Molecular and biological characterization of a recombinant isolate of potato virus Y from Mexico.

An isolate of potato virus Y (PVY), PVY-M3, was subjected to biological characterization on potato indicators and to whole-genome sequencing. PVY-M3 induced a local and systemic hypersensitive resistance (HR) response in potato cultivar Maris Bard expressing the Nz gene while inducing no HR in potato cultivars Desiree and King Edward, carrying Ny and Nc genes, respectively. These HR responses, combined with a lack of vein necrosis in tobacco, clearly defined PVY-M3 as an isolate of the PVY(Z) strain. Recombination analysis demonstrated that PVY-M3 had a typical European PVY(NTN) genome with three recombinant junctions, and PVY(N) and PVY(O) were identified as parents.

The helper component proteinase cistron of Potato virus Y induces hypersensitivity and resistance in Potato genotypes carrying dominant resistance genes on chromosome IV.

The Nc(tbr) and Ny(tbr) genes in Solanum tuberosum determine hypersensitive reactions, characterized by necrotic reactions and restriction of the virus systemic movement, toward isolates belonging to clade C and clade O of Potato virus Y (PVY), respectively. We describe a new resistance from S. sparsipilum which possesses the same phenotype and specificity as Nc(tbr) and is controlled by a dominant gene designated Nc(spl). Nc(spl) maps on potato chromosome IV close or allelic to Ny(tbr). The helper component proteinase (HC-Pro) cistron of PVY was shown to control necrotic reactions and resistance elicitation in plants carrying Nc(spl), Nc(tbr), and Ny(tbr). However, inductions of necrosis and of resistance to the systemic virus movement in plants carrying Nc(spl) reside in different regions of the HC-Pro cistron. Also, genomic determinants outside the HC-Pro cistron are involved in the systemic movement of PVY after induction of necroses on inoculated leaves of plants carrying Ny(tbr). These results suggest that the Ny(tbr) resistance may have been involved in the recent emergence of PVY isolates with a recombination breakpoint near the junction of HC-Pro and P3 cistrons in potato crops. Therefore, this emergence could constitute one of the rare examples of resistance breakdown by a virus which was caused by recombination instead of by successive accumulation of nucleotide substitutions.

Identification of the molecular make-up of the Potato virus Y strain PVY(Z): genetic typing of PVY(Z)-NTN.

Potato virus Y (PVY) strains were originally defined by interactions with different resistance genes in standard potato cultivars. Five distinct strain groups are defined that cause local or systemic hypersensitive responses (HRs) in genetic background with a corresponding N gene: PVY(O), PVY(N), PVY(C), PVY(Z), and PVY(E). The nucleotide sequences of multiple isolates of PVY(O) and PVY(N) differ from each other by ≈8% along their genomes. Additionally, complete genome sequences of multiple recombinant isolates are composed of segments of parental PVY(O) and PVY(N) sequences. Here, we report that recombinant isolate PVY-L26 induces an HR in potato 'Maris Bard' carrying the putative Nz gene, and is not recognized by two other resistance genes, Nc and Ny(tbr). These genetic responses in potato, combined with the inability of PVY-L26 to induce vein necrosis in tobacco, clearly define it as an isolate from the PVY(Z) strain group and provide the first information on genome structure and sequence of PVY(Z). The genome of PVY-L26 displays typical features of European NTN-type isolates with three recombinant junctions (PVY(EU-NTN)), and the PVY-L26 is named PVY(Z)-NTN. Three typical PVY(NTN) isolates and two PVY(N) isolates, all inducing vein necrosis in tobacco, were compared with PVY-L26. One PVY(NTN) isolate elicited HR reactions in Maris Bard, similar to PVY-L26, while two induced a severe systemic HR-like reaction quite different from the quasi-symptomless reaction induced by two PVY(N) isolates. 'Yukon Gold' potato from North America produced HR against several PVY(NTN) isolates, including PVY-L26, but only late and limited systemic necrosis against one PVY(N) isolate. Consequently, according to symptoms in potato indicators, both PVY(Z) and PVY(NTN) isolates appeared biologically very close and clearly distinct from PVY(O) and PVY(N) strain groups.

Genetic diversity of the ordinary strain of Potato virus Y (PVY) and origin of recombinant PVY strains.

The ordinary strain of Potato virus Y (PVY), PVY(O), causes mild mosaic in tobacco and induces necrosis and severe stunting in potato cultivars carrying the Ny gene. A novel substrain of PVY(O) was recently reported, PVY(O)-O5, which is spreading in the United States and is distinguished from other PVY(O) isolates serologically (i.e., reacting to the otherwise PVY(N)-specific monoclonal antibody 1F5). To characterize this new PVY(O)-O5 subgroup and address possible reasons for its continued spread, we conducted a molecular study of PVY(O) and PVY(O)-O5 isolates from a North American collection of PVY through whole-genome sequencing and phylogenetic analysis. In all, 44 PVY(O) isolates were sequenced, including 31 from the previously defined PVY(O)-O5 group, and subjected to whole-genome analysis. PVY(O)-O5 isolates formed a separate lineage within the PVY(O) genome cluster in the whole-genome phylogenetic tree and represented a novel evolutionary lineage of PVY from potato. On the other hand, the PVY(O) sequences separated into at least two distinct lineages on the whole-genome phylogenetic tree. To shed light on the origin of the three most common PVY recombinants, a more detailed phylogenetic analysis of a sequence fragment, nucleotides 2,406 to 5,821, that is present in all recombinant and nonrecombinant PVY(O) genomes was conducted. The analysis revealed that PVY(N:O) and PVY(N-Wi) recombinants acquired their PVY(O) segments from two separate PVY(O) lineages, whereas the PVY(NTN) recombinant acquired its PVY(O) segment from the same lineage as PVY(N:O). These data suggest that PVY(N:O) and PVY(N-Wi) recombinants originated from two separate recombination events involving two different PVY(O) parental genomes, whereas the PVY(NTN) recombinants likely originated from the PVY(N:O) genome via additional recombination events.

A multiple single nucleotide polymorphisms interrogation assay for reliable Potato virus Y group and variant characterization.

The complex Potato virus Y classification, including groups (PVYN and PVYO) and variants (PVYNTN and PVYN-W), is based mainly on biological properties of isolates. Published PVY detection tools targeting markers not associated with biological properties could fail to assign correctly isolates in the current classification. To improve PVY detection tools, a single nucleotide polymorphism (SNaPshot) detection assay was developed. The technique was adapted to target the T/C9259, A/C2271, G/C8573 and A/G2213 PVY polymorphic nucleotides. The "TAGA", "CCCG", "CACA" and "CAGA" four-digit codes associated with tested samples allowed identification of PVYN, PVYO, PVYN-W and PVYNTN isolates, respectively. The PVY SNaPshot procedure is efficient and reliable for PVY detection and characterization in samples containing as few as 10(2) viral RNA copies. Moreover, PVY group assignment is possible for fractions containing only 10 copies of a PVY RNA genome. Finally, the SNaPshot assay allows PVY(N)/PVYO dual characterization for mixed samples containing PVYN/PVYO quantity ratios in the range of 0.1-10. This innovative SNaPshot tool improved clearly PVY diagnostic assays described previously by targeting simultaneously major functional markers and sequence unlinked to biological properties used separately in PVY detection tools available currently.

Improvement of Potato virus Y (PVY) detection and quantitation using PVY(N)- and PVY(O)-specific real-time RT-PCR assays.

A Potato virus Y (PVY) single nucleotide polymorphism (A/G(2213)), recently identified as a molecular determinant of the tobacco leaf necrosis symptom induced by PVY(N) isolates, has been used as a target to develop two PVY group-specific (PVY(N) and PVY(O)) fluorescent (TaqMan-based) real-time RT-PCR assays. These procedures allow detection, characterisation, and quantitation of a wide range of PVY isolates in samples containing 10(3)-10(8) viral transcripts. Moreover, the high specificity of these two new assays make the simultaneous detection and the reliable quantitation of PVY(N) and PVY(O) isolates in mixed solutions, regardless of the Y(N)/Y(O) ratio, feasible. The high sensitivity (threshold of 10(3) copies per reaction) and the PVY group specificity of these two new PVY detection tools clearly improve previously published PVY detection tests and offer new opportunities for PVY research programs.

A single nucleotide polymorphism-based technique for specific characterization of YO and YN isolates of Potato virus Y (PVY).

One of the most important properties used to classify Potato virus Y (PVY) isolates is their ability to induce (PVY(N)) or not (PVY(O)) veinal necrosis symptoms on the indicator host plant Nicotiana tabacum cv. Xanthi. As an alternative to biological assays, several serological and molecular detection tools have been developed for PVY detection and characterization and these have evolved as our knowledge of PVY has improved. However, the assays that have been previously published are all based on the use of neutral markers (antigenic determinants, sequence data, recombination sites or restriction enzyme cleavage sites), which are unlinked to the biological property being characterized (e.g. veinal necrosis). Using the recently identified molecular determinants of the tobacco leaf necrosis symptom induced by PVY(N) isolates, a one-step fluorescent [TaqMan] RT-PCR assay, based on a single nucleotide polymorphism (SNP) linked to the necrosis property of PVY isolates, has been designed. This assay reliably detects and distinguishes PVY(N) and PVY(O) isolates. The method is simple (leaf soak extraction process, gel-free, no post-PCR manipulations), rapid (96 tests in less than 3h from plants sampling to diagnostic results), sensitive (threshold in a range of 10(4)-10(5) PVY copies), reliable (correctly assigns 42 PVY isolates in their respective group) and allows co-detection of mixed samples containing close to equivalent PVY(N) and PVY(O) quantities. All these characteristics suggest that the newly developed SNP assay could be used to reliably classify PVY isolates, as a substitute for biological assays performed on N. tabacum cv. Xanthi.

Complementations and exclusions between mutated versions of a potato virus Y genotype during mixed infections of Nicotiana hosts.

Understanding the processes that have led to the recent prevalence of necrotic genotypes in PVY populations is an important challenge for research programs studying this virus. Non-necrotic PVY(O)-139, necrotic PVY(N)-605 and point mutated versions of PVY(N)-605 (PVY(KRED), PVY(KR) and PVY(ED)), were used in mixtures to inoculate two Nicotiana hosts which express (N. tabacum cv. Xanthi) or not (N. clevelandii) necrosis symptoms in response to infection by PVY(N) group members. The comparison during serial passage experiments of proportions of PVY genotypes produced in mixed infected plants with those of the inocula was used to describe: (i) complementation between PVY(KR) and PVY(N) and between PVY(KRED) and PVY(O) genotypes; (ii) exclusion of the PVY(KRED) genotype, previously described as fitter, during mixed infections in the presence of one of the less fit PVY(N), PVY(ED) and PVY(KR) genotypes and (iii) the prevalence of the non-necrotic PVY(KR) genotype in the presence of PVY(N) parental sequence. These results indicate that the role of both A/G(2213) and A/C(2271) nucleotides in the fitness of PVY genotypes depends on other genetic information in the viral genome that has not yet been identified. Moreover, the collected data indicate that mutation of the nucleotide 2213 in the PVY(N)-605 sequence could lead to the prevalence, both in N. tabacum cv. Xanthi and in N. clevelandii, of the non-necrotic PVY(KR) genotype.

The acquisition of molecular determinants involved in potato virus Y necrosis capacity leads to fitness reduction in tobacco plants.

The prevalence of necrotic potato virus Y (PVY) in natural populations could reflect increased fitness of necrotic isolates. In this paper, the effects of the acquisition of molecular determinants (A/G(2213) and A/C(2271)) involved in necrosis capacity on both the number of progeny produced and the competitiveness of PVY were characterized. The relationship between necrosis and fitness was tested using (i) Nicotiana tabacum cv. Xanthi and Nicotiana clevelandii, (ii) necrotic PVY(N)-605 and non-necrotic PVY(O)-139 isolates, (iii) single-mutated (PVY(KR) and PVY(ED)) and double-mutated (PVY(KRED)) versions of PVY(N)-605 and (iv) three quantitative PCR assays specific for nt A(2213), G(2213) and A(2271) of the PVY genome. The data demonstrated effects of both the genetic background and nt 2213 and 2271 on the fitness of PVY. Quantification of PVY RNA in singly infected plants revealed that both the PVY(N)-605 genetic background and the acquisition of necrotic capacity resulted in a decrease in the number of progeny produced. Competition experiments revealed that the genetic background of PVY(N) had a positive impact on competitiveness. In contrast, nucleotides involved in necrotic properties were associated with decreased fitness. Finally, in the host that did not respond to infection with necrosis, the benefit associated with the PVY(N)-605 genetic background was higher than the cost associated with the acquisition of molecular determinants involved in necrosis capacity. The opposite result was obtained in the host responding to the infection with necrosis. These results indicate that the emergence of necrotic isolates from a non-necrotic population is unlikely in tobacco.

Characterization of Potato virus Y (PVY) molecular determinants involved in the vein necrosis symptom induced by PVYN isolates in infected Nicotiana tabacum cv. Xanthi.

Viral molecular determinant(s) involved in the tobacco vein necrosis (TVN) symptom induced by necrotic isolates of Potato virus Y (PVY) on Nicotiana tabacum cv. Xanthi leaves remain undetermined. Reference isolates belonging to PVY(N) (infectious PVY(N)-605 clone) and PVY(O) (PVY(O)-139) were used to produce PVY chimeric genomes by using reverse-genetic techniques. These chimeric clones were inoculated biolistically onto Nicotiana clevelandii plants to establish the clone, prior to being tested on N. tabacum for their ability to induce TVN symptoms. Comparison between sequence data and symptoms observed for each mutated PVY construct shows that the C-terminal part of the multifunctional HC-Pro protein includes two residues (K(400) and E(419)) that are involved in TVN induced by PVY(N) isolates. Site-directed mutagenesis was used to confirm that these two HC-Pro residues are involved in the TVN phenotype.