Viroid RNA Research Articles

Resistance of the CRISPR-Cas13a Gene-Editing System to Potato Spindle Tuber Viroid Infection in Tomato and Nicotiana benthamiana.

Gene-editing technology, specifically the CRISPR-Cas13a system, has shown promise in breeding plants resistant to RNA viruses. This system targets RNA and, theoretically, can also combat RNA-based viroids. To test this, the CRISPR-Cas13a system was introduced into tomato plants via transient expression and into Nicotiana benthamiana through transgenic methods, using CRISPR RNAs (crRNAs) targeting the conserved regions of both sense and antisense genomes of potato spindle tuber viroid (PSTVd). In tomato plants, the expression of CRISPR-Cas13a and crRNAs substantially reduced PSTVd accumulation and alleviated disease symptoms. In transgenic N. benthamiana plants, the PSTVd levels were lower as compared to wild-type plants. Several effective crRNAs targeting the PSTVd genomic RNA were also identified. These results demonstrate that the CRISPR-Cas13a system can effectively target and combat viroid RNAs, despite their compact structures.

Composting and fermentation: mitigating hop latent viroid infection risk in hop residues

Hop cultivation, integral to the brewing industry, faces challenges from viroids, especially the citrus bark cracking viroid (CBCVd) but also the hop latent viroid (HLVd) influences hop cone quality. We focused on the degradation kinetics of HLVd thereby covering compost, silage, and digestate made from hop residues. In this study, HLVd serves as a model for understanding CBCVd, which causes significant stunting and yield losses in European hop crops. Composting experiments revealed that although composting significantly lowers HLVd levels, complete degradation within 7 weeks is not guaranteed, with loose compost showing a more rapid reduction than compacted variants. Infectivity experiments conducted using inocula obtained from HLVd-infected hop plant residues exposed to composting, ensiling, and biogas digestate did not result in the transmission of HLVd to viroid-free plants. Also extracting and analyzing the soil-root mixture of plants inoculated with HLVd-infected hop residues did not show evidence for viroid persistence. Degradation experiments further differentiated between the physiochemical and biological influences on viroid and viroid-like random RNA stability, showing that higher temperatures of 50 °C enhance degradation over 40 °C, and pH levels of 5 or 7 are slowing degradation. In contrast deionized water or a pH of 4 or 9 enhances viroid degradation. Adding extracts from digestate accelerated the process indicating a role of biological activity. Interestingly, a viroid-like random RNA with similar physiochemical properties, showed to degrade faster compared to HLVd, suggesting high robustness of the actual viroid secondary structure. These findings offer valuable insights into managing HLVd in hops and potentially other crops, highlighting effective strategies to mitigate viroid spread, and contributing to broader understanding of RNA degradation in agriculture.

RNA three-dimensional structure drives the sequence organization of potato spindle tuber viroid quasispecies.

RNA viruses and viroids exist and evolve as quasispecies due to error-prone replication. Quasispecies consist of a few dominant master sequences alongside numerous variants that contribute to genetic diversity. Upon environmental changes, certain variants within quasispecies have the potential to become the dominant sequences, leading to the emergence of novel infectious strains. However, the emergence of new infectious variants remains unpredictable. Using mutant pools prepared by saturation mutagenesis of selected stem and loop regions, our study of potato spindle tuber viroid (PSTVd) demonstrates that mutants forming local three-dimensional (3D) structures similar to the wild type (WT) are more likely to accumulate in PSTVd quasispecies. The selection mechanisms underlying this biased accumulation are likely associated with cell-to-cell movement and long-distance trafficking. Moreover, certain trafficking-defective PSTVd mutants can be spread by functional sister genomes in the quasispecies. Our study reveals that the RNA 3D structure of stems and loops constrains the evolution of viroid quasispecies. Mutants with a structure similar to WT have a higher likelihood of being maintained within the quasispecies and can potentially give rise to novel infectious variants. These findings emphasize the potential of targeting RNA 3D structure as a more robust approach to defend against viroid infections.

Exogenous Application of dsRNA for Protection against Tomato Leaf Curl New Delhi Virus.

Tomato leaf curl New Delhi virus (ToLCNDV) is an emerging plant pathogen, fast spreading in Asian and Mediterranean regions, and is considered the most harmful geminivirus of cucurbits in the Mediterranean. ToLCNDV infects several plant and crop species from a range of families, including Solanaceae, Cucurbitaceae, Fabaceae, Malvaceae and Euphorbiaceae. Up to now, protection from ToLCNDV infection has been achieved mainly by RNAi-mediated transgenic resistance, and non-transgenic fast-developing approaches are an urgent need. Plant protection by the delivery of dsRNAs homologous to a pathogen target sequence is an RNA interference-based biotechnological approach that avoids cultivating transgenic plants and has been already shown effective against RNA viruses and viroids. However, the efficacy of this approach against DNA viruses, particularly Geminiviridae family, is still under study. Here, the protection induced by exogenous application of a chimeric dsRNA targeting all the coding regions of the ToLCNDV DNA-A was evaluated in zucchini, an important crop strongly affected by this virus. A reduction in the number of infected plants and a delay in symptoms appearance, associated with a tendency of reduction in the viral titer, was observed in the plants treated with the chimeric dsRNA, indicating that the treatment is effective against geminiviruses but requires further optimization. Limits of RNAi-based vaccinations against geminiviruses and possible causes are discussed.

Cell-cell communication and initial population composition shape the structure of potato spindle tuber viroid quasispecies.

RNA viruses and viroids replicate with high mutation rates, forming quasispecies, population of variants centered around dominant sequences. The mechanisms governing quasispecies remain unclear. Plasmodesmata regulate viroid movement and were hypothesized to impact viroid quasispecies. Here, we sequenced the progeny of potato spindle tuber viroid intermediate (PSTVd-I) strain from mature guard cells lacking plasmodesmal connections and from in vitro-cultivated mesophyll cell protoplasts from systemic leaves of early-infected tomato (Solanum lycopersicum) plants. Remarkably, more variants accumulated in guard cells compared to whole leaves. Similarly, after extended cell culture, we observed more variants in cultivated mesophyll protoplasts. Coinfection and single-cell sequencing experiments demonstrated that the same plant cell can be infected multiple times by the same or different PSTVd sequences. To study the impact of initial population composition on PSTVd-I quasispecies, we conducted coinfections with PSTVd-I and variants. Two inoculum ratios (10:1 or 1:10) established quasispecies with or without PSTVd-I as the master sequence. In the absence of the master sequence, the percentage of novel variants initially increased. Moreover, a 1:1 PSTVd-I/variant RNA ratio resulted in PSTVd-I dominating (>50%), while the variants reached 20%. After PSTVd-I-only infection, the variants reached around 10%, while after variant-only infection, the variants were significantly more than 10%. These results emphasize the role of cell-to-cell communication and initial population composition in shaping PSTVd quasispecies.

The Secondary Structure of Potato Spindle Tuber Viroid Determines Its Infectivity in Nicotiana benthamiana.

The function of RNAs is determined by their structure. However, studying the relationship between RNA structure and function often requires altering RNA sequences to modify the structures, which leads to the neglect of the importance of RNA sequences themselves. In our research, we utilized potato spindle tuber viroid (PSTVd), a circular-form non-coding infectious RNA, as a model with which to investigate the role of a specific rod-like structure in RNA function. By generating linear RNA transcripts with different start sites, we established 12 PSTVd forms with different secondary structures while maintaining the same sequence. The RNA secondary structures were predicted using the mfold tool and validated through native PAGE gel electrophoresis after in vitro RNA folding. Analysis using plant infection assays revealed that the formation of a correct rod-like structure is crucial for the successful infection of PSTVd. Interestingly, the inability of PSTVd forms with non-rod-like structures to infect plants could be partially compensated by increasing the amount of linear viroid RNA transcripts, suggesting the existence of additional RNA secondary structures, such as the correct rod-like structure, alongside the dominant structure in the RNA inoculum of these forms. Our study demonstrates the critical role of RNA secondary structures in determining the function of infectious RNAs.

Diagnosis of Peach latent mosaic viroid (PLMVd) and Hop stunt viroid (HSVd) by RT-PCR using different extraction protocols

RT-PCR method was performed using four different nucleic acid extraction methods to identify viroids; peach latent mosaic viroid (PLMVd) and hop stunt viroid (HSVd) which causes serious damage to stone fruits. Leaf samples were collected from fruit orchards showing viroid-like symptoms in İzmir province. Silica capture, citric buffer, lithium chloride, and ames buffer methods were used to extract total nucleic acids. The four extraction methods were compared using samples collected during the vegetation period from naturally infected trees (plum, apricot, and peach). They were evaluated with RT-PCR tests. In 64 stone fruit tree samples, only the silica capture method gave reliable and accurate results in RT-PCR molecular tests for the detection of PLMVd and HSVd. The other wielded nucleic acid extraction methods were found to be ineffective for the isolation of the viroid RNAs.

Small Heat Shock Protein (sHsp22.98) from Trialeurodes vaporariorum Plays Important Role in Apple Scar Skin Viroid Transmission.

Trialeurodes vaporariorum, commonly known as the greenhouse whitefly, severely infests important crops and serves as a vector for apple scar skin viroid (ASSVd). This vector-mediated transmission may cause the spread of infection to other herbaceous crops. For effective management of ASSVd, it is important to explore the whitefly's proteins, which interact with ASSVd RNA and are thereby involved in its transmission. In this study, it was found that a small heat shock protein (sHsp) from T. vaporariorum, which is expressed under stress, binds to ASSVd RNA. The sHsp gene is 606 bp in length and encodes for 202 amino acids, with a molecular weight of 22.98 kDa and an isoelectric point of 8.95. Intermolecular interaction was confirmed through in silico analysis, using electrophoretic mobility shift assays (EMSAs) and northwestern assays. The sHsp22.98 protein was found to exist in both monomeric and dimeric forms, and both forms showed strong binding to ASSVd RNA. To investigate the role of sHsp22.98 during ASSVd infection, transient silencing of sHsp22.98 was conducted, using a tobacco rattle virus (TRV)-based virus-induced gene silencing system. The sHsp22.98-silenced whiteflies showed an approximate 50% decrease in ASSVd transmission. These results suggest that sHsp22.98 from T. vaporariorum is associated with viroid RNA and plays a significant role in transmission.

Tobacco mosaic virus movement protein complements a Potato spindle tuber viroid RNA mutant impaired for mesophyll entry but not mutants unable to enter the phloem.

Tobacco mosaic virus movement protein (TMV MP) is essential for virus spread between cells. To accomplish its task, TMV MP binds viral RNA, interacts with components of the cytoskeleton, and increases the size exclusion limit (SEL) of plasmodesmata. Plasmodesmata are gated intercellular channels that allow passage of small molecules and macromolecules, including RNA and protein, between plant cells. Moreover, plasmodesmata are diverse and those connecting different cell types appear to have unique mechanisms to regulate macromolecular trafficking, which likely contributes to the establishment of distinct cell boundaries. Consequently, TMV MP might be competent to mediate RNA transport through some but not all plasmodesmal gates. Due to a lack of viral mutants defective for movement between specific cell types, the ability of TMV MP in this regard is incompletely understood. In contrast, a number of trafficking impaired Potato spindle tuber viroid (PSTVd) mutants have been identified. PSTVd is a systemically infectious non-coding RNA that nevertheless can perform all functions required for replication as well as cell-to-cell and systemic spread. Previous studies have shown that PSTVd employs different structure and sequence elements to move between diverse cell types in host plants, and mutants defective for transport between specific cell types have been identified. Therefore, PSTVd may serve as a tool to analyze the functions of MPs of viral and cellular origin. To probe the RNA transport activity of TMV MP, transgenic plants expressing the protein were inoculated with PSTVd mutants. Remarkably, TMV MP complemented a PSTVd mutant defective for mesophyll entry but could not support two mutants impaired for phloem entry, suggesting it fails to productively interface with plasmodesmata at the phloem boundary and that additional viral and host factors may be required. Consistent with this idea, TMV co-infection, but not the combination of MP and coat protein (CP) expression, was able to complement one of the phloem entry mutants. These observations suggest that phloem loading is a critical impediment to establishing systemic infection that could involve the entire ensemble of TMV proteins. They also demonstrate a novel strategy for analysis of MPs.

Extreme resilience of avocado sunblotch viroid RNA in sampled avocado leaves and fruit

Extreme resilience of avocado sunblotch viroid RNA in sampled avocado leaves and fruit

On the early identification and characterization of pear blister canker viroid, apple dimple fruit viroid, peach latent mosaic viroid and chrysanthemum chlorotic mottle viroid

In the 90′s, pear blister canker viroid (PBCVd), apple dimple fruit viroid (ADFVd), peach latent mosaic viroid (PLMVd) and chrysanthemum chlorotic mottle viroid (CChMVd) were identified and characterized in the Ricardo Flores’ laboratory. In these studies, the autonomous replication of these infectious RNAs and their involvement in the elicitation of diseases in their natural hosts were also shown. Their discovery was achieved by classical approaches based on the physical purification of the viroid RNAs from polyacrylamide gels followed by the sequencing of their genomic RNAs and by bioassays to assess their autonomous replication and the fulfillment of Koch's postulates. The molecular characterization of these four viroids, including the study of their sequence variability, contributed to the establishment of the concept of quasispecies for viroids and to the development of reliable molecular diagnostic methods that have facilitated the control of the diseases they caused. Most importantly, some of these viroids became valuable experimental model systems that are still used nowadays to study structural-functional relationships in RNAs and to dissect evolutionary and pathogenic pathways underlying plant-viroid interaction. The differences between early viroid discovery strategies, relying on biological and pathogenic issues, and the current high-throughput sequencing-based approaches, that frequently allow the discovery of new viroids and viroid-like RNAs in symptomless hosts, is also discussed, clarifying why the traditional molecular and biological studies mentioned above are still required to conclusively define the nature of any novel viroid-like RNA.

A Novel Self-Cleaving Viroid-Like RNA Identified in RNA Preparations from a Citrus Tree Is Not Directly Associated with the Plant.

Viroid and viroid-like satellite RNAs are infectious, circular, non-protein coding RNAs reported in plants only so far. Some viroids (family Avsunviroidae) and viroid-like satellite RNAs share self-cleaving activity mediated by hammerhead ribozymes (HHRzs) endowed in both RNA polarity strands. Using a homology-independent method based on the search for conserved structural motifs of HHRzs in reads and contigs from high-throughput sequenced RNAseq libraries, we identified a novel small (550 nt) viroid-like RNA in a library from a Citrus reticulata tree. Such a viroid-like RNA contains a HHRz in both polarity strands. Northern blot hybridization assays showed that circular forms of both polarity strands of this RNA (tentatively named citrus transiently-associated hammerhead viroid-like RNA1 (CtaHVd-LR1)) exist, supporting its replication through a symmetric pathway of the rolling circle mechanism. CtaHVd-LR1 adopts a rod-like conformation and has the typical features of quasispecies. Its HHRzs were shown to be active during transcription and in the absence of any protein. CtaHVd-LR1 was not graft-transmissible, and after its first identification, it was not found again in the original citrus source when repeatedly searched in the following years, suggesting that it was actually not directly associated with the plant. Therefore, the possibility that this novel self-cleaving viroid-like RNA is actually associated with another organism (e.g., a fungus), in turn, transiently associated with citrus plants, is proposed.

A remodeled RNA polymerase II complex catalyzing viroid RNA-templated transcription.

Viroids, a fascinating group of plant pathogens, are subviral agents composed of single-stranded circular noncoding RNAs. It is well-known that nuclear-replicating viroids exploit host DNA-dependent RNA polymerase II (Pol II) activity for transcription from circular RNA genome to minus-strand intermediates, a classic example illustrating the intrinsic RNA-dependent RNA polymerase activity of Pol II. The mechanism for Pol II to accept single-stranded RNAs as templates remains poorly understood. Here, we reconstituted a robust in vitro transcription system and demonstrated that Pol II also accepts minus-strand viroid RNA template to generate plus-strand RNAs. Further, we purified the Pol II complex on RNA templates for nano-liquid chromatography-tandem mass spectrometry analysis and identified a remodeled Pol II missing Rpb4, Rpb5, Rpb6, Rpb7, and Rpb9, contrasting to the canonical 12-subunit Pol II or the 10-subunit Pol II core on DNA templates. Interestingly, the absence of Rpb9, which is responsible for Pol II fidelity, explains the higher mutation rate of viroids in comparison to cellular transcripts. This remodeled Pol II is active for transcription with the aid of TFIIIA-7ZF and appears not to require other canonical general transcription factors (such as TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, and TFIIS), suggesting a distinct mechanism/machinery for viroid RNA-templated transcription. Transcription elongation factors, such as FACT complex, PAF1 complex, and SPT6, were also absent in the reconstituted transcription complex. Further analyses of the critical zinc finger domains in TFIIIA-7ZF revealed the first three zinc finger domains pivotal for RNA template binding. Collectively, our data illustrated a distinct organization of Pol II complex on viroid RNA templates, providing new insights into viroid replication, the evolution of transcription machinery, as well as the mechanism of RNA-templated transcription.

A nuclear import pathway exploited by pathogenic noncoding RNAs.

The prevailing view of intracellular RNA trafficking in eukaryotic cells is that RNAs transcribed in the nucleus either stay in the nucleus or cross the nuclear envelope, entering the cytoplasm for function. However, emerging evidence illustrates that numerous functional RNAs move in the reverse direction, from the cytoplasm to the nucleus. The mechanism underlying RNA nuclear import has not been well elucidated. Viroids are single-stranded circular noncoding RNAs that infect plants. Using Nicotiana benthamiana, tomato (Solanum lycopersicum), and nuclear-replicating viroids as a model, we showed that cellular IMPORTIN ALPHA-4 (IMPa-4) is likely involved in viroid RNA nuclear import, empirically supporting the involvement of Importin-based cellular pathway in RNA nuclear import. We also confirmed the involvement of a cellular protein (viroid RNA-binding protein 1 [VIRP1]) that binds both IMPa-4 and viroids. Moreover, a conserved C-loop in nuclear-replicating viroids serves as a key signal for nuclear import. Disrupting C-loop impairs VIRP1 binding, viroid nuclear accumulation, and infectivity. Further, C-loop exists in a subviral satellite noncoding RNA that relies on VIRP1 for nuclear import. These results advance our understanding of subviral RNA infection and the regulation of RNA nuclear import.

Emerging value of the viroid model in molecular biology and beyond

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.

Adaptation of a filter paper method for RNA template preparation for the detection of avocado sunblotch viroid by reverse transcription qPCR

An easy, rapid and inexpensive method of preparing RNA template for a reverse transcription qPCR assay for avocado sunblotch viroid (ASBVd) is described. This method depends on the principle of reversible binding of viroid RNA to filter paper under different concentrations of monovalent cation. Lysis buffers containing either sodium chloride or lithium chloride were compared, and 1.5 M lithium chloride was shown to be optimal for the adsorption of the viroid RNA to the filter paper. The extraction method was validated using field samples and equivalent yields of viroid RNA were obtained using this method and either a commercial RNA extraction kit or a dsRNA chromatography method. The filter paper method of RNA extraction is ideally suited for the large-scale surveillance for ASBVd.

An Improved Method for the Extraction of Nucleic Acids from Plant Tissue without Grinding to Detect Plant Viruses and Viroids.

Gene amplification techniques such as polymerase chain reaction (PCR) are widely used for the diagnosis of plant diseases caused by viruses and viroids. It is preferable that sample preparation methods for PCR or reverse transcription (RT) PCR are rapid, straightforward, and inexpensive. We previously reported a method for the extraction of nucleic acids without mechanical tissue grinding using a buffer containing potassium ethyl xanthogenate (PEX) to detect viroid RNAs. In the present report, the previous PEX method was improved and simplified. In the simplified PEX (SPEX) method, the process of PEX buffer treatment for plant cell wall disruption is improved to one step of incubation at 80 °C for 10 min, instead of three steps that took more than 26 min at 65 °C in the previous method. Total nucleic acids could be extracted from fresh, frozen, or dried leaves of a cultivar or wild species of tobacco, tomato, citron, hop plants, and pericarps of persimmon fruits by the SPEX method. Several RNA viruses and viroids were successfully detected from the extracted nucleic acids together with an internal mRNA by RT-PCR. The SPEX method may be useful for detecting not only viruses and viroids, but also other plant pathogens.

Detection of Viroids by RT-PCR.

Reverse transcription-polymerase chain reaction (RT-PCR) is an effective method for detecting the presence of viroids in plant tissue. Viroid RNA is converted to cDNA and amplified to detectable levels, making it a fast and useful detection tool, even when the viroid is present at low levels. Methods of viroid detection using conventional RT-PCR are described in this chapter.

Detection of Viroids Using RT-qPCR.

Viroids are the smallest known infectious pathogens. They are nonprotein-encoding, single-stranded, circular, naked RNA molecules that can cause several diseases in economically important crops. With the advent of thermal cyclers incorporating fluorescent detection, reverse transcription coupled to the quantitative polymerase chain reaction (RT-qPCR) has transformed the way the viroids are detected. The method involves using sequence-specific primers that anneal to the viroid RNA of interest. The viroid RNA serves as a template during reverse transcription, in which the enzyme reverse transcriptase generates a cDNA copy of a portion of the target RNA molecule. After first-strand cDNA synthesis, RNA template from cDNA:RNA hybrid molecule is removed by digestion with RNase H to improve the sensitivity of PCR step. This cDNA is then be used as a template for amplification of viroid sequence in PCR.

Extraction and Purification of Viroids from Herbaceous Hosts.

Protocols for extraction and purification of viroid RNAs from the tissues of infected herbaceous plant hosts are numerous. They range from lengthy, traditional protocols that require large amounts of starting tissue and take several days to perform to those based on column chromatography which is more efficient and can be performed with smaller amounts of infected tissue. The goal of all protocols is to enrich for RNA fractions that contain viroid RNAs, and the RNA extraction procedure is chosen and adjusted for the downstream method used for detection and characterization. Removal of inhibitors/impurities is generally not an issue for herbaceous hosts unless they contain and inordinate amounts of polysaccharides, tannins, and phenols. Subsequent purification of viroid circular and linear RNAs is performed using denaturing polyacrylamide gel electrophoresis. In this chapter, a specific method routinely used for viroid purification from herbaceous hosts and problems that may be encountered is described and is intended as a reference for beginners in the field.