Transfer of resistance to potato leafroll virus, potato virus Y and potato virus X from Solarium brevidens to S. tuberosum through symmetric and designed asymmetric somatic hybridisation

Resistance to potato leafroll virus (PLRV), potato virus Y (PVY^o) and potato virus X (PVX) was studied in symmetric and asymmetric somatic hybrids produced by electrofusion between Solanum brevidens (2n=2×=24) and dihaploid S. tuberosum (2n=2×=24), and also in regenerants (B-hybrids) derived through protoplast culture from a single somatic hybrid (chromosome number 48). All of the somatic hybrids between 5. brevidens and the two dihaploid lines of potato cv. Pito were extremely resistant to PLRV and PVY^oand moderately resistant to PVX, irrespective of their chromosome number and ploidy level (tetraploid or hexaploid). Most (56%) of the asymmetric hybrids of irradiated S. brevidens and the dihaploid line of potato cv. Pentland Crown (PDH40) had high titres of PVY^osimilar to those of PDH40, whereas the rest of the hybrids had PVY^otitres less than a tenth of those in PDH40. Three B-hybrids had a highly reduced chromosome number (27, 30 and 34), but were however as resistant to PLRV, PVY^oand PVX as 5. brevidens. Two asymmetric hybrids and one B-hybrid were extremely resistant to PLRV but susceptible to both PVY and PVX. The results suggested that resistance to PLRV in 5. brevidens is controlled by a gene or genes different from those controlling resistance to PVY and PVX, and the gene(s) for resistance to PVY and PVX are linked in S. brevidens.     

Virulence determines beneficial trade‐offs in the response of virus‐infected plants to drought via induction of salicylic acid

It has been hypothesized that plants can get beneficial trade‐offs from viral infections when grown under drought conditions. However, experimental support for a positive correlation between virus‐induced drought tolerance and increased host fitness is scarce. We investigated whether increased virulence exhibited by the synergistic interaction involving Potato virus X (PVX) and Plum pox virus (PPV) improves tolerance to drought and host fitness in Nicotiana benthamiana and Arabidopsis thaliana. Infection by the pair PPV/PVX and by PPV expressing the virulence protein P25 of PVX conferred an enhanced drought‐tolerant phenotype compared with single infections with either PPV or PVX. Decreased transpiration rates in virus‐infected plants were correlated with drought tolerance in N. benthamiana but not in Arabidopsis. Metabolite and hormonal profiles of Arabidopsis plants infected with the different viruses showed a range of changes that positively correlated with a greater impact on drought tolerance. Virus infection enhanced drought tolerance in both species by increasing salicylic acid accumulation in an abscisic acid‐independent manner. Viable offspring derived from Arabidopsis plants infected with PPV increased relative to non‐infected plants, when exposed to drought. By contrast, the detrimental effect caused by the more virulent viruses overcame potential benefits associated with increased drought tolerance on host fitness.     

Genetically engineered resistance to potato virus X in four commercial potato cultivars

The genes for the capsid protein (CP) and the 8K movement protein of PVX were introduced into potato (Solanum tuberosum L.) and expressed under the control of CaMV 35S promoter using a binary vector andAgrobacterium tumefaciens. Four commercial potato cultivars (Russet Burbank, Shepody, Desirée and Bintje) have been efficiently transformed. Eleven independent transgenic clones, with CP expression levels higher than 0.05% of the soluble leaf proteins, were analyzed for resistance to inoculation with PVX (5 and 50µg/ml). The resistance of the transgenic plants to PVX was observed with the lower titer of virus inoculation (5 µg/ml) but not with higher titer (50 µg/ml). A significant reduction in the accumulation of virus in the inoculated transgenic potato plants has been observed under greenhouse and field conditions. Furthermore, the CP gene is very stable and is transferred to new plants originated from stem cuttings or from tubers. The transgenic plants appeared to be phenotypically identical to the nontransformed controls.Abbreviations BAP benzyl-aminopurine - BCIP 5-bromo-4-chloro-3-indolylphosphate p-Toluidine salt - CaMV cauliflower mosaic virus - CP capsid protein - GA₃ gibberellic acid - Kbp kilobase pair - NAA naphthalene acetic acid - NBT nitroblue tetrazolium chloride - NOS nopaline synthase - NPT II neomycin phosphotransferase II - PMSF phenyl methyl sulfonyl fluoride - PVX potato virus X - PVY potato virus Y     

Interactions of the Solanum spp. of the Etuberosa group and nine potato-infecting viruses and a viroid

All 26 accessions of Solanum brevidens, one accession of S. etuberosum and one accession of S. fernandezianum tested were all extremely resistant to potato leafroll virus (PLRV) and potato viruses Y (PVY) and A (PVA). S. brevidens and S. etuberosum were also resistant to Andean potato mottle virus (APMV) and moderately resistant to potato virus X (PVX), whereas S. fernandezianum was susceptible to these viruses. Additionally, S. brevidens was resistant to sap-inoculated potato viruses M (PVM) and S (PVS). All the Etuberosa accessions were susceptible by graft-inoculation to PVM, PVS, potato virus T (PVT) and Andean potato latent virus (APLV). Infections by the above mentioned viruses were symptomless in all of the Etuberosa spp. S. etuberosum and S. fernandezianum were infected by mechanical inoculation with potato spindle tuber viroid, S. etuberosum developing severe stunting and leaf-curl symptoms, but S. brevidens was infected only by graft-inoculation. The genes conferring resistance to PVY and PVX in S. brevidens and S. etuberosum appeared to be different from those currently utilised by plant breeders.     

A chimeric Potato virus X encoding a heterologous peptide affects Nicotiana benthamiana chloroplast structure

Abstract

The cytopathology of a Potato virus X (PVX) recombinant variant (encoding as fusion of an epitope of immunological interest with the N‐terminus of the coat protein, PVXSmaP18DD) has been compared with that induced by the wild‐type virus (PVX wt) in Nicotiana benthamiana plants. Both PVX wt and PVXSmaP18DD caused similar ultrastructural alterations, characterized by the presence of laminated inclusion components and bulk virus accumulations in mesophyll cells. However, some striking differences were observed not only in the morphology of these accumulations (typically ordered in PVX wt infection and disordered in PVXSmaP18DD infection) but also because the chimeric virus caused peculiar alterations in chloroplasts structure.

Abbreviations: CP, coat protein; d.p.i., days post inoculation; LIC, laminated inclusion components; PVX, Potato virus X     

Complete Genome Sequence,Phylogenetic Relationships and Molecular Diagnosis of an Indian Isolate of Potato Virus X

The complete genome of a Potato virus X (PVX) isolate from India (ptDel‐9), which occurred symptomlessly in potato but induced ringspots on Nicotiana tabacum cv. Xanthi and necrotic mosaic on Nicotiana benthamiana, was sequenced. The genome was 6435 nucleotides long ( JF430080 ) and contained five open reading frames. The isolate was closely related to those reported from the Eurasian region (95.1–97.1% sequence similarity) and distantly related to those reported from South America (77.2–77.9%). The CP gene was expressed in Escherichia coli as a 76‐kDa fusion protein with maltose‐binding protein and used to generate polyclonal antibodies, which successfully detected PVX in field samples of potato by ELISA. In 20% of field samples, for which ELISA failed, the virus was successfully detected by RT‐PCR. This is the first report of molecular characterization of PVX occurring in India.     

Virions and the Coat Protein of the Potato Virus X Interact with Microtubules and Induce Tubulin Polymerization In Vitro

A study was made of the in vitro interactions of virions and the coat protein (CP) of the potato virus X (PVX) with microtubules (MT). Both virions and CP cosedimented with taxol-stabilized MT. In the presence of PVX CP, tubulin polymerized to produce structures resistant to chilling. Electron microscopy revealed the aberrant character of the resulting tubulin polymers (protofilaments and their sheets), which differed from MT assembled in the presence of cell MAP2. In contrast, PVX virions induced the assembly of morphologically normal MT sensitive to chilling. Virions were shown to compete with MAP2 for MT binding, suggesting an overlap for the MT sites interacting with MAP2 and with PVX virions. It was assumed that PVX virions interact with MT in vivo and that, consequently, cytoskeleton elements participate in intracellular compartmentalization of the PVX genome.     

Characterization of Viruses Infecting Potato Plants from a Single Location in Shetland,an Isolated Scottish Archipelago

Sequence data were obtained from 29 isolates of Potato virus A (PVA), Potato virus S (PVS), Potato virus V (PVV) and Potato virus X (PVX) infecting nine tubers from Shetland, one of the most remote inhabited islands in the United Kingdom. These isolates were sequenced in the coat protein region, as were 29 Scottish mainland isolates of the same four potato virus species, and these 58 isolates were compared to previously published sequence data. This has allowed the characterization of viruses from a relatively isolated location, where there is little production of ware potatoes and no seed potato production. Phylogenetic homogeneity of the Shetland isolates of PVS and PVV was apparent. PVX was more heterogeneous, and Shetland isolates cluster with the Scottish isolates in a group which includes Asian and European isolates. For PVA, the majority of the Shetland and Scottish mainland isolates formed a predominantly Scottish grouping, with the remaining Shetland and Scottish mainland isolates clustering with a previously characterized Scottish isolate. There were three main groups of PVA, of which the Scottish grouping was the only one which did not have a fully characterized representative. To extend the characterization of PVA, the nucleotide sequence of the full polyprotein region encoding all the gene products of an isolate from Shetland was determined.     

Simultaneous detection of potato viruses Y and X by DAC-ELISA using polyclonal antibodies raised against fused coat proteins expressed in Escherichia coli

Polyclonal antibodies were raised against the bacterial expressed fused coat proteins (CPs) of Potato virus Y (PVY) and Potato virus X (PVX). Truncated CP sequences of PVY (~246 bp) and PVX (~243 bp) were amplified by PCR, cloned into T&A cloning vector and subsequently mobilized in a protein expression vector pET-28b (+). The recombinant CP was expressed as a fusion protein (~20 kDa) with His-tag and purified from E. coli BL21 (DE3) using His-Bind resin. The specificity of the recombinant protein was confirmed by Western blot using previously made polyclonal antibodies against each virus. Polyclonal antibodies developed against the fused CPs in rabbit detected natural infection of PVY and PVX in potato leaf samples collected from IARI experimental farm, by direct antigen-coated enzyme-linked immunosorbent assay (DAC-ELISA).     

A potato hypersensitive resistance gene against potato virus X maps to a resistance gene cluster on chromosome 5

 The dominant Nb gene of potato confers strain-specific hypersensitive resistance against potato virus X (PVX). A population segregating for Nb was screened for resistance by inoculating with PVX strain CP2, which is sensitive to Nb. Through a combination of bulked segregant analysis and selective restriction fragment amplification, several amplified fragment length polymorphism (AFLP) markers linked to Nb were identified. These were cloned and converted into dominant cleaved amplified polymorphic sequence (CAPS) markers. The segregation of these markers in a Lycopersicon esculentum×L. pennellii mapping population suggested that Nb is located on chromosome 5. This was confirmed by examining resistant and susceptible potato individuals with several tomato and potato chromosome-5-specific markers. Nb maps to a region of chromosome 5 where several other resistance genes– including R1, a resistance gene against Phytophthora infestans, Gpa, a locus that confers resistance against Globodera pallida, and Rx2, a gene that confers extreme resistance against PVX–have previously been identified. Received: 2 January 1997/Accepted: 7 February 1997     

The coat protein of potato virus X is a strain-specific elicitor of Rx1-mediated virus resistance in potato

The Rx1 gene in potato confers extreme resistance to potato virus X (PVX). To investigate the mechanism and elicitation of Rx resistance, protoplasts of potato cv. Cara (Rx1 genotype) and Maris Bard (rx1 genotype) were inoculated with PVX and tobacco mosaic virus (TMV). At 24 h post-inoculation in Maris Bard protoplasts there was at least 100-fold more PVX RNA than in protoplasts of Cara. TMV RNA accumulated to the same level in both types of protoplast. However, when the TMV was inoculated together with PVX the accumulation of TMV RNA was suppressed in the Cara (Rx1 genotype) protoplasts to the same extent as PVX. The Rx1 resistance also suppressed accumulation of a recombinant TMV in which the coat protein gene was replaced with the coat protein gene of PVX. It is therefore concluded that Rx1-mediated resistance is elicited by the PVX coat protein, independently of any other proteins encoded by PVX. The domain of the coat protein with elicitor activity was localized by deletion and mutation analysis to the structural core of a non-virion form of the coat protein.     

Disruption of the methionine cycle and reduced cellular gluthathione levels underlie potex–potyvirus synergism in Nicotiana benthamiana

Infection caused by the synergistic interaction of two plant viruses is typically manifested by severe symptoms and increased accumulation of either virus. In potex–potyviral synergism, the potyviral RNA silencing suppressor helper component proteinase (HCPro) is known to enhance the pathogenicity of the potexvirus counterpart. In line with this, Potato virus X (PVX; genus Potexvirus) genomic RNA (gRNA) accumulation and gene expression from subgenomic RNA (sgRNA) are increased in Nicotiana benthamiana by Potato virus A (PVA; genus Potyvirus) HCPro expression. Recently, we have demonstrated that PVA HCPro interferes with the host cell methionine cycle by interacting with its key enzymes S‐adenosyl‐l ‐methionine synthetase (SAMS) and S‐adenosyl‐l ‐homocysteine hydrolase (SAHH). To study the involvement of methionine cycle enzymes in PVX infection, we knocked down SAMS and SAHH. Increased PVX sgRNA expression between 3 and 9 days post‐infiltration (dpi) and upregulation of (–)‐strand gRNA accumulation at 9 dpi were observed in the SAHH‐silenced background. We found that SAMS and SAHH silencing also caused a significant reduction in glutathione (GSH) concentration, specifically in PVX‐infected plants between 2 and 9 dpi. Interestingly, HCPro expression in PVX‐infected plants caused an even stronger reduction in GSH levels than did SAMS + SAHH silencing and a similar level of reduction was also achieved by knocking down GSH synthetase. PVX sgRNA expression was increased in the GSH synthetase‐silenced background. GSH is a major antioxidant of plant cells and therefore GSH shortage may explain the strong oxidative stress and severe symptoms observed during potex–potyvirus mixed infection.     

Properties of a resistance-breaking strain of potato virus X

During indexing of a potato germplasm collection from Bolivia, a strain of potato virus X (PVX), X_HB, which failed to cause local lesions in inoculated leaves of Gomphrena globosa was found in 7% of the clones. X_HB was transmitted by inoculation of sap to 56 species from 11 families out of 64 species from 12 families tested. It was best propagated in Nicotiana glutinosa or N. debneyi; Montia perfolia and Petunia hybrida were useful as local lesion hosts. Inoculated leaves of G. globosa plants kept at 10°, 14°, 18°, 22°, or 26 °C after inoculation were always infected symptomlessly. X_HB caused a mild mosaic, systemic chlorotic blotching or symptomless infection in 16 wild potato species and eight Andean potato cultivars, systemic necrotic symptoms in clone A6 and cultivar Mi Peru, and bright yellow leaf markings in cultivar Renacimiento. It caused necrotic local lesions in inoculated leaves of British potato cultivars with the PVX hypersensitivity gene Nb but then invaded the plants systemically without causing further necrosis; with gene Nx systemic invasion occurred but no necrotic symptoms developed. These reactions resemble those of PVX strain group four. X_HB differed from other known strains of PVX in readily infecting PVX-immune clones 44/1016/10, G. 4298.69 and USDA 41956, cultivars Saphir and Saco, and Solanum acaule PI 230554. X_HB had slightly flexuous filamentous particles with a normal length of 516 nm. It was transmitted readily by plant contact and it partially protected G. globosa leaves from infection with X_CP, a group two strain of PVX. Sap from infected N. glutinosa was infective after dilution to 10^--⁶ but not 10^--⁷ after 10 min at 75° but not 80 °C and after 1 yr at 20 °C. X_HB was readily purified from infected N. debneyi leaves by precipitation with polyethylene glycol followed by differential centrifugation. Microprecipitin tests showed that X_HB and X_CP are closely related serologically.     

NbALY916 is involved in potato virus X P25-triggered cell death in Nicotiana benthamiana

Systemic necrosis often occurs during viral infection of plants and is thought mainly to be the result of long-term stress induced by viral infection. Potato virus X (PVX) encodes the P25 pathogenicity factor that triggers a necrotic reaction during PVX-potato virus Ysynergistic coinfection. In this study, we discovered that NbALY916, a multifunctional nuclear protein, could interact with P25. When NbALY916 expression was reduced by tobacco rattle virus (TRV)-based virus-induced gene silencing, the accumulation of P25 was increased, which would be expected to cause more severe necrosis. However, silencing of NbALY916 reduced the extent of cell death caused by P25. Furthermore, we found that overexpression of NbALY916 increased the accumulation of H₂O₂ and triggered more extensive cell death when coexpressed with P25, even though accumulation of P25 was itself reduced by the increased expression of NbALY916. Furthermore, transient expression of P25 specifically induced the expression of NbALY916 mRNA, but not the mRNAs of three other ALYs in Nicotiana benthamiana. In addition, we showed that silencing of NbALY916 or transient overexpression of NbALY916 affected the infection of PVX in N. benthamiana. Our results reveal that NbALY916 has an antiviral role that, in the case of PVX, operates by inducing the accumulation of H₂O₂ and mediating the degradation of P25.     

Occurrence of potato virus X strain group 1 in seed stocks of potato cultivars lacking resistance genes

Potato virus X (PVX) isolates were obtained from a simple seed potato production scheme or from ware potatoes produced by seed potatoes obtained from it. In this scheme, PVX infection is widespread in seed stocks and most of the potatoes grown lack PVX resistance genes. Thirteen PVX isolates were typed to strain group by inoculation to potato cultivars containing different combinations of hypersensitivity genes Nx and Nb. Six failed to overcome either gene and therefore belonged to strain group 1, four overcame Nb only and were placed in strain group 3 and three were mixtures of the two. All 13 isolates failed to overcome extreme resistance/immunity gene Rx. Naturally infected cultivars of genotype nx.nb contained strain group 1 alone or strain groups 1 and 3, while those of genotype nx:Nb contained only strain group 3. The widespread occurrence of strain group 1 contrasts with the predominant occurrence of strain group 3 in potatoes in the UK. However, it resembles the UK situation before sophisticated seed potato production schemes were introduced and before PVX hypersensitivity genes Nx and Nb were deliberately exploited in potato breeding. Prior infection with potato leafroll virus (PLRV) did not affect expression of hypersensitivity to PVX in inoculated leaves of an nx:Nb genotype.