The tomato calcium-permeable channel 4.1 (SlOSCA4.1) is a susceptibility factor for pepino mosaic virus.

The hyperosmolality-gated calcium permeable channel 4.1 (OSCA4.1) belongs to an evolutionarily conserved small family of mechano-sensitive channels. OSCA members may represent key players in plant resistance to drought and to pathogen infection but are scarcely studied. After screening for resistance to pepino mosaic virus (PepMV) a collection of 1000 mutagenized tomato families, we identified a mutant showing no symptoms and reduced virus accumulation. Resistance was mapped to chromosome 2 between positions 46 309 531 to 47 044 163, where a missense mutation caused the putative truncation of the OSCA4.1 protein. A CRISPR/Cas9 slosca4.1 mutant was resistant to PepMV, but not to tobacco mosaic virus or potato virus X. Inoculation of mutant and wild type tomato protoplasts showed that resistance was expressed in single cells, suggesting a role for SlOSCA4.1 in early viral function(s); congruently, SlOSCA4.1 re-localized to structures reminiscent of viral replication complexes. We propose that SlOSCA4.1 contributes to the correct regulation of the Ca2+ homeostasis necessary for optimal PepMV infection. PepMV is a pandemic virus that causes significant losses in tomato crops worldwide. In spite of its importance, no tomato-resistant varieties have been deployed yet; the mutant identified here has great potential to breed tomato varieties resistant to PepMV.

Potato virus X: A global potato-infecting virus and type member of the Potexvirus genus.

TAXONOMY: Potato virus X is the type-member of the plant-infecting Potexvirus genus in the family Alphaflexiviridae. PHYSICAL PROPERTIES: Potato virus X (PVX) virions are flexuous filaments 460-480 nm in length. Virions are 13 nm in diameter and have a helical pitch of 3.4 nm. The genome is approximately 6.4 kb with a 5' cap and 3' poly(A) terminus. PVX contains five open reading frames, four of which are essential for cell-to-cell and systemic movement. One protein encodes the viral replicase. Cellular inclusions, known as X-bodies, occur near the nucleus of virus-infected cells. HOSTS: The primary host is potato, but it infects a wide range of dicots. Diagnostic hosts include Datura stramonium and Nicotiana tabacum. PVX is transmitted in nature by mechanical contact. USEFUL WEBSITE: https://talk.ictvonline.org/ictv-reports/ictv_online_report/positive-sense-rna-viruses/w/alphaflexiviridae/1330/genus-potexvirus.

Evolution of Potato virus X.

Potato virus X (PVX) is the type potexvirus of economic significance. The pathogen is distributed worldwide, threatening solanaceous plants in particular. Based on the coat protein (CP) gene, PVX isolates are classified into two major genotypes (I and II). To gain more insights into the molecular epidemiology and evolution of PVX, recombination analyses were conducted and significant signals were detected. Bayesian coalescent method was then applied to the time-stamped entire CP sequences. According to the estimates, the global subtype I-1 went into expansion in the 20th century and was evolving at a moderate rate. Based on the CP phylogenies, a divergence scenario was proposed for PVX. Surveys of codon usage variation showed that PVX genes had additional bias independent of compositional constraint. In codon preference, PVX was both similar to and different from the three major hosts, potato (Solanum tuberosum), tobacco (Nicotiana tabacum), and tomato (S. lycopersicum). Moreover, the suppression of CpG and UpA dinucleotide frequencies was observed in PVX.

One-step real-time multiplex reverse transcription-polymerase chain reaction assay with melt curve analysis for detection of potato leafroll virus, potato virus S, potato virus X, and potato virus Y.

BACKGROUND: Certification of seed potato as free of viruses is essential for stable potato production. Among more than 30 virus species infecting potato, potato leafroll virus (PLRV), potato virus S (PVS), potato virus X (PVX), and potato virus Y (PVY) predominate worldwide and should be the targets of a high-throughput detection protocol for seed potato quarantine. RESULTS: We developed an assay based on one-step real-time multiplex reverse transcription-polymerase chain reaction (mRT-PCR) with melt curve analysis for the four viruses and one internal control, potato elongation factor 1 alpha gene (EF1α). Virus-specific primers were derived from conserved regions among randomly selected representatives considering viral genomic diversity. Our assay simultaneously detected representative Japanese isolates of PLRV, O lineage of PVS, PVX, and NTN strain of PVY. The variability of melting temperature (Tm) values for each virus was confirmed using Japanese isolates, and virus species could be identified by the values of 87.6 for PLRV, 85.9 for PVX, 82.2 (Ordinary lineage) to 83.1 (Andean lineage) for PVS, and 79.4 (NA-N strain) to 80.5 (O strain and NTN strain) for PVY on average. The reliability of calculation was validated by comparing the calculated Tm values and measured Tm values and the values had a strong linear correlation (correlation of determination: R2 = 0.9875). Based on the calculated Tm values, representative non-Japanese isolates could also be identified by our assay. For removing false positives, two criteria were set for the evaluation of result; successful amplification was considered as 30.0 ≥ threshold cycle value, and the virus-specific peak higher than the EF1α-specific peak was considered as positive. According to these criteria, our assay could detect PLRV and PVS from 100-fold dilution of potato leaf homogenate and PVX and PVY from 1000-fold in a model assay. CONCLUSION: This new high-throughput detection protocol using one-step real-time mRT-PCR was sensitive enough to detect viruses in a 100-fold dilution of singly-virus contaminated homogenate in a model assay. This protocol can detect the four viruses in one assay and yield faster results for a vast number of samples, and greatly save the labor for seed potato quarantine and field surveys.

Rapid and sensitive detection of potato virus X by one-step reverse transcription-recombinase polymerase amplification method in potato leaves and dormant tubers.

Potato virus X (PVX), is a serious threat to global potato production. A simple and rapid detection method is imperative for PVX diagnosis and early management. In this study, an isothermal one-step reverse transcription-recombinase polymerase amplification (RT-RPA) method was optimized for the quick and convenient detection of PVX in potato leaves and tubers. Our results revealed that this one-step RT-RPA method was highly efficient than the conventional reverse transcription-polymerase chain reaction (RT-PCR). The amplification reaction was free from cross-reactivity with other common potato viruses and completed within 30 min. Moreover, this RT-RPA assay did not require a thermocycler based specific temperature phase amplification and can be easily performed using a simple heating block or water bath at a temperature range of 39-42 °C. The sensitivity assay demonstrated that the developed one-step RT-RPA method was 100 times more sensitive than a routine one-step RT-PCR. Initially, the purified total RNA as the template isolated from infected leaves of potato was used for the detection of PVX. One-step RT-RPA was later performed using cellular disc paper-based simple RNA extract as a template that could detect the virus more efficiently than purified total RNA. The performance of the one-step RT-RPA assay was further evaluated using 500 field samples of leaves and tubers representing different cultivars and geographical regions. To our knowledge, this is the first report of rapid, sensitive, and reliable detection of PVX infection by one-step RT-RPA using cellular disc paper-based simple RNA extract from leaves and dormant tubers of potato. It is superior to the common RT-PCR assay in terms of its versatility, quickness, and independence of highly purified RNA template and can be adopted as a substitute to RT-PCR as an effective technique for seed potato certification, quarantine, breeding, and field surveys.

Transcriptional Regulatory Networks Associate with Early Stages of Potato Virus X Infection of Solanum tuberosum.

Potato virus X (PVX) belongs to genus Potexvirus. This study characterizes the cellular transcriptome responses to PVX infection in Russet potato at 2 and 3 days post infection (dpi). Among the 1242 differentially expressed genes (DEGs), 268 genes were upregulated, and 37 genes were downregulated at 2 dpi while 677 genes were upregulated, and 265 genes were downregulated at 3 dpi. DEGs related to signal transduction, stress response, and redox processes. Key stress related transcription factors were identified. Twenty-five pathogen resistance gene analogs linked to effector triggered immunity or pathogen-associated molecular pattern (PAMP)-triggered immunity were identified. Comparative analysis with Arabidopsis unfolded protein response (UPR) induced DEGs revealed genes associated with UPR and plasmodesmata transport that are likely needed to establish infection. In conclusion, this study provides an insight on major transcriptional regulatory networked involved in early response to PVX infection and establishment.

The Phylogeography of Potato Virus X Shows the Fingerprints of Its Human Vector.

Potato virus X (PVX) occurs worldwide and causes an important potato disease. Complete PVX genomes were obtained from 326 new isolates from Peru, which is within the potato crop's main domestication center, 10 from historical PVX isolates from the Andes (Bolivia, Peru) or Europe (UK), and three from Africa (Burundi). Concatenated open reading frames (ORFs) from these genomes plus 49 published genomic sequences were analyzed. Only 18 of them were recombinants, 17 of them Peruvian. A phylogeny of the non-recombinant sequences found two major (I, II) and five minor (I-1, I-2, II-1, II-2, II-3) phylogroups, which included 12 statistically supported clusters. Analysis of 488 coat protein (CP) gene sequences, including 128 published previously, gave a completely congruent phylogeny. Among the minor phylogroups, I-2 and II-3 only contained Andean isolates, I-1 and II-2 were of both Andean and other isolates, but all of the three II-1 isolates were European. I-1, I-2, II-1 and II-2 all contained biologically typed isolates. Population genetic and dating analyses indicated that PVX emerged after potato's domestication 9000 years ago and was transported to Europe after the 15th century. Major clusters A-D probably resulted from expansions that occurred soon after the potato late-blight pandemic of the mid-19th century. Genetic comparisons of the PVX populations of different Peruvian Departments found similarities between those linked by local transport of seed potato tubers for summer rain-watered highland crops, and those linked to winter-irrigated crops in nearby coastal Departments. Comparisons also showed that, although the Andean PVX population was diverse and evolving neutrally, its spread to Europe and then elsewhere involved population expansion. PVX forms a basal Potexvirus genus lineage but its immediate progenitor is unknown. Establishing whether PVX's entirely Andean phylogroups I-2 and II-3 and its Andean recombinants threaten potato production elsewhere requires future biological studies.

Construction of an infectious full-length cDNA clone of potato aucuba mosaic virus.

Potato aucuba mosaic virus (PAMV), a positive single-strand RNA virus, has one of the longest genomes of the viruses in the genus Potexvirus. In 2019, potato samples with mottle and crinkling symptoms from Huzhou, Zhejiang province, China, were identified to be infected with PAMV, potato virus X (PVX), and potato virus Y (PVY) by transcriptome sequencing. To study the effects of single infection by PAMV, the full-length sequence of PAMV from Huzhou (MT193476) was determined and an infectious full-length cDNA clone was constructed. This cDNA clone was infectious by agro-infiltration, leading to systemic symptoms in Nicotiana benthamiana, tomato, pepper, and potato.

Molecular Viral Diagnosis and Sanitation of Yam Genetic Resources: Implications for Safe Yam Germplasm Exchange.

Yam (Dioscorea spp.) is an important crop in tropical and subtropical regions. Many viruses have been recently identified in yam, hampering genetic conservation and safe international exchanges of yam germplasm. We report on the implementation of reliable and cost-effective PCR-based detection tools targeting eight different yam-infecting viruses. Viral indexing of the in vitro yam collection maintained by the Biological Resources Center for Tropical Plants (BRC-TP) in Guadeloupe (French West Indies) unveiled a high prevalence of potyviruses, badnaviruses, Dioscorea mosaic associated virus (DMaV) and yam asymptomatic virus 1 (YaV1) and a high level of coinfections. Infected yam accessions were subjected to a combination of thermotherapy and meristem culture. Sanitation levels were monitored using PCR-based and high-throughput sequencing-based diagnosis, confirming the efficacy and reliability of PCR-based detection tools. Sanitation rates were highly variable depending on viruses. Sixteen accessions were successfully sanitized, paving the way to safe yam germplasm exchanges and the implementation of clean seed production programs worldwide.

RNA silencing-related genes contribute to tolerance of infection with potato virus X and Y in a susceptible tomato plant.

BACKGROUND: In plants, the RNA silencing system functions as an antiviral defense mechanism following its induction with virus-derived double-stranded RNAs. This occurs through the action of RNA silencing components, including Dicer-like (DCL) nucleases, Argonaute (AGO) proteins, and RNA-dependent RNA polymerases (RDR). Plants encode multiple AGOs, DCLs, and RDRs. The functions of these components have been mainly examined in Arabidopsis thaliana and Nicotiana benthamiana. In this study, we investigated the roles of DCL2, DCL4, AGO2, AGO3 and RDR6 in tomato responses to viral infection. For this purpose, we used transgenic tomato plants (Solanum lycopersicum cv. Moneymaker), in which the expression of these genes were suppressed by double-stranded RNA-mediated RNA silencing. METHODS: We previously created multiple DCL (i.e., DCL2 and DCL4) (hpDCL2.4) and RDR6 (hpRDR6) knockdown transgenic tomato plants and here additionally did multiple AGO (i.e., AGO2 and AGO3) knockdown plants (hpAGO2.3), in which double-stranded RNAs cognate to these genes were expressed to induce RNA silencing to them. Potato virus X (PVX) and Y (PVY) were inoculated onto these transgenic tomato plants, and the reactions of these plants to the viruses were investigated. In addition to observation of symptoms, viral coat protein and genomic RNA were detected by western and northern blotting and reverse transcription-polymerase chain reaction (RT-PCR). Host mRNA levels were investigated by quantitative RT-PCR. RESULTS: Following inoculation with PVX, hpDCL2.4 plants developed a more severe systemic mosaic with leaf curling compared with the other inoculated plants. Systemic necrosis was also observed in hpAGO2.3 plants. Despite the difference in the severity of symptoms, the accumulation of PVX coat protein (CP) and genomic RNA in the uninoculated upper leaves was not obviously different among hpDCL2.4, hpRDR6, and hpAGO2.3 plants and the empty vector-transformed plants. Moneymaker tomato plants were asymptomatic after infection with PVY. However, hpDCL2.4 plants inoculated with PVY developed symptoms, including leaf curling. Consistently, PVY CP was detected in the uninoculated symptomatic upper leaves of hpDCL2.4 plants through western blotting. Of note, PVY CP was rarely detected in other asymptomatic transgenic or wild-type plants. However, PVY was detected in the uninoculated upper leaves of all the inoculated plants using reverse transcription-polymerase chain reactions. These findings indicated that PVY systemically infected asymptomatic Moneymaker tomato plants at a low level (i.e., no detection of CP via western blotting). CONCLUSION: Our results indicate that the tomato cultivar Moneymaker is susceptible to PVX and shows mild mosaic symptoms, whereas it is tolerant and asymptomatic to systemic PVY infection with a low virus titer. In contrast, in hpDCL2.4 plants, PVX-induced symptoms became more severe and PVY infection caused symptoms. These results indicate that DCL2, DCL4, or both contribute to tolerance to infection with PVX and PVY. PVY CP and genomic RNA accumulated to a greater extent in DCL2.4-knockdown plants. Hence, the contribution of these DCLs to tolerance to infection with PVY is at least partly attributed to their roles in anti-viral RNA silencing, which controls the multiplication of PVY in tomato plants. The necrotic symptoms observed in the PVX-infected hpAGO2.3 plants suggest that AGO2, AGO3 or both are also distinctly involved in tolerance to infection with PVX.

Utilizing Potato Virus X to Monitor RNA Movement.

Mobility assays coupled with RNA profiling have revealed the presence of hundreds of full-length non-cell-autonomous messenger RNAs that move through the whole plant via the phloem cell system. Monitoring the movement of these RNA signals can be difficult and time consuming. Here we describe a simple, virus-based system for surveying RNA movement by replacing specific sequences within the viral RNA genome of potato virus X (PVX) that are critical for movement with other sequences that facilitate movement. PVX is a RNA virus dependent on three small proteins that facilitate cell-to-cell transport and a coat protein (CP) required for long-distance spread of PVX. Deletion of the CP blocks movement, whereas replacing the CP with phloem-mobile RNA sequences reinstates mobility. Two experimental models validating this assay system are discussed. One involves the movement of the flowering locus T RNA that regulates floral induction and the second involves movement of StBEL5, a long-distance RNA signal that regulates tuber formation in potato.

Potato Virus X-Based microRNA Silencing (VbMS) In Potato.

Virus-based microRNA silencing (VbMS) is a rapid and efficient tool for functional characterization of microRNAs (miRNAs) in plants. The VbMS system has been developed and applied for various plant species including Nicotiana benthamiana, tomato, Arabidopsis, cotton, and monocot plants such as wheat and maize. Here, we describe a detailed protocol using PVX-based VbMS vectors to silence endogenous miRNAs in potato. To knock down the expression of a specific miRNA, target mimic (TM) molecules of miRNA of interest are designed, integrated into plant virus vectors, and expressed in potato by Agrobacterium infiltration to bind directly to the endogenous miRNA of interest and block its function.

Small RNA-Omics for Virome Reconstruction and Antiviral Defense Characterization in Mixed Infections of Cultivated Solanum Plants.

In plants, RNA silencing-based antiviral defense generates viral small RNAs (sRNAs) faithfully representing the viral genomes. We employed sRNA sequencing and bioinformatics (sRNA-omics) to characterize antiviral defense and to reconstruct the full genomic sequences and their variants in the evolving viral quasispecies in cultivated solanaceous plants carrying mixed infections. In naturally infected Solanum tuberosum (potato), one case study revealed a virome comprising Potato virus Y (genus Potyvirus) and Potato virus X (genus Potexvirus), which was reconstructed by de novo-assembling separate genome-size sRNA contigs. Another case study revealed a virome comprising NTN and O strains of Potato virus Y, whose sRNAs assembled in chimeric contigs, which could be disentangled on the basis of reference genome sequences. Both viromes were stable in vegetative potato progeny. In a cross-protection trial of Solanum lycopersicum (tomato), the supposedly protective mild strain CH2 of Pepino mosaic virus (genus Potexvirus) was tested for protection against strain LP of the same virus. Reciprocal mechanical inoculations eventually resulted in co-infection of all individual plants with CH2 and LP strains, reconstructed as separate sRNA contigs. LP invasions into CH2-preinfected plants and vice versa were accompanied by alterations of consensus genome sequences in viral quasispecies, indicating a potential risk of cross-protection measures. Additionally, the study also revealed, by reconstruction from sRNAs, the presence of the mechanically nontransmissible Southern tomato virus (genus Amalgavirus) in some plants. Our in-depth analysis of sRNA sizes, 5'-nucleotide frequencies and hotspot maps revealed similarities in sRNA-generating mechanisms in potato and tomato, differential silencing responses to virome components and potential for sRNA-directed cross-targeting between viral strains which could not, however, prevent the formation of stable viromes.

Allexiviruses may have acquired inserted sequences between the CP and CRP genes to change the translation reinitiation strategy of CRP.

Allexiviruses are economically important garlic viruses that are involved in garlic mosaic diseases. In this study, we characterized the allexivirus cysteine-rich protein (CRP) gene located just downstream of the coat protein (CP) gene in the viral genome. We determined the nucleotide sequences of the CP and CRP genes from numerous allexivirus isolates and performed a phylogenetic analysis. According to the resulting phylogenetic tree, we found that allexiviruses were clearly divided into two major groups (group I and group II) based on the sequences of the CP and CRP genes. In addition, the allexiviruses in group II had distinct sequences just before the CRP gene, while group I isolates did not. The inserted sequence between the CP and CRP genes was partially complementary to garlic 18S rRNA. Using a potato virus X vector, we showed that the CRPs affected viral accumulation and symptom induction in Nicotiana benthamiana, suggesting that the allexivirus CRP is a pathogenicity determinant. We assume that the inserted sequences before the CRP gene may have been generated during viral evolution to alter the termination-reinitiation mechanism for coupled translation of CP and CRP.

Combining abilities for agronomic traits and marker-assisted selection for Potato virus X and Potato virus Y resistance.

Disease-resistant potato cultivars with good tuber appearance and desirable agronomic traits are essential for meeting the demands of producers and the market. Attaining these cultivars is the focus of potato breeding programs whose aim is to benefit the productive chain. The purpose of this study was to estimate combining abilities and evaluate potato clones based on tuber appearance, yield, and resistance to the PVY and PVX viruses. Crosses between four commercial cultivars of potato with good tuber appearance were performed, using eight clones with proven resistance to PVY and PVX from the breeding program of UFLA. The clones obtained were evaluated for agronomic traits, tuber appearance, and the presence of both Ryadg and Rx1 alleles, which confer extreme resistance to the PVY and PVX viruses, respectively. The independent culling level method was used to select genotypes of commercial interest, as well as to estimate the combining abilities of the parents. We identified clones carrying the Ryadg and Rx1 alleles with agronomic traits suitable for the fresh market and for processing. The BRS Ana cultivar and CMA-399 and CMA-385 clones showed positive effects on general combining ability (GCA) for tuber yield, while the Monalisa cultivar showed positive effects on GCA for general tuber appearance.

Complete genome sequences of new divergent potato virus X isolates and discrimination between strains in a mixed infection using small RNAs sequencing approach.

Potato virus X (PVX; genus Potexvirus, family Alphaflexiviridae, order Tymovirales) is one of the most widespread and intensively studied viruses of potato. However, little is known about its diversity in its likely center of radiation, the Andean region of South America. To fill this gap, the strategy of Illumina deep sequencing of small RNAs was used to obtain complete or near complete genome sequence of PVX from 5 symptomatically infected greenhouse and 3 field samples (Solanum tuberosum) from Peru. PVX sequences determined in this study were assigned into three different phylogenetic groups of isolates. Notably, a complete genome sequence of a representative of a new PVX phylogenetic lineage was obtained, which shows a high level of sequence dissimilarity to other completely sequenced isolates (∼17%). The new PVX genotype was detected in greenhouse and field samples. One of the field samples was infected with the mixture of two PVX strains, which were efficiently discriminated using small RNA sequencing approach. The study confirms the utility of small RNAs deep sequencing for successful viral strain differentiation and discovery of new viral strains and indicates a high diversity of PVX in the Andean region of South America, a pattern which may be expected also for other potato pathogens.

Agroinfiltration and PVX agroinfection in potato and Nicotiana benthamiana.

Agroinfiltration and PVX agroinfection are two efficient transient expression assays for functional analysis of candidate genes in plants. The most commonly used agent for agroinfiltration is Agrobacterium tumefaciens, a pathogen of many dicot plant species. This implies that agroinfiltration can be applied to many plant species. Here, we present our protocols and expected results when applying these methods to the potato (Solanum tuberosum), its related wild tuber-bearing Solanum species (Solanum section Petota) and the model plant Nicotiana benthamiana. In addition to functional analysis of single genes, such as resistance (R) or avirulence (Avr) genes, the agroinfiltration assay is very suitable for recapitulating the R-AVR interactions associated with specific host pathogen interactions by simply delivering R and Avr transgenes into the same cell. However, some plant genotypes can raise nonspecific defense responses to Agrobacterium, as we observed for example for several potato genotypes. Compared to agroinfiltration, detection of AVR activity with PVX agroinfection is more sensitive, more high-throughput in functional screens and less sensitive to nonspecific defense responses to Agrobacterium. However, nonspecific defense to PVX can occur and there is a risk to miss responses due to virus-induced extreme resistance. Despite such limitations, in our experience, agroinfiltration and PVX agroinfection are both suitable and complementary assays that can be used simultaneously to confirm each other's results.

Simultaneous detection and identification of four viruses infecting pepino by multiplex RT-PCR.

Potato virus M (PVM), pepino mosaic virus (PepMV), tomato mosaic virus (ToMV), and potato virus S (PVS) infect pepino and cause serious crop losses. In this study, a multiplex RT-PCR method was developed for simultaneous detection and differentiation of PVS, ToMV, PepMV and PVM. The method was highly reliable and sensitive; validation was accomplished by testing pepino samples collected from different regions of China. In this survey, PVM, ToMV and PVS were detected in 37.0 %, 31.0 % and 5.5 % of samples tested, respectively, confirming the widespread occurrence of these three viruses in China. PepMV was not detected in any of the samples, which indicated that this virus may not be prevalent in China. The results suggest that the new multiplex RT-PCR method has potential to be used routinely for surveys of pepino for virus infection.

[NBS-ARC domain variability of Rx1 homologues of cultivated potato and related wild species].

In the present work NBS-ARC domain sequences of Rx1 homologues of ten accessions of cultivated and wild potato species which differ in susceptibility to potato virus X were obtained and studied. Within the NBS-ARC domain different indels and nucleotide/amino acid substitutions, including substitutions in the conservative motives of the domain were detected. There were no direct associations between the mutational changes found in the conservative motives of the NBS-ARC domain and the susceptibility of the studied accessions to X virus.

In vitro template-dependent synthesis of Pepino mosaic virus positive- and negative-strand RNA by its RNA-dependent RNA polymerase.

Pepino mosaic virus (PepMV)-infected tomato plants were used to develop an in vitro template-dependent system for the study of viral RNA synthesis. Differential sedimentation and sucrose-gradient purification of PepMV-infected tomato extracts resulted in fractions containing a transcriptionally active membrane-bound RNA-dependent RNA polymerase (RdRp). In the presence of Mg(2+) ions, (32)P-labelled UTP and unlabelled ATP, CTP, GTP, the PepMV RdRp catalysed the conversion of endogenous RNA templates into single- and double-stranded (ds) genomic RNAs and three 3'-co-terminal subgenomic dsRNAs. Hybridisation experiments showed that the genomic ssRNA was labelled only in the plus strand, the genomic dsRNA mainly in the plus strand and the three subgenomic dsRNAs equally in both strands. Following removal of the endogenous templates from the membrane-bound complex, the purified template-dependent RdRp could specifically catalyse transcription of PepMV virion RNA, in vitro-synthesized full-length plus-strand RNA and the 3'-termini of both the plus- and minus-strand RNAs. Rabbit polyclonal antibodies against an immunogenic epitope of the PepMV RdRp (anti-RdRp) detected a protein of approximately 164kDa in the membrane-bound and template-dependent RdRp preparations and exclusively inhibited PepMV RNA synthesis when added to the template-dependent in vitro transcription system. The 300 nucleotides long 3'-terminal region of the PepMV genome, containing a stretch of at least 20 adenosine (A) residues, was an adequate exogenous RNA template for RdRp initiation of the minus-strand synthesis but higher transcription efficiency was observed as the number of A residues increased. This observation might indicate a role for the poly(A)-tail in the formation and stabilisation of secondary structure(s) essential for initiation of transcription. The template-dependent specific RdRp system described in this article will facilitate identification of RNA elements and host components required for PepMV RNA synthesis.