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.

Knockout of SlDCL2b attenuates the resistance of tomato to potato spindle tuber viroid infection.

RNA interference, or RNA silencing, is an important defence mechanism against viroid infection in plants. Plants encode multiple DICER-LIKE (DCL) proteins that are key components of the RNA silencing pathway. However, the roles of different DCLs in defence responses against viroid infection remain unclear. Here, we determined the function of tomato DCL2b (SlDCL2b) in defence responses against potato spindle tuber viroid (PSTVd) infection using SlDCL2b loss-of-function tomato mutant plants. Compared with wild-type plants, mutant plants were more susceptible to PSTVd infection, developing more severe symptoms earlier and accumulating higher levels of PSTVd RNAs. Moreover, we verified the feedback mechanism for the regulation of SlDCL2b expression by miR6026. Functional blocking of tomato miR6026, by expressing its target mimics, can enhance resistance to PSTVd infection in tomato plants. These findings deepen the current understanding of RNAi-based resistance against viroid infection and provide a potentially new strategy for viroid control.

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.

First Detection of Potato Spindle Tuber Viroid in Natural Isolates of Potato Blight Agent Phytophthora infestans.

Phytophthora infestans is the oomycete that causes potato blight, an important disease. The potato spindle tuber viroid (PSTVd) is a dangerous pathogen of many plants, including potato. We have previously shown that PSTVd can be transmitted from infected potato plants into the Ph. infestans mycelium, replicated within the mycelium, and then transmitted to other potato plants upon their infection with Ph. infestans in laboratory conditions. The objective of this work was to check the hypothesis that PSTVd transmission, preservation, and replication in Ph. infestans are possible to occur in natural conditions during long-term coevolution of the host and pathogen in the Solanum spp.-Ph. infestans system. A screening test for PSTVd was performed in 111 natural Ph. infestans isolates obtained from potato plants, which represented various cultivars, had signs of potato blight, and were collected from industrial potato fields of the Moscow, Vologda, and Bryansk regions and breeding and variety test plots of the St. Petersburg and Moscow regions in 2020 and 2022. Using RT-PCR with PSTVd-specific primers, 42 Ph. infestans isolates collected in 2020 were tested after five passages and 69 Ph. infestans isolates collected in 2022, after a single passage on rye agar. Diagnostic amplicons were detected in 8 and 50 isolates, respectively. Some of the amplicons were visually assessed as minor amplification products, apparently resulting from nonspecific priming on a host Ph. infestans gene, which codes for a hypothetical protein-coding mRNA in Ph. infestans and other oomycetes. Eight amplicons were sequenced to verify the PSTVd presence in Ph. infestans isolates. Three amplicons corresponded to the complete PSTVd genome and five, to its part (~260 bp). The nucleotide sequences of cloned amplification products were identified to species in the BLAST system and deposited in GenBank. The amplicons obtained with the PSTVd-specific primers were identified as PSTVd sequences in all Ph. infestans isolates examined. The majority of the nucleotide sequences were phylogenetically related to BLAST sequences of PSTVd strains originating from Russia; several strains showed similarity to strains from other countries (France, China, and West African countries). The results demonstrate that PSTVd was for the first time detected in natural (field) Ph. infestans isolates and offer new opportunities for studying the intricate multilevel host-parasite interactions.

Down-regulation of tomato STEROL GLYCOSYLTRANSFERASE 1 perturbs plant development and facilitates viroid infection.

Potato spindle tuber viroid (PSTVd) is a plant pathogen naturally infecting economically important crops such as tomato (Solanum lycopersicum). Here, we aimed to engineer tomato plants highly resistant to PSTVd and developed several S. lycopersicum lines expressing an artificial microRNA (amiRNA) against PSTVd (amiR-PSTVd). Infectivity assays revealed that amiR-PSTVd-expressing lines were not resistant but instead hypersusceptible to the viroid. A combination of phenotypic, molecular, and metabolic analyses of amiRNA-expressing lines non-inoculated with the viroid revealed that amiR-PSTVd was accidentally silencing the tomato STEROL GLYCOSYLTRANSFERASE 1 (SlSGT1) gene, which caused late developmental and reproductive defects such as leaf epinasty, dwarfism, or reduced fruit size. Importantly, two independent transgenic tomato lines each expressing a different amiRNA specifically designed to target SlSGT1 were also hypersusceptible to PSTVd, thus demonstrating that down-regulation of SlSGT1 was responsible for the viroid-hypersusceptibility phenotype. Our results highlight the role of sterol glycosyltransferases in proper plant development and indicate that the imbalance of sterol glycosylation levels favors viroid infection, most likely by facilitating viroid movement.

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.

Effect of potato spindle tuber viroid variants and infection stage on seed transmission through pollen.

Viroids are small, proteinless single-stranded circular RNAs. In plants, they can be transmitted via infected pollen and seeds. The effectiveness of viroid transmission through pollen depends on both the viroid and host species. It is, however, unclear whether viroid variant type or infection stage influences seed transmission through pollen. In the present study, we collected pollen from petunia infected with nine different variants of the potato spindle tuber viroid (PSTVd) at various stages after inoculation and used the material to pollinate healthy plants. Five and eight PSTVd variants were transmitted by pollen at 3 and 6 mpi respectively. All variants were pollen-transmissible at 9 mpi. The foregoing results indicated that seed transmission of PSTVd through pollen collected from infected donor plants may depend on the time elapsed since inoculation. For variant no. EU862231, however, the rate of seed transmission via pollen may depend on the pollen viroid titre. Nevertheless, there was no apparent correlation between the transmission rate and the pollen viroid titre in the U23058 or V01465 variant. Hence, the relationship between the viroid transmission rate and the pollen viroid titre may depend on the viroid variant type.

A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of plum viroid I (PlVd-I).

Plum viroid I (PlVd-I) is found in marbling and corky flesh diseased plum trees in South Africa. In this study a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the high-throughput detection of PlVd-I was developed. This assay can be performed on crude extracts and detection can either be a pH dependent colorimetric reaction or a real-time fluorescent signal reaction. The false discovery rate was shown to be low and no decrease in sensitivity was detected compared to RT-PCR. The RT-LAMP assay allows for the fast and cost-effective detection of PlVd-I that will curtail the distribution of infected plant material.

Polyclonal antibodies against potato spindle tuber viroid RNA.

Ribonucleic acid (RNA) can act as a hapten in the direct immunization of animals. For antigen synthesis, 65 mg of viroid RNA were obtained by in vitro transcription of the recombinant DNA. We received a reasonable immune response in mice and rabbits with synthesized conjugate viroid RNA-lysozyme. Analyses of polyclonal mouse and rabbit antisera as well as estimates of antibody specificity were performed by dot-Enzyme Linked Immunosorbent Assay (ELISA), sandwich ELISA, and northern immunoblotting. Antiserum obtained showed strong cross-reactions with cellular RNA. The viroid polyclonal antibody cross-reactions with cellular RNAs were depleted via titration antibodies by the plant cellular or commercial yeast RNA. We successfully used antibodies against the viroid RNA-lysozyme antigen to detect the wild-type potato viroid and diagnose potato viroid infection. We presume that intrinsic cross-reactions of RNA antibodies are potentially dangerous after nucleic acid vaccination. Research into the specificity of antibodies against viral RNAs is underway.

The Splicing Variant TFIIIA-7ZF of Viroid-Modulated Transcription Factor IIIA Causes Physiological Irregularities in Transgenic Tobacco and Transient Somatic Depression of "Degradome" Characteristic for Developing Pollen.

Viroids are small, non-coding, pathogenic RNAs with a significant ability of adaptation to several basic cellular processes in plants. TFIIIA-7ZF, a splicing variant of transcription factor IIIA, is involved in replication of nuclear-replicating viroids by DNA-dependent polymerase II. We overexpressed NbTFIIIA-7ZF from Nicotiana benthamiana in tobacco (Nicotiana tabacum) where it caused morphological and physiological deviations like plant stunting, splitting of leaf petioles, pistils or apexes, irregular branching of shoots, formation of double-blade leaves, deformation of main stems, and modification of glandular trichomes. Plant aging and senescence was dramatically delayed in transgenic lines. Factors potentially involved in viroid degradation and elimination in pollen were transiently depressed in transgenic leaves. This depressed "degradome" in young plants involved NtTudor S-like nuclease, dicers, argonoute 5, and pollen extracellular nuclease I showing expression in tobacco anthers and leaves. Analysis of the "degradome" in tobacco leaves transformed with either of two hop viroids confirmed modifications of the "degradome" and TFIIIA expression. Thus, the regulatory network connected to TFIIIA-7ZF could be involved in plant pathogenesis as well as in viroid adaptation to avoid its degradation. These results support the hypothesis on a significant impact of limited TFIIIA-7ZF on viroid elimination in pollen.

Modes of Viroid Transmission.

Studies on the ways in which viroids are transmitted are important for understanding their epidemiology and for developing effective control measures for viroid diseases. Viroids may be spread via vegetative propagules, mechanical damage, seed, pollen, or biological vectors. Vegetative propagation is the most prevalent mode of spread at the global, national and local level while further dissemination can readily occur by mechanical transmission through crop handling with viroid-contaminated hands or pruning and harvesting tools. The current knowledge of seed and pollen transmission of viroids in different crops is described. Biological vectors shown to transmit viroids include certain insects, parasitic plants, and goats. Under laboratory conditions, viroids were also shown to replicate in and be transmitted by phytopathogenic ascomycete fungi; therefore, fungi possibly serve as biological vectors of viroids in nature. The term "mycoviroids or fungal viroids" has been introduced in order to denote these viroids. Experimentally, known sequence variants of viroids can be transmitted as recombinant infectious cDNA clones or transcripts. In this review, we endeavor to provide a comprehensive overview of the modes of viroid transmission under both natural and experimental situations. A special focus is the key findings which can be applied to the control of viroid diseases.

Highly divergent isolates of chrysanthemum virus B and chrysanthemum virus R infecting chrysanthemum in Russia.

BACKGROUND: Chrysanthemum is a popular ornamental and medicinal plant that suffers from many viruses and viroids. Among them, chrysanthemum virus B (CVB, genus Carlavirus, family Betaflexiviridae) is widespread in all chrysanthemum-growing regions. Another carlavirus, chrysanthemum virus R (CVR), has been recently discovered in China. Information about chrysanthemum viruses in Russia is very scarce. The objective of this work was to study the prevalence and genetic diversity of CVB and CVR in Russia. METHODS: We surveyed the chrysanthemum (Chrysanthemum morifolium Ramat.) germplasm collection in the Nikita Botanical Gardens, Yalta, Russia. To detect CVB and CVR, we used RT-PCR with virus-specific primers. To reveal the complete genome sequences of CVB and CVR isolates, metatransciptomic analysis of the cultivars Ribonette, Fiji Yellow, and Golden Standard plants, naturally co-infected with CVB and CVR, was performed using Illumina high-throughput sequencing. The recombination detection tool (RDP4) was employed to search for recombination in assembled genomes. RESULTS: A total of 90 plants of 23 local and introduced chrysanthemum cultivars were surveyed. From these, 58 and 43% plants tested positive for CVB and CVR, respectively. RNA-Seq analysis confirmed the presence of CVB and CVR, and revealed tomato aspermy virus in each of the three transcriptomes. Six near complete genomes of CVB and CVR were assembled from the RNA-Seq reads. The CVR isolate X21 from the cultivar Golden Standard was 92% identical to the Chinese isolate BJ. In contrast, genomes of the CVR isolates X6 and X13 (from the cultivars Ribonette and Fiji Yellow, respectively), were only 76% to 77% identical to the X21 and BJ, and shared 95% identity to one another and appear to represent a divergent group of the CVR. Two distantly related CVB isolates, GS1 and GS2, were found in a plant of the cultivar Golden Standard. Their genomes shared from 82% to 87% identity to each other and the CVB genome from the cultivar Fiji Yellow (isolate FY), as well as to CVB isolates from Japan and China. A recombination event of 3,720 nucleotides long was predicted in the replicase gene of the FY genome. It was supported by seven algorithms implemented in RDP4 with statistically significant P-values. The inferred major parent was the Indian isolate Uttar Pradesh (AM765837), and minor parent was unknown. CONCLUSION: We found a wide distribution of CVB and CVR in the chrysanthemum germplasm collection of the Nikita Botanical Gardens, which is the largest in Russia. Six near complete genomes of CVR and CVB isolates from Russia were assembled and characterized for the first time. This is the first report of CVR in Russia and outside of China thus expanding the information on the geographical distribution of the virus. Highly divergent CVB and CVR isolates have been identified that contributes the better understanding the genetic diversity of these viruses.

Changes in metabolism modulate induced by viroid infection in the orchid Dendrobium officinale.

Dendrobium officinale is an important traditional Chinese medicinal herb, and the stem tissue is the main medicinal that is harvested from D. officinale. Recently, the first viroid was identified from D. officinale in China, and it has been named Dendrobium viroid (DVd). Whether DVd interferes with metabolic pathways in dendrobium plants and affects the medicinal value of the host is unknown. In this study, metabolomics data from stem tissues supported by transcriptome studies were used to investigate how metabolism modulate of D. officinale is altered by DVd infection. Our results show that metabolism of D. officinale is reprogrammed in many ways during DVd infection, and this is reflected by significant changes in the levels of flavonoids, alkaloids, and phenolic acids. Furthermore, we found that DVd infection significantly decreased the accumulation of flavonoids and alkaloid metabolites in infected stems, and the decreases in these metabolites appears to affect the medicinal components of the infected plants, weakening the host antiviral immune response as well. Conversely, phenolic acids occupy a larger proportion of the up-regulated metabolites from DVd infection in comparison with the mock-inoculated control, and the increase in the total phenolic acids may reflect the activation of the pathogen defense response in D. officinale. Taken together, our results provide an interesting overview and give a better understanding of the relationship between metabolism and DVd infection in the orchid D. officinale.

Detection and Characterization of Viroids via Biological Assays on Herbaceous Hosts.

The characterization of the elusive disease agent of the potato spindle tuber disease, potato spindle tuber viroid (PSTVd), was aided by the ability to obtain large amounts of infected tomato tissue in a simple bioassay where PSTVd was easily mechanically transmissible to an alternate herbaceous host in which it thrived and produced dramatic symptoms in a relatively short period (Diener, Viroids. Handbook of plant virus infections: comparative diagnosis. Elsevier/North-Holland, Amsterdam, pp 913-934, 1981; Diener, Virology 45:411-428, 1971; Raymer and O'Brien, Am Pot J, 39:401-408, 1962). Reactions in the primary, or secondary, herbaceous indicator host can range from asymptomatic to severe depending upon the viroid strain, host species, and environmental conditions and can provide evidence of a viroid infection, but do not permit identification of the viroid in question. Further characterization by molecular hybridization, RT-PCR, and sequence analysis is used to determine the etiology of the disease agent. In this chapter, methods are described for mechanical inoculation of viroids to herbaceous hosts to determine the viroid nature of diseases and the experimental host range of the viroid or to shorten the time required for obtaining relatively large amounts of viroid for subsequent purification and characterization.

A Simplified Dot-Blot Hybridization Protocol for Potato spindle tuber viroid Detection in Solanaceae.

A simplified dot-blot hybridization protocol for Potato spindle tuber viroid (PSTVd) detection in Solanaceae species is described here. The protocol uses an RNA DIG-labeled probe and a simplified extraction procedure that avoids the use of hazardous chemicals. PSTVd was detected in composite tomato leaf samples in a ratio of up to 1:15 of PSTVd-infected to non-infected tissue and in composite potato tuber samples in a ratio up to 1:5 of PSTVd-infected to non-infected tissue. In Brugmansia spp., PSTVd was detected solely in the standard sample extract preparation. The method is suitable for a reliable, large-scale sample screening especially where cost is a limiting factor.

Detection of Avocado Sunblotch and Other Viroids Using RNA Filter Paper Capture and RT-PCR.

This method originated due to the need to quickly and sensitively detect Avocado sunblotch viroid (ASBVd) in nursery and field trees in California. Optimum sampling protocols were developed for leaf collection from different sized trees based on size and branching as well as for fruit. An ethanol containing buffered extract from 1 g of ground leaf tissue was used as the source of RNA. The extract was absorbed onto small pieces (disks) of Whatman No. 1 filter paper which were then washed and dried. RNA was eluted from the filter paper using sterile water and used as a template in a standard single-tube RT-PCR reaction. The RNA adsorbed on the filter paper disks was quite stable, and the disks could be stored for over 1 year and shipped worldwide at ambient temperature with no noticeable decline in the quality or quantity of the resulting RT-PCR products. The filter paper capture method was expanded to the detection of other viroids including Potato spindle tuber viroid, Peach latent mosaic viroid, and Chrysanthemum stunt viroid and was tested with some viruses as well with minor modifications of the standard protocol.

Comparison of two highly sensitive methods to detect potato spindle tuber viroid in Dahlia using quantitative-reverse transcription-polymerase chain reaction assays.

Potato spindle tuber viroid (PSTVd) belongs to the Pospiviroidae family and is the type species for the genus Pospiviroid. In 2011, PSTVd was first detected in dahlias in Japan. Since that time, unregistered PSTVd isolates have been identified in seven field-grown dahlia cultivars. None of the infected dahlias showed disease symptoms during the early stages of infection, however, growth suppression occasionally occurred during later stages. Therefore, in dahlia, diagnosing PSTVd by the external appearance of plants is difficult, and the threat of new PSTVd isolates spreading to other susceptible hosts still remains. In this study, we developed an efficient inspection method using several dahlia plant tissues and organs including dried bulbs. This developed method will be useful for inspecting seedlings to prevent the invasion of PSTVd at the border.

Effect of VIRP1 Protein on Nuclear Import of Citrus Exocortis Viroid (CEVd).

Before replicating, Pospiviroidae viroids must move into the plant nucleus. However, the mechanisms of viroid nuclear import are not entirely understood. To study the nuclear import of viroids, we established a nuclear import assay system using onion cell strips and observed the import of Alexa Fluor-594-labeled citrus exocortis viroid (CEVd). To identify the plant factors involved in the nuclear import of viroids, we cloned the Viroid RNA-binding Protein 1 (VIRP1) gene from a tomato cultivar, Seokwang, and heterologously expressed and purified the VIRP1 protein. The newly prepared VIRP1 protein had alterations of amino acid residues at two points (H52R, A277G) compared with a reference VIRP1 protein (AJ249595). VIRP1 specifically bound to CEVd and promoted its nuclear import. However, it is still uncertain whether VIRP1 is the only factor required for the nuclear import of CEVd because CEVd entered the plant nuclei without VIRP1 in our assay system. The cause of the observed nuclear accumulation of CEVd in the absence of VIRP1 needs to be further clarified.

Integrated Proteo-Transcriptomic Analyses Reveal Insights into Regulation of Pollen Development Stages and Dynamics of Cellular Response to Apple Fruit Crinkle Viroid (AFCVd)-Infection in Nicotiana tabacum.

Tobacco (Nicotiana tabacum) pollen is a well-suited model for studying many fundamental biological processes owing to its well-defined and distinct development stages. It is also one of the major agents involved in the transmission of infectious viroids, which is the primary mechanism of viroid pathogenicity in plants. However, some viroids are non-transmissible and may be possibly degraded or eliminated during the gradual process of pollen development maturation. The molecular details behind the response of developing pollen against the apple fruit crinkle viroid (AFCVd) infection and viroid eradication is largely unknown. In this study, we performed an integrative analysis of the transcriptome and proteome profiles to disentangle the molecular cascade of events governing the three pollen development stages: early bicellular pollen (stage 3, S3), late bicellular pollen (stage 5, S5), and 6 h-pollen tube (PT6). The integrated analysis delivered the molecular portraits of the developing pollen against AFCVd infection, including mechanistic insights into the viroid eradication during the last steps of pollen development. The isobaric tags for label-free relative quantification (iTRAQ) with digital gene expression (DGE) experiments led us to reliably identify subsets of 5321, 5286, and 6923 proteins and 64,033, 60,597, and 46,640 expressed genes in S3, S5, and PT6, respectively. In these subsets, 2234, 2108 proteins and 9207 and 14,065 mRNAs were differentially expressed in pairwise comparisons of three stages S5 vs. S3 and PT6 vs. S5 of control pollen in tobacco. Correlation analysis between the abundance of differentially expressed mRNAs (DEGs) and differentially expressed proteins (DEPs) in pairwise comparisons of three stages of pollen revealed numerous discordant changes in mRNA/protein pairs. Only a modest correlation was observed, indicative of divergent transcription, and its regulation and importance of post-transcriptional events in the determination of the fate of early and late pollen development in tobacco. The functional and enrichment analysis of correlated DEGs/DEPs revealed the activation in pathways involved in carbohydrate metabolism, amino acid metabolism, lipid metabolism, and cofactor as well as vitamin metabolism, which points to the importance of these metabolic pathways in pollen development. Furthermore, the detailed picture of AFCVd-infected correlated DEGs/DEPs was obtained in pairwise comparisons of three stages of infected pollen. The AFCVd infection caused the modulation of several genes involved in protein degradation, nuclear transport, phytohormone signaling, defense response, and phosphorylation. Intriguingly, we also identified several factors including, DNA-dependent RNA-polymerase, ribosomal protein, Argonaute (AGO) proteins, nucleotide binding proteins, and RNA exonucleases, which may plausibly involve in viroid stabilization and eradication during the last steps of pollen development. The present study provides essential insights into the transcriptional and translational dynamics of tobacco pollen, which further strengthens our understanding of plant-viroid interactions and support for future mechanistic studies directed at delineating the functional role of candidate factors involved in viroid elimination.

Identification and Molecular Mechanisms of Key Nucleotides Causing Attenuation in Pathogenicity of Dahlia Isolate of Potato Spindle Tuber Viroid.

While the potato spindle tuber viroid (PSTVd) variant, PSTVd-Dahlia (PSTVd-D or PSTVd-Dwt) induces very mild symptoms in tomato cultivar 'Rutgers', PSTVd-Intermediate (PSTVd-I or PSTVd-Iwt) induces severe symptoms. These two variants differ by nine nucleotides, of which six mutations are located in the terminal left (TL) to the pathogenicity (P) domains. To evaluate the importance of mutations located in the TL to the P domains, ten types of point mutants were created by swapping the nucleotides between the two viroid variants. Bioassay in tomato plants demonstrated that two mutants created on PSTVd-Iwt at positions 42 and 64 resulted in symptom attenuation. Phenotypic and RT-qPCR analysis revealed that mutation at position 42 of PSTVd-Iwt significantly reduced disease severity and accumulation of the viroid, whereas mutation at position 64 showed a significant reduction in stunting when compared to the PSTVd-Iwt infected plant. RT-qPCR analysis on pathogenesis-related protein 1b1 and chalcone synthase genes showed a direct correlation with symptom severity whereas the expansin genes were down-regulated irrespective of the symptom severity. These results indicate that the nucleotides at positions 42 and 64 are in concert with the ones at positions 43, 310, and 311/312, which determines the slower and stable accumulation of PSTVd-D without eliciting excessive host defense responses thus contributing in the attenuation of disease symptom.