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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Cell-to-Cell and Long-distance Trafficking of RNA Functions in Multiple Processes
  5. Viroid Infection: a Model System to Investigate the Regulation of Systemic RNA Trafficking
  6. Viroids Traffic through Plasmodesmata and the Phloem
  7. Selective Trafficking of Viroids into Shoot Apical Organs
  8. Presence of a Viroid Motif can Mediate Unidirectional Trafficking across a Cellular Boundary
  9. A Viroid Motif Mediates Bundle Sheath-to-Phloem Trafficking
  10. Genomic Map of PSTVd Trafficking Motifs
  11. Diversity in Viroid Motifs Controlling RNA Trafficking Reveals Complexity in Host Machinery
  12. Future Directions of Research
  13. Agricultural Implications of Systemic RNA Trafficking
  14. Acknowledgements
  15. References

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.

inline imageBiao Ding (Corresponding author)

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Cell-to-Cell and Long-distance Trafficking of RNA Functions in Multiple Processes
  5. Viroid Infection: a Model System to Investigate the Regulation of Systemic RNA Trafficking
  6. Viroids Traffic through Plasmodesmata and the Phloem
  7. Selective Trafficking of Viroids into Shoot Apical Organs
  8. Presence of a Viroid Motif can Mediate Unidirectional Trafficking across a Cellular Boundary
  9. A Viroid Motif Mediates Bundle Sheath-to-Phloem Trafficking
  10. Genomic Map of PSTVd Trafficking Motifs
  11. Diversity in Viroid Motifs Controlling RNA Trafficking Reveals Complexity in Host Machinery
  12. Future Directions of Research
  13. Agricultural Implications of Systemic RNA Trafficking
  14. Acknowledgements
  15. References

Mechanisms of gene regulation have long been studied and illustrated mostly at the level of individual cells. With increasing findings of cell-to-cell and long-distance trafficking of RNAs and proteins, some of which have been shown to regulate plant development, mechanisms of gene regulation including final cellular destination of certain gene products must now be considered at the whole plant level (Lucas and Lee 2004; Lough and Lucas 2006; Ding and Itaya 2007a; Kehr and Buhtz 2008; Lucas et al. 2009; Turgeon and Wolf 2009). Besides its role in plant developmental processes, cell-to-cell and/or long-distance trafficking of RNA molecules and/or proteins is crucial to the establishment of systemic infection by viruses and viroids (Boevink and Oparka 2005; Flores et al. 2005; Scholthof 2005; Lucas 2006; Taliansky et al. 2008; Tsagris et al. 2008; Ding 2009) and to systemic plant defense responses (Ding and Voinnet 2007; Díaz-Pendón and Ding 2008; Kalantidis et al. 2008). The study of how gene expression and metabolism in individual cells within a plant are integrated, through RNA and protein trafficking, to enable development, internal function and response to the environment is rapidly emerging as a new frontier of plant biology.

The plasmodesmata and phloem form a symplasmic network of channels for cell-to-cell and long-distance trafficking of RNAs, proteins, viruses, viroids as well as photoassimilates from sources where they are generated to various sink organs (Figure 1). This review addresses cell-to-cell and long-distance trafficking of RNA, with a focus on the use of viroids as models to probe mechanistic and evolutionary questions. Mechanisms and functions of cellular RNA trafficking are covered by Hannapel in this special issue (Hannapel 2010). Here, we first briefly summarize examples of RNA trafficking to show its importance and then devote most of the discussion on research findings from viroids that have contributed to advance our understanding of the trafficking mechanisms. We further use viroid examples to illustrate the potentially enormous diversity of trafficking machinery plants have evolved and the great promise for new discoveries for the coming years. 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.

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Figure 1. Conceptualized integration of cell-to-cell and long-distance symplasmic transport pathways for proteins, RNAs, viruses, viroids as well as photoassimilates within a plant body. (A) Molecules generated within a source leaf that are destined to remote sink organs are transported through plasmodesmata across various cell layers (blue arrows; cell layers are not illustrated for simplicity) to enter the phloem for long-distance transport to the sink organs (red arrows). (B) Schematic of a plasmodesma that comprises the plasma membrane (PM) surrounding a cylinder of modified endoplasmic reticulum (ER) that create a cytoplasmic connection between two neighboring cells. The cytoplasmic sleeve (CS) forms microchannels for intercellular transport. (C) An idealized vascular bundle in which a layer of bundle sheath encloses the xylem, identified by the tracheary elements, and phloem, identified by the sieve elements and companion cells. The sieve elements are interconnected end to end to form sieve tubes for transport.

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Cell-to-Cell and Long-distance Trafficking of RNA Functions in Multiple Processes

  1. Top of page
  2. Abstract
  3. Introduction
  4. Cell-to-Cell and Long-distance Trafficking of RNA Functions in Multiple Processes
  5. Viroid Infection: a Model System to Investigate the Regulation of Systemic RNA Trafficking
  6. Viroids Traffic through Plasmodesmata and the Phloem
  7. Selective Trafficking of Viroids into Shoot Apical Organs
  8. Presence of a Viroid Motif can Mediate Unidirectional Trafficking across a Cellular Boundary
  9. A Viroid Motif Mediates Bundle Sheath-to-Phloem Trafficking
  10. Genomic Map of PSTVd Trafficking Motifs
  11. Diversity in Viroid Motifs Controlling RNA Trafficking Reveals Complexity in Host Machinery
  12. Future Directions of Research
  13. Agricultural Implications of Systemic RNA Trafficking
  14. Acknowledgements
  15. References

Infectious agents such as viroids and viruses must traffic throughout a plant in order to establish a systemic infection. All viroids and many viruses have RNA genomes, so their systemic infection represents classical examples of cell-to-cell and long-distance trafficking of RNA (Flores et al. 2005; Scholthof 2005; Lucas 2006; Ding and Itaya 2007b; Ding 2009). Research over the past decade has experimentally established that many cellular RNAs also traffic between cells and between organs as a normal plant function, starting with the demonstration of KNOTTED1 mRNA trafficking in the presence of its protein (Lucas et al. 1995). Analyses of phloem sap collected from various plant species revealed the trafficking capacity of many mRNA species (Sasaki et al. 1998; Ruiz-Medrano et al. 1999; Doering-Saad et al. 2006; Omid et al. 2007; Deeken et al. 2008) and small RNAs, including microRNAs (miRNAs) and short interfering RNAs (siRNAs) (Yoo et al. 2004; Buhtz et al. 2008).

Grafting experiments have demonstrated that some mRNA species are transported over long distances in the phloem to regulate developmental processes, such as leaf morphogenesis in tomato (Kim et al. 2001; Haywood et al. 2005) and tuber formation in potato (Banerjee et al. 2006). On a physiological note, trafficking of an miRNA from shoot to root has been implicated in the regulation of a gene involved in phosphate homeostasis in Arabidopsis (Lin et al. 2008; Pant et al. 2008). Gene silencing signals, likely consisting of an RNA component (including miRNA or siRNA), traffic within a plant to cause systemic silencing as a means of gene regulation and antiviral defense (Ding and Voinnet 2007; Kalantidis et al. 2008). RNA trafficking also occurs between some parasitic plants and their hosts, with biological functions yet to be understood (Roney et al. 2007; David-Schwartz et al. 2008).

Based on the well-understood structural motif-mediated intracellular RNA trafficking in animal and fungal cells, it was suggested that cell-to-cell trafficking of plant RNAs might also be mediated by special motif signals (Ding et al. 1999; Citovsky and Zambryski 2000; Lucas et al. 2001). In support of this notion, several studies on viral and cellular RNAs have indicated that they contain sequence or structural information to potentiate trafficking. A cis element in the 5′ untranslated region (UTR) of potexviral RNA, which plays a role in replication (Miller et al. 1998), mediates cell-to-cell trafficking of the fused green fluorescent protein (GFP) reporter RNA (Lough et al. 2006). In another example, the UTRs of potato StBEL5 mRNA appear to be important for long-distance trafficking (Banerjee et al. 2006). The demonstration that Brome mosaic virus RNAs can traffic long-distance in the absence of replication suggests that these RNAs have structural elements directly recognized by cellular factors for trafficking (Gopinath and Kao 2007). A cis-acting element in nucleotides 1–102 of the Arabidopsis FLOWERING LOCUS T mRNA can mediate trafficking of a fused GFP mRNA or a modified viral RNA (Li et al. 2009). Motifs in the coding sequences and 3′ UTR as well as the secondary structure of the GIBBERELLIC ACID-INSENSITIVE RNA can mediate trafficking of a fused GFP mRNA (Huang and Yu 2009). The precise structural nature of the trafficking motifs in these RNAs remains to be further elucidated. In major advances in dissecting the plant RNA trafficking machinery, cellular proteins involved in the trafficking of some endogenous RNAs have been identified (Xoconostle-Cázares et al. 1999; Yoo et al. 2004; Ham et al. 2009).

In summary, there is now a significant body of experimental evidence in support of the hypothesis that systemic RNA trafficking, through plasmodesmata and the phloem, is a normal and crucial plant function underlying gene regulation at the whole plant level, infection by viroids and viruses, and plant defense responses. Figure 1 illustrates the basic plant structural components of this long-distance trafficking pathway. In the next section, we describe the utility of viroid infection as a model system with which to dissect the RNA structural motifs that regulate trafficking between specific cellular boundaries.

Viroid Infection: a Model System to Investigate the Regulation of Systemic RNA Trafficking

  1. Top of page
  2. Abstract
  3. Introduction
  4. Cell-to-Cell and Long-distance Trafficking of RNA Functions in Multiple Processes
  5. Viroid Infection: a Model System to Investigate the Regulation of Systemic RNA Trafficking
  6. Viroids Traffic through Plasmodesmata and the Phloem
  7. Selective Trafficking of Viroids into Shoot Apical Organs
  8. Presence of a Viroid Motif can Mediate Unidirectional Trafficking across a Cellular Boundary
  9. A Viroid Motif Mediates Bundle Sheath-to-Phloem Trafficking
  10. Genomic Map of PSTVd Trafficking Motifs
  11. Diversity in Viroid Motifs Controlling RNA Trafficking Reveals Complexity in Host Machinery
  12. Future Directions of Research
  13. Agricultural Implications of Systemic RNA Trafficking
  14. Acknowledgements
  15. References

Viroids are single-stranded, circular and non-coding RNAs that infect plants (Flores et al. 2005; Tsagris et al. 2008; Ding 2009). With genome sizes ranging from 250 to 400 nucleotides, viroids are the smallest nucleic acid-based infectious agents and also the smallest self-replicating genetic elements known to date.

The approximately 30 species of known viroids are classified into two families, Avsunviroidae (type member Avocado sunblotch viroid, ASBVd) and Pospiviroidae (type member Potato spindle tuber viroid, PSTVd), based on several distinguishing features. The members of the Avsunviroidae fold into a quasi-rod-like secondary structure with branches (Figure 2) and replicate within the chloroplast. This raises the interesting question as to how the viroid RNA moves across the chloroplast membranes. Interestingly, these viroid RNAs also possess intrinsic ribozyme activities and share similar structures in the hammerhead ribozyme region, but differ in sequence and structure elsewhere. Members of the Pospiviroidae fold into a rod-like secondary structure (Figure 2) and replicate within the nucleus; in this case, movement is through the nuclear pore complex with mechanisms still yet to be understood. In contrast to the Avsunviroidae, these viroid RNA molecules lack ribozyme activity and some species share conserved sequences/secondary structures in the central and terminal regions, but overall all viroid species in the family have significant differences in sequence and structure. As will be discussed later, these sequence/structural conservations and variations among viroids make them useful model systems to investigate the evolution of common and unique mechanisms of RNA trafficking in plants.

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Figure 2. Secondary structural comparisons between representative members from the two viroid families. Avsunviroidae, ASBVd (Avocado sunblotch viroid); Pospiviroidae, PSTVd (Potato spindle tuber viroid). CEVd (Citrus exocortis viroid) and HSVd (Hop stunt viroid). PSTVd trafficking motifs are highlighted in green and red colors. The red colored motifs of PSTVd are conserved in CEVd. These motifs are identified in Zhong et al. (2007, 2008).

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Viroids usually have a relatively narrow host range under field conditions, and there is evidence for the continuing expansion of host range for some species (Ding and Zhong 2009). Although some viroids can cause devastating diseases in certain plant hosts, there are others whose infection does not cause any visible signs of disease (Flores et al. 2005; Ding and Itaya 2007b; Owens and Hammond 2009). Whether there are situations in which the presence of a viroid can be beneficial to plant function remains an intriguing question.

Both families of viroids replicate via an RNA–RNA rolling circle mechanism without a DNA intermediate. The incoming circular, genomic RNA molecules are transcribed by the host cellular DNA-dependent RNA polymerases into concatemeric, linear RNA molecules. These molecules then undergo cleavage to produce unit-length molecules that are circularized. The detailed aspects of this replication process, including the RNA structures and cellular factors involved as well as the common and unique features for the two families, have recently been reviewed (Ding 2009; Flores et al. 2009). What is important for trafficking studies, from a technical point of view, is that the small, compact viroid genome is unlikely to accommodate additional RNA sequences without disrupting replication functions. This feature makes it difficult to follow viroid systemic trafficking by using a reporter gene, such as GFP, as has been used so successfully for tracing viral or plant RNA movement. However, as viroid molecules generally accumulate to high levels in the infected cells, their presence can readily be detected by in situ hybridization techniques.

Establishment of systemic infection, when a viroid is inoculated onto a leaf, involves the following processes: (i) import into the nucleus (pospiviroid) or chloroplast (avsunviroid) in an initially infected cell for replication to begin; (ii) export of progeny RNA out of the nucleus/chloroplast; (iii) trafficking from cell to cell; and (iv) long-distance trafficking to distant sink organs (the upper parts of shoots and the roots) to establish new sites of infection (Ding 2009; Ding and Wang 2009).

Viroid systemic infection has many distinct advantages as a model to study the mechanisms of RNA trafficking (Di Serio and Flores 2008; Ding and Wang 2009). First, without encoding proteins, a viroid RNA likely has evolved structural motifs that interact with the host's pre-existing cellular machinery to accomplish all trafficking functions. Therefore, elucidating viroid trafficking mechanisms has relevance to understanding the general mechanistic principles underlying cellular RNA trafficking. Second, the structural simplicity and noncoding nature of viroid RNA molecules makes them simpler models with which to dissect the RNA motifs that mediate trafficking. In particular, the secondary structures of some viroids are among the best understood of all RNAs as a result of extensive computational, biophysical and chemical studies (reviewed in Ding and Wang 2009). Third, the relatively small size of the viroid genome makes it easier to carry out saturation mutagenesis studies to genetically identify functional motifs.

Viroids Traffic through Plasmodesmata and the Phloem

  1. Top of page
  2. Abstract
  3. Introduction
  4. Cell-to-Cell and Long-distance Trafficking of RNA Functions in Multiple Processes
  5. Viroid Infection: a Model System to Investigate the Regulation of Systemic RNA Trafficking
  6. Viroids Traffic through Plasmodesmata and the Phloem
  7. Selective Trafficking of Viroids into Shoot Apical Organs
  8. Presence of a Viroid Motif can Mediate Unidirectional Trafficking across a Cellular Boundary
  9. A Viroid Motif Mediates Bundle Sheath-to-Phloem Trafficking
  10. Genomic Map of PSTVd Trafficking Motifs
  11. Diversity in Viroid Motifs Controlling RNA Trafficking Reveals Complexity in Host Machinery
  12. Future Directions of Research
  13. Agricultural Implications of Systemic RNA Trafficking
  14. Acknowledgements
  15. References

The first experimental evidence that viroids traffic between cells through plasmodesmata came from microinjection experiments (Ding et al. 1997). Here, fluorescently labeled in vitro transcripts of PSTVd were injected into either tobacco leaf mesophyll cells that are interconnected by plasmodesmata, or mature guard cells that lack plasmodesmal connections to their neighboring epidermal cells. The injected transcripts were observed to traffic rapidly from cell to cell in mesophyll, but failed to traffic out of the mature guard cells, consistent with the hypothesis that plasmodesmata function as the pathway for cell-to-cell trafficking of PSTVd. Furthermore, the PSTVd transcripts could mediate trafficking of vector RNA sequences, which otherwise did not move between cells by themselves, suggesting that the PSTVd RNA has motifs that impart the capacity for cell-to-cell trafficking (Ding et al. 1997). However, to date, direct localization of viroid RNA within plasmodesmata has not been reported.

Palukaitis (1987) provided the first evidence that viroid long-distance trafficking from one plant organ to another occurs through the phloem. He inoculated a tomato leaf with PSTVd and then followed the systemic spread, by RNA blots, into different plant organs. The data showed that PSTVd accumulated in the root and leaves above the inoculated leaf, but not in leaves below the inoculated leaf. This pattern is similar to the distribution of photoassimilates from source to sink organs, thereby implicating the phloem as the pathway for long-distance trafficking of PSTVd. Direct visualization of PSTVd in the phloem came later from in situ hybridization experiments (Zhu et al. 2001).

Selective Trafficking of Viroids into Shoot Apical Organs

  1. Top of page
  2. Abstract
  3. Introduction
  4. Cell-to-Cell and Long-distance Trafficking of RNA Functions in Multiple Processes
  5. Viroid Infection: a Model System to Investigate the Regulation of Systemic RNA Trafficking
  6. Viroids Traffic through Plasmodesmata and the Phloem
  7. Selective Trafficking of Viroids into Shoot Apical Organs
  8. Presence of a Viroid Motif can Mediate Unidirectional Trafficking across a Cellular Boundary
  9. A Viroid Motif Mediates Bundle Sheath-to-Phloem Trafficking
  10. Genomic Map of PSTVd Trafficking Motifs
  11. Diversity in Viroid Motifs Controlling RNA Trafficking Reveals Complexity in Host Machinery
  12. Future Directions of Research
  13. Agricultural Implications of Systemic RNA Trafficking
  14. Acknowledgements
  15. References

After long-distance transport to the shoot apex, viroids may or may not further enter the region of the shoot apical meristem (SAM). By using in situ hybridization, Zhu et al. (2001) and Qi and Ding (2003) showed that PSTVd was absent from the SAM in infected tomato and Nicotiana benthamiana plants. In contrast, studies by Rodio et al. (2007) showed the presence of Peach latent mosaic viroid (PLMVd), which is a member of the family Avsunviroidae, in the SAM of infected peach trees. Such differences may be attributed to particular viroid-host combinations, but it will be interesting to determine whether they could also be a distinguishing feature for the two viroid families.

The mechanistic basis for these differences remains to be elucidated. It is possible that PSTVd does not have structural motifs recognizable by plant factors that are required for entry into the SAM of tomato and N. benthamiana. Alternatively, RNA silencing may play a role to prevent the entry of PSTVd RNA in the SAM (Di Serio and Flores 2008). There is evidence that RNA silencing can function as a surveillance mechanism to prevent viral entry into the SAM (Foster et al. 2002; Schwach et al. 2005). Recent work by Flores and colleagues has provided experimental evidence that RNA silencing may indeed play a role in preventing PSTVd from infecting the SAM region of N. benthamiana (R Flores, pers. comm., 2009). They showed that in transgenic N. benthamiana plants with downregulated expression of RNA-dependent RNA polymerase 6 (RDR6), an enzyme that has a prominent role in RNA silencing against viral infection (Foster et al. 2002; Qu et al. 2005; Schwach et al. 2005; Qu et al. 2008; Vaistij and Jones, 2009), there was an abundance of PSTVd in the SAM of these infected plants.

An intriguing question is why the presence of RDR6 in N. benthamiana plants mainly contributes to repress PSTVd infection in the SAM, but not in other organs. Furthermore, it will be of interest to ascertain whether RNA silencing functions to inhibit the replication of incoming viroids in the SAM, or to actively block viroid RNA trafficking into the SAM. How PLMVd overcomes the RNA silencing system in peach to invade the SAM warrants further investigation. In addition to the role of RNA silencing, other possible mechanisms for preventing viroid infection of SAM cannot be excluded. These include selective degradation of viroid RNA by nucleases, absence of critical cellular factors to support replication, or the presence of factors that repress replication. Further studies on these issues in more viroid-host combinations should contribute valuable insights into the relative contributions of RNA silencing, other RNA turnover pathways and motif-cellular factor interactions during RNA trafficking.

Analyses of PSTVd infection in tomato and N. benthamiana indicated the presence of PSTVd in sepals but not in the other floral organs, such as petals, stamen and pistils (Zhu et al. 2001; Zhu et al. 2002). Because PSTVd could replicate in all floral organs when they were directly “inoculated” by transgenic expression of a PSTVd cDNA that produced primary transcripts to initiate RNA-RNA replication, the differential presence of PSTVd in the floral organs in mechanically inoculated plants can best be explained by the selective transport of PSTVd into sepals but not into the other floral organs (Zhu et al. 2002). Such selective trafficking into different sink organs has also been reported for some plant mRNAs (Haywood et al. 2005; Banerjee et al. 2006) and even proteins (Aoki et al. 2005), indicating its broad biological significance. Elucidating the specific mechanisms underlying such selective trafficking will be critical to furthering our understanding of systemic gene regulation and the spread of infectious agents within the body of the plant.

How viroids traffic into roots of an infected plant has not been explored. Hence, filling this gap in our research efforts may provide additional insights into systemic regulation of RNA trafficking at the whole-plant level.

Presence of a Viroid Motif can Mediate Unidirectional Trafficking across a Cellular Boundary

  1. Top of page
  2. Abstract
  3. Introduction
  4. Cell-to-Cell and Long-distance Trafficking of RNA Functions in Multiple Processes
  5. Viroid Infection: a Model System to Investigate the Regulation of Systemic RNA Trafficking
  6. Viroids Traffic through Plasmodesmata and the Phloem
  7. Selective Trafficking of Viroids into Shoot Apical Organs
  8. Presence of a Viroid Motif can Mediate Unidirectional Trafficking across a Cellular Boundary
  9. A Viroid Motif Mediates Bundle Sheath-to-Phloem Trafficking
  10. Genomic Map of PSTVd Trafficking Motifs
  11. Diversity in Viroid Motifs Controlling RNA Trafficking Reveals Complexity in Host Machinery
  12. Future Directions of Research
  13. Agricultural Implications of Systemic RNA Trafficking
  14. Acknowledgements
  15. References

The existence of a PSTVd motif(s) for cell-to-cell trafficking was inferred from microinjection experiments with PSTVd-vector RNA fusions (Ding et al. 1997). Studies on two PSTVd strains, PSTVdNT and PSTVdNB, with experimental protocols to separate replication and trafficking functions, provided conclusive experimental evidence for the direct role of an RNA motif in mediating cell-to-cell trafficking (Qi et al. 2004). PSTVdNT was derived from the tomato isolate PSTVdKF440–2 by the spontaneous nucleotide substitution C259U that converted PSTVdKF440–2 from a noninfectious to an infectious RNA species in tobacco (Wassenegger et al. 1996). PSTVdNB further evolved from PSTVdNT through five spontaneous nucleotide substitutions (G201U, A309U, A47U/U313A and U315C) in cuttings propagated vegetatively from tobacco plants originally infected with PSTVdNT.

When inoculated onto a young tobacco leaf, both PSTVdNB and PSTVdNT replicated to similar levels. However, in systemically infected young leaves, PSTVdNB accumulated to a higher level than PSTVdNT. To find out the basis for this difference, in situ hybridization was used to localize these strains in systemically infected leaves. Such analysis showed that in young systemic leaves, PSTVdNB was present in all cell types, whereas PSTVdNT was only detected in the vascular and bundle sheath cells (Qi et al. 2004). These findings indicated that in a young systemic leaf PSTVdNT cannot traffic from the bundle sheath into the mesophyll, although it can traffic from the mesophyll into bundle sheath in the inoculated leaf. Hence, trafficking of PSTVd at the bundle sheath-mesophyll boundary must be regulated differently in the opposing directions. This implies that different plasmodesmal and/or cytosolic factors are involved in regulating the bidirectional trafficking across the same cellular boundary. Such a mechanism may well operate for the trafficking of viral and cellular RNAs. Elucidating the specifics of this mechanism is an important goal for future studies.

Nucleotide swapping between PSTVdNB and PSTVdNT identified four of the five PSTVdNB-specific nucleotides that were each required and collectively are sufficient to potentiate trafficking from the bundle sheath into the mesophyll, thereby establishing the first bipartite RNA trafficking motif (Qi et al. 2004). Importantly, the requirement for this motif appears to depend on plant development, because PSTVdNB could traffic from the bundle sheath into the mesophyll during leaf maturation.

Besides demonstrating the feasibility of combining genetic and cellular approaches to dissect the trafficking motif in a viroid RNA, these findings provided solid evidence in support of the hypothesis that viroids have evolved motifs that are recognized by the host machinery for cell-to-cell trafficking. In view of this fact, viroids should prove invaluable for the identification of the endogenous plant RNA trafficking machinery (Ding et al. 1999, 2005). Clearly, the PSTVdNB and PSTVdNT example raises the distinct possibility that new viroid trafficking motifs are still evolving in nature, especially when a viroid comes into contact with a potential new host plant. Given that viroids have the highest mutation rate among different types of nucleic acids during replication in vivo (Gago et al. 2009), they are ideal models for probing the plant trafficking machinery through gain-of-function studies of newly evolved trafficking motifs on a relatively short time scale. Such studies will be even more productive when efficient protocols have been developed to identify the host factors that recognize these RNA motifs.

A Viroid Motif Mediates Bundle Sheath-to-Phloem Trafficking

  1. Top of page
  2. Abstract
  3. Introduction
  4. Cell-to-Cell and Long-distance Trafficking of RNA Functions in Multiple Processes
  5. Viroid Infection: a Model System to Investigate the Regulation of Systemic RNA Trafficking
  6. Viroids Traffic through Plasmodesmata and the Phloem
  7. Selective Trafficking of Viroids into Shoot Apical Organs
  8. Presence of a Viroid Motif can Mediate Unidirectional Trafficking across a Cellular Boundary
  9. A Viroid Motif Mediates Bundle Sheath-to-Phloem Trafficking
  10. Genomic Map of PSTVd Trafficking Motifs
  11. Diversity in Viroid Motifs Controlling RNA Trafficking Reveals Complexity in Host Machinery
  12. Future Directions of Research
  13. Agricultural Implications of Systemic RNA Trafficking
  14. Acknowledgements
  15. References

Although the gain-of-function genetic approach is useful for exploring the evolution of new RNA trafficking motifs, it cannot be used to identify trafficking motifs already existing in a viroid RNA sequence. To identify these motifs requires loss-of-function genetic analyses. This approach was first used to identify a PSTVd motif required for trafficking from the bundle sheath into the phloem in N. benthamiana leaves (Zhong et al. 2007). As shown in Figure 3, closing the U43/C318 loop by a U43G or C318A substitution resulted in failure of PSTVd to establish systemic infection, but did not affect its replication at the cellular level. Reverse transcription polymerase chain reaction analysis showed that the mutant RNA molecules were absent from the petioles of inoculated leaves, indicating that they failed to traffic out of the inoculated leaves. In situ hybridization studies localized these mutants in epidermal, mesophyll and bundle sheath cells in the inoculated leaves, in contrast to the localization of wild-type viroid in all cell types. These studies establish that the U43/C318 loop represents a motif required for trafficking from the bundle sheath into the phloem. Whether this motif functions alone or together with another yet-to-be-identified RNA sequence/motif remains to be determined.

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Figure 3. A Potato spindle tuber viroid (PSTVd) motif required for trafficking from the bundle sheath into phloem of an inoculated Nicotiana benthamiana leaf. (A) A portion of PSTVd secondary structure showing nucleotide substitutions (U43G and C318A) that each close the U43/C318 loop to create an extended local helix. (B) Northern blot showing the presence of the wild type (WT) and absence of the two mutants (C318A and U43G) of PSTVd in a systemic leaf. (C) Northern blot showing accumulations of the wild type (WT) and two mutants (C318A and U43G) of PSTVd in an inoculated leaf. (D)In situ hybridization localizing PSTVd mutant U43G in the mesophyll (MS) and bundle sheath (BS) cells but not in the phloem (Ph). (E) Superimposition of a U/C water-inserted helix on a canonical A/U helix with matching flanking base pairs in stereo views, as visualized from the side (upper panel) and top (lower panel) of the helix. The U/C opens up for interaction with a water-molecule (orange sphere). (F) Base pairs corresponding to those illustrated in (E). The orange-colored base pairs are G/C (top), U/C (middle), and C/G (bottom), with overall sequence GUC…GCC. The blue-colored base pairs are G/C (top), A/U (middle), and C/G (bottom), with overall sequence GAC…GUC. (Images B–F are reproduced from Zhong et al. (2007) with permission from Nature Publishing Group). Scale bars, 10 μm.

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To gain insights into potential mechanisms of U43/C318 function, a tertiary structural model for this motif was inferred by comparisons with X-ray crystal structures of similar motifs in rRNAs. This model predicts cis Watson-Crick/Watson-Crick base pairing with water insertion for U43/C318 (Figure 3). Analyses of all other possible base pair combinations (a total of 15) to replace the U/C pair showed that all combinations that maintain trafficking function have a distorted local helix by water insertion, whereas all combinations that form canonical cis Watson-Crick/Watson-Crick base pairs (i.e., without water insertion) lose trafficking (and sometimes replication) functions. Given that the U/C and similar motifs in rRNAs serve as a protein-binding site, it is possible that the PSTVd U/C motif interacts with a host factor(s) to potentiate viriod RNA trafficking (Zhong et al. 2007).

Genomic Map of PSTVd Trafficking Motifs

  1. Top of page
  2. Abstract
  3. Introduction
  4. Cell-to-Cell and Long-distance Trafficking of RNA Functions in Multiple Processes
  5. Viroid Infection: a Model System to Investigate the Regulation of Systemic RNA Trafficking
  6. Viroids Traffic through Plasmodesmata and the Phloem
  7. Selective Trafficking of Viroids into Shoot Apical Organs
  8. Presence of a Viroid Motif can Mediate Unidirectional Trafficking across a Cellular Boundary
  9. A Viroid Motif Mediates Bundle Sheath-to-Phloem Trafficking
  10. Genomic Map of PSTVd Trafficking Motifs
  11. Diversity in Viroid Motifs Controlling RNA Trafficking Reveals Complexity in Host Machinery
  12. Future Directions of Research
  13. Agricultural Implications of Systemic RNA Trafficking
  14. Acknowledgements
  15. References

Following the successful identification of the PSTVd motifs for trafficking from the bundle sheath into mesophyll and phloem, respectively, Zhong et al. (2008) used a loss-of-function approach to conduct a genome-wide search for additional PSTVd motifs involved in systemic trafficking as well as replication. Some 25 mutants were generated, each carrying an obliterated loop by deletions/base substitutions that were predicted not to affect the overall RNA secondary structure. Each viroid mutant was then tested for replication in single cells (protoplasts) and trafficking in the host plant. This analysis identified multiple loops in the PSTVd secondary structure that are essential for systemic trafficking. These findings, together with previous work (Zhong et al. 2007), yielded a genomic map of PSTVd trafficking (and replication) motifs for infection in N. benthamiana (Zhong et al. 2008) (Figure 2; the replication motifs are not shown for simplicity).

Based on these mapping experiments it is clear that additional studies should be conducted to determine: (i) the specific cellular boundary at which each of the trafficking motifs functions; (ii) whether one or more motif is responsible for trafficking through a particular boundary; and (iii) the tertiary structures of these motifs. In addition, such trafficking motifs and the corresponding mutants also provide invaluable tools to assist in the biochemical and functional characterization of the cognate host cellular factors.

Diversity in Viroid Motifs Controlling RNA Trafficking Reveals Complexity in Host Machinery

  1. Top of page
  2. Abstract
  3. Introduction
  4. Cell-to-Cell and Long-distance Trafficking of RNA Functions in Multiple Processes
  5. Viroid Infection: a Model System to Investigate the Regulation of Systemic RNA Trafficking
  6. Viroids Traffic through Plasmodesmata and the Phloem
  7. Selective Trafficking of Viroids into Shoot Apical Organs
  8. Presence of a Viroid Motif can Mediate Unidirectional Trafficking across a Cellular Boundary
  9. A Viroid Motif Mediates Bundle Sheath-to-Phloem Trafficking
  10. Genomic Map of PSTVd Trafficking Motifs
  11. Diversity in Viroid Motifs Controlling RNA Trafficking Reveals Complexity in Host Machinery
  12. Future Directions of Research
  13. Agricultural Implications of Systemic RNA Trafficking
  14. Acknowledgements
  15. References

The identification of motifs in the PSTVd genome that regulate RNA trafficking and comparative analysis with other viroids demonstrates the likely complexity in the host machinery that has evolved to mediate the trafficking of RNA in the plant kingdom. Such complexity likely has relevance for studying trafficking mechanisms of viral and cellular RNAs. This level of complexity can be examined from two broad perspectives.

First, the identification of multiple trafficking motifs in PSTVd allows comparative analyses to deduce, in a qualitative manner, the extent of conservation of these motifs among viroids. A number of motifs are indeed conserved in some closely related viroids, but most are unique to PSTVd (Zhong et al. 2007, 2008). As an example shown in Figure 2, three out of 11 PSTVd motifs involved in RNA trafficking are present in the Citrus exocortis viroid (CEVd) genome. Furthermore, none of the identified PSTVd motifs were found to have counterparts in the Hop stunt viroid (HSVd). Whether the conserved motifs function similarly in the trafficking of individual viroid species in their hosts remains to be tested. Experimental identification of motifs essential for different viroid RNA trafficking will allow a more comprehensive comparative analysis. Viroids in the family Avsunviroidae differ in sequences and secondary structures from those in the family Pospiviroidae (see Figure 2 for a comparison between ASBVd and the other viroids) and structural motifs involved in trafficking from this family have yet to be identified. If we assume that, similar to PSTVd, other viroids will have multiple motifs involved in mediating systemic trafficking, the enormous diversity in trafficking mechanisms becomes all too apparent. Experimental exploration of this diversity is technically feasible with the established protocols (Zhong et al. 2007, 2008). If we also assume that RNA motif-host cellular factor interactions are a general rule to dictate trafficking, we can postulate the involvement of numerous cellular proteins in viroid trafficking and, further, the tremendous repertoire of trafficking mechanisms in the plant kingdom.

Second, certain viroids can infect the same plant species, yet they appear to have very limited similarities in terms of sequence or structural motifs. One example is that of PSTVd and HSVd, which differ significantly in sequence and secondary structural details (Figure 2), yet both infect systemically N. benthamiana (Hu et al. 1997; Gómez and Pallás 2007). It is possible that HSVd uses a different set of motifs to interact with host cellular factors to mediate systemic trafficking in this plant. Alternatively, the specific sets of motifs in these two viroids may well interact with a common set of host factors associated with cell-to-cell trafficking of RNA (Figure 4). It is also possible that these viroids traffic via mechanisms more complicated than a simple motif-protein interaction mode. Further adding to this complexity is the fact that the same viroid can infect different host plants. For instance, PSTVd can infect N. benthamiana and tomato, while HSVd can infect cucumber (Gómez and Pallás 2001; Owens et al. 2001) as well as N. benthamiana. Clearly, the question is whether a particular viroid uses the same or different motifs to direct trafficking in different plant hosts.

image

Figure 4. Schematic showing two viroids that use distinct motifs to traffic from the upper epidermis (Ep) into palisade parenchyma (Pa), spongy mesopyll (Sp), bundle sheath (BS) cells and the phloem (Ph). The different motifs used for trafficking across each cellular boundary and for trafficking into the phloem are represented by red and blue colors located at different positions along the viroid RNAs. Trafficking motifs for crossing other cellular boundaries are not drawn. These hypothetical examples illustrate the diversity of viroid trafficking motifs, which presumably interact with different cellular factors (not depicted). The principles of this model may also apply to the control of viral and plant RNA trafficking. Xy, xylem.

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Here, we should point out that our analysis of motif conservation and diversity is merely based on comparisons of sequence/secondary structural features. It is important to bear in mind that, in RNA, different nucleotides may interact to form a similar or identical tertiary structure having the same function, as has been shown for two PSTVd motifs and their variants (Zhong et al. 2006, 2007). One example is shown in Figure 2 for the PSTVd U43/C318 motif and the equivalent C43/C328 motif in CEVd. Therefore, it will be essential to carry out experimental and structural studies in order to gain greater insights into the diversity of RNA motifs that have evolved to mediate trafficking within the body of the plant. Nonetheless, this analysis of a few specific examples suffices to show that our current knowledge of viroid trafficking has merely scratched the surface of a vast hidden universe of biological principles regulating RNA trafficking. Identification of the trafficking motifs in additional viroid species over a wider range of plant hosts will provide important insights into the principles governing RNA movement within the plant kingdom.

Future Directions of Research

  1. Top of page
  2. Abstract
  3. Introduction
  4. Cell-to-Cell and Long-distance Trafficking of RNA Functions in Multiple Processes
  5. Viroid Infection: a Model System to Investigate the Regulation of Systemic RNA Trafficking
  6. Viroids Traffic through Plasmodesmata and the Phloem
  7. Selective Trafficking of Viroids into Shoot Apical Organs
  8. Presence of a Viroid Motif can Mediate Unidirectional Trafficking across a Cellular Boundary
  9. A Viroid Motif Mediates Bundle Sheath-to-Phloem Trafficking
  10. Genomic Map of PSTVd Trafficking Motifs
  11. Diversity in Viroid Motifs Controlling RNA Trafficking Reveals Complexity in Host Machinery
  12. Future Directions of Research
  13. Agricultural Implications of Systemic RNA Trafficking
  14. Acknowledgements
  15. References

It is now well established that plants evolved mechanisms to allow for the cell-to-cell and long-distance transport of RNA species. The significance of the research findings that have laid this foundation is that, in the plant kingdom, RNA can function as a non-cell-autonomous agent. Furthermore, these findings clearly reveal that there is still a great deal to be discovered in terms of the wide range of pathways upon which RNA appears to move. In this regard, the importance for individual viroid species to use unique motifs for trafficking in different hosts may be uncertain when they are analyzed individually, but becomes compelling when they are considered together in the context of addressing the evolution of complex trafficking mechanisms in plants. Therefore, studying the conserved and non-conserved mechanisms for viroid trafficking should yield important insights into plant evolution as well as the co-evolution of plant-infectious agent interactions.

To understand the mechanisms of viroid trafficking requires knowledge of both the trafficking motifs and their cognate cellular interacting factors. While we have made significant progress in the identification of the trafficking motifs and will be able to continue this trend with the established methods, we expressly need to develop effective approaches to characterize the host interacting factors. Genetic identification of such factors is currently not feasible because all known viroid hosts are not amenable to efficient mutagenesis screening. Biochemical identification of viroid-interacting proteins, followed by functional studies using targeted gene knockout or knockdown strategies will likely remain a major approach in the perceivable future (Takeda and Ding 2009).

Biochemical approaches have identified a number of phloem mobile proteins that interact with various viroids, including the cucumber phloem lectin, CsPP2 that interacts with HSVd (Gómez and Pallás 2001, 2004; Owens et al. 2001) and the melon phloem lectin, CmmLec17 that interacts with ASBVd (Gómez et al. 2005). To provide conclusive evidence for the role of these proteins in viroid trafficking requires a demonstration that the knockout or knockdown of each protein negatively impacts the systemic movement of the viroid in question. The availability of a series of Apple latent spherical virus vectors enabling efficient RNA interference-mediated gene silencing in cucumber and other cucurbits (Igarashi et al. 2009) may greatly facilitate such studies. In another development, Ham et al. (2009) isolated a phloem-mobile ribonucleoprotein complex from pumpkin that mediates in the selective trafficking of specific mRNA molecules. It will be of great interest to test whether this complex, or some component(s) of this complex, plays a role in viroid trafficking.

In order to achieve a comprehensive picture of RNA trafficking mechanisms and functions in plants, we eventually must integrate findings from studies on viroid, viral and plant RNA species. We need to know what trafficking motifs or higher level of structural features are common and distinct among all trafficking RNAs and what cellular factors are shared or are unique to each mobile RNA species. Besides elucidating how systemic RNA trafficking integrates cellular processes at the whole plant level (Figure 1), we will ultimately need to map out the evolutionary patterns of the RNA trafficking machinery by incorporating phylogenetic analyses. This may sound like a daunting task, but will certainly be a motivating force behind our unwavering efforts to pursue one of the big secrets of life.

Agricultural Implications of Systemic RNA Trafficking

  1. Top of page
  2. Abstract
  3. Introduction
  4. Cell-to-Cell and Long-distance Trafficking of RNA Functions in Multiple Processes
  5. Viroid Infection: a Model System to Investigate the Regulation of Systemic RNA Trafficking
  6. Viroids Traffic through Plasmodesmata and the Phloem
  7. Selective Trafficking of Viroids into Shoot Apical Organs
  8. Presence of a Viroid Motif can Mediate Unidirectional Trafficking across a Cellular Boundary
  9. A Viroid Motif Mediates Bundle Sheath-to-Phloem Trafficking
  10. Genomic Map of PSTVd Trafficking Motifs
  11. Diversity in Viroid Motifs Controlling RNA Trafficking Reveals Complexity in Host Machinery
  12. Future Directions of Research
  13. Agricultural Implications of Systemic RNA Trafficking
  14. Acknowledgements
  15. References

In addition to its importance to facilitate discoveries of fundamental principles underlying gene regulation and pathogen infection at the whole plant level, knowledge of the molecular mechanisms controlling cell-to-cell and long-distance trafficking of RNA has practical implications in many aspects of agriculture in long terms. Targeted modifications of RNA trafficking pathways may create new patterns of plant development and physiology for enhanced quantity and quality of plant products as well as resistance to pathogens. For instance, it may be possible to boost potato tuber production via enhanced long-distance trafficking of StBEL5 mRNA from the shoot to stolon tips (Banerjee et al. 2006). These improvements in plant performance are vital to the long-term sustainability of food security, biofuel production and environment protection. Furthermore, an improved understanding of the mechanisms of cellular, viral and viroid RNA trafficking may enable development of novel vectors for producing specific nutritional and biomedical products in plants. For instance, technologies to enhance viral spread and to subdue systemic RNA silencing may enable mass production of a useful protein encoded by a modified viral vector in an infected plant. The nuclear and chloroplastic localization of viroids may also be exploited to produce artificial miRNA/siRNAs from modified viroids that can spread systemically for manipulating the expression of nuclear or chloroplastic genes for crop improvements.

(Co-Editor: William J. Lucas)

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Cell-to-Cell and Long-distance Trafficking of RNA Functions in Multiple Processes
  5. Viroid Infection: a Model System to Investigate the Regulation of Systemic RNA Trafficking
  6. Viroids Traffic through Plasmodesmata and the Phloem
  7. Selective Trafficking of Viroids into Shoot Apical Organs
  8. Presence of a Viroid Motif can Mediate Unidirectional Trafficking across a Cellular Boundary
  9. A Viroid Motif Mediates Bundle Sheath-to-Phloem Trafficking
  10. Genomic Map of PSTVd Trafficking Motifs
  11. Diversity in Viroid Motifs Controlling RNA Trafficking Reveals Complexity in Host Machinery
  12. Future Directions of Research
  13. Agricultural Implications of Systemic RNA Trafficking
  14. Acknowledgements
  15. References