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Potato mop-top virus (PMTV) RNA3 contains a triple gene block (TGB) encoding viral movement proteins and an open reading frame for a putative 8 kDa cysteine-rich protein (CRP). In this study, PMTV CRP was shown to be expressed in the course of virus infection, and a PMTV CRP-specific subgenomic RNA was mapped. CRP has previously been shown to be dispensable for infection of PMTV in Nicotiana benthamiana. In this study, PMTV CRP was found to increase the severity of disease symptoms when expressed from Potato virus X or Tobacco mosaic virus in N. benthamiana and Nicotiana tabacum, suggesting that the protein affects virulence of the virus or might suppress a host defence mechanism. However, PMTV CRP did not show RNA silencing suppression activity in three assays. Host responses to the PMTV CRP expression from different viral genomes ranged from an absence of response to extreme resistance at a single cell level and were dependent on the viral genome. These findings emphasized involvement of viral proteins and/or virus-induced cell components in the plant reaction to CRP. PMTV CRP was predicted to possess a transmembrane segment. CRP fused to the green fluorescent protein was associated with endoplasmic reticulum-derived membranes and induced dramatic rearrangements of the endoplasmic reticulum structure, which might account for protein functions as a virulence factor of the virus.

Full-length genomic cDNA clones of the Swedish isolate of Potato mop-top virus (PMTV) were transcribed in vitro using T7 RNA polymerase. The combination of RNA 1, 2 and 3 synthesized in the presence of m(7)GpppG cap analogue was infectious when inoculated onto Nicotiana benthamiana plants. Also, the combination of RNA 1 (encodes the viral replicase) with RNA 3 [encodes the triple gene block proteins and a small cysteine-rich protein (CRP)] was infectious and both RNAs moved systemically in N. benthamiana plants in the absence of RNA 2, which encodes the coat protein (CP). However, the yellow mosaic symptoms that typically developed following PMTV infection with all three RNAs were not observed in plants infected with RNA 1+RNA 3. Site-directed mutagenesis experiments revealed that expression of the putative CRP was not required for systemic infection and symptom induction in N. benthamiana. These data show that PMTV represents an example of a multipartite virus capable of establishing systemic infection without the CP-encoding RNA, and also without the putative CRP.

The necrotic symptoms (spraing) in tubers caused byPotato mop-top pomovirus (PMTV) have become a severe problem for potato production in Scandinavia. PMTV is transmitted by the powdery scab pathogen (Spongospora subterranea) and no methods for control are currently available. Two breeding lines (NY99 and NY103) from the Cornell University potato breeding program, twotuberosum ×acaule lines (TA3.8.3.3 and TA3.5.3.7) from the International Potato Center (CIP), and one line (IvP35) ofSolarium phureja were tested for resistance to PMTV in field trials in Sweden in 1998 and 1999. In both years, NY99 had a low incidence of PMTV infection in tubers, and the infected tubers showed a low accumulation of PMTV, indicating that NY99 is a promising new source of resistance to PMTV for breeding programs. Furthermore, it may be suitable for cultivation in Scandinavia, which, however, needs to be evaluated in more extensive field trials.

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.

Resistance tests were made on seedlings of transformed lines of Nicotiana benthamiana which contain a transgene encoding the coat protein (CP) gene of a Scottish isolate of potato mop-top virus (PMTV). This transgene has been reported to confer strong resistance to the PMTV isolate from which the transgene sequence was derived and also to a second Scottish isolate. Plants of lines of the transgenic N. benthamiana were as resistant to two Swedish and two Danish PMTV isolates as to a Scottish isolate, and of five lines tested, greater than 93.5% of transgenic plants were immune. The coat protein gene sequences of these four Scandinavian isolates were very similar to those of the two Scottish isolates. The greatest divergence between the isolates was three amino acid changes and there was less than 2% change in CP gene nucleotide sequence. It is concluded that the PMTV CP transgene used in these experiments could confer resistance against isolates from different geographical areas because it is becoming apparent that the CP genes of PMTV isolates are highly conserved.

The occorrence of potato mop-top virus (PMTV) was assessed by two different soil sampling methods. PMTV was prevalent in southwestern Sweden but was not detected in the northern part of the country. In storage trials more symptoms developed in tubers kept at 9°C than 4 °C. irrespective of the cultivar. Cutting the tubers enhanced post storage symptom develoment in some cultivars. Of 14 cultivars grown in Sweden, those used for crisp products were more inclined to develop spraing than the others. A comparison of the severity of PMTV symptoms in field trials in Sweden. Denmark and Finland indicated substantial geographic variation in the relative susceptibility of different cultivars suggesting variation among isolates of the virus. From glasshouse experiments it was found that inoculations done at flower bud development or at flowering resulted in most spraing. Cultivar trials in pots produced a low level of spraing symptoms in general.

In trials with potato tubers infected with tobacco rattle virus (TRV), symptoms of spraing in cvs Bellona, King Edward, Maris Bard, Matilda, Sv 82146 and Sv 82149 increased during storage when the tubers were cut. Storage of intact tubers at a constant temperature of 9°C or at fluctuating temperatures (2 weeks at 18°C, 2 weeks at 9°C and 2 weeks at 18°C) did not increase the frequency of symptoms.

TGBp1, TGBp2, and TGBp3, three plant virus movement proteins encoded by the “triple gene block” (TGB), may act in concert to facilitate cell-to-cell transport of viral RNA genomes. Transient expression of Potato mop-top virus (genus Pomovirus) movement proteins was used as a model to reconstruct interactions between TGB proteins. In bombarded epidermal cells of Nicotiana benthamiana, green fluorescent protein (GFP)-TGBp1 was distributed uniformly. However, in the presence of TGBp2 and TGBp3, GFP-TGBp1 was directed to intermediate bodies at the cell periphery, and to cell wall-embedded punctate bodies. Moreover, GFP-TGBp1 migrated into cells immediately adjacent to the bombarded cell. These data suggest that TGBp2 and TGBp3 mediate transport of GFP-TGBp1 to and through plasmodesmata. Mutagenesis of TGBp1 suggested that the NTPase and helicase activities of TGBp1 were not required for its transport to intermediate bodies directed by TGBp2 and TGBp3, but these activities were essential for the protein association with cell wall-embedded punctate bodies and translocation of TGBp1 to neighboring cells. The C-terminal region of TGBp1 was critical for trafficking mediated by TGBp2 and TGBp3. Mutation analysis also suggested an involvement of the TGBp2 C-terminal region in interactions with TGBp1.

Molecular data on Potato mop-top virus (PMTV), genus Pomovirus, is currently mostly based on analysis of two Scottish isolates, PMTV-S and PMTV-T. Here we report the complete sequence of "the coat protein (CP) encoding RNA" of an isolate of PMTV obtained from the field in Sweden. Our data show that this RNA (3134 nt) is the second largest of the three RNA species in the tripartite PMTV genome, and it should, therefore, be referred to as RNA 2. This nomenclature is consistent with other pomoviruses. The sequence of the readthrough domain (RT) of RNA 2 was determined also in two additional field isolates of PMTV from Finland and Denmark. All three isolates contained a novel, 109 nucleotides long sequence at the 3'-end of the RT, which has not been found in PMTV-S and PMTV-T. Hence, our data suggest that the RNA 2 sequences previously described for the isolates PMTV-T and PMTV-S may represent deletion derivatives. The C-proximal half of RT contained many amino acid (aa) differences among the isolates, in contrast to only few aa differences in the N-proximal part of RT. Deletion variants of RNA 2 were generated from the Nordic isolates in potato tubers infected in the field, and in the mechanically inoculated test plants. All deletions started within a short region (18 nt) and removed 558-940 nt from the 3'-end of RT region. This study for the first time describes the full-length sequence of the "CP-encoding RNA" (RNA2) of PMTV, and reveals considerable aa variability and occurrence of deletion variants of RT in the field isolates of PMTV.

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 (genusPomovirus), 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.

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.

Potato mop-top virus (PMTV) is the type member of the genus Pomovirus characterized by a tripartite, single-stranded, positive-sense RNA genome. However, PMTV differs from the other pomoviruses by having an open reading frame (ORF) for a 8-kDa cysteine-rich protein (CRP) on RNA 3, downstream the triple gene block. Site-directed mutagenesis of infectious cDNA clones of PMTV showed that the CRP ORF is not needed for the systemic infection in Nicotiana benthamiana and several other hosts, but it has an effect on the symptom development of PMTV. CRP of PMTV was not capable of RNA silencing suppression in agroinfiltration tests. However, the expression of CRP using vectors based on Potato virus X (PVX) or Tobacco mosaic virus (TMV) resulted in necrotic symptoms on N. benthamiana and tobacco plants, respectively. Sequencing of PMTV isolates obtained from the field revealed significant sequence variability of the CRP. These data suggest that the functions of CRP may be expressed only in certain hosts or at certain phases of the PMTV infection cycle.