary

The plasmodesmata and phloem form a symplasmic network that mediates direct cell-cell communication and transport throughout a plant. Selected endogenous RNAs, viral RNAs, and viroids traffic between specific cells or organs via this network. Whether an RNA itself has structural motifs to potentiate trafficking is not well understood. We have used mutational analysis to identify a motif that the noncoding Potato spindle tuber viroid RNA evolved to potentiate its efficient trafficking from the bundle sheath into mesophyll that is vital to establishing systemic infection in tobacco (Nicotiana tabacum). Surprisingly, this motif is not necessary for trafficking in the reverse direction (i.e., from the mesophyll to bundle sheath). It is not required for trafficking between other cell types either. We also found that the requirement for this motif to mediate bundle sheath-to-mesophyll trafficking is dependent on leaf developmental stages. Our results provide genetic evidence that (1) RNA structural motifs can play a direct role in mediating trafficking across a cellular boundary in a defined direction, (2) the bundle sheath-mesophyll boundary serves as a novel regulatory point for RNA trafficking between the phloem and nonvascular tissues, and (3) the symplasmic network remodels its capacity to traffic RNAs during plant development. These findings may help further studies to elucidate the interactions between RNA motifs and cellular factors that potentiate directional trafficking across specific cellular boundaries.     

A genomic map of viroid RNA motifs critical for replication and systemic trafficking

RNA replication and systemic trafficking play significant roles in developmental regulation and host-pathogen interactions. Viroids are the simplest noncoding eukaryotic RNA pathogens and genetic units that are capable of autonomous replication and systemic trafficking and offer excellent models to investigate the role of RNA structures in these processes. Like other RNAs, the predicted secondary structure of a viroid RNA contains many loops and bulges flanked by double-stranded helices, the biological functions of which are mostly unknown. Using Potato spindle tuber viroid infection of Nicotiana benthamiana as the experimental system, we tested the hypothesis that these loops/bulges are functional motifs that regulate replication in single cells or trafficking in a plant. Through a genome-wide mutational analysis, we identified multiple loops/bulges essential or important for each of these biological processes. Our results led to a genomic map of viroid RNA motifs that mediate single-cell replication and systemic trafficking, respectively. This map provides a framework to enable high-throughput studies on the tertiary structures and functional mechanisms of RNA motifs that regulate viroid replication and trafficking. Our model and approach should also be valuable for comprehensive investigations of the replication and trafficking motifs in other RNAs.     

Cell-to-Cell Trafficking of Macromolecules through Plasmodesmata Potentiated by the Red Clover Necrotic Mosaic Virus Movement Protein

Direct evidence is presented for cell-to-cell trafficking of macromolecules via plasmodesmata in higher plants. The fluorescently labeled 35-kD movement protein of red clover necrotic mosaic virus (RCNMV) trafficked rapidly from cell to cell when microinjected into cowpea leaf mesophyll cells. Furthermore, this protein potentiated rapid cell-to-cell trafficking of RCNMV RNA, but not DNA. Electron microscopic studies demonstrated that the 35-kD movement protein does not unfold the RCNMV RNA molecules. Thus, if unfolding of RNA is necessary for cell-to-cell trafficking, it may well involve participation of endogenous cellular factors. These findings support the hypothesis that trafficking of macromolecules is a normal plasmodesmal function, which has been usurped by plant viruses for their cell-to-cell spread.     

A long-distance translocatable phloem protein from cucumber forms a ribonucleoprotein complex in vivo with Hop stunt viroid RNA

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.     

Influence of viral genes on the cell-to-cell spread of RNA silencing

The turnip crinkle virus-based vector TCV-GFP Delta CP had been devised previously to study cell-to-cell and long-distance spread of virus-induced RNA silencing. TCV-GFP Delta CP, which had been constructed by replacing the coat protein (CP) gene with a green fluorescent protein (GFP) coding sequence, was able to induce RNA silencing in single epidermal cells, from which RNA silencing spread from cell-to-cell. Using this unique local silencing assay together with mutagenesis analysis, two TCV genes, p8 and p9, which were involved in the intercellular spread of virus-induced RNA silencing, were identified. TCV-GFP Delta CP and its p8- or p9-mutated derivatives, TCVmp8-GFP Delta CP and TCVmp9-GFP Delta CP, replicated efficiently but were restricted to single Nicotiana benthamiana epidermal cells. TCV-GFP Delta CP, TCVmp8-GFP Delta CP, or TCVmp9-GFP Delta CP was able to initiate RNA silencing that targeted and degraded recombinant viral RNAs in inoculated leaves of the GFP-expressing N. benthamiana line 16c. However, cell-to-cell spread of silencing to form silencing foci was triggered only by TCV-GFP Delta CP. Non-replicating TCVmp88-GFP Delta CP and TCVmp28mp88-GFP Delta CP with dysfunctional replicase genes, and single-stranded gfp RNA did not induce RNA silencing. Transient expression of the TCV p9 protein could effectively complement TCVmp9-GFP Delta CP to facilitate intercellular spread of silencing. These data suggest that the plant cellular trafficking machinery could hijack functional viral proteins to permit cell-to-cell movement of RNA silencing.     

Vacuoles and fungal biology

Fungal vacuoles have long been recognised as versatile organelles, involved in many aspects of protein turnover, cellular homeostasis, membrane trafficking, signalling and nutrition. Recent research has also revealed an expanding repertoire of physiological functions for fungal vacuoles that are vital for fungal growth, differentiation, symbiosis and pathogenesis. Vacuole-mediated long-distance nutrient transporting systems have been shown to facilitate mycelial foraging and long-distance communication in saprophytes and mycorrhizal fungi. Some hyphae of plant and human fungal pathogens can grow under severely nutrient-limited conditions by expanding the vacuolar space rather than synthesising new cytoplasm and organelles. Autophagy has been recognised as a crucial process in plant pathogens for the initiation of appressorium formation. These studies demonstrate the importance of fungal vacuoles as organelles that are essential for many of the attributes that define the activities and roles of fungi in their natural environments.     

Non-cell Autonomous RNA Trafficking and Long-Distance Signaling

A wide range of proteins and RNA molecules in plants have been recently identified as non-cell autonomous, phloem-mobile molecules and suggested to play important roles in physiological and developmental processes. Systemic movement of both protein-coding mRNAs and non-coding small RNAs is shown to correlate with the epigenetic changes brought about across grafting junctions, supporting their potential roles as long-distance signaling molecules. Plants appear to have evolved this unique RNA-based signaling mechanism to control systemic regulation of various responses to environmental stimuli and challenges such as photoperiods, nutrient availabilities, and pathogen attacks. This mechanism may have been exploited by viroids, non-coding RNA pathogens, to spread infection cell to cell and through phloem. A model describing potential molecular mechanisms by which the systemic RNA trafficking occurs will be presented.     

Long distance transport and movement of RNA through the phloem

Cell-to-cell communication is essential for plant development and adaptation to environmental changes. As a strategy for efficient intercellular communication, plants have evolved a plant-specific symplasmic network connected via plasmodesmata that allows a locally restricted information exchange from cell to cell. A rapid information transfer over long distances is enabled via the phloem transport tubes that pervade the complete plant and thus connect even the most distant organs. While communication by small molecules like metabolites and phytohormones is comparably well studied, the intercellular trafficking of proteins and RNAs has only recently emerged as a novel mechanism of cell-to-cell and long-distance signalling in plants. In particular the non-cell-autonomous and systemic transport pathway for specific RNAs seems to play a key role in the co-ordination of important physiological processes, including virus defence, gene silencing, regulation of development, and nutrient allocation. This review is a summary of the current knowledge on RNAs contained in the phloem long-distance transport system, their transport mechanism, and their potential functions.