Application of Biotechniques for In Vitro Virus and Viroid Elimination in Pome Fruit Crops.

Sustainable production of pome fruit crops is dependent upon having virus-free planting materials. The production and distribution of plants derived from virus- and viroid-negative sources is necessary not only to control pome fruit viral diseases but also for sustainable breeding activities, as well as the safe movement of plant materials across borders. With variable success rates, different in vitro-based techniques, including shoot tip culture, micrografting, thermotherapy, chemotherapy, and shoot tip cryotherapy, have been employed to eliminate viruses from pome fruits. Higher pathogen eradication efficiencies have been achieved by combining two or more of these techniques. An accurate diagnosis that confirms complete viral elimination is crucial for developing effective management strategies. In recent years, considerable efforts have resulted in new reliable and efficient virus detection methods. This comprehensive review documents the development and recent advances in biotechnological methods that produce healthy pome fruit plants. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Effects of a Potato Spindle Tuber Viroid Tomato Strain on the Symptoms, Biomass, and Yields of Classical Indicator and Currently Grown Potato and Tomato Cultivars.

The Chittering strain of potato spindle tuber viroid (PSTVd) infects solanaceous crops and wild plants in the subtropical Gascoyne Horticultural District of Western Australia. Classical PSTVd indicator hosts tomato cultivar Rutgers (R) and potato cultivar Russet Burbank (RB) and currently widely grown tomato cultivars Petula (P) and Swanson (S) and potato cultivars Nadine (N) and Atlantic (A) were inoculated with this strain to study its pathogenicity, quantify fruit or tuber yield losses, and establish whether tomato strains might threaten potato production. In potato foliage, infection caused spindly stems, an upright growth habit, leaves with ruffled margins and reduced size, and upward rolling and twisting of terminal leaflets (RB, A, and N); axillary shoot proliferation (A); severe plant stunting (N and RB); and necrotic spotting of petioles and stems (RB). Tubers from infected plants were tiny (N) or small and "spindle shaped" with (A) or without (RB) cracking. Potato foliage dry weight biomass was decreased by 30 to 44% in A and RB and 37% in N, whereas tuber yield was diminished by 50 to 89% in A, 69 to 71% in RB, and 90% in N. In tomato foliage, infection caused epinasty and rugosity in apical leaves, leaf chlorosis, and plant stunting (S, P, and N); cupped leaves (S and P); and reduced leaf size, flower abortion, and necrosis of midribs, petioles, and stems (R). Mean tomato fruit size was greatly decreased in all three cultivars. Tomato foliage dry weight biomass was diminished by 40 to 53% (P), 42% (S), and 37 to 51% (R). Tomato fruit yield was decreased by 60 to 76% (P), 52% (S), and 64 to 89% (R), respectively. Thus, the tomato strain studied was highly pathogenic to classical indicator and representative current tomato and potato cultivars, causing major losses in fruit and tuber yields. Tomato PSTVd strains, therefore, pose a threat to tomato and potato industries worldwide.

Surveys in the Chrysanthemum Production Areas of Brazil and Colombia Reveal That Weeds Are Potential Reservoirs of Chrysanthemum Stunt Viroid.

The stunting disease, incited by chrysanthemum stunt viroid (CSVd), has become a serious problem in chrysanthemum production areas worldwide. Here we identified 46 weed species from chrysanthemum fields in two producing regions of the State of São Paulo, Brazil. The mechanical inoculation of these weeds with a Brazilian CSVd isolate revealed that this viroid was able to infect 17 of these species, in addition to chrysanthemum, tomato and potato. Plants of Oxalis latifolia and chrysanthemum naturally infected with CSVd were found in chrysanthemum fields in Colombia, which is the first CSVd report in that country. Therefore, weeds have the potential to act as reservoirs of CSVd in the field. These results are the first reports of experimental CSVd infection in the following species: Amaranthus viridis, Cardamine bonariensis, Chamaesyce hirta, Conyza bonariensis, Digitaria sanguinalis, Gomphrena globosa, Helianthus annuus, Lupinus polyphyllus, Mirabilis jalapa, Oxalis latifolia, Portulaca oleracea and Catharanthus roseus. The phylogenetic analyses of the CSVd variants identified herein showed three groups with Brazilian CSVd variants distributed in them all, which suggests that Brazilian CSVd isolates may have different origins through successive introductions of infected germplasm of chrysanthemum in Brazil.

Influence of the terminal left domain on horizontal and vertical transmissions of tomato planta macho viroid and potato spindle tuber viroid through pollen.

Viroids can be transmitted vertically and/or horizontally by pollen. Tomato planta macho viroid (TPMVd) has a high rate of horizontal transmission by pollen, whereas potato spindle tuber viroid (PSTVd) does not. To specify the domain(s) involved in horizontal transmission, four viroid chimeras were created by exchanging the terminal left (TL) and/or pathogenicity (P) domains between PSTVd and TPMVd. PSTVd-based chimeras containing TPMVd-TL and P, or TPMVd-TL alone, displayed a high rate of horizontal transmission. TPMVd-based chimeras containing PSTVd-TL and P lost infectivity, and those containing PSTVd-TL alone displayed a low rate of horizontal transmission. In addition, the vertical transmission rate was also higher in the mutants containing TPMVd-TL than in the others. These findings indicate that the sequences or structures in the TL and P (although the role is limited) domains are important not only for horizontal but also for vertical transmission by pollen.

Vertical and Horizontal Transmission of Pospiviroids.

Viroids are highly structured, single-stranded, non-protein-coding circular RNA pathogens. Some viroids are vertically transmitted through both viroid-infected ovule and pollen. For example, potato spindle tuber viroid, a species that belongs to Pospiviroidae family, is delivered to the embryo through the ovule or pollen during the development of reproductive tissues before embryogenesis. In addition, some of Pospiviroidae are also horizontally transmitted by pollen. Tomato planta macho viroid in pollen infects to the ovary from pollen tube during pollen tube elongation and eventually causes systemic infection, resulting in the establishment of horizontal transmission. Furthermore, fertilization is not required to accomplish the horizontal transmission. In this review, we will overview the recent research progress in vertical and horizontal transmission of viroids, mainly by focusing on histopathological studies, and also discuss the impact of seed transmission on viroid dissemination and seed health.

Role of microRNA (miRNA) and Viroids in Lethal Diseases of Plants and Animals. Potential Contribution to Human Neurodegenerative Disorders.

Both plants and animals have adopted a common strategy of using ~18-25-nucleotide small non-coding RNAs (sncRNAs), known as microRNAs (miRNAs), to transmit DNA-based epigenetic information. miRNAs (i) shape the total transcriptional output of individual cells; (ii) regulate and fine-tune gene expression profiles of cell clusters, and (iii) modulate cell phenotype in response to environmental stimuli and stressors. These miRNAs, the smallest known carriers of gene-encoded post-transcriptional regulatory information, not only regulate cellular function in healthy cells but also act as important mediators in the development of plant and animal diseases. Plants possess their own specific miRNAs; at least 32 plant species have been found to carry infectious sncRNAs called viroids, whose mechanisms of generation and functions are strikingly similar to those of miRNAs. This review highlights recent remarkable and sometimes controversial findings in miRNA signaling in plants and animals. Special attention is given to the intriguing possibility that dietary miRNAs and/or sncRNAs can function as mobile epigenetic and/or evolutionary linkers between different species and contribute to both intra- and interkingdom signaling. Wherever possible, emphasis has been placed on the relevance of these miRNAs to the development of human neurodegenerative diseases, such as Alzheimer's disease. Based on the current available data, we suggest that such xeno-miRNAs may (i) contribute to the beneficial properties of medicinal plants, (ii) contribute to the negative properties of disease-causing or poisonous plants, and (iii) provide cross-species communication between kingdoms of living organisms involving multiple epigenetic and/or potentially pathogenic mechanisms associated with the onset and pathogenesis of various diseases.

Potato Spindle Tuber Viroid RNA-Templated Transcription: Factors and Regulation.

Viroids are circular noncoding RNAs that infect plants. Without encoding any protein, these noncoding RNAs contain the necessary genetic information for propagation in hosts. Nuclear-replicating viroids employ DNA-dependent RNA polymerase II (Pol II) for replication, a process that makes a DNA-dependent enzyme recognize RNA templates. Recently, a splicing variant of transcription factor IIIA (TFIIIA-7ZF) was identified as essential for Pol II to replicate potato spindle tuber viroid (PSTVd). The expression of TFIIIA-7ZF, particularly the splicing event, is regulated by a ribosomal protein (RPL5). PSTVd modulates its expression through a direct interaction with RPL5 resulting in optimized expression of TFIIIA-7ZF. This review summarizes the recent discoveries of host factors and regulatory mechanisms underlying PSTVd-templated transcription processes and raises new questions that may help future exploration in this direction. In addition, it briefly compares the machinery and the regulatory mechanism for PSTVd with the replication/transcription system of human hepatitis delta virus.

Potato Spindle Tuber Viroid Modulates Its Replication through a Direct Interaction with a Splicing Regulator.

Viroids are circular noncoding RNAs (ncRNAs) that infect plants. Despite differences in the genetic makeup and biogenesis, viroids and various long ncRNAs all rely on RNA structure-based interactions with cellular factors for function. Viroids replicating in the nucleus utilize DNA-dependent RNA polymerase II for transcription, a process that involves a unique splicing form of transcription factor IIIA (TFIIIA-7ZF). Here, we provide evidence showing that potato spindle tuber viroid (PSTVd) interacts with a TFIIIA splicing regulator (ribosomal protein L5 [RPL5]) in vitro and in vivo PSTVd infection compromises the regulatory role of RPL5 over splicing of TFIIIA transcripts, while ectopic expression of RPL5 reduces TFIIIA-7ZF expression and attenuates PSTVd accumulation. Furthermore, we illustrate that the RPL5 binding site on the PSTVd genome resides in the central conserved region critical for replication. Together, our data suggest that viroids can regulate their own replication and modulate specific regulatory factors leading to splicing changes in only one or a few genes. This study also has implications for understanding the functional mechanisms of ncRNAs and elucidating the global splicing changes in various host-pathogen interactions.IMPORTANCE Viroids are the smallest replicons among all living entities. As circular noncoding RNAs, viroids can replicate and spread in plants, often resulting in disease symptoms. Potato spindle tuber viroid (PSTVd), the type species of nuclear-replicating viroids, requires a unique splicing form of transcription factor IIIA (TFIIIA-7ZF) for its propagation. Here, we provide evidence showing that PSTVd directly interacts with a splicing regulator, RPL5, to favor the expression of TFIIIA-7ZF, thereby promoting viroid replication. This finding provides new insights to better understand viroid biology and sheds light on the noncoding RNA-based regulation of splicing. Our discovery also establishes RPL5 as a novel negative factor regulating viroid replication in the nucleus and highlights a potential means for viroid control.

Time-Course Microarray Analysis Reveals Differences between Transcriptional Changes in Tomato Leaves Triggered by Mild and Severe Variants of Potato Spindle Tuber Viroid.

Viroids are small non-capsidated non-coding RNA replicons that utilize host factors for efficient propagation and spread through the entire plant. They can incite specific disease symptoms in susceptible plants. To better understand viroid-plant interactions, we employed microarray analysis to observe the changes of gene expression in "Rutgers" tomato leaves in response to the mild (M) and severe (S23) variants of potato spindle tuber viroid (PSTVd). The changes were analyzed over a time course of viroid infection development: (i) the pre-symptomatic stage; (ii) early symptoms; (iii) full spectrum of symptoms and (iv) the so-called 'recovery' stage, when stem regrowth was observed in severely affected plants. Gene expression profiles differed depending on stage of infection and variant. In S23-infected plants, the expression of over 3000 genes was affected, while M-infected plants showed 3-fold fewer differentially expressed genes, only 20% of which were specific to the M variant. The differentially expressed genes included many genes related to stress; defense; hormone metabolism and signaling; photosynthesis and chloroplasts; cell wall; RNA regulation, processing and binding; protein metabolism and modification and others. The expression levels of several genes were confirmed by nCounter analysis.

Differences in dynamics of horizontal transmission of Tomato planta macho viroid and Potato spindle tuber viroid after pollination with viroid-infected pollen.

For viroids, pollen transmission is an important transmission pathway to progeny seeds and new hosts. In the current study, we found that Tomato planta macho viroid (TPMVd)-but not Potato spindle tuber viroid (PSTVd)-was horizontally transmitted by pollen from petunia plants. Using tissue-printing hybridization to track the changes in viroid distribution after pollination, we noted that TPMVd was present in petunia stigma, styles, and eventually ovaries, whereas PSTVd was detected in the stigma and upper style but not the ovary. These findings suggest that horizontal transmission of viroids depends on the infection of the lower style and ovary during the elongation of pollen tubes after pollination. Additionally, TPMVd was transmitted horizontally, leading to systematic infection, when we used TPMVd-infected petunia pollen to pollinate the flowers of healthy tomato plants. Fertilization typically does not occur after heterologous pollination and thus likely is not required to accomplish horizontal transmission of viroids.

The effect of viroid infection of citrus trees on the amoebicidal activity of 'Maltese half-blood' (Citrus sinensis) against trophozoite stage of Acanthamoeba castellanii Neff.

In order to promote a local Tunisian product, this study was designed to examine, for the first time, the anti-Acanthamoeba activity (Acanthamoeba castellanii Neff) of the essential oils of Tunisian Citrus sinensis peels (Maltese half-blood) and the effect of viroid plant infection on this activity. To do so, three samples of peels' essential oils were studied: from a healthy plant (Control), a plant inoculated with Citrus exocortis viroid (CEVd) and one inoculated with hot stunt cachexia viroid (HSVd). The samples were extracted by hydrodistillation from dried peels and characterized by GC-MS. Limonene was the major component with a percentage ranging from 90.76 to 93.34% for (CEVd) sample and (Control), respectively. Anti-Acanthamoeba activity of the tested oils was determined by the Alamar Blue® assay. Primary results showed a strong potential anti-Acanthamoeba activity with an IC50 ranging from 36.6 to 54.58 µg/ml for (HSVd) and (CEVd) samples, respectively. In terms of the effect of viroid infection, a strong positive correlation was observed between different chemical classes and anti-Acanthamoeba activity.

Phylogenetic and functional analyses of a plant protein related to human B-cell receptor-associated proteins.

Human B-cell receptor-associated protein BAP31 (HsBAP31) is the endoplasmic reticulum-resident protein involved in protein sorting and transport as well as pro-apoptotic signaling. Plant orthologs of HsBAP31 termed 'plant BAP-like proteins' (PBL proteins) have thus far remained unstudied. Recently, the PBL protein from Nicotiana tabacum (NtPBL) was identified as an interactor of Nt-4/1, a plant protein known to interact with plant virus movement proteins and affect the long-distance transport of potato spindle tuber viroid (PSTVd) via the phloem. Here, we have compared the sequences of PBL proteins and studied the biochemical properties of NtPBL. Analysis of a number of fully sequenced plant genomes revealed that PBL-encoding genes represent a small multigene family with up to six members per genome. Two conserved motifs were identified in the C-terminal region of PBL proteins. The NtPBL C-terminal hydrophilic region (NtPBL-C) was expressed in bacterial cells, purified, and used for analysis of its RNA binding properties in vitro. In gel shift experiments, NtPBL-C was found to bind several tested RNAs, showing the most efficient binding to microRNA precursors (pre-miRNA) and less efficient interaction with PSTVd. Mutational analysis suggested that NtPBL-C has a composite RNA-binding site, with two conserved lysine residues in the most C-terminal protein region being involved in binding of pre-miRNA but not PSTVd RNA. Virus-mediated transient expression of NtPBL-C in plants resulted in stunting and leaf malformation, developmental abnormalities similar to those described previously for blockage of miRNA biogenesis/function. We hypothesize that the NtPBL protein represents a previously undiscovered component of the miRNA pathway.

Replication of a pathogenic non-coding RNA increases DNA methylation in plants associated with a bromodomain-containing viroid-binding protein.

Viroids are plant-pathogenic molecules made up of single-stranded circular non-coding RNAs. How replicating viroids interfere with host silencing remains largely unknown. In this study, we investigated the effects of a nuclear-replicating Potato spindle tuber viroid (PSTVd) on interference with plant RNA silencing. Using transient induction of silencing in GFP transgenic Nicotiana benthamiana plants (line 16c), we found that PSTVd replication accelerated GFP silencing and increased Virp1 mRNA, which encodes bromodomain-containing viroid-binding protein 1 and is required for PSTVd replication. DNA methylation was increased in the GFP transgene promoter of PSTVd-replicating plants, indicating involvement of transcriptional gene silencing. Consistently, accelerated GFP silencing and increased DNA methylation in the of GFP transgene promoter were detected in plants transiently expressing Virp1. Virp1 mRNA was also increased upon PSTVd infection in natural host potato plants. Reduced transcript levels of certain endogenous genes were also consistent with increases in DNA methylation in related gene promoters in PSTVd-infected potato plants. Together, our data demonstrate that PSTVd replication interferes with the nuclear silencing pathway in that host plant, and this is at least partially attributable to Virp1. This study provides new insights into the plant-viroid interaction on viroid pathogenicity by subverting the plant cell silencing machinery.

Effects of potato spindle tuber viroid infection on tomato metabolic profile.

Viroids are the smallest plant pathogens consisting of a single stranded circular RNA molecule with a strong secondary structure, lacking a coat protein or any other proteins. The mechanism of viroid pathogenicity has remained unclear. Recent advances in instrumentation and data mining have made it possible to study the effects of various stresses on primary and secondary metabolisms. Here, we have utilized metabolic profiling approach to show how PSTVd infection alters tomato metabolic profile and the related pathways. Three terminal leaflets of third true leaf of 20-day-old tolerant tomato cultivar 'Moneymaker' were mechanically inoculated by PSTVd intermediate variant cDNAs and samples were taken from eighth leaf, 19days post-inoculation. Metabolites were extracted and analyzed by gas chromatography/mass spectrometry (GC/MS) and subjected to statistical data analysis. Affected pathways were identified by Pathway Tools program and were compared with microarray data previously reported. The study showed that 79 metabolites changed significantly and 23 pathways were identified in relation to these metabolites. Fourteen of these pathways were similar to those reported in other works. The altered pathways in PSTVd infected tomato leaves included, eight cutin and wax biosynthesis, seven pathways that produce defense related compounds, two energy generator pathways, three hormone biosynthesis pathways, two signal transduction pathways, and one nucleotide biosynthesis pathway. Our data on up/down-regulation of pathways supported the data produced on their corresponding gene(s) up/down-regulation.

Digestion of chrysanthemum stunt viroid by leaf extracts of Capsicum chinense indicates strong RNA-digesting activity.

KEY MESSAGE: CSVd could not infect Nicotiana benthamiana when the plants were pretreated with crude leaf extract of Capsicum chinense 'Sy-2'. C. chinense leaves were revealed to contain strong RNA-digesting activity. Several studies have identified active antiviral and antiviroid agents in plants. Capsicum plants are known to contain antiviral agents, but the mechanism of their activity has not been determined. We aimed to elucidate the mechanism of Capsicum extract's antiviroid activity. Chrysanthemum stunt viroid (CSVd) was inoculated into Nicotiana benthamiana plants before or after treating the plants with a leaf extract of Capsicum chinense 'Sy-2'. CSVd infection was determined using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) 3 weeks after inoculation. When Capsicum extract was sprayed or painted onto N. benthamiana before inoculation, it was effective in preventing infection by CSVd. To evaluate CSVd digestion activity in leaf extracts, CSVd was mixed with leaf extracts of Mirabilis, Phytolacca, Pelargonium and Capsicum. CSVd-digesting activities were examined by quantifying undigested CSVd using qRT-PCR, and RNA gel blotting permitted visualization of the digested CSVd. Only Capsicum leaf extract digested CSVd, and in the Capsicum treatment, small digested CSVd products were detected by RNA gel blot analysis. When the digesting experiment was performed for various cultivars and species of Capsicum, only cultivars of C. chinense showed strong CSVd-digesting activity. Our observations indicated that Capsicum extract contains strong RNA-digesting activity, leading to the conclusion that this activity is the main mechanism for protection from infection by CSVd through spraying or painting before inoculation. To our knowledge, this is the first report of a strong RNA-digesting activity by a plant extract.

Insight on Genes Affecting Tuber Development in Potato upon Potato spindle tuber viroid (PSTVd) Infection.

Potato (Solanum tuberosum L) is a natural host of Potato spindle tuber viroid (PSTVd) which can cause characteristic symptoms on developing plants including stunting phenotype and distortion of leaves and tubers. PSTVd is the type species of the family Pospiviroidae, and can replicate in the nucleus and move systemically throughout the plant. It is not well understood how the viroid can affect host genes for successful invasion and which genes show altered expression levels upon infection. Our primary focus in this study is the identification of genes which can affect tuber formation since viroid infection can strongly influence tuber development and especially tuber shape. In this study, we used a large-scale method to identify differentially expressed genes in potato. We have identified defence, stress and sugar metabolism related genes having altered expression levels upon infection. Additionally, hormone pathway related genes showed significant up- or down-regulation. DWARF1/DIMINUTO, Gibberellin 7-oxidase and BEL5 transcripts were identified and validated showing differential expression in viroid infected tissues. Our study suggests that gibberellin and brassinosteroid pathways have a possible role in tuber development upon PSTVd infection.

Distribution of potato spindle tuber viroid in reproductive organs of petunia during its developmental stages.

Embryo infection is important for efficient seed transmission of viroids. To identify the major pattern of seed transmission of viroids, we used in situ hybridization to histochemically analyze the distribution of Potato spindle tuber viroid (PSTVd) in each developmental stage of petunia (flowering to mature seed stages). In floral organs, PSTVd was present in the reproductive tissues of infected female × infected male and infected female × healthy male but not of healthy female × infected male before embryogenesis. After pollination, PSTVd was detected in the developed embryo and endosperm in all three crosses. These findings indicate that PSTVd is indirectly delivered to the embryo through ovule or pollen during the development of reproductive tissues before embryogenesis but not directly through maternal tissues as cell-to-cell movement during embryogenesis.

Biological and molecular analysis of the pathogenic variant C3 of potato spindle tuber viroid (PSTVd) evolved during adaptation to chamomile (Matricaria chamomilla).

Viroid-caused pathogenesis is a specific process dependent on viroid and host genotype(s), and may involve viroid-specific small RNAs (vsRNAs). We describe a new PSTVd variant C3, evolved through sequence adaptation to the host chamomile (Matricaria chamomilla) after biolistic inoculation with PSTVd-KF440-2, which causes extraordinary strong ('lethal') symptoms. The deletion of a single adenine A in the oligoA stretch of the pathogenicity (P) domain appears characteristic of PSTVd-C3. The pathogenicity and the vsRNA pool of PSTVd-C3 were compared to those of lethal variant PSTVd-AS1, from which PSTVd-C3 differs by five mutations located in the P domain. Both lethal viroid variants showed higher stability and lower variation in analyzed vsRNA pools than the mild PSTVd-QFA. PSTVd-C3 and -AS1 caused similar symptoms on chamomile, tomato, and Nicotiana benthamiana, and exhibited similar but species-specific distributions of selected vsRNAs as quantified using TaqMan probes. Both lethal PSTVd variants block biosynthesis of lignin in roots of cultured chamomile and tomato. Four 'expression markers' (TCP3, CIPK, VSF-1, and VPE) were selected from a tomato EST library to quantify their expression upon viroid infection; these markers were strongly downregulated in tomato leaf blades infected by PSTVd-C3- and -AS1 but not by PSTVd-QFA.

Elimination of hop latent viroid upon developmental activation of pollen nucleases.

Hop latent viroid (HLVd) is not transmissible through hop generative tissues and seeds. Here we describe the process of HLVd elimination during development of hop pollen. HLVd propagates in uninucleate hop pollen, but is eliminated at stages following first pollen mitosis during pollen vacuolization and maturation. Only traces of HLVd were detected by RT-PCR in mature pollen after anthesis and no viroid was detectable in in vitro germinating pollen, suggesting complete degradation of circular and linear HLVd forms. The majority of the degraded HLVd RNA in immature pollen included discrete products in the range of 230-100 nucleotides and therefore did not correspond to siRNAs. HLVd eradication from pollen correlated with developmental expression of a pollen nuclease and specific RNAses. Activity of the pollen nuclease HBN1 was maximal during the vacuolization step and decreased in mature pollen. Total RNAse activity increased continuously up to the final steps of pollen maturation. HBN1 mRNA, which is abundant at the uninucleate microspore stage, encodes a protein of 300 amino acids (34.1 kDa, isoeletric point 5.1). Sequence comparisons revealed that HBN1 is a homolog of S1-like bifunctional plant endonucleases. The developmentally activated HBN1 and pollen ribonucleases could participate in the mechanism of HLVd recognition and degradation.

Tertiary structural and functional analyses of a viroid RNA motif by isostericity matrix and mutagenesis reveal its essential role in replication.

RNA-templated RNA replication is essential for viral or viroid infection, as well as for regulation of cellular gene expression. Specific RNA motifs likely regulate various aspects of this replication. Viroids of the Pospiviroidae family, as represented by the Potato spindle tuber viroid (PSTVd), replicate in the nucleus by utilizing DNA-dependent RNA polymerase II. We investigated the role of the loop E (sarcin/ricin) motif of the PSTVd genomic RNA in replication. A tertiary-structural model of this motif, inferred by comparative sequence analysis and comparison with nuclear magnetic resonance and X-ray crystal structures of loop E motifs in other RNAs, is presented in which core non-Watson-Crick base pairs are precisely specified. Isostericity matrix analysis of these base pairs showed that the model accounts for the reported natural sequence variations and viable experimental mutations in loop E motifs of PSTVd and other viroids. Furthermore, isostericity matrix analysis allowed us to design disruptive, as well as compensatory, mutations of PSTVd loop E. Functional analyses of such mutants by in vitro and in vivo experiments demonstrated that loop E structural integrity is crucial for replication, specifically during transcription. Our results suggest that the PSTVd loop E motif exists and functions in vivo and provide loss-of-function genetic evidence for the essential role of a viroid RNA three-dimensional motif in rolling-circle replication. The use of isostericity matrix analysis of non-Watson-Crick base pairing to rationalize mutagenesis of tertiary motifs and systematic in vitro and in vivo functional assays of mutants offers a novel, comprehensive approach to elucidate the tertiary-structure-function relationships for RNA motifs of general biological significance.