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.     

Ultrastructural events during hypersensitive response of potato cv. Rywal infected with necrotic strains of potato virus Y

Potato plants cv. Rywal with hypersensitivity gene Ny-1 infected with PVY^N or PVY^NTN reacted in local necroses 3 days after infection. Potato virus Y (PVY) particles were found in epidermis, mesophyll, phloem and xylem cells in inoculated leaves. Noncapsidated virus particles (without capsid protein) were observed already 10 h after infection by using electron microscopy in situ. Capsid protein on one terminus of noncapsidated virus particles was located 5 days after inoculation with the use of immunogold labeling method. Whereas cytoplasmic inclusions were observed for the first time 24 days after infection during hypersensitive response. Ultrastructural studies showed that ER may take part in PVY RNA replication and capsidation of Potyvirus particles. Observed cytopathological changes and virus particles indicate that cell nucleus and mitochondrion might participate in PVY life cycle. During hypersensitive response PVY particles were found in plasmodesmata as well as in phloem and xylem.     

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.     

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     

Multiple hormone analysis indicates involvement of jasmonate signalling in the early defence of potato to potato virus YNTN

The involvement of plant hormones in the very early response of plants to virus infection was studied in potato plants (Solanum tuberosum L.) infected with potato virus Y^NTN (PVY^NTN). Endogenous plant hormones and compounds mediating a stress response (JA-jasmonic acid, OPDA-12-oxo phytodienoic acid, SA-salicylic acid, IAA-indole-3-acetic acid, ABA-abscisic acid) were simultaneously quantified in susceptible cv. Désirée and resistant cv. Santé, one and three hours after inoculation. Of the hormones analysed, only the contents of endogenous JA and its precursor OPDA changed in a way that could be clearly connected with the early resistant response. In comparison to susceptible cultivar, a much more pronounced increase of JA was detected in virus-inoculated leaves of resistant cultivar at both time points. The same trend of changes was also observed with OPDA. However, there were no significant changes of JA and its precursor in upper intact systemic leaves and roots, at either time point. These findings implicate the jasmonate signalling pathway in a very early local but not systemic resistant defence of potato to PVY^NTN.     

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.     

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.     

Recombination of strain O segments to HCpro‐encoding sequence of strain N of Potato virus Y modulates necrosis induced in tobacco and in potatoes carrying resistance genes Ny or Nc

Hypersensitive resistance (HR) to strains O and C of Potato virus Y (PVY, genus Potyvirus) is conferred by potato genes Ny_tbr and Nc_tbr, respectively; however, PVY N strains overcome these resistance genes. The viral helper component proteinases (HCpro, 456 amino acids) from PVY^N and PVY^O are distinguished by an eight‐amino‐acid signature sequence, causing HCpro to fold into alternative conformations. Substitution of only two residues (K269R and R270K) of the eight‐amino‐acid signature in PVY^N HCpro was needed to convert the three‐dimensional (3D) model of PVY^N HCpro to a PVY^O‐like conformation and render PVY^N avirulent in the presence of Ny_tbr, whereas four amino acid substitutions were necessary to change PVY^O HCpro to a PVY^N‐like conformation. Hence, the HCpro conformation rather than other features ascribed to the sequence were essential for recognition by Ny_tbr. The 3D model of PVY^C HCpro closely resembled PVY^O, but differed from PVY^N HCpro. HCpro of all strains was structurally similar to β‐catenin. Sixteen PVY^N605‐based chimeras were inoculated to potato cv. Pentland Crown (Ny_tbr), King Edward (Nc_tbr) and Pentland Ivory (Ny_tbr/Nc_tbr). Eleven chimeras induced necrotic local lesions and caused no systemic infection, and thus differed from both parental viruses that infected King Edward systemically, and from PVY^N605 that infected Pentland Crown and Pentland Ivory systemically. These 11 chimeras triggered both Ny_tbr and Nc_tbr and, in addition, six induced veinal necrosis in tobacco. Further, specific amino acid residues were found to have an additive impact on necrosis. These results shed new light on the causes of PVY‐related necrotic symptoms in potato.     

Capacity assessment of <Emphasis Type="Italic">Myzus persicae,Aphis gossypii</Emphasis> and <Emphasis Type="Italic">Aphis spiraecola</Emphasis> (Hemiptera: Aphididae) to acquire and retain PVY<Superscript>NTN</Superscript> in Tunisia

The study was carried out to investigate the ability of three aphids, Myzus persicae, Aphis gossypii and Aphis spiraecola, to acquire and retain the Potato Virus Y (PVY) isolate, PVY^NTN. Tobacco plants, Nicotiana tabacum var. Xanthi, were used as test plant for the virus inoculation and aphid acquisition. The serological test double-antibody sandwich enzyme-linked immunosorbent assay was applied for virus detection on the test plants and aphids. Furthermore, virus retention by aphids was also assessed using a monoclonal anti-PVY^N. Although a duration of 2 min was enough for the virus acquisition, the three tested aphids showed different capacities to retain PVY^NTN. The retention of PVY^NTN was 3 h for M. persicae and A. spiraecola, and 2 h for A. gossypii. This study provides basic information of the virus retention by potato-colonizing aphid species, which may increase our understanding of PVY epidemiology in Tunisia.     

Linking belowground and aboveground phenology in two boreal forests in Northeast China

Plants are frequently attacked by both pathogens and insects, and an attack from one can induce plant responses that affect resistance to the other. However, we currently lack a predictive framework for understanding how pathogens, their vectors, and other herbivores interact. To address this gap, we have investigated the effects of a viral infection in the host plant on both its aphid vector and non-vector herbivores. We tested whether the infection by three different strains of Potato virus Y (PVY^NTN, PVY^NO and PVY^O) on tomato plants affected: (1) the induced plant defense pathways; (2) the abundance and fecundity of the aphid vector (Macrosiphum euphorbiae); and (3) the performance of two non-vector species: a caterpillar (Trichoplusia ni) and a beetle (Leptinotarsa decemlineata). While infection by all three strains of PVY induced the salicylate pathway, PVY^NTN induced a stronger and longer response. Fecundity and density of aphids increased on all PVY-infected plants, suggesting that the aphid response is not negatively associated with salicylate induction. In contrast, the performance of non-vector herbivores positively correlated with the strength of salicylate induction. PVY^NTN infection decreased plant resistance to both non-vector herbivores, increasing their growth rates. We also demonstrated that the impact of host plant viral infection on the caterpillar results from host plant responses and not the effects of aphid vector feeding. We propose that pathogens chemically mediate insect–plant interactions by activating the salicylate pathway and decreasing plant resistance to chewing insects, which has implications for both disease transmission and insect community structure.     

Expression of the PVYO coat protein (CP) under the control of the PVX CP gene leader sequence: protection under greenhouse and field conditions against PVYO and PVYN infection in three potato cultivars

Coat protein-mediated resistance (CPMR), resistance conferred as a result of the expression of viral coat proteins in transgenic plants, has been illustrated to be an effective way of protecting plants against several plant viruses. Nonetheless, consistent protection has not been achieved for transgenic plants expressing the coat protein of potato virus Y (PVY), the type member of the potyvirus family. In this report, three different potato cultivars were transformed with a chimeric construct consisting of the capsid protein (CP) coding sequences of PVY flanked by the AUG codon and the translational enhancer from the coat protein gene of potato virus X (PVX). These cultivars were shown to express high levels of PVY CP and confer a high degree of protection against PVY^o and PVY^N under both greenhouse and field conditions. In addition, transgenic plants infected with potato virus A (PVA), a related potyvirus, exhibited a delay in virus accumulation, which could be easily overcome with increasing virus concentrations. Received: 26 October 1995 / Accepted: 14 June 1996     

Optimum Conditions for the Storage of Potato Virus YNTN Strain

The effect of storage conditions on the serological activity of two isolates of potato virus Y^NTN strain (PVY^NTN) was studied by ELISA. Purified virus, intact and homogenized infected leaves stored freeze-dried and frozen at various temperatures were tested. Purified virus was the most stable at +4 °C and non-purified virus was best preserved as a freeze-dried leaf homogenate at –20 °C. Their serological activity did not change after three months of storage.     

The role of volunteer potatoes in the spread of potato virus YN in ware crops of cv. Record

Surveys were made for the presence of potato virus Y (PVY) in the planted seed and harvested tubers in ware potato crops of cv. Record grown at three sites in England in 1994 (survey 1) and seven sites in 1995 (survey 2). PVY was not found in samples of planted seed, but high levels of infection were found in many, but not all, harvested crops. However, plants of volunteer potatoes (VP) (i.e. plants arising from tubers or true seed derived from previous crops and surviving in the soil) were frequently found to be infected. Infection in tubers harvested from crops in the first survey ranged from 2–52%. In 1995, VP were collected from two of the three English sites where potato crops had been grown the previous season and also from a site in Scotland where PVY infection in an experimental crop of cv. Record had been monitored in 1994. The percentages of infected VP ranged from 2–54%. PVY^N was the predominant strain found in sampled VP, with only two plants (out of 300 infected) containing PVY^O. In the second survey, VP were assessed within the 1995 ware crops and were found at four sites, at which they comprised between 4–8% of emerged potato plants. Between 31–93% of VP were infected. Again, PVY^N was the predominant strain with one plant containing PVY^O and another PVY^C (out of 189 infected). A sample of harvested tubers from each site was also tested for PVY. At those sites which had many infected VP, the harvested crop contained a large percentage of infected tubers, ranging from 60–97%. Two sites which had not previously been used for cropping potatoes had no VP and a very low incidence of PVY infection in the harvested tubers (1% and 2%). However, although no VP were found at one site, 31% of harvested tubers were infected, suggesting that alternative inoculum sources may be important.     

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.     

β-1,3-glucanase class III promotes spread of PVYNTN and improves in planta protein production

Glucanases are enzymes regulating the size exclusion limit and permeability of plasmodesmata and play a role in biotic stress. In plant genomes, they are encoded as relatively large gene families divided into four classes. Most studies of plant virus interactions have focused on glucanases from classes I and II. In our study, we have evaluated the role of the β-1,3-glucanase class III (Glu-III) gene in the potato–potato virus Y^NTN (PVY^NTN) interaction and implemented the findings to plant biotechnology application. Potato cultivars Désirée and Santé, which are tolerant and extremely resistant to PVY^NTN, respectively, were stably transformed with Agrobacterium tumefaciens harbouring constructs for Glu-III overexpression. Localization of Glu-III protein in patches within the cell wall was determined by tagging the Glu-III protein with green fluorescent protein. Transgenic and non-transgenic plants were challenged with PVY^NTN and its multiplication and spreading was followed. Differences in viral spread were observed between transgenic lines overexpressing Glu-III and non-transgenic lines, with stronger and faster viral spread in transgenic Désirée, and some multiplication in transgenic Santé. In addition, the ability of Glu-III to improve in planta protein production after agroinfiltration was tested. The results have shown that Glu-III overexpression enables faster spreading of vectors between cells and better protein production, which could be beneficial in improving in planta protein production system using viral vectors.