The participation of plant cell organelles in compatible and incompatible potato virus Y-tobacco and -potato plant interaction

Potyviruses replicate and express their genomes in the cytoplasm in closely related membranous structures such as the endoplasmic reticulum or in the vicinity of the ER. The present research demonstrates the participation of plant cell organelles based on ultrastructural examination of compatible and incompatible interactions in tobacco- and potato-potato virus Y (PVY) necrotic strains. In two interaction types, PVY^N Wi and PVY^NTN particles were documented inside cell nuclei. Virus cytoplasmic inclusions and particles were associated with nuclear envelope pore complexes. Moreover, the PVY capsid protein was immunolocalised in the cell nucleus and nucleolus. Our results for the first time show PVY particles and capsid proteins inside the mitochondrion in compatible interactions, whereas in hypersensitive responses these interactions were identified inside chloroplasts. The PVY particles attached to mitochondria caused association groups of these organelles. The ultrastructural analysis clearly demonstrated both the dynamics of the endoplasmatic reticulum in two types of PVY interactions and connections between PVY cytoplasmic inclusions and particles with its membranous structures. Moreover, we demonstrated strongly localised immunodetection of the PVY capsid protein on the surface and in the vicinity of ER in cases of hypersensitive response as well as in compatible interaction.     

Strong resistance to potato tuber necrotic ringspot disease in potato induced by transformation with coat protein gene sequences from an NTN isolate of Potato virus Y

The potato cv. Igor is susceptible to infection with Potato virus Y (PVY) and in Slovenia it has been so severely affected with NTN isolates of PVY causing potato tuber necrotic ringspot disease (PTNRD) that its cultivation has ceased. Plants of cv. Igor were transformed with two transgenes that contained coat protein gene sequence of PVY^NTN. Both transgenes used PVY sequence in a sense (+) orientation, one in native translational context (N‐CP), and one with a frame‐shift mutation (FS‐CP). Although most transgenic lines were susceptible to infection with PVY^NTN and PVY^O, several lines showed resistance that could be classified into two types. Following manual or graft inoculation, plants of partially resistant lines developed some symptoms in foliage and tubers, and virus titre in the foliage, estimated by ELISA, was low or undetectable. In highly resistant (R) lines, symptoms did not develop in foliage and on tubers, and virus could not be detected in foliage by ELISA or infectivity assay. Four lines from 34 tested (two N‐CP and two FS‐CP) were R to PVY^NTN and PVY^O and one additional line was R to PVY^O. When cv. Spey was transformed with the same constructs, they did not confer strong resistance to PVY^O.     

Ny-1 and Ny-2 genes conferring hypersensitive response to potato virus Y (PVY) in cultivated potatoes: mapping and marker-assisted selection validation for PVY resistance in potato breeding

Potato virus Y (PVY) is one of the most important viruses affecting potato (Solanum tuberosum) production. In this study, a novel hypersensitive response (HR) gene, Ny-2, conferring resistance to PVY was mapped on potato chromosome XI in cultivar Romula. In cultivars Albatros and Sekwana, the Ny-1 gene was mapped on chromosome IX. In cv. Romula, the local lesions appeared in leaves inoculated with the PVY^N-Wi isolate at 20 and 28 °C; PVY systemic infections were only occasionally observed at the higher temperature. In cvs. Albatros and Sekwana, expression of the necrotic reaction to virus infection was temperature-dependent. PVY^N-Wi was localized at 20 °C; at 28 °C, the systemic, symptomless infection was observed. We developed the B11.6₁₆₀₀ marker co-segregating with Ny-2 and the S1d11 marker specific for the Ny-1 gene. Fifty potato cultivars were tested with markers B11.6 and S1d11 and marker SC895 linked to the Ny-1 gene in cv. Rywal. These results indicated the utility of these markers for marker-assisted selection of HR-like PVY resistance in potato breeding programs.     

The novel gene Ny-1 on potato chromosome IX confers hypersensitive resistance to Potato virus Y and is an alternative to Ry genes in potato breeding for PVY resistance

Hypersensitive resistance (HR) is an efficient defense strategy in plants that restricts pathogen growth and can be activated during host as well as non-host interactions. HR involves programmed cell death and manifests itself in tissue collapse at the site of pathogen attack. A novel hypersensitivity gene, Ny-1, for resistance to Potato virus Y (PVY) was revealed in potato cultivar Rywal. This is the first gene that confers HR in potato plants both to common and necrotic strains of PVY. The locus Ny-1 mapped on the short arm of potato chromosome IX, where various resistance genes are clustered in Solanaceous genomes. Expression of HR was temperature-dependent in cv. Rywal. Strains PVY^O and PVY^N, including subgroups PVY^NW and PVY^NTN, were effectively localized when plants were grown at 20°C. At 28°C, plants were systemically infected but no symptoms were observed. In field trials, PVY was restricted to the inoculated leaves and PVY-free tubers were produced. Therefore, the gene Ny-1 can be useful for potato breeding as an alternative donor of PVY resistance, because it is efficacious in practice-like resistance conferred by Ry genes.     

Comparison of transmission efficiency of various isolates of Potato virus Y among three aphid vectors

Potato virus Y (PVY) strains are transmitted by different aphid species in a non‐persistent, non‐circulative manner. Green peach aphid (GPA), Myzus persicae Sulzer, is the most efficient vector in laboratory studies, but potato aphid (PA), Macrosiphum euphorbiae Thomas (both Hemiptera: Aphididae, Macrosiphini), and bird cherry‐oat aphid (BCOA), Rhopalosiphum padi L. (Hemiptera: Aphididae, Aphidini), also contribute to PVY transmission. Studies were conducted with GPA, PA, and BCOA to assess PVY transmission efficiency for various isolates of the same strain. Treatments included three PVY strains (PVY^O, PVY^N:O, PVY^NTN) and two isolates of each strain (Oz and NY090031 for PVY^O; Alt and NY090004 for PVY^N:O; N4 and NY090029 for PVY^NTN), using each of three aphid species as well as a sham inoculation. Virus‐free tissue‐cultured plantlets of potato cv. Russet Burbank were used as virus source and recipient plants. Five weeks post inoculation, recipient plants were tested with quantitative DAS‐ELISA to assess infection percentage and virus titer. ELISA‐positive recipient plants were assayed with RT‐PCR to confirm presence of the expected strains. Transmission efficiency (percentage infection of plants) was highest for GPA, intermediate for BCOA, and lowest for PA. For all aphid species, transmission efficiency did not differ significantly between isolates within each strain. No correlations were found among source plant titer, infection percentage, and recipient plant titer. For both GPA and BCOA, isolates of PVY^NTN were transmitted with greatest efficiency followed by isolates of PVY^O and PVY^N:O, which might help explain the increasing prevalence of necrotic strains in potato‐growing regions. Bird cherry‐oat aphid transmitted PVY with higher efficiency than previously reported, suggesting that this species is more important to PVY epidemiology than has been considered.     

Generation of transgenic potato plants highly resistant to potato virus Y (PVY) through RNA silencing

In this study we applied RNA silencing to engineer potato plants that are resistant to potato virus Y (PVY). We expressed double-stranded (ds) RNA derived from the 3 terminal part of the coat protein gene of PVY, which is highly conserved in sequence amongst different PVY isolates, in transgenic potatoes of the commercial variety Spunta. Transgenic plants were analyzed for generation of transgene-derived short interfering RNAs (siRNAs) prior to virus inoculation. Twelve of fifteen transgenic lines produced siRNAs and were highly resistant to three strains of PVY, each belonging to three different subtypes of the virus (PVY^N, PVY^O and PVY^NTN). Infection of transgenic plants with Potato virus X (PVX) simultaneously or prior to the challenge with PVY did not interfere with PVY-resistance.Anastasia Missiou: M.A. and K.K. have contributed equally to this workKriton Kalantidis: M.A. and K.K. have contributed equally to this work     

Expression of Coat Protein of an Ordinary Strain of Potato virus Y in Escherichia coli and Production of Polyclonal Antibodies for Diagnosis

The coat protein gene (CP) of an ordinary strain of Potato virus Y (PVY^O) was cloned into the expression vector, pET‐28a(+). The insert was sequenced and analysis showed that the CP gene was in frame with intact N‐terminal 6X histidine tags. An approximately 35 kDa recombinant fusion protein was observed in inclusion bodies of induced Escherichia coli BL21 cells. This fusion protein was purified and used as antigen to raise polyclonal antibodies in rabbits. In Western blot and dot blot immuno‐binding assay (DIBA), both PVY^O‐CP IgG and PVY^O IgG strongly reacted with the recombinant CP. The PVY^O‐CP IgG could detect PVY^O in infected samples up to 1 : 3200 dilutions. A PVY^O‐CP ELISA kit was prepared and compared with conventional ELISA kit based on purified virus particles (PVY^O ELISA kit). The PVY^O‐CP ELISA kit consistently detected the PVY^O in DAS‐ELISA of field samples and was as effective as PVY^O ELISA kit.     

Primary Metabolism,Phenylpropanoids and Antioxidant Pathways Are Regulated in Potato as a Response to Potato virus Y Infection

Potato production is one of the most important agricultural sectors, and it is challenged by various detrimental factors, including virus infections. To control losses in potato production, knowledge about the virus—plant interactions is crucial. Here, we investigated the molecular processes in potato plants as a result of Potato virus Y (PVY) infection, the most economically important potato viral pathogen. We performed an integrative study that links changes in the metabolome and gene expression in potato leaves inoculated with the mild PVY^N and aggressive PVY^NTN isolates, for different times through disease development. At the beginning of infection (1 day post-inoculation), virus-infected plants showed an initial decrease in the concentrations of metabolites connected to sugar and amino-acid metabolism, the TCA cycle, the GABA shunt, ROS scavangers, and phenylpropanoids, relative to the control plants. A pronounced increase in those metabolites was detected at the start of the strong viral multiplication in infected leaves. The alterations in these metabolic pathways were also seen at the gene expression level, as analysed by quantitative PCR. In addition, the systemic response in the metabolome to PVY infection was analysed. Systemic leaves showed a less-pronounced response with fewer metabolites altered, while phenylpropanoid-associated metabolites were strongly accumulated. There was a more rapid onset of accumulation of ROS scavengers in leaves inoculated with PVY^N than those inoculated with PVY^NTN. This appears to be related to the lower damage observed for leaves of potato infected with the milder PVY^N strain, and at least partially explains the differences between the phenotypes observed.     

Comparison of resistance to potyviruses within Solanaceae: infection of potatoes with tobacco etch potyvirus and peppers with potato A and Y potyviruses

The reaction of several cultivated potato varieties (Solarium tuberosum L.) to three strains of tobacco etch potyvirus (TEV-F, TEV-Mex21 and TEV-ATCC) and the reaction of several pepper lines (Capsicum annuum L. and C. chinense L.) to two strains of potato Y potyvirus (PVY^O and PVY^N) and one strain of potato A potyvirus (PVA-M) was tested. The potato varieties included in this study carried resistance genes against PVY, PVA and potato V potyvirus, but all were susceptible to TEV and developed mottle and mosaic symptoms. TEV was readily transmitted by mechanical inoculation from tobacco and potato to potato, whereas transmission from pepper to potato occurred infrequently. TEV was transmitted through potato tubers, and from pepper to potato plants by aphids. Lack of detectable systemic infection following graft-inoculation indicated extreme resistance to PVY^O and PVA in several pepper lines. No pepper line was systemically infected with PVY^N following mechanical inoculation (graft-inoculation was not carried out with PVY^N). The development of necrotic lesions following mechanical and graft-inoculation indicated hypersensitive response to PVY^O in several pepper lines which resembled the resistance responses to these potyvirus strains in potato. Results of this study together with previous work indicate that C. annuum cv. Avelar is resistant to four potyviruses [PVY, PVA, pepper mottle potyvirus (PepMoV) and some isolates of TEV]; C. annuum cv. Criollo de Morelos and C. chinense PI 152225 and PI 159236 are resistant to three potyviruses (PVY, PepMoV and PVA; and PVY, PepMoV and TEV, respectively); C. annuum 9093–1 and 92016–1 are resistant to PVY and PepMoV; and C. annuum cv. Jupiter and C. annuum cv. RNaky are resistant to PVY^N and PVA.     

Tobacco plants transformed with the potato virus YN coat protein gene are protected against different PVY isolates and against aphid-mediated infection

Tobacco plant lines transformed with the coat protein (CP) gene of the tobacco veinal necrosis strain of potato virus Y (PVY^N), and previously shown to be protected against mechanical inoculation with the virus, have now been tested for specificity and protection against virus infection mediated by viruliferous aphids. To determine the specificity of virus protection, two transgenic tobacco lines, A30 and A80, were challenged with several isolates of distinct PVY strains (PVY^N, PVY^O and PVY^C) by mechanical inoculation. Clear levels of protection against the PVY^O-isolates tested were maintained in the transgenic plants, although these levels were slightly lower than the protection against the homologous PVY^N strain from which the CP gene was derived. Interestingly, no protection against mechanical virus inoculation with the Gladblaadje isolate of PVY^C could be observed. To assess the levels of protection against aphid-mediated virus infection, two transgenic plant lines, A30 and D25, showing respective levels of protection of 95 and 80% against mechanical virus inoculation, were challenged using PVY^N viruliferousMyzus persicae. Virus inoculation using six aphids per plant, resulted in similar levels of protection in both transgenic lines as found previously for mechanical inoculation. Protection was maintained in both lines, even when as many as 60 viruliferous aphids were used per plant in the inoculation experiments.     

The Genetic Structure of Populations of Potato virus Y in Japan; Based on the Analysis of 20 Full Genomic Sequences

The genetic structure of Potato virus Y (PVY) populations in Japan was analysed using 20 isolates; five were retrieved from the public DNA sequence databases, and an additional 15 complete genomic sequences were determined using field samples collected in Japan. Recombination and phylogenetic analyses of a total of 149 isolates from Japan and other countries showed that PVY has three major lineages (C, N and O); at least one, two and six sublineages in C, N and O lineages, respectively. One recombination pattern was newly found among Japanese PVY^NTN strain isolates, which was most closely related to the PVY^NTN strain isolates previously found in Europe and North America. On the other hand, PVY^O was a complex of several divergent lineages, and there were at least three non‐recombinant subpopulations in Japan. Studies on nucleotide diversities of populations and phylogenetic relationships of the isolates in the PVY sequences showed that Japanese PVY populations were in part distinct from the European and North American populations.     

Expression of genes for resistance to potato virus Y in potato plants and protoplasts

Plants of several potato clones with major gene resistance to potato virus Y (PVY) developed necrotic local lesions and systemic necrosis after manual inoculation with common (PVY_o) or veinal necrosis (PVY_N) strains of the virus. The clones reacted similarly, although their resistance genes are thought to be derived from four different wild species of Solarium. Mesophyll protoplasts from each clone became infected when inoculated with RNA of PVY_o by the polyethylene glycol method. The proportion of protoplasts infected, assessed by staining with fluorescent antibody to virus particles, was similar to that of protoplasts of susceptible potato cultivars. In contrast, plants of potato cultivars Corine and Pirola, which possess gene Ry from S. stoloniferum, developed few or no symptoms when manually inoculated or grafted with PVY_o. Moreover, only very few protoplasts of these cultivars produced virus particle antigen after inoculation with PVY_o RNA. The extreme resistance to PVY of cvs Corine and Pirola was therefore expressed by inoculated protoplasts whereas the resistance of the necrotic-reacting potato clones was not.     

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.     

Determination of aphid transmission efficiencies for N, NTN and Wilga strains of Potato virus Y

Potato virus Y (PVY, genus Potyvirus, family Potyviridae) causes high economic losses worldwide, especially in the production of seed potatoes (Solanum tuberosum). PVY control systems rely on measuring virus pressure and vector pressure in the field. Calculation of the vector pressure is based on the relative efficiency factors (REFs) of aphid species. These REFs express the transmission efficiency of aphid species in relation to the transmission efficiency of Myzus persicae, the most efficient vector of PVY. In this paper, we report on the determination of aphids' relative transmission efficiency factors (REFs) for isolates of the PVY strains PVY^N, PVY^NTN and PVY^N-Wi. Biotype Mp2 of M. persicae was tested for its transmission efficiency for six PVY isolates (one PVY^N, three PVY^NTN and two PVY^N-Wi isolates) and showed comparable average transmission efficiencies for all isolates. The transmission rate of this biotype for the six PVY isolates was set to 1 and Mp2 was used as an internal control in transmission experiments to determine the REFs of three other biotypes of M. persicae and 16 other aphid species (three biotypes per species when available) for the six PVY isolates. Comparing the calculated REFs for PVY^N with the REFs reported in the previous century for PVY^N, we observe overall comparable REFs, except for Aphis fabae, Aphis spp., Hyperomyzus lactucae, Macrosiphum euphorbiae and Rhopalosiphum padi, which have a lower REF in our experiments, and Aphis frangulae and Phorodon humuli, which have now a higher REF. Comparing the new REFs found for the PVY^NTN strains with the new REFs for PVY^N, we observe that they are overall comparable, except for A. frangulae (0.17 compared with 0.53) and Schizaphis graminum (0.05 compared with 0.00). Comparing the REFs calculated for PVY^N-Wi with those calculated for PVY^N, we can observe six aphid species with higher REFs (Acyrthosiphon pisum, A. fabae, Aphis nasturtii, Aphis spp., P. humuli and R. padi). Only the species A. frangulae shows a lower REF for PVY^N-Wi compared with the transmission efficiency of PVY^N. Three aphid species (Aulacorthum solani, Myzus ascalonicus and S. graminum) for which no REF was determined earlier were found to be capable to transmit PVY and their REFs were determined.     

Hypersensitive response to Potato virus Y in potato cultivar Sárpo Mira is conferred by the Ny-Smira gene located on the long arm of chromosome IX

Potato virus Y (PVY, Potyvirus) is the fifth most important plant virus worldwide in terms of economic and scientific impact. It infects members of the family Solanaceae and causes losses in potato, tomato, tobacco, pepper and petunia production. In potato and its wild relatives, two types of resistance genes against PVY have been identified. While Ry genes confer symptomless extreme resistance, Ny genes cause a hypersensitive response visible as local necrosis that may also be able to prevent the virus from spreading under certain environmental conditions. The potato cultivar Sárpo Mira originates from Hungary and is highly resistant to PVY, although the source of this resistance remains unknown. We show that cv. Sárpo Mira reacts with a hypersensitive response leading to necrosis after PVY^NTN infection in detached leaf, whole plant and grafting assays. The hypersensitivity to PVY^NTN segregated amongst 140 individuals of tetraploid progeny of cvs. Sárpo Mira × Maris Piper in a 1:1 ratio, indicating that it was conferred by a single, dominant gene in simplex. Moreover, we identified five DNA markers linked to this trait and located the underlying locus (Ny-Smira) to the long arm of potato chromosome IX. This position corresponds to the location of the Ry _chc and Ny-1 genes for PVY resistance. A simple PCR marker, located 1 cM from the Ny-Smira gene, can be recommended for selection of PVY-resistant progeny of cv. Sárpo Mira.     

Production of monoclonal antibodies for the detection of potato virus Y

Monoclonal antibodies (McAb) were obtained to potato virus Y (PVY) after immunisation of BALB/c mice with purified PVY, tobacco necrotic strain (PVY_n). Spleen cells from a mouse showing a high serum titre were used for fusion with X63NS1 myeloma cells. Hybridomas were selected in medium containing HAT. Culture supernatants were screened for antibody production against PVY, ordinary strain (PVY₀) and PVY_n using indirect ELISA. Clones of interest were further cross-reacted with 12 isolates each of PVY₀ and PVY_n and two isolates of potato virus A (PVA) and healthy sap. For further trials, two clones which reacted specifically with PVY_n isolates and one which detected all PVY isolates except two of potato virus C (PVC) were selected.