Viroid Structure Research Articles

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.

Phylogenetic analysis and molecular characterization of chrysanthemum chlorotic mottle viroid and chrysanthemum stunt viroid from chrysanthemum in Thailand

AbstractChrysanthemum chlorotic mottle viroid (CChMVd) and chrysanthemum stunt viroid (CSVd) are limiting factors for chrysanthemum cultivation and cause economic losses throughout the world. Chrysanthemum leaves were collected from Chiang Mai and Chiang Rai provinces of Thailand during 2019–2021. Both viroids were detected by using reverse transcription polymerase chain reaction (RT‐PCR) and molecularly characterized. Six CChMVd and five CSVd amplicons were detected, cloned and sequenced. BLAST analysis revealed that CChMVd shared 93%–99% identical and CSVd shared 95%–99% identical with the representative species retrieved from GenBank, Moreover, the nucleotides at the position 82–85 of the tetraloop region were keys for CChMVd clusters resulting to the distinct clade of either symptomatic or non‐symptomatic strains. The phylogenetic analysis of individual viroid represented that six CChMVd variants were closely related to Korea and China variants and clustered in the symptomatic clade of the Asia cluster. For CSVd, five variants in the present study were grouped with CSVd from Dahlia spp. of Japan. Predicted secondary structure of both viroids showed the distribution of sequence variations. The predicted secondary structure showed sequence variants of CChMVd were distributed on most of the secondary structure, whereas sequence variants of CSVd were frequently found in pathogenic, variable and terminal right domains. This study provides phylogenetic relationship of CChMVd and CSVd Thai variants along with their sequence variability.

Conserved Motifs and Domains in Members of Pospiviroidae.

In 1985, Keese and Symons proposed a hypothesis on the sequence and secondary structure of viroids from the family Pospiviroidae: their secondary structure can be subdivided into five structural and functional domains and “viroids have evolved by rearrangement of domains between different viroids infecting the same cell and subsequent mutations within each domain”; this article is one of the most cited in the field of viroids. Employing the pairwise alignment method used by Keese and Symons and in addition to more recent methods, we tried to reproduce the original results and extent them to further members of Pospiviroidae which were unknown in 1985. Indeed, individual members of Pospiviroidae consist of a patchwork of sequence fragments from the family but the lengths of fragments do not point to consistent points of rearrangement, which is in conflict with the original hypothesis of fixed domain borders.

Apple chlorotic fruit spot viroid: a putative new pathogenic viroid on apple characterized by next-generation sequencing

Viroid-like symptoms were observed in 2016 on apple fruits of the cultivar “Ilzer Rose” in southern Burgenland, Austria. Preliminary molecular biological investigations indicated that the symptoms were caused by a new unknown viroid. Therefore, new primers were designed, and the whole genome sequence of the viroid (354 nt) was determined by next-generation amplicon sequencing using the Illumina MiSeq® platform (San Diego, California, USA). The viroid secondary structure has a rod-like conformation and contains conserved regions (the TCR, CCR upper strand, and CCR lower strand) that are characteristic of members of the genus Apscaviroid. Based on our results and the demarcation criteria for viroids, the tentatively named “apple chlorotic fruit spot viroid” should be considered a putative new member of the genus Apscaviroid.

Classification of the Pospiviroidae based on their structural hallmarks.

The simplest known plant pathogens are the viroids. Because of their non-coding single-stranded circular RNA genome, they depend on both their sequence and their structure for both a successful infection and their replication. In the recent years, important progress in the elucidation of their structures was achieved using an adaptation of the selective 2’-hydroxyl acylation analyzed by primer extension (SHAPE) protocol in order to probe viroid structures in solution. Previously, SHAPE has been adapted to elucidate the structures of all of the members of the family Avsunviroidae, as well as those of a few members of the family Pospiviroidae. In this study, with the goal of providing an entire compendium of the secondary structures of the various viroid species, a total of thirteen new Pospiviroidae members were probed in solution using the SHAPE protocol. More specifically, the secondary structures of eleven species for which the genus was previously known were initially elucidated. At this point, considering all of the SHAPE elucidated secondary structures, a classification system for viroids in their respective genera was proposed. On the basis of the structural classification reported here, the probings of both the Grapevine latent viroid and the Dahlia latent viroid provide sound arguments for the determination of their respective genera, which appear to be Apscaviroid and Hostuviroid, respectively. More importantly, this study provides the complete repertoire of the secondary structures, mapped in solution, of all of the accepted viroid species reported thus far. In addition, a classification scheme based on structural hallmarks, an important tool for many biological studies, is proposed.

Dissecting the secondary structure of the circular RNA of a nuclear viroid in vivo: A “naked” rod-like conformation similar but not identical to that observed in vitro

ABSTRACTWith a minimal (250–400 nt), non-protein-coding, circular RNA genome, viroids rely on sequence/structural motifs for replication and colonization of their host plants. These motifs are embedded in a compact secondary structure whose elucidation is crucial to understand how they function. Viroid RNA structure has been tackled in silico with algorithms searching for the conformation of minimal free energy, and in vitro by probing in solution with RNases, dimethyl sulphate and bisulphite, and with selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE), which interrogates the RNA backbone at single-nucleotide resolution. However, in vivo approaches at that resolution have not been assayed. Here, after confirming by 3 termodynamics-based predictions and by in vitro SHAPE that the secondary structure adopted by the infectious monomeric circular (+) RNA of potato spindle tuber viroid (PSTVd) is a rod-like conformation with double-stranded segments flanked by loops, we have probed it in vivo with a SHAPE modification. We provide direct evidence that a similar, but not identical, rod-like conformation exists in PSTVd-infected leaves of Nicotiana benthamiana, verifying the long-standing view that this RNA accumulates in planta as a “naked” form rather than tightly associated with protecting host proteins. However, certain nucleotides of the central conserved region, including some of the loop E involved in key functions such as replication, are more SHAPE-reactive in vitro than in vivo. This difference is most likely due to interactions with proteins mediating some of these functions, or to structural changes promoted by other factors of the in vivo habitat.

Structure and Associated Biological Functions of Viroids.

Mature viroids consist of a noncoding, covalently closed circular RNA that is able to autonomously infect respective host plants. Thus, they must utilize proteins of the host for most biological functions such as replication, processing, transport, and pathogenesis. Therefore, viroids can be regarded as minimal parasites of the host machinery. They have to present to the host machinery the appropriate signals based on either their sequence or their structure. Here, we summarize such sequence and structural features critical for the biological functions of viroids.

Prediction of Secondary Structure of Citrus Viroids Reported from Southern Iran

Abstract Viroids are smallest, single-stranded, circular, highly structured plant pathogenic RNAs that do not code for any protein. Viroids belong to two families, the Avsunviroidae and the Pospiviroidae. Members of the Pospiviroidae family adopt a rod-like secondary structure. In this study the most stable secondary structures of citrus viroid variants that reported from Fars province were drawn. The most stable secondary structures of these viroid variants were a classical rod-like structure and adopted cruciform structure including various additional small hairpins. Comparison of secondary structures of these viroid variants with other viroid variants indicates their highly similarities in the rod-like structures, number of loops and free energies and it’s obvious to result these closest variants of the Pospiviroidae family. HSVd-cit1 and CVd-III-1 differed from under study variants in the stability and number of secondary structure branches. Because of relationship between secondary structure and pathogenicity of viroids, it is supposed that these two variants possibly will have high risk for citrus cultivations.

Comprehensive secondary structure elucidation of four genera of the family Pospiviroidae.

Viroids are small, circular, single stranded RNA molecules that infect plants. Since they are non-coding, their structures play a critical role in their life cycles. To date, little effort has been spend on elucidating viroid structures in solution due to both the experimental difficulties and the time-consuming nature of the methodologies implicated. Recently, the technique of high-throughput selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) was adapted for the probing of the members of family Avsunviroidae, all of whom replicate in the chloroplast and demonstrate ribozyme activity. In the present work, twelve viroid species belonging to four different genera of the family Pospiviroidae, whose members are characterized by the presence of a central conserved region (CCR) and who replicate in nucleus of the host, were probed. Given that the structures of five distinct viroid species from the family Pospiviroidae have been previously reported, an overview of the different structural characteristics for all genera and the beginning of a manual classification of the different viroids based on their structural features are presented here.

Elucidation of the structures of all members of the Avsunviroidae family.

Viroids are small single-stranded RNA pathogens which cause significant damage to plants. As their nucleic acids do not encode for any proteins, they are dependant solely on their structure for their propagation. The elucidation of the secondary structures of viroids has been limited because of the exhaustive and time consuming nature of classic approaches. Here, the method of high-throughput selective 2'-hydroxyl acylation analysed by primer extension (hSHAPE) has been adapted to probe the viroid structure. The data obtained using this method were then used as input for computer-assisted structure prediction using RNA structure software in order to determine the secondary structures of the RNA strands of both (+) and (–) polarities of all Avsunviroidae members, one of the two families of viroids. The resolution of the structures of all of the members of the family provides a global view of the complexity of these RNAs. The structural differences between the two polarities, and any plausible tertiary interactions, were also analysed. Interestingly, the structures of the (+) and (–) strands were found to be different for each viroid species. The structures of the recently isolated grapevine hammerhead viroid-like RNA strands were also solved. This species shares several structural features with the Avsunviroidae family, although its infectious potential remains to be determined.To our knowledge, this article represents the first report of the structural elucidation of a complete family of viroids.

Raman characterization of Avocado Sunblotchviroid and its response to external perturbations and self-cleavage

BackgroundViroids are the smallest pathogens of plants. To date the structural and conformational details of the cleavage of Avocado sunblotch viroid (ASBVd) and the catalytic role of Mg2+ ions in efficient self-cleavage are of crucial interest.ResultsWe report the first Raman characterization of the structure and activity of ASBVd, for plus and minus viroid strands. Both strands exhibit a typical A-type RNA conformation with an ordered double-helical content and a C3′-endo/anti sugar pucker configuration, although small but specific differences are found in the sugar puckering and base-stacking regions. The ASBVd(-) is shown to self-cleave 3.5 times more actively than ASBVd(+). Deuteration and temperature increase perturb differently the double-helical content and the phosphodiester conformation, as revealed by corresponding characteristic Raman spectral changes. Our data suggest that the structure rigidity and stability are higher and the D2O accessibility to H-bonding network is lower for ASBVd(+) than for ASBVd(-). Remarkably, the Mg2+-activated self-cleavage of the viroid does not induce any significant alterations of the secondary viroid structure, as evidenced from the absence of intensity changes of Raman marker bands that, however exhibit small but noticeable frequency downshifts suggesting several minor changes in phosphodioxy, internal loops and hairpins of the cleaved viroids.ConclusionsOur results demonstrate the sensitivity of Raman spectroscopy in monitoring structural and conformational changes of the viroid and constitute the basis for further studies of its interactions with therapeutic agents and cell membranes.

Evolution of microRNA (miRNA) Structure and Function in Plants and Animals: Relevance to Aging and Disease.

Our concepts of the structure and function of sncRNA, including miRNA and vsRNA and their regulatory mechanisms in development and aging, and how they fit into the fascinating realm of dysfunctional RNA metabolism in diseases of plants and animals, and more specifically human neurological diseases such as AD, continues to evolve. Similarities in the molecular-genetics of miRNA and viroid generation, structure and function suggest that some very basal and conserved mechanisms of disease processes have been preserved across the evolution of multiple and diverse plant and animal species. Indeed, pathogenically up-regulated miRNAs and sncRNAs can be considered as an ancient, epigenetic system used to down-regulate specific mRNAs and their expression, and this elegant regulatory system has been maintained across a plant and animal species divergence that occurred some 1.5 billion years ago [13,14,30,31,69]. Significantly up-regulated miRNAs in neurodegenerative disorders such as AD may help explain the large number of brain gene mRNAs, essential for homeostatic brain activity, that are observed to be progressively down-regulated in AD susceptible anatomical regions as AD advances [62,70,74–77]. In the case of AD, and perhaps other progressive age-related neurological diseases, miRNA- and/or anti-miRNA-directed therapeutics represent an obvious choice for future pharmacological treatment strategies, and these would enable the diseased CNS to regain more normalized and homeostatic gene expression functions.

In vitro system for studying interactions between Citrus exocortis viroid and Gynura aurantiaca (Blume) DC. metabolism and growing conditions

Citrus exocortis viroid (CEVd) is an economically important plant pathogen, which infects a broad range of hosts. In order to investigate complex interactions among viroid, host-growing conditions, and plant secondary metabolism, we setup an in vitro system for the cultivation of CEVd-infected and viroid-free Gynura aurantiaca (Blume) DC. shoots. Both basal Murashige and Skoog (MS) medium and MS medium supplemented with cytokinines supported the growth of shoots, but viroid-free shoots performed better than infected ones. The addition of cytokinines induced an increase in the concentration of purple-reddish pigment, cyanidin tetra-glucoside (GAA), which did not depend on the presence of viroid. The differences in single strand conformation polymorphism patterns between CEVd sequence variants obtained from plants grown on medium supplemented with cytokinines and those obtained from plants grown on basal MS medium or in the greenhouse, indicated that cytokinines may influence not only the plant metabolism, but also the viroid population structure.

Comprehensive diversity analysis of viroids infecting grapevine in China and Japan

To date, several viroid species have been shown to infect grapevine, including Hop stunt viroid (HpSVd), Citrus exocortis viroid (CEVd), Australian grapevine viroid (AGVd), Grapevine yellow speckle viroid-1 (GYSVd-1), Grapevine yellow speckle viroid-2 (GYSVd-2) and a tentative new species, Grapevine yellow speckle viroid-3 (GYSVd-3). Here, we identified and analyzed the distribution, genetic diversity, and molecular properties of viroids infecting grapevine cultivated in China and Japan, including old grapevines. The analysis showed that all the five known viroids and a tentative species GYSVd-3 infecting grapevine exist in China, and three of them (HpSVd, GYSVd-1 and GYSVd-3) exist in Japan. The contrast in diversity of viroid species in old grapevines from China and Japan may reflect different history of viticulture between the two countries. In general, the species of viroids infecting grapevine in China, as well as those in Iran and Australia, were more diverse than in the other countries. The population structure of viroids infecting grapevine in China and Japan showed species-dependency; i.e., HpSVd shared similar population structures in both countries, but GYSVd-1, GYSVd-2, and AGVd showed regional disparity even within the same country, although the role of sequence diversity in the biology of viroids infecting grapevine, such as the pathogenicity and evolution, still needs further study.

Predicting the Structure of a Viroid : Structure, Structure Distribution, Consensus Structure, and Structure Drawing.

Viroids are small non-coding RNAs that require a special sequence and structure to be replicated and transported by the host machinery. Many of these features can be predicted and later experimentally verified. Here, we will present workflows to predict viroid structures and draw the predicted structures in a pleasing and descriptive way using recently developed software.

The Biology of Viroid-Host Interactions

Viroids are single-stranded, circular, and noncoding RNAs that infect plants. They replicate in the nucleus or chloroplast and then traffic cell-to-cell through plasmodesmata and long distance through the phloem to establish systemic infection. They also cause diseases in certain hosts. All functions are mediated directly by the viroid RNA genome or genome-derived RNAs. I summarize recent advances in the understanding of viroid structures and cellular factors enabling these functions, emphasizing conceptual developments, major knowledge gaps, and future directions. Newly emerging experimental systems and research tools are discussed that are expected to enable significant progress in a number of key areas. I highlight examples of groundbreaking contributions of viroid research to the development of new biological principles and offer perspectives on using viroid models to continue advancing some frontiers of life science.

Robert Henry Symons 1934 - 2006

Bob Symons died in Adelaide on 4 October 2006 after a long illness. He was distinguished through his contributions to our knowledge of the structure, function and replication of plant viruses, viroids and virusoids. His research culminated in the discovery of the hammerhead folding of the RNA chain and its role as a ribozyme in self-cleavage of the RNA in some of these plant pathogens. He was a leader in his field and was responsible for commercial applications of his research and the establishment in Adelaide of the first Australian company to produce and market molecular biologicals for research.

Robert Henry Symons. 20 March 1934 — 4 October 2006

Bob Symons died in Adelaide on 4 October 2006 after a long illness. He was distinguished through his contributions to our knowledge of the structure, function and replication of plant viruses, viroids and virusoids. His research culminated in the discovery of the hammer head folding of the RNA chain and its role as a ribozyme in self–cleavage of the RNA in some of these plant pathogens. He was a leader in his field and was responsible for commercial applications of his research and the establishment in Adelaide of the first Australian company to produce and market molecular biologicals for research.

Analysis of thermal stress-mediated PSTVd variation and biolistic inoculation of progeny of viroid ?thermomutants? to tomato and Brassica species

Thermal stress of PSTVd-infected Nicotiana benthamiana led to appearance of a broad PSTVd sequence distribution, where most of mutations accumulated in the left half of the viroid's secondary structure including the “pathogenicity” domain. A similar effect had been reported for hop latent viroid [Virology 287 (2001) 349]. The pool of viroid “thermomutants” progenies was transcribed into cDNA and used for biolistic inoculation of Raphanus sativa , where the PSTVd infection was detectable by reverse transcription and polymerase chain reaction (RT-PCR). Newly generated inoculum from R. sativa was used for biolistic transfer to Arabidopsis thaliana wild-type and silencing-deficient mutants bearing one of sde1 , sde2 , and sde3 locuses. Irrespective to A. thaliana silencing mutants, viroid levels in Brasicaceae species infected with mutated PSTVd variants were of approximately 300 times lower than it is expected for tomato. At the same time, no systemic infection of A. thaliana was achieved with the wild-type PSTVd. In Arabidopsis , a population of PSTVd, consisting of frequent and minor variants, was present and the sequence distribution differed from that of the original viroid “thermomutants”; that is, mutations were not predominantly restricted to the left half of viroid's secondary structure. At least 65% of viroid sequences from Arabidopsis library accumulated mutations in the upper conserved central region (UCCR). In addition, mutants having changes in “hairpin II” domain (C→A transition at position 229) and in the conserved internal loop element in the left part of viroid structure (single insertion of G at position 39) were detected. All those mutants were inoculated biolistically to tomato and promoted infection especially after prolonged period of plant cultivation (50–80 days pi) when infection reached 70–90%. However, the sequence variants were unstable and reverted to the wild type and to other sequence variants stable in tomato. Our results demonstrate that heat stress-mediated production of viroid quasi-species could be of significance for viroid adaptations.

Analysis of thermal stress-mediated PSTVd variation and biolistic inoculation of progeny of viroid “thermomutants” to tomato and Brassica species

Thermal stress of PSTVd-infected Nicotiana benthamiana led to appearance of a broad PSTVd sequence distribution, where most of mutations accumulated in the left half of the viroid's secondary structure including the “pathogenicity” domain. A similar effect had been reported for hop latent viroid [Virology 287 (2001) 349]. The pool of viroid “thermomutants” progenies was transcribed into cDNA and used for biolistic inoculation of Raphanus sativa, where the PSTVd infection was detectable by reverse transcription and polymerase chain reaction (RT-PCR). Newly generated inoculum from R. sativa was used for biolistic transfer to Arabidopsis thaliana wild-type and silencing-deficient mutants bearing one of sde1, sde2, and sde3 locuses. Irrespective to A. thaliana silencing mutants, viroid levels in Brasicaceae species infected with mutated PSTVd variants were of approximately 300 times lower than it is expected for tomato. At the same time, no systemic infection of A. thaliana was achieved with the wild-type PSTVd. In Arabidopsis, a population of PSTVd, consisting of frequent and minor variants, was present and the sequence distribution differed from that of the original viroid “thermomutants”; that is, mutations were not predominantly restricted to the left half of viroid's secondary structure. At least 65% of viroid sequences from Arabidopsis library accumulated mutations in the upper conserved central region (UCCR). In addition, mutants having changes in “hairpin II” domain (C→A transition at position 229) and in the conserved internal loop element in the left part of viroid structure (single insertion of G at position 39) were detected. All those mutants were inoculated biolistically to tomato and promoted infection especially after prolonged period of plant cultivation (50–80 days pi) when infection reached 70–90%. However, the sequence variants were unstable and reverted to the wild type and to other sequence variants stable in tomato. Our results demonstrate that heat stress-mediated production of viroid quasi-species could be of significance for viroid adaptations.