Revisiting the non-coding nature of pospiviroids

Abstract : Viroids are small, circular, highly structured pathogens that infect a broad range of plants, causing economic losses. Since their discovery in the 1970s, they have been considered as non-coding pathogens. In the last few years, the discovery of other RNA entities, similar in terms of size and structure, that were shown to be translated (e.g., cirRNAs, precursors of miRNA, RNA satellites) as well as studies showing that some viroids are located in ribosomes, have reignited the idea that viroids may be translated. In this study, we used advanced bioinformatic analysis, in vitro experiments and LC-MS/MS to search for small viroid peptides of the PSTVd. Our results suggest that in our experimental conditions, even though the circular form of PSTVd is found in ribosomes, no produced peptides were identified. This indicates that the presence of PSTVd in ribosomes is most probably not related to peptide production but rather to another unknown function that requires further study

Impact of nucleic acid sequencing on viroid biology

Abstract: The early 1970s marked two breakthroughs in the field of biology: (i) The development of nucleotide sequencing technology; and, (ii) the discovery of the viroids. The first DNA sequences were obtained by two-dimensional chromatography which was later replaced by sequencing using electrophoresis technique. The subsequent development of fluorescence-based sequencing method which made DNA sequencing not only easier, but many orders of magnitude faster. The knowledge of DNA sequences has become an indispensable tool for both basic and applied research. It has shed light biology of viroids, the highly structured, circular, single-stranded non-coding RNA molecules that infect numerous economically important plants. Our understanding of viroid molecular biology and biochemistry has been intimately associated with the evolution of nucleic acid sequencing technologies. With the development of the next-generation sequence method, viroid research exponentially progressed, notably in the areas of the molecular mechanisms of viroids and viroid diseases, viroid pathogenesis, viroid quasi-species, viroid adaptability, and viroid–host interactions, to name a few examples. In this review, the progress in the understanding of viroid biology in conjunction with the improvements in nucleotide sequencing technology is summarized. The future of viroid research with respect to the use of third-generation sequencing technology is also briefly envisaged

Viroid-derived small RNA induces early flowering in tomato plants by RNA silencing

Abstract: Viroid infection often leads to early flowering in the host plant. This report describes the targeting of the FRIGIDA-like protein 3 (FRL3) mRNA in tomato plants by a small RNA derived from the conserved left terminal region of the potato spindle tuber viroid (PSTVd). This targeting leads to the silencing of the FRL3 mRNA. Viroid infection assays using a severe variant of PSTVd induced early flowering in tomato plants by the down-regulation of greater amounts of the target than did a mild PSTVd variant. The targeting of the FRL3 mRNA by RNA silencing was validated by both an artificial microRNA experiment transiently expressing viroid-derived small RNAs in tomato plants, and by 5′ RNA ligase- mediated rapid amplification of cDNA ends (RACE). These data unambiguously demonstrated the role of small RNAs in the early flowering seen in viroid-infected plants

Citrus exocortis viroid causes ribosomal stress in tomato plants

Abstract: Viroids are naked RNAs that do not code for any known protein and yet are able to infect plants causing severe diseases. Because of their RNA nature, many studies have focused on the involvement of viroids in RNA-mediated gene silencing as being their pathogenesis mechanism. Here, the alterations caused by the Citrus exocortis viroid (CEVd) on the tomato translation machinery were studied as a new aspect of viroid pathogenesis. The presence of viroids in the ribosomal fractions of infected tomato plants was detected. More precisely, CEVd and its derived viroid small RNAs were found to co-sediment with tomato ribosomes in vivo, and to provoke changes in the global polysome profiles, particularly in the 40S ribosomal subunit accumulation. Additionally, the viroid caused alterations in ribosome biogenesis in the infected tomato plants, affecting the 18S rRNA maturation process. A higher expression level of the ribosomal stress mediator NAC082 was also detected in the CEVd-infected tomato leaves. Both the alterations in the rRNA processing and the induction of NAC082 correlate with the degree of viroid symptomatology. Taken together, these results suggest that CEVd is responsible for defective ribosome biogenesis in tomato, thereby interfering with the translation machinery and, therefore, causing ribosomal stress

Current overview on viroid–host interactions

Abstract: Viroids are one of the most enigmatic highly structured, circular, single-stranded RNA phytopathogens. Although they are not known to code for any peptide, viroids induce visible symptoms in susceptible host plants that resemble those associated with many plant viruses. It is known that viroids induce disease symptoms by direct interaction with host factors; however, the precise mechanism by which this occurs remains poorly understood. Studies on the host's responses to viroid infection, host susceptibility and nonhost resistance have been underway for several years, but much remains to be done in order to fully understand the complex nature of viroid–host interactions. Recent progress using molecular biology techniques combined with computational algorithms, in particular evidence of the role of viroid-derived small RNAs in the RNA silencing pathways of a disease network, has widened the knowledge of viroid pathogenicity. The complexity of viroid–host interactions has been revealed in the past decades to include, but not be limited to, the involvement of host factors, viroid structural complexity, and viroid-induced ribosomal stress, which is further boosted by the discovery of long noncoding RNAs (lncRNAs). In this review, the current understanding of the viroid–host interaction has been summarized with the goal of simplifying the complexity of viroid biology for future research

3' RNA ligase mediated rapid amplification of cDNA ends for validating viroid induced cleavage at the 3' extremity of the host mRNA

Abstract : 5′ RNA ligase-mediated rapid amplification of cDNA ends (5′ RLM-RACE) is a widely-accepted method for the validation of direct cleavage of a target gene by a microRNA (miRNA) and viroid-derived small RNA (vd-sRNA). However, thi\s method cannot be used if cleavage takes place in the 3′ extremity of the target RNA, as this gives insufficient sequence length to design nested PCR primers for 5′ RLM RACE. To overcome this hurdle, we have developed 3′ RNA ligase-mediated rapid amplification of cDNA ends (3′ RLM RACE). In this method, an oligonucleotide adapter having 5′ adenylated and 3′ blocked is ligated to the 3′ end of the cleaved RNA followed by PCR amplification using gene specific primers. In other words, in 3′ RLM RACE, 3′ end is mapped using 5′ fragment instead of small 3′ fragment. The method developed here was verified by examining the bioinformatics predicted and parallel analysis of RNA ends (PARE) proved cleavage sites of chloride channel protein CLC-b-like mRNA in Potato spindle tuber viroid infected tomato plants. The 3′ RLM RACE developed in this study has the potential to validate the miRNA and vd-sRNA mediated cleavage of mRNAs at its 3′ untranslated region (3′ UTR)

Potato spindle tuber viroid infection triggers degradation of chloride channel protein CLC-b-like and Ribosomal protein S3a-like mRNAs in tomato plants

Abstract : It is well established that viroid derived small RNA (vd-sRNA) induces RNA silencing of endogenous mRNA. However, it remains not clear how exactly viroid infections can lead to severe symptom induction given the fact that fewer vd-sRNAs binding the specific target mRNAs were recovered from the infected plants. To answer this question, the two least expressed (+) and (−) strand vd-sRNAs of potato spindle tuber viroid (PSTVd) binding to both the 3′ UTR and the coding region of tomato mRNAs were analyzed by infecting tomato plants with two variants of PSTVd. As products of these putative target mRNAs are involved in plant phenotype, the effect of this viroid on these genes were analyzed by infecting tomato plants with two variants of PSTVd. The direct interaction between the vd-sRNAs and putative mRNAs was validated by artificial microRNA experiments in a transient expression system and by RNA ligase-mediated rapid amplification of cDNA ends. Parallel analysis of RNA ends of viroid infected plants revealed the widespread cleavage of the target mRNAs in locations other than the vd-sRNA binding site during the viroid infection implying the viroid-infection induced vd-sRNA independent degradation of endogenous mRNAs during viroid infection

Elucidation of the structures of all members of the Avsunviroidae family

Abstract: 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 timeconsuming 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 RNAstructure 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

Small RNA derived from the virulence modulating region of the Potato spindle tuber viroid silences callose synthase genes of tomato plants

Abstract: The tomato (Solanum lycopersicum) callose synthase genes CalS11-like and CalS12-like encode proteins that are essential for the formation of callose, a major component of pollen mother cell walls; these enzymes also function in callose formation during pathogen infection. This article describes the targeting of these callose synthase mRNAs by a small RNA derived from the virulence modulating region of two Potato spindle tuber viroid variants. More specifically, viroid infection of tomato plants resulted in the suppression of the target mRNAs up to 1.5-fold, depending on the viroid variant used and the gene targeted. The targeting of these mRNAs by RNA silencing was validated by artificial microRNA experiments in a transient expression system and by RNA ligase-mediated rapid amplification of cDNA ends. Viroid mutants incapable of targeting callose synthase mRNAs failed to induce typical infection phenotypes, whereas a chimeric viroid obtained by swapping the virulence modulating regions of a mild and a severe variant of Potato spindle tuber viroid greatly affected the accumulation of viroids and the severity of disease symptoms. These data provide evidence of the silencing of multiple genes by a single small RNA derived from a viroid

Citrus exocortis viroid causes ribosomal stress in tomato plants

[EN] Viroids are naked RNAs that do not code for any known protein and yet are able to infect plants causing severe diseases. Because of their RNA nature, many studies have focused on the involvement of viroids in RNA-mediated gene silencing as being their pathogenesis mechanism. Here, the alterations caused by the Citrus exocortis viroid (CEVd) on the tomato translation machinery were studied as a new aspect of viroid pathogenesis. The presence of viroids in the ribosomal fractions of infected tomato plants was detected. More precisely, CEVd and its derived viroid small RNAs were found to co-sediment with tomato ribosomes in vivo, and to provoke changes in the global polysome profiles, particularly in the 40S ribosomal subunit accumulation. Additionally, the viroid caused alterations in ribosome biogenesis in the infected tomato plants, affecting the 18S rRNA maturation process. A higher expression level of the ribosomal stress mediator NAC082 was also detected in the CEVd-infected tomato leaves. Both the alterations in the rRNA processing and the induction of NAC082 correlate with the degree of viroid symptomatology. Taken together, these results suggest that CEVd is responsible for defective ribosome biogenesis in tomato, thereby interfering with the translation machinery and, therefore, causing ribosomal stress.Spanish Ministry of Science, Innovation and Universities [BIO2009-11818, BIO2015-70483-R to A.F.]; Spanish Ministry of Science, Innovation and Universities [BFU2009-11958]; Generalitat Valenciana (Valencia, Spain) [AICO/2017/048]; Natural Sciences and Engineering Research Council of Canada [155219-17 to J.-P.P.]; The RNA group is supported by a grant from the Universite de Sherbrooke; J.-P.P. holds the Research Chair of the Universite de Sherbrooke in RNA Structure and Genomics, and is a member of the Centre de Recherche du CHUS; B.B.-P. was a recipient of a VALi+d postdoctoral contract of the Generalitat Valenciana [APOSTD/2017/039]; Schleiff group is funded through the Deutsche Forschungsgemeinschaft [SFB 902]. Funding for open access charge: Spanish Ministry of Science, Innovation and Universities.Cottilli, P.; Belda-Palazón, B.; Adkar-Purushothama, CR.; Perreault, J.; Schleiff, E.; Rodrigo Bravo, I.; Ferrando Monleón, AR.... (2019). Citrus exocortis viroid causes ribosomal stress in tomato plants. Nucleic Acids Research. 47(16):8649-8661. https://doi.org/10.1093/nar/gkz679S86498661471