Potato Spindle Tuber Viroid Variants Research Articles

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.

Mechanism underlying potato spindle tuber viroid affecting tomato (Solanum lycopersicum): loss of control over reactive oxygen species production

Severe infection of the tomato cultivar ‘Rutgers’ with potato spindle tuber viroid (PSTVd) variants resulted in systemic stunting, leaf malformation, and vein necrosis; accompanied by a rapid increase in reactive oxygen species (ROS) and abnormally high expression of the stress-responsive microRNAs, miR398 and miR398a-3p. The severity of the symptoms, PSTVd accumulation, the high expression of miR398 and miR398a-3p, and the high level of ROS production were positively correlated with the pathogenesis of the PSTVd variants. In contrast, the cytosolic and chloroplast-localized Cu/Zn-superoxide dismutase genes SlSOD4 and SlSOD3, respectively that encode the ROS scavenging enzymes were down-regulated and negatively correlated with the severity of the symptoms. Conversely, in the PSTVd-infected, PSTVd-tolerant (almost asymptomatic) tomato cultivar ‘Moneymaker’ in which PSTVd accumulation was suppressed, the ROS production was as low as in the control, miR398 and miR398a-3p were down-regulated, and almost all the SOD genes were up-regulated. The results indicated that in the PSTVd-sensitive tomato cultivar ‘Rutgers’, infection with the severe PSTVd variant caused suppression of the cytosolic and chloroplast-localized Cu/Zn-SOD genes via PSTVd-induced overexpression of miR398 and miR398a-3, respectively. This condition significantly reduced or eliminated the normal ROS scavenging function and an excess of harmful ROS in cells and tissues seem to have caused severe pathological symptoms accompanied by necrosis.

Differences in gene expression profiles at the early stage of Solanum lycopersicum infection with mild and severe variants of potato spindle tuber viroid

Viroids with small, non-coding circular RNA genome can induce diseases in many plant species. The extend of infection symptoms depends on environmental conditions, viroid strain, and host plant species and cultivar. Pathogen recognition leads to massive transcriptional reprogramming to favor defense responses over normal cellular functions. To better understand the interaction between plant host and potato spindle tuber viroid (PSTVd) variants that differ in their virulence, comparative transcriptomic analysis was performed by an RNA-seq approach. The changes of gene expression were analyzed at the time point when subtle symptoms became visible in plants infected with the severe PSTVd-S23 variant, while those infected with the mild PSTVd-M variant looked like non-infected healthy plants. Over 3000 differentially expressed genes (DEGs) were recognized in both infections, but the majority of them were specific for infection with the severe variant. In both infections recognized DEGs were mainly related to biotic stress, hormone metabolism and signaling, transcription regulation, protein degradation, and transport. The DEGs related to cell cycle and microtubule were uniquely down-regulated only in the PSTVd-S23-infected plants. Similarly, expression of transcription factors from C2C2-GATA and growth-regulating factor (GRF) families was only altered upon infection with the severe variant. Both PSTVd variants triggered plant immune response; however expression of genes encoding crucial factors of this process was markedly more changed in the plants infected with the severe variant than in those with the mild one.

Root Transcriptomic Analysis Reveals Global Changes Induced by Systemic Infection of Solanum lycopersicum with Mild and Severe Variants of Potato Spindle Tuber Viroid.

Potato spindle tuber viroid (PSTVd) causes systemic infection in plant hosts. There are many studies on viroid-host plant interactions, but they have predominantly focused on the aboveground part of the plant. Here, we investigated transcriptomic profile changes in tomato roots systemically infected with mild or severe PSTVd variants using a combined microarray/RNA-seq approach. Analysis indicated differential expression of genes related to various Gene Ontology categories depending on the stage of infection and PSTVd variant. A majority of cell-wall-related genes were down-regulated at early infection stages, but at the late stage, the number of up-regulated genes increased significantly. Along with observed alterations of many lignin-related genes, performed lignin quantification indicated their disrupted level in PSTVd-infected roots. Altered expression of genes related to biosynthesis and signaling of auxin and cytokinin, which are crucial for lateral root development, was also identified. Comparison of both PSTVd infections showed that transcriptional changes induced by the severe variant were stronger than those caused by the mild variant, especially at the late infection stage. Taken together, we showed that similarly to aboveground plant parts, PSTVd infection in the underground tissues activates the plant immune response.

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

SummaryViroid 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.

Viroid quasispecies revealed by deep sequencing

ABSTRACTViroids are non-coding single-stranded circular RNA molecules that replicate autonomously in infected host plants causing mild to lethal symptoms. Their genomes contain about 250–400 nucleotides, depending on viroid species. Members of the family Pospiviroidae, like the Potato spindle tuber viroid (PSTVd), replicate via an asymmetric rolling-circle mechanism using the host DNA-dependent RNA-Polymerase II in the nucleus, while members of Avsunviroidae are replicated in a symmetric rolling-circle mechanism probably by the nuclear-encoded polymerase in chloroplasts. Viroids induce the production of viroid-specific small RNAs (vsRNA) that can direct (post-)transcriptional gene silencing against host transcripts or genomic sequences. Here, we used deep-sequencing to analyze vsRNAs from plants infected with different PSTVd variants to elucidate the PSTVd quasipecies evolved during infection. We recovered several novel as well as previously known PSTVd variants that were obviously competent in replication and identified common strand-specific mutations. The calculated mean error rate per nucleotide position was less than , quite comparable to the value of reported for a member of Avsunviroidae. The resulting error threshold allows the synthesis of longer-than-unit-length replication intermediates as required by the asymmetric rolling-circle mechanism of members of Pospiviroidae.

Occurrence and molecular characterization of Potato spindle tuber viroid (PSTVd) isolates from potato plants in North China

AbstractChina is the largest potato producing country worldwide, with this crop representing the fourth largest staple food crop in China. However, the steady presence of Potato spindle tuber viroid (PSTVd) over the past five decades has a significant economic impact on potato production. To determine why PSTVd control measures have been ineffective in China, more than 1 000 seed potatoes collected between 2009 and 2014 were subjected to PSTVd detection at the Supervision and Testing Center for Virus-free Seed Potatoes Quality, Ministry of Agriculture, China. A high PSTVd infection rate (6.5%) was detected among these commercial seed potatoes. Some breeding lines of potato collected from 2012 to 2015 were also tested for PSTVd infection, revealing a high rate of PSTVd contamination in these potato propagation materials. Furthermore, comparison of the full-length sequences of 71 different Chinese PSTVd isolates revealed a total of 74 predominant PSTVd variants, which represented 42 different sequence variants of PSTVd. Comparative sequence analysis revealed 30 novel PSTVd sequence variants specific to China. Comprehensive phylogenetic analysis uncovered a close relationship between the Chinese PSTVd sequence variants and those isolated from Russia. It is worth noting that three intermediate strains and six mild strains were identified among these variants. These results have important implications for explaining the ineffective control of PSTVd in China and thus could serve as a basic reference for designing more effective measures to eliminate PSTVd from China in the future.

Small RNA Derived from the Virulence Modulating Region of the Potato spindle tuber viroid Silences callose synthase Genes of Tomato Plants.

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.

Analysis of small RNA production patterns among the two potato spindle tuber viroid variants in tomato plants.

In order to analyze the production of small RNA (sRNA) by viroids upon infecting the plants, the tomato plants (Solanum lycopersicum cultivar Rutgers) were inoculated with the variants of Potato spindle tuber viroid (PSTVd). After 21-days of postinoculation, total RNA was extracted and subjected for deep-sequencing using Illumina HiSeq platform. The primers were trimmed and only 21- to 24-nt long sRNAs were filtered after quality check of the raw data. The filtered sRNA population was then mapped against both the genomic (+) and antigenomic (−) strands of the respective PSTVd variants using standard pattern-matching algorithm. The profiling of viroid derived sRNA (vd-sRNA) revealed that the viroids are susceptible to host RNA silencing mechanism. High-throughput sequence data linked to this project have been deposited in the Gene Expression Omnibus (GEO) database under accession number GSE69225.

Expression of SANT/HTH Myb mRNA, a plant morphogenesis-regulating transcription factor, changes due to viroid infection

Potato spindle tuber viroid (PSTVd) belongs to plant-pathogenic, circular, non-coding RNAs. Its propagation is accompanied by (mis)regulation of host genes and induction of pathogenesis symptoms including changes of leaf morphogenesis depending on the strength of viroid variant. We found strong genotype-dependent suppression of tomato morphogenesis-regulating transcription factor SANT/HTH-Myb (SlMyb) due to viroid pathogenesis. Its relative mRNA level was found to be significantly decreased in PSTVd-sensitive tomato (cvs Rutgers and Heinz 1706) due to degradation processes, but increased in PSTVd-tolerant (cv. Harzfeuer). In heterologous system of Nicotiana benthamiana, we observed a SlMyb-associated necrotic effect in agroinfiltrated leaf sectors during ectopic overexpression. Leaf sector necroses were accompanied by activation of nucleolytic enzymes but were suppressed by a strongly pathogenic PSTVd variant. Contrary to that, PSTVd's effect was inhibited by the silencing suppressor p19. It was found that in both, Solanum lycopersicum leaves and N. benthamiana leaf sectors, SlMyb mRNA degradation was significantly stronger in viroid-infected tissues. Necroses induction as well as gene silencing experiments using the SANT/HTH-Myb homologues revealed involvement of this Myb in physiological changes like distortions in flower morphogenesis and growth suppression.

Detection of Potato spindle tuber viroid sequence variants derived from PSTVd-infected Phelipanche ramosa in flower organs of tomato plants

Potato spindle tuber viroid (PSTVd) is an infectious small, circular, non-coding single-stranded RNA that induces disease on many crop species, ornamental plants, weeds and parasitic plants. PSTVd propagate in their host as a population of closely related but non-identical RNA variants referred to as quasispecies. Recently, we have described three de novo arising PSTVd variants in the parasitic plant Phelipanche ramosa after mechanical inoculation with the PSTVd KF440-2 isolate. These P. ramosa derived mutants were designated as G241-C, C208-U and C227-U PSTVd variants. Each of these variants carries a single-nucleotide substitution compared to the PSTVd KF440-2 sequence from which they are considered to have evolved. Here we complement our previous studies on these mutants by exploring their potential to infect the floral organs of tomato plants. We found that the PSTVd G241-C and C208-U variants were able to replicate in systemic leaves and floral organs of tomato plants, while the PSTVd C227-U variant did not develop systemic infection. Furthermore, we analysed the progeny of these PSTVd variants in sepals and petals of tomato plants for retention of the specific mutations.

Characterization of Potato spindle tuber viroid (PSTVd) incidence and new variants from ornamentals

We analyzed the spreading and persistence of PSTVd variants in several ornamentals in the territory of the Czech Republic. The pool of PSTVd variants detected in Solanum jasminoides, S. muricatum, Datura sp. and Brugmansia sp. was biolistically transferred to Matricaria chamomilla, Argyranthemum frutescens and Diascia sp., species which we found as sensitive hosts for PSTVd from ornamentals. The PSTVd pool showed sequence changes and increased variation after its transfer to potato, suggesting a wide adaptation potential of PSTVd in this crop. Potato exhibited genotype-dependent leaf and spindle tuber symptoms, when inoculated with the sap from S. jasminoides infected with the predominant and sequence-stable PSTVd-S1.

Secondary structures of Potato spindle tuber viroid variants detected in Slovenia

A survey of Potato spindle tuber viroid was initiated in Slovenia in 2006. Until the end of 2010, 100 samples of ornamental plants were found to be infected. Analysis of 96 complete master sequences of Slovene samples revealed new genetic variants. Our sequence variants showed considerable variation in their predicted secondary structure. The variability was observed in the pathogenic, central and variable domains. In several cases even single mutations were sufficient for the change in predicted secondary structure.

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.

Use of randomly mutagenized genomic cDNA banks of potato spindle tuber viroid to screen for viable versions of the viroid genome

In an effort to study sequence space allowing the recovery of viable potato spindle tuber viroid (PSTVd) variants we have developed an in vivo selection (Selex) method to produce and bulk-inoculate by agroinfiltration large PSTVd cDNA banks in which a short stretch of the genome is mutagenized to saturation. This technique was applied to two highly conserved 6 nt-long regions of the PSTVd genome, the left terminal loop (TL bank) and part of the polypurine stretch in the upper strand of pre-melting loop 1 (PM1 bank). In each case, PSTVd accumulation was observed in a large fraction of bank-inoculated tomato plants. Characterization of the progeny molecules showed the recovery of the parental PSTVd sequence in 89 % (TL bank) and 18 % (PM1 bank) of the analysed plants. In addition, viable and genetically stable PSTVd variants with mutations outside of the known natural variability of PSTVd were recovered in both cases, although at different rates. In the case of the TL region, mutations were recovered at five of the six mutagenized positions (357, 358, 359, 1 and 3 of the genome) while for the PM1 region mutations were recovered at all six targeted positions (50-55), providing significant new insight on the plasticity of the PSTVd genome.

Russian Isolates of Potato spindle tuber viroid Exhibit Low Sequence Diversity.

Potato spindle tuber viroid (PSTVd) is currently widespread in seed potatoes grown in Russia. Characterization of 39 PSTVd isolates collected over a 15-year period from widely separated areas in Russia revealed the presence of 17 different sequence variants, all but one of which were previously unknown. Most variants were recovered only once, but two were more widely distributed; one of these was a mild variant previously isolated in Germany, the second was a novel variant inducing symptoms similar to those of the type strain in tomato. Despite this apparent lack of population diversity, several informative PSTVd variants were recovered. Sequence changes in the pathogenicity and variable domains were particularly common, but previously unknown changes were also detected within the loop E motif in the central domain, a structural motif known to play a key role in PSTVd replication and host range determination.

Recovery of Four Novel Potato spindle tuber viroid Sequence Variants from Russian Seed Potatoes.

First described in the early 1930s, the limited distribution of potato "gothic" disease made it of little economic significance in European Russia until the early 1970s when meristem-tip culture was widely adopted throughout the former USSR to increase production of virus-free seed potatoes. Shortly thereafter, the yield and quality of Russian seed potatoes began a dramatic decline. Symptoms of potato "gothic" resemble those of Potato spindle tuber viroid (PSTVd) (3), and initial suspicions that in vitro plantlets and seed potatoes might be viroid-infected were later proved correct when Kastalyeva et al. (2) showed that approximately 50 to 70% of in vitro plantlets and tubers collected from different regions of Russia as well as the in vitro germplasm collection maintained by the All-Russian Potato Research Institute (ARPRI) were infected with PSTVd. Measures have since been taken to reduce the incidence of PSTVd infection, and numerous PSTVd isolates were collected from territories of the former USSR; however, none of these isolates have been characterized at the molecular level. Overlapping reverse transcription (RT)-PCR products (1) were generated from four PSTVd isolates maintained in field-grown tubers at the VNIIF using two pairs of primers; PSTVd180F (5'-TCACCCTTCCTTTCTTCGGGTGTC-3') + PSTVd179R (5'-AAACCCTGTTTCGGCGGGAATTAC-3') and PSTVd112F (5'-ACT GGCAAAAAAGGACGGTGGGGA-3') + PSTVd359R (5'-AGGAACC AACTGCGGTTCCAAGGG-3'). Automated sequence analysis of the resulting uncloned PCR products revealed the presence of four previously unknown PSTVd variants (GenBank Accession Nos. EF044302-EF044305). All four tubers were also infected with Potato virus M and Potato virus Y and one tuber also contained Potato virus S. ELISA tests for Potato leaf roll virus were negative. Each isolate appeared to contain only a single 358-359 nt variant differing from PSTVd-intermediate strain (GenBank Accession No. V01465) at 2-5 positions. The three closely related variants originating from Leningradskaya Province (Northwest Russia) contain two to three changes in the variable domain and central conserved region and induced intermediate symptoms in Rutgers tomato. The fourth variant originating from Samarskaya Province (Volga River Region) contains additional changes in the pathogenicity domain and induced mild symptoms. Minor differences among the Leningradskaya variants may represent sequence drift during extended (9 to 11 year) tuber passage. The presence of additional sequence changes in the variant from Samarskaya is consistent with independent origin and/or prolonged separation. Additional studies with a wider range of Russian isolates of PSTVd are currently underway to develop diagnostic methods suitable for future large-scale screening programs.

Co-inoculation with two non-infectious cDNA copies of potato spindle tuber viroid (PSTVd) leads to the appearance of novel fully infectious variants.

Potato spindle tuber viroid (PSTVd) is one of the smallest (about 360 nt) infectious plant agents. It is composed of a single-stranded circular non-coding RNA molecule. In the course of previous passage experiments with two intermediate PSTVd variants I2 and I4, three non-infectious clones (I2-50, I4-37 and I4 VI-17) were found. When inoculated separately as cDNAs on tomato "Rutgers" test plants these variants did not induce any visible disease symptoms and did not produce progeny. The presence of such non-infectious variants raises several questions about their origin and biology and to answer them, mixed co-infections with cDNA copies of two non-infectious variants (I2-50, I4-37) were performed. PSTVd infection was observed in seven out of 30 inoculated plants. The progeny isolated from three separate plants contained novel variants, together with the parental I2 and I4 sequences. It is conceivable that the appearance of repaired PSTVd molecules, clearly capable of cell-to-cell movement leading to the systemic infection, results from recombination events. An analysis of the recombinant molecules and comparison with databases identified the specific sites responsible for the restricted infectivity of the I2-50 and I4-37 PSTVd variants. In parallel experiments in which (+) strand PSTVd infectious transcripts were used, no recombinants were observed, and the original I2-50 and I4-37 non-infectious sequences were not detected in the progeny.

Co-inoculation with two non-infectious cDNA copies of potato spindle tuber viroid (PSTVd) leads to the appearance of novel fully infectious variants.

Potato spindle tuber viroid (PSTVd) is one of the smallest (about 360 nt) infectious plant agents. It is composed of a single-stranded circular non-coding RNA molecule. In the course of previous passage experiments with two intermediate PSTVd variants I2 and I4, three non-infectious clones (I2-50, I4-37 and I4 VI-17) were found. When inoculated separately as cDNAs on tomato "Rutgers" test plants these variants did not induce any visible disease symptoms and did not produce progeny. The presence of such non-infectious variants raises several questions about their origin and biology and to answer them, mixed co-infections with cDNA copies of two non-infectious variants (I2-50, I4-37) were performed. PSTVd infection was observed in seven out of 30 inoculated plants. The progeny isolated from three separate plants contained novel variants, together with the parental I2 and I4 sequences. It is conceivable that the appearance of repaired PSTVd molecules, clearly capable of cell-to-cell movement leading to the systemic infection, results from recombination events. An analysis of the recombinant molecules and comparison with databases identified the specific sites responsible for the restricted infectivity of the I2-50 and I4-37 PSTVd variants. In parallel experiments in which (+) strand PSTVd infectious transcripts were used, no recombinants were observed, and the original I2-50 and I4-37 non-infectious sequences were not detected in the progeny.

Inhibition of cell growth and shoot development by a specific nucleotide sequence in a noncoding viroid RNA.

Viroids are small noncoding and infectious RNAs that replicate autonomously and move systemically throughout an infected plant. The RNAs of the family Pospiviroidae contain a central conserved region (CCR) that has long been thought to be involved in replication. Here, we report that the CCR of Potato spindle tuber viroid (PSTVd) also plays a role in pathogenicity. A U257A change in the CCR converted the intermediate strain PSTVd(Int) to a lethal strain that caused severe growth stunting and premature death of infected plants. PSTVd with nucleotide U257 changed to C or G did not cause such symptoms. The pathogenic effect of the U257A substitution was abolished by a C259U substitution in the same RNA. Analyses of the pathogenic effects of the U257A substitution in three other PSTVd variants established A257 as a new pathogenicity determinant that functions independently and synergistically with the classic pathogenicity domain. The U257A substitution did not alter PSTVd secondary structure, replication levels, or tissue tropism. The stunted growth of PSTVd(Int)U257A-infected tomato plants resulted from restricted cell expansion but not cell division or differentiation. This was correlated positively with the downregulated expression of an expansin gene, LeExp2. Our results demonstrate that specific nucleotides in a noncoding, pathogenic RNA have a profound effect in altering distinct cellular responses, which then lead to well-defined alterations in plant growth and developmental patterns. The feasibility of correlating viroid RNA sequence/structure with the altered expression of specific host genes, cellular processes, and developmental patterns makes viroid infection a valuable system in which to investigate host factors for symptom expression and perhaps also to characterize the mechanisms of RNA regulation of gene expression in plants.