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Published in final edited form as: Virus Res. 2022 Mar 6;313:198730. doi: 10.1016/j.virusres.2022.198730

Emerging value of the viroid model in molecular biology and beyond

Junfei Ma a,1, Shachinthaka D Dissanayaka Mudiyanselage a,1, Ying Wang a,*
PMCID: PMC8976779  NIHMSID: NIHMS1787764  PMID: 35263622

Abstract

Viroids are single-stranded circular noncoding RNAs that infect plants. Research in the past five decades has deciphered the viroid genome structures, viroid replication cycles, numerous host factors for viroid infection, viroid motifs for intracellular and intercellular trafficking, interactions with host defense machinery, etc. In this review, we mainly focus on some significant questions that remain to be tackled, centered around 1) how the RNA polymerase II machinery performs transcription on RNA templates of nuclear-replicating viroids, 2) how viroid RNAs coordinate multiple structural elements for diverse functions, and 3) how viroid RNAs activate plant immunity. Research on viroids has led to seminal discoveries in biology, and we expect the research directions outlined in this review to continue providing key knowledge inspiring other areas of biology.

Keywords: Viroid, RNA-templated transcription, Pol II, RNA trafficking, Plant immunity

1. Introduction

The first viroid, potato spindle tuber viroid (PSTVd), was discovered and named in 1971 (Diener, 1971; Diener, 2018). Since then, about 40 viroids have been reported and categorized into two families, Pospiviroidae and Avsunviroidae (Di Serio et al., 2018; Di Serio et al., 2021). All the known viroids are circular noncoding RNAs that infect plants (Flores et al., 2016; Navarro et al., 2021; Wang, 2021). Members of the family Pospiviroidae possess rod-shaped genome structures and replicate in the nucleus. Transcription from their circular genome to multimeric (−) intermediates is known to harness DNA-dependent RNA polymerase II (Pol II) (Mühlbach and Sänger, 1979; Rackwitz et al., 1981). An RNA-specific transcription factor (TFIIIA-7ZF) is critical for redirecting Pol II to use the RNA genome of PSTVd, a representative species of Pospiviroidae (Wang et al., 2016). Viroids of the family Avsunviroidae all replicate in chloroplasts and adapt a highly branched portion in their RNA genomes (Flores et al., 2016; Navarro et al., 2021; Wang, 2021). They rely on nuclear-encoded polymerase (NEP) for RNA-templated transcription (Navarro et al., 2000).

Viroids can spread systematically after replication in compatible host plants. Studies on PSTVd structural motifs have identified a series of RNA 3-dimensional (3D) motifs regulating RNA movement across various cellular boundaries (Wang et al., 2018; Zhong et al., 2008). A recent finding also identifies a critical, and likely conserved, RNA 3D motif regulating the nuclear import of viroids of the family Pospiviroidae (Ma et al., 2021). These discoveries support a model that viroids harness their RNA 3D motifs for intracellular, intercellular and systemic trafficking.

While numerous reports described symptomless viroid infection in some hosts (Daros, 2016; Li et al., 2021a; Verhoeven et al., 2013), many viroids are well-known pathogens to economically important crops (Flores et al., 2020; Flores et al., 2016; Navarro et al., 2012). Recent comprehensive transcriptome analyses from multiple groups showed that viroid infection can elicit host immune responses (Kappagantu et al., 2017; Thibaut and Claude, 2018; Zheng et al., 2017), in addition to the well-known viroid interaction with host RNA silencing machinery (Ding, 2009; Navarro et al., 2021). These insights provide a framework to understand the pathogenesis that is caused by foreign RNA alone.

In this review, we mainly discuss some of the emerging questions in plant-viroid interactions and provide testable hypotheses for future investigations, particularly revolving around the composition and mechanism of Pol II machinery on viroid RNA templates, the functional RNA 3D motifs in regulating RNA trafficking in plants, as well as plant defense against viroids from the innate immunity perspective.

2. Viroid RNA-templated transcription in the nucleus

Mounting evidence supports that viroids of the family Pospiviroidae use host Pol II to catalyze the transcription from circular genome to linear multimeric (−) intermediates (Mühlbach and Sänger, 1979; Rackwitz et al., 1981). In contrast to Pol I, Pol III and NEP, Pol II has a specialized carboxy-terminal domain (CTD) that supports co-transcriptional regulation (Bentley, 2014). While Pol II-catalyzed cellular mRNA synthesis is proceeding, a G cap is attached to nascent transcripts and introns are removed. This is feasible because the conserved “YSPTSPS” heptapeptide repeats in the Pol II CTD domain can recruit distinct factors based on the different phosphorylation statuses (Corden et al., 1985; Egloff et al., 2012). Viroids clearly do not exploit the whole set of co-transcription machinery for their own replication, therefore providing opportunities to understand the regulation of transcription on RNA templates with limited co-transcriptional regulation.

2.1. Viroid RNAs do not possess a G cap

The G cap attachment can be a simultaneous process during the transcription of many cellular mRNAs (Bentley, 2014). When nascent transcripts reach a length of ~20-30 nt, these short transcripts become capped near the RNA exit channel of the Pol II complex (Rasmussen and Lis, 1993). The phosphorylation of Ser5 in the heptapeptide repeats is critical to recruiting nuclear capping enzyme and guanine-7-methyltransferase for co-transcriptional capping (Cho et al., 1997; Ho and Shuman, 1999; McCracken et al., 1997).

The capping process coincides with the promoter-proximal pause during the transcription of many metazoan genes (Adelman and Lis, 2012; Lenasi et al., 2011; Rasmussen and Lis, 1993). During this transcriptional pause, the nuclear capping enzyme interacts with the elongation factor SPT4/5. Phosphorylation of SPT4/5 releases pausing to resume productive elongation (Mandal et al., 2004; Pei and Shuman, 2002; Wen and Shatkin, 1999). The promoter-proximal pause serves as a checkpoint for coupling the G cap addition and elongation as well as allows the preparation of permissive DNA templates.

To date, there is no evidence showing that viroid transcripts possess any G cap. In addition, transcriptional pausing has not been clearly demonstrated in the RNA-templated transcription either. These observations prompt the question of whether the phosphorylation codes of the Pol II CTD domain are necessary for RNA-templated transcription. Notably, Pol II-dependent RNA-templated transcription of B2 ncRNA and the human hepatitis delta virus (HDV) genomic/antigenomic RNAs do not possess G cap either (Taylor, 2015; Wagner et al., 2013). The HDAg mRNA from HDV with a defined G cap accounts for an extremely low percentage of total HDV RNAs (Gudima et al., 2000), but it is unclear whether the G cap in HDAg mRNA is added through co-transcriptional activity or other activities such as through cytosolic G cap enzyme. Therefore, the replication of viroids and HDV genomes relies on Pol II but has no known co-transcriptional regulation. Future studies on viroid replication may provide insights into the composition and modus operandi of Pol II complex on RNA templates.

2.2. Splicing and nuclear export

Intron splicing can occur co-transcriptionally or post-transcriptionally (Aslanzadeh et al., 2018; Bentley, 2014; Drexler et al., 2020). The assembly of spliceosomes on nascent transcripts regulates the co-transcriptional splicing (Herzel et al., 2017). Importantly, splicing and nuclear export are coupled via the TREX complex, which is conserved in metazoans and contains both splicing factors and nuclear export factors (Strasser et al., 2002). Hence, splicing is a necessary step for the nuclear export of most cellular mRNAs.

Nuclear-replicating viroids do not harbor any intron, despite their dependence on Pol II for replication. Moreover, unlike the cellular mRNAs that are transported to the cytoplasm directly from their synthesis sites in the nucleoplasm, PSTVd appears to be transported into the nucleolus from the production site before nuclear export (Qi and Ding, 2003). Therefore, viroids probably harness another route for nuclear export. One hypothesis is that viroids can use the 5S rRNA nuclear export pathway through transcription factor IIIA (the full-length protein with nine zinc fingers; TFIIIA-9ZF) (Pieler and Rudt, 1997). Evidence showed that TFIIIA-9ZF interacts with PSTVd in vivo, but the functional significance of this interaction awaits future investigation (Wang et al., 2016). Alternatively, viroid nuclear export may rely on another pathway yet-to-be-elucidated.

2.3. Transcription termination

It is well accepted that cleavage at the poly(A) site (i.e., AAUAAA) leads to the addition of the poly(A) tail to mRNAs and the exonuclease-involved transcription termination (Proudfoot and Brownlee, 1976; Shi and Manley, 2015; Tian and Graber, 2012). However, the transcripts using the AAUAAA signal for termination only account for ~10% in plants (Wu et al., 2011). Recently, a specific histone deacetylase (HDA6) has been shown to regulate the usage of poly(A) sites other than AAUAAA in Arabidopsis, possibly through modifying the epigenetic hallmarks on DNA templates surrounding the AAUAAA sites (Lin et al., 2020). This observation indicates that transcriptional termination may be regulated via multiple means in plants.

The regulation of Pol II termination on RNA templates has not been fully understood, except for the HDV HDAg gene that contains a functional AAUAAA element (Hsieh et al., 1990). Early studies found that the replication intermediates from circular (+) viroid templates have relatively unique size ranges based on viroid species. For example, PSTVd (−) intermediates can be tetramers (Branch et al., 1981; Spiesmacher et al., 1983) or hexamers (Muhlbach et al., 1983); citrus exocortis viroid (CEVd) (−) intermediates are roughly 6-8 times monomer in size (Hutchins et al., 1985); and hop stunt viroid (HSVd) (−) intermediates are dominantly dimers or tetramers (Ishikawa et al., 1984). While it is intuitive to consider the template run-off theme that Pol II leaves templates after several rounds of transcription on circular RNA templates, other possibilities involving regulatory factors cannot be ruled out. The relatively species-specific consistency in the lengths of (−) intermediate implies the existence of regulation over transcription termination when Pol II uses viroid genomic RNA as templates for transcription. Interestingly, reports showed that HSVd interacts with HDA6 directly and affects its cellular function (Castellano et al., 2016). Whether HDA6 is involved in regulating transcription termination on RNA templates may be clarified in future investigations.

3. RNA structure-mediated viroid trafficking

Due to their noncoding nature, viroids rely on their RNA structures to harness host factors for infection. Genome-wide analysis on PSTVd local motifs found that many RNA loop structures are critical for either replication or systemic trafficking (Zhong et al., 2008). Detailed analyses have uncovered the function of those trafficking-related loops in regulating nuclear import and spreading across various cellular boundaries.

3.1. RNA structure-mediated nuclear import

Although it is well known that members of Pospiviroidae enter the nucleus for replication (Ding, 2009), the regulatory mechanism for nuclear imports remains unclear. Recently, we identified a critical C-loop for viroid nuclear imports. C-loop is the binding site for Virp1, a known host factor for viroid infection (Martínez de Alba et al., 2003). Previous studies suggest that Virp1 recognizes at least one of the two RY motifs in PSTVd (Gozmanova et al., 2003). RY motif appears to be conserved in members of Pospiviroidae. We found the PSTVd C-loop partially overlaps with the RY motif closer to the right terminus (Ma et al., 2021). Point mutations in PSTVd C-loop strongly impair PSTVd infectivity, nuclear accumulation, and interaction with Virp1. We also found a C-loop in HSVd, which is not overlapping with the described RY motif therein. Point mutations in HSVd C-loop also strongly impair HSVd infectivity and interaction with Virp1, supporting that C-loop is the bona fide binding site of Virp1 (Ma et al., 2021). C-loop can be found in nearly all, except one, former members of Pospiviroidae (Ma et al., 2021) and even in a satellite RNA of cucumber mosaic virus that relies on Virp1 for nuclear import (Chaturvedi et al., 2014). Altogether, C-loop is probably a conserved signal regulating the nuclear imports of plant subviral RNAs.

3.2. RNA structure-mediated systemic infection

In a simplified view, viroids need to move from leaf epidermis, through palisade mesophyll and spongy mesophyll, to cross bundle sheath and enter phloem for systemic trafficking. Viroids will also need to cross bundle sheath and invade mesophyll and epidermis in systemic leaves (Wang and Ding, 2010). Strikingly, PSTVd possesses at least one RNA motif regulating the trafficking across most of these tissues.

The right terminal loop is critical for movement from epidermis to mesophyll (Wu et al., 2019). Loops 6 and 19 both regulate trafficking from palisade mesophyll to spongy mesophyll (Jiang et al., 2017; Takeda et al., 2011). Loop 7 dictates the phloem entry from bundle sheath (Zhong et al., 2007). A bipartite motif controls phloem exiting to bundle sheath in systemic leaves (Qi et al., 2004). An emerging model from these data outlines that distinct RNA structural motifs contain the necessary information for crossing various checkpoints between diverse tissue types.

3.3. Organizing multiple functional elements in one RNA

The presence of the bipartite motif regulating the bundle sheath exit of PSTVd is intriguing (Qi et al., 2004). It suggests that viroid genomic RNAs will alter the rod-shaped structures to achieve certain functions in plants. Perhaps an alternative possibility to explain the functional redundancy of loop 6 and loop 19 is that they form some sort of bipartite element as well. Another intriguing question is how viroids organize so many elements for diverse functions without interfering with each other. For instance, how do viroids coordinate 1) nuclear import and nuclear export as well as 2) nuclear import and intercellular trafficking? Investigations on these questions may help elucidate the coordination of diverse functions of a single RNA molecule through organizing its structure in the dynamic cellular environment, which may advance the understanding of RNA structure-function relationships to a deeper level.

4. Viroid and host defense

It becomes clear that plant defense against viroid infection relies on both RNA silencing and innate immunity (Flores et al., 2020; Navarro et al., 2021). Viroid infection generates small RNAs (sRNAs) ranging from 20-24 nt in size. These viroid-derived sRNAs (vd-sRNAs) likely play an inhibitory role in viroid replication. Viroid infection also triggers host immune responses that reprogram host gene expression to activate ROS signaling, cell wall fortification, and hormonal pathways related to defense. In general, the activation of immune responses often has impacts on plant signaling and metabolism that lead to cytopathic effects and alterations in morphology.

4.1. Viroid interaction with host RNA silencing machinery

Based on the current model, the replication of viroids will generate double-stranded intermediates that will be cleaved by various Dicer-like proteins (DCLs) in plants (Ding, 2010). Noteworthy is that the PSTVd RNA genome without replication can also be a target of DCLs purified from plants (Itaya et al., 2007). In general, DCL2 and DCL3 synergistically suppress PSTVd infection, whereas DCL4 somehow positively regulates PSTVd replication in Nicotiana benthamiana (Dadami et al., 2013; Katsarou et al., 2016). Viroid-derived sRNAs can be loaded into Argonaute proteins (AGOs) for function (Itaya et al., 2007; Minoia et al., 2014). Specifically, Agrobacterium tubefaciens-mediated transient expression of Arabidopsis AGOs followed by RNA-immunoprecipitation revealed that plant AGO1, AGO2, AGO3, AGO4, AGO5, AGO8, and AGO9 all have the ability to recruit vd-sRNAs. AGO1, AGO2, and AGO3 favor the binding of 21- and 22-nt vd-sRNAs, while AGO4, AGO5, and AGO9 enriched a good portion of 24-nt vd-sRNAs. Interestingly, ectopic expression of AGO1, AGO2, AGO4, or AGO5 attenuated PSTVd titers in infected N. benthamiana, supporting their roles in plant-viroid inetractions (Minoia et al., 2014).

RNA-dependent RNA polymerase 6 (RDR6), a critical player in the RNA silencing pathway, plays a role in preventing PSTVd from invading shoot apical meristem in host plants (i.e., N. benthamiana and tomato) (Di Serio et al., 2010; Naoi et al., 2020). Importantly, RDR6 also modulates HSVd-triggered pathogenicity in plants (Gomez et al., 2008). The detailed mechanism underlying the role of RDR6 in controlling PSTVd tissue tropism and pathogenicity remains to be determined. A recent report also shows that perturbing the expression of RDR1 affects PSTVd infectivity (Li et al., 2021b). However, RDR1 expression remains unchanged in PSTVd- and CEVd-infected plants based on reported RNA-Seq data (Thibaut and Claude, 2018; Zheng et al., 2017), rendering it questionable whether RDR1 is a bona fide defense gene against viroid.

4.2. Viroid interaction with plant innate immunity

Plants generally deploy a two-layer immunity defending various pathogens, namely pathogen-associated molecular patterns (PAMPs)-triggered immunity (PTI) and effector-triggered immunity (ETI) (Jones and Dangl, 2006). PTI functions mainly at cell surface, whereas ETI largely occurs within cells. The orchestrated PTI and ETI activities are essential for plant survival.

The framework that the presence of viroids can trigger host immune responses has been established recently (Kappagantu et al., 2017; Thibaut and Claude, 2018; Zheng et al., 2017). How can plant cells sense the presence of foreign RNAs (i.e., viroids) and activate the innate immune system remains obscure. Previously it was thought that plants utilize PKV (protein kinase, viroid-induced), a double-stranded-RNA-binding protein kinase, to sense viroid RNAs and triggers defense signaling (Hammond and Zhao, 2000; Hammond and Zhao, 2009). However, this PKV appears to be a pseudogene based on comprehensive RNA-Seq analyses (Thibaut and Claude, 2018; Zheng et al., 2017). Viroids may not trigger PTI response because they enter host cells mainly through wounding or, to a lesser extent based on current knowledge, insect vectors (Matsushita et al., 2018). Within the infected plants, viroids move through plasmodesmata (Ding et al., 1997). Therefore, viroids are rarely present on cell surface to elicit PTI. Whether viroids can activate ETI is a puzzle because no known R gene can specifically sense viroids. If viroid does not trigger ETI response, then the immune responses elicited by viroids might be attributable to damage-associated molecular pattern-triggered immunity that was elicited by the emission of cell damage related signal molecules yet-to-be-identified (Boutrot and Zipfel, 2017; Erb and Reymond, 2019; Hou et al., 2019). Interestingly, a recent study showed that PSTVd replication can lead to up-regulation of miR398-regulated production of reactive oxygen species (Fujibayashi et al., 2021; Suzuki et al., 2019), which may link the activity of RNA silencing and innate immunity in defending viroids. It will be critical to elucidate the detailed mechanism underlying the regulation of miR398 and the detailed events along this regulatory cascade in viroid-infected plants. Undoubtedly, the efforts to understand the viroid-triggered immune response will shed light on the mechanism for plants to perceive the presence of foreign RNAs.

5. Concluding remarks

As a peculiar group of subviral agents, viroids can serve as a productive model to tackle basic questions in virology and beyond. PSTVd is the first known natural RNA redirecting a DNA-dependent RNA polymerase (DdRP) for transcription. To date, intrinsic RNA-dependent RNA polymerase (RdRP) activity has been found in many DdRPs (Dissanayaka Mudiyanselage et al., 2018). These observations imply a missing link in the molecular evolution of transcription, where RNA templates emerged first before DNA templates (Lehmann et al., 2007). The viroid model can help dissect the needed factors for redirecting DdRPs to use RNA templates as well as distinguish the differences in regulating efficient transcription on DNA and RNA templates. This line of research is highly related to studies on HDV, which also relies on the host RdRP activity of DdRPs (Taylor, 2015). Viroids also serve as a productive model to delineate RNA structure-function relationships. The function of many PSTVd RNA motifs has been illustrated, and some of them have been studied in detail in terms of structures and cognate factors. It is particularly interesting to expand similar analyses to other viroids, and even RNA viruses, to establish a general view of RNA motif organizations for effective infection. Last but not the least, it is an exciting area to study the interaction between viroids and host innate immunity. RNAs represent a universal component in all pathogens, yet their direct interactions with plant innate immunity are often overlooked. The viroid model can be useful to advance our understanding of plant innate immunity in terms of sensing foreign RNAs, including but not limited to viruses and viroids.

Highlights.

  • This review reasons the emerging value of the viroid model in understanding RNA-templated transcription, RNA structure-function relationship, and plant innate immunity.

  • This review outlines some unresolved yet significant questions in viroid biology and provides testable hypotheses.

Acknowledgements

This review is in dedication to the memory of late professor Dr. Ricardo Flores, a true leader and a caring colleague in the field of viroid research. This work was supported by the National Institutes of Health [1R15GM135893 to YW] and the US National Science Foundation [MCB-1906060 and MCB-2145967 to YW]. We apologize to colleagues whose work was not cited in this review due to the page limit.

Footnotes

Declaration of Competing Interest

The authors declare that they have no conflict of interest.

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