Potato Mop-top Virus Research Articles

Production of Polyclonal Antibodies to a Recombinant Coat Protein of Potato mop‐top virus

AbstractThe coat protein (CP) coding regions of two Czech Potato mop‐top virus (PMTV) isolates were sequenced and shown to be identical. One, the Korneta isolate CP gene, was cloned in several expression vectors. The recombinant PMTV‐CP was expressed in Escherichia coli and the purified recombinant protein was used to produce PMTV‐specific polyclonal antibodies. The antiserum had a titre of 1 : 2000 in an indirect enzyme‐linked immunosorbent assay (ELISA) and reacted specifically in immunoblotting and IPTA‐ ELISA (indirect plate‐trapped antigen (PTA)‐ELISA).

한국에서의 감자 바이러스병 발생과 그 연구에 대한 고찰

The occurrence of potato(Sotanum tuberosum) viral diseases caused by Potato virus X(PVX), Potato virus Y (PVY), Potato leafroll virus(PLRV), Potato vims S(PVS), Potato virus M(PVM), Potato virus A(PVA), Potato virus T(PVT), Alfalfa mosic virus(AIMV), Tobacco mosic virus(TMV), Potato mop top virus(PMTV) Tobacco rattle virus(TRV) and Potato spindle tuber viroid(PSTVd), potato witches` broom phytoplasma, have been identified so far in Korea. Major viral diseases such as PVX, PVY and PLRV had been studied more deeply, however, the others are just identified and only partially characterized since the first study on the relation between PVX nucleic acid and virus protein by Kim in 1961. The most studies on potato viral diseases are mainly focused on the problems of seed potato production. The National Alpine Agricultural Experiment Station(NAAES), since it began its activities in 1961, has given special attention to this problem by doing studies to identify, characterize and control potato virus diseases. This effort resulted in the development of new potato virus detection methods as a basis for elaborating new method of control, such as the production of seed potato free of virus and the selection of new virus-resistant transgenic potatoes. The further studies of potato viral diseases required would be fallowings: the continuous monitoring for the occurrence of identified or not identified potato viruses in Korea, the isolation of resistant viral genes, the development of control method for the non-persistently transmitted viruses like PVY, special vectors such as nematode and fungus transmitted viruses, TRV and PMTV and the development of control methods against potato viral diseases by viral cross protection, therapy, transgenic plant, and the use of the agents or molecules, such as virus inhibitors and antiviral proteins, etc., blocking viral replication.

Identification of two nucleotide sequence sub-groups within Potato mop-top virus.

To evaluate the variation of Potato mop-top pomovirus from potato fields, 21 isolates were collected from different Danish locations. Reverse transcription-polymerase chain reaction-restriction fragment length polymorphism (RT-PCR-RFLP) of regions of RNA2 was performed for all 21 isolates resulting in the establishment of two sub-groups of isolates. The nucleotide sequence of a region encoding part of the 'readthrough protein' of RNA2 was compared for 9 of these isolates. This sequence analysis confirmed the RT-PCR-RFLP grouping. The isolates were tested for symptom expression in indicator plants and grouped according to symptom development. No correlation between grouping based on symptom development and genotype was observed.

Detection and Quantification of Spongospora subterranea in Soil, Water and Plant Tissue Samples Using Real-Time PCR

A sensitive real-time polymerase chain reaction (PCR) assay was developed for the quantification of Spongospora subterranea, the cause of powdery scab and root galling in potato, and the vector of Potato mop top virus. A specific primer pair and a fluorogenic TaqMan® probe were designed to perform a quantitative assay for the detection of S. subterranea in soil, water and plant tissue samples. The assay was tested using DNA from cystosori, zoospores, plasmodia and zoosporangia of the pathogen. DNA was extracted directly from cystosori suspended in water and from clay soil with varying levels of added cystosori. DNA obtained from zoospores released into nutrient solution by cystosori in the presence of tomato bait plants was also tested, as was DNA from plasmodia and zoosporangia in infected tomato roots. In many cases, detection was successful even at low inoculum levels. This specific quantitative assay could therefore be a useful tool for studying the biology of S. subterranea, and for the optimisation of disease avoidance and control measures.

Genetic variability between different isolates of Potato mop-top virus (PMTV)

We determined partial nucleotide sequences of several different field isolates of PMTV with well-characterized biological properties from the Czech Republic and Denmark. Subsequently we were trying to elucidate the correlation between nucleotide sequences of various isolates, their biological properties and geographical origin. From the comparison with other known sequences we can conclude, that PMTV genome is relatively conserved.

Occurrence of Potato Powdery Scab Caused by Spongospora subterranea f. sp. subterranea in Costa Rica.

Powdery scab of potatoes, caused by Spongospora subterranea (Wallr.) Lagerheim f. sp. subterranea Tomlinson, is important worldwide due to its effect on tuber quality and transmission of Potato mop-top virus. Although powdery scab-like lesions have been observed on potato in Costa Rica (1), the presence of the pathogen has not been confirmed. During a survey in 2001, powdery scab-on was observed from a field and a greenhouse in the high elevation zone of the main potato-producing area of Costa Rica. Commercial potatoes with scab-like lesions were also obtained at a farmers' market. Scraping the lesions, and observing spore balls or cystosori with a honey-comb-like structure under light microscopy confirmed the identity of S. subterranea. The identity of the pathogen was also confirmed by enzyme-linked immunosorbent assay using monoclonal antibodies specific for S. subterranea (BioReba Ag, Reinach, Switzerland). Pathogenicity of S. subterranea was confirmed by a bioassay on tomato plants grown in nutrient solution culture (2). Tomato cv. Supermarmande plants were grown from seed in pots filled with quartz and watered with nutrient solution. Three weeks after planting, the roots were trimmed to 60 mm, and the plants were transferred to the nutrient solution for additional growth. After growing for 1 week in the nutrient solution, tomato seedlings were inoculated by replacing the nutrient solution with nutrient solution containing cystosori (20 mg/liter, wt/vol) that were scraped from the scab lesions. Zoosporangia of S. subterranea were observed in root hairs and epidermal cells of the seedlings 2 weeks after inoculation. To our knowledge, this is the first report that confirms the presence of S. subterranea on potato in Costa Rica.

Subcellular Localisation, Protein Interactions, and RNA Binding of Potato mop-top virus Triple Gene Block Proteins

Subcellular localisation, protein interactions, and RNA binding of the triple gene block proteins (TGBp) of Potato mop-top virus (PMTV) were studied. The 13-kDa (TGBp2) and 21-kDa (TGBp3) proteins with or without green fluorescent protein fused to their N-terminus, and the 51-kDa protein (TGBp1) were expressed individually from a recombinant Tobacco mosaic virus (TMV) vector. Fluorescent images and Western immunoblotting experiments of recombinant TMV-infected Nicotiana benthamiana cells suggested that TGBp2 and TGBp3 were associated with cellular endomembranes and that TGBp3 was associated with the cell wall, possibly located close to plasmodesmata. In Western blots, TGBp1 was detected in fractions containing the cell wall and those enriched for organelles and membranous structures. Self-interactions were demonstrated with all three proteins in yeast two-hybrid experiments, and a heterologous interaction was found between TGBp2 and TGBp3. No additional heterologous interactions were discovered between the different TGBp and none were detected in an in vitro binding assay. TGBp1 and TGBp2 but not TGBp3 were shown to bind ssRNA in a sequence nonspecific manner. The results support the model where TGBp2 and TGBp3 facilitate delivery and localisation of the ribonucleoprotein complex to the plasmodesmata. However, the process is facilitated by RNA–protein rather than protein:protein interactions between the TGBp1 in complex with viral RNA and membrane-localised TGBp2.

Initial infection of roots and leaves reveals different resistance phenotypes associated with coat protein gene-mediated resistance to Potato mop-top virus.

Resistance to the pomovirus Potato mop-top virus (PMTV) was studied in potato (Solanum tuberosum cv. Saturna) and Nicotiana benthamiana transformed with the coat protein (CP) gene of PMTV. The incidence of PMTV infections was reduced in tubers of the CP-transgenic potatoes grown in the field in soil infested with the viruliferous vector, Spongospora subterranea. However, in those tubers that were infected, all three virus RNAs were detected and virus titres were high. The CP-transgenic N. benthamiana plants were inoculated with PMTV using two methods. Following mechanical inoculation of leaves, no RNA 3 (the CP-encoding RNA homologous to the transgene) was detected in leaves, but in some plants low amounts of RNA 3 were detected in roots; RNA 2 was readily detected in leaves and roots of several plants. Inoculation of roots using viruliferous S. subterranea resulted in infection of roots in all plants and the three PMTV RNAs were detected. However, no systemic movement of PMTV from roots to the above-ground parts was observed, indicating a novel expression of resistance. These data indicate that the CP gene-mediated resistance to PMTV specifically restricts accumulation of PMTV RNA 3, and is more effective in leaves than roots. Furthermore, expression of resistance is different depending on whether leaves or roots are inoculated. Data do not exclude the possibility that both a protein-mediated and an RNA-mediated resistance mechanism are involved.

Alternative hosts for potato mop-top virus, genus Pomovirus and its vectorSpongospora subterranea f.sp. subterranea

Seventeen weed species common in the potato fields in Denmark were grown in a hydroponic system infested with viruliferous zoospores ofSpongospora subterranea f.sp.subterranea carrying potato mop-top virus (PMTV). The plants were examined for infection with PMTV andS.s.s. using DAS-ELISA and based on visible symptoms. Only two weed species were found to be infected with PMTV,Chenopodium album andSolanum nigrum, whereas 13 became infected withS.s.s. C. album was infected with PMTV by mechanical inoculation were the infection became systemically and the leaves showed necrotic spots.S. nigrum became local infected with PMTV by mechanical inoculation. By inoculation with viruliferousS.s.s. the roots ofS. nigrum became infected but in three weeks PMTV had not spread into the top of the plants.Nicotiana benthamiana was used as a control susceptible toS.s.s. and PMTV.

Molecular studies of Potato mop-top virus (PMTV) in transgenic N. benthamiana and S. tuberosum

Resistance to Potato mop-top virus (PMTV) was studied in Nicotiana benthamiana and potato (Solanum tuberosum cv.<br />Saturna) transformed with the coat protein (CP) gene of PMTV. In N. benthamiana plants mechanically inoculated with<br />PMTV, RNA2 was detected in leaves and roots in several plants in the absence of RNA3. When N. benthamiana was<br />grown in infested soil, viral RNA was detected in roots, but no systemic movement of PMTV to above-ground parts was<br />observed. The incidence of PMTV infections was reduced in the CP-transgenic potato plants grown in an infested field<br />in Sweden. However, in infected tubers, all three virus RNAs were detected.

Reduction of potato mop-top virus accumulation and incidence in tubers of potato transformed with a modified triple gene block gene of PMTV

Potato mop-top virus (PMTV) RNA 2 contains a triple gene block (TGB) that consists of a set of three overlapping genes. These genes encode three proteins necessary for viral cell-to-cell movement. The gene encoding for the second triple gene block protein, TGB2, was mutated in a region that is highly conserved among TGB2 proteins from different viruses. The mutated TGB2 gene, under transcriptional control of the 35S promoter from cauliflower mosaic virus, was transformed into potato (Solanum tuberosum cv. Hulda) by Agrobacterium-mediated transformation. Ten of the transgenic lines obtained, all of which were shown to transcribe the introduced gene, were evaluated for resistance to PMTV in a field trial. Tubers from the field were analysed for virus content by ELISA. In seven of the transgenic lines analysed there was a reduction in ELISA values relative to a non-transgenic control line. In the three lines that had the lowest ELISA readings this reduction was manifested both as a reduction in the proportion of infected tubers and as a reduction in virus levels in infected tubers. In the line showing the highest level of resistance, the total ELISA value was reduced by 79%. These results demonstrate that the introduction of a mutated TGB2 gene into potato can confer increased resistance to PMTV.

Detection of Potato mop top virus and Tobacco rattle virus Using a Multiplex Real-Time Fluorescent Reverse-Transcription Polymerase Chain Reaction Assay

Tobacco rattle virus (TRV) and Potato mop top virus (PMTV) are important diseases of potato that are difficult to diagnose reliably by visual symptoms. Effective control strategies rely on accurate diagnosis. This paper describes the development of a multiplex assay for the detection of TRV and PMTV directly from potato tubers and leaves by polymerase chain reaction (PCR) combined with in-tube fluorescent product detection (TaqMan). This technology obviates any post-PCR manipulations and has many advantages including reducing contamination risks, eliminating the need for ethidium bromide staining, and removing the time and cost of gel running. The new assay also allows the replacement of the two separate tests (a TRV reverse-transcription-PCR and a PMTV enzyme-linked immuno-sorbent assay) currently used with a single-tube multiplex format. In addition to greatly simplifying the detection of these two viruses, the multiplex TaqMan assay was also shown to be more sensitive than either of the tests that it replaces, allowing 100- and 10,000-fold increases in sensitivity for TRV and PMTV detection, respectively. The test reliably detected over 40 different isolates of TRV and PMTV obtained from a wide range of different cultivars and geographical locations, including some samples in which existing tests failed to detect virus. The use of an assay of this kind in routine diagnosis helps to speed up and streamline the diagnostic laboratory; in addition, more reliable diagnosis should help in the control of this damaging disease.

Potato mop-top virus RNA can move long distance in the absence of coat protein: evidence from resistant, transgenic plants.

Transgenic expression of a translatable version of the Potato mop-top virus (PMTV) coat protein (CP) gene (encoded by RNA 3) in Nicotiana benthamiana prevented production of symptoms and infective virus particles. RNAs 1 and 2 accumulated in inoculated and systemic leaves but, apart from small amounts of CP transgene RNA transcript, no genomic-length RNA 3 was found. Crude leaf extracts from inoculated plants were not infective. However, when RNA extracts from such transgenic plants were inoculated to nontransgenic N. benthamiana and N. clevelandii, RNA 1 and RNA 2 replicated in systemic leaves of both species in the absence of RNA 3 and virus particles, but symptoms did not develop. We suggest that the triple-gene block proteins of PMTV (encoded by RNA 2) represent a class of long-distance RNA movement factors.

Complete sequence of RNA 1 and the presence of tRNA-like structures in all RNAs of Potato mop-top virus, genus Pomovirus.

The complete nucleotide sequence (6043 nt) of RNA 1 from Potato mop-top virus (PMTV-Sw), the type member of the genus Pomovirus, was determined. The first (5'-terminal) open reading frame (ORF 1) encodes a predicted protein of 148 kDa. ORF 2 extends through the opal stop codon of ORF 1 producing a predicted readthrough protein of 206 kDa which resembles the RNA-dependent RNA polymerases (RdRp) of other fungal-transmitted viruses. It includes a methyltransferase, a helicase and a GDD RdRp motif, respectively. Phylogenetic analyses of RdRps indicated that PMTV is most closely related to Beet soil-borne virus (genus Pomovirus), Broad bean necrosis virus (genus Pomovirus) and Soil-borne wheat mosaic virus (genus Furovirus), and is more distantly related to the other viruses of the former furovirus group. The 5' and 3' termini of RNA 1 in PMTV contained untranslated regions (UTR) of 114 nt and 489 nt, respectively. The 3'-UTR of RNA 1 contained a tRNA-like structure, which has previously been reported in the 3'-UTR of RNA 2 but not RNA 3. However, in this study, the tRNA-like structure was also found in the 3'-UTR of RNA 3, which confirms its presence in the 3'-UTRs of all three RNAs of PMTV.

A naturally occurring deleted form of RNA 2 of Potato mop-top virus

A spontaneous deletion in RNA 2 of Potato mop-top virus (PMTV) was identified by RT-PCR. The deletion occurred reproducibly during manual passage of two isolates of PMTV and during fungal inoculation of plants with viruliferous soil. The borders of the deletion were conserved in all instances and sequence analyses showed that a contiguous segment of 2113 nucleotides was deleted internally from the genomic RNA 2, leaving the 5'- and 3'-terminal sequences. RT-PCR experiments also showed that the deletion was present in preparations of PMTV particles.

Association of sequences in the coat protein/readthrough domain of potato mop-top virus with transmission by Spongospora subterranea.

A monofungal culture of Spongospora subterranea was unable to acquire and transmit the T isolate of potato mop-top pomovirus (PMTV-T), which has been maintained by manual transmission in the laboratory for 30 years. A recently obtained field isolate (PMTV-S) was efficiently acquired and transmitted by the same fungus culture. Sequence analysis of the readthrough (RT) protein-coding region of PMTV-S showed the presence of an additional 543 nt in the 3' half of the coding region relative to that of PMTV-T. These additional nucleotides preserved the reading frame of the RT protein and inserted 181 amino acids into the RT protein. This was confirmed by a comparison by immunoblotting of the sizes of the RT protein of PMTV-T and other recent isolates of PMTV.

Factors affecting the detection of potato mop‐top virus in potato tubers and improvement of test procedures for more reliable assays

Summary.Progeny tubers were harvested from plants of five potato cultivars that had been grown in potato mop‐top virus (PMTV) infested soil. The tubers were tested for virus infection by methods based on reverse transcriptase‐polymerase chain reaction (RT‐PCR) or enzyme‐linked immunosorbent assay (ELISA) incorporating monoclonal antibodies. Both RT‐PCR and ELISA detected PMTV in extracts from tuber flesh. However, when the assay results were compared for individual tubers, there was an incomplete correlation between the two methods, and more infected tubers were detected by ELISA than by RT‐PCR. PMTV was detected in some samples but not in others taken from different parts of the same tuber, and this uneven distribution may explain the discrepancy between the results of the ELISA and RT‐PCR tests. Storage of tubers of two cvs at an elevated temperature (20oC) for 4 wk prior to testing, nearly doubled the number of sample sites at which PMTV was detected by ELISA. Such pre‐treatment may improve the accuracy of tuber tests. Most tubers of cvs Saturna and Pentland Crown were symptomlessly infected indicating that PMTV may easily escape detection in tubers of these cvs if tests are based solely on presence of visual symptoms. The application of post‐harvest tuber testing, particularly to cvs that do not display obvious symptoms, and the subsequent elimination of contaminated tubers will help to provide effective control of spread, and the establishment of PMTV at new sites.

Transformation of Nicotiana benthamiana with the Potato Mop-Top Virus Coat Protein Gene Produces a Novel Resistance Phenotype Mediated by the Coat Protein

Transformed lines of Nicotiana benthamiana expressing a translatable transgene (CPtrans) encoding the coat protein (CP) gene of potato mop-top virus (PMTV) differed considerably in steady-state levels of transgene RNA transcript and CP that were positively correlated. A mean of 88% of transgenic T1 plants from seven transgenic lines were resistant to manual PMTV inoculation. Resistance was identified by failure to develop symptoms in plants and inability to recover infectious virus with a sensitive infectivity assay: many of the 12% of transgenic plants that gave a positive infectivity assay did not produce symptoms and virus was not detected in repeat assays on the same plants. Resistant plants did not express a “recovery” phenotype and resistance was not overcome by use of RNA inoculum. Strong resistance was expressed in all CPtrans lines irrespective of the amount of transcript RNA or CP detected, and there was no evidence that resistance was mediated by a “transgene silencing” mechanism. Lines transformed with PMTV CP gene sequence in a nontranslatable form (CPnontrans) also contained differing steady-state levels of RNA transcript, but were not resistant to PMTV, although in some lines symptoms were slightly milder and were slower to develop than those in nontransgenic control plants. These results suggest that CPtrans is unique because, although it depends on CP translation to be effective, it mediates very strong resistance in 10 independent transformed lines (tested here and in other work).

The genome organization of the broad bean necrosis virus (BBNV).

The genome of the broad bean necrosis virus Oita-isolate (BBNV-O) [RNA1 (6.0 kb), RNA2 (2.8 kb) and RNA3 (2.4 kb)] was cloned and sequenced. Computer analysis indicates that methyltransferase, helicase and RNA-dependent RNA polymerase (RdRp) motifs are present in RNA1. The viral capsid protein (CP) cistron is located at the 5' terminal end of RNA2 and the Mr of CP (20 K) is close to that determined by SDS-PAGE analysis. An ochre codon (UAA) in the CP cistron is thought to be partially suppressed to produce a large readthrough protein. RNA3 possesses typical motifs of triple gene block proteins, which are also reported in several other plant viruses. The furovirus genome organization and phylogenetic analysis using RdRp and CP amino acid sequences suggest that BBNV is closely related to potato mop-top virus (PMTV), but is relatively distantly related to other furoviruses. The data also suggest that the genus Furovirus should be separated into several genera: the prototypical genus Furovirus, which excludes the following viruses: the PMTV group including BBNV; the beet necrotic yellow vein virus (BNYVV) group; and the peanut clump virus (PCV) group.

Transfer RNA Mimicry in a New Group of Positive-Strand RNA Plant Viruses, the Furoviruses: Differential Aminoacylation between the RNA Components of One Genome

Recent sequencing of the genomes of several furoviruses—fungus-transmitted rod-shaped positive-strand plant viruses—has suggested the presence of tRNA-like structures (TLSs) at the 3′ ends of the genomic RNAs. We show here that the genomic RNAs of soil-borne wheat mosaic virus (SBWMV), beet soil-borne virus (BSBV), potato mop-top virus (PMTV), peanut clump virus (PCV), and Indian peanut clump virus (IPCV) all possess functional TLSs that are capable of high-efficiency valylation. While the SBWMV, BSBV, and PMTV TLSs are similar to those found in tymoviruses, the PCV and IPCV TLSs harbor an insertion of about 40 nucleotides between the two halves of the TLS. The valylated SBWMV and BSBV RNAs formed tight complexes with wheat germ EF-1α · GTP (Kd= 2 to 11 nM), whereas valylated PMTV, PCV, and IPCV RNAs bound EF-1α · GTP weakly (Kd≥ 50 nM). The TLS of PCV RNA2 differs from PCV RNA1 in lacking the major valine identity nucleotide in the anticodon and consequently is capable of only very inefficient valylation. This is the first case of differential aminoacylation between the RNA components of one genome.