Infection Dynamics of Potato Virus Y Isolate Combinations in Three Potato Cultivars.

The United States potato industry has recently experienced a strain shift; recombinant potato virus Y (PVY) strains (e.g., PVYNTN) have emerged as the predominant strains over the long dominant ordinary strain (PVYO), yet both are often found as single infections within the same field and as mixed infections within individual plants. To understand mixed infection dynamics in potato plants and in daughter tubers, three potato varieties varying for PVY resistance, 'Red Maria', 'CalWhite', and 'Pike', were mechanically inoculated either at the pre- or postflowering stage with all possible heterologous isolate combinations of two PVYO and two PVYNTN isolates. Virus titer was determined from leaves collected at different positions on the plant at different times, and tuber-borne infection was determined for two successive generations. PVYNTN accumulated to higher levels than PVYO at nearly all sampling time points in 'Pike' potato. However, both virus strains accumulated to similar amounts in 'Red Maria' and 'CalWhite' potato early in the infection when inoculated preflowering; however, PVYNTN dominated at later stages and in plants inoculated postflowering. Regardless of inoculation time, both virus strains were transmitted to daughter plants raised from the tubers for most isolate combinations. The relative titer of PVYNTN and PVYO isolates at the later stages of mother plant development was indicative of what was found in the daughter plants. Although virus titer differed among cultivars depending on their genetics and virus isolates, it did not change the strain outcome in tuber-borne infection in subsequent generations. Differential virus accumulation in these cultivars suggests isolate-specific resistance to PVY accumulation.

Virus and helper component interactions favour the transmission of recombinant potato virus Y strains.

In recent years, several recombinant strains of potato virus Y, notably PVYNTN and PVYN:O have displaced the ordinary strain, PVYO, and emerged as the predominant strains affecting the USA potato crop. Previously we reported that recombinant strains were transmitted more efficiently than PVYO when they were acquired sequentially, regardless of acquisition order. In another recent study, we showed that PVYNTN binds preferentially to the aphid stylet over PVYO when aphids feed on a mixture of PVYO and PVYNTN. To understand the mechanism of this transmission bias as well as preferential virus binding, we separated virus and active helper component proteins (HC), mixed them in homologous and heterologous combinations, and then fed them to aphids using Parafilm sachets. Mixtures of PVYO HC with either PVYN:O or PVYNTN resulted in efficient transmission. PVYN:O HC also facilitated the transmission of PVYO and PVYNTN, albeit with reduced efficiency. PVYNTN HC failed to facilitate transmission of either PVYO or PVYN:O. When PVYO HC or PVYN:O HC was mixed with equal amounts of the two viruses, both viruses in all combinations were transmitted at high efficiencies. In contrast, no transmission occurred when combinations of viruses were mixed with PVYNTN HC. Further study evaluated transmission using serial dilutions of purified virus mixed with HCs. While PVYNTN HC only facilitated the transmission of the homologous virus, the HCs of PVYO and PVYN:O facilitated the transmission of all strains tested. This phenomenon has likely contributed to the increase in the recombinant strains affecting the USA potato crop.

Affinity Purification-Mass Spectrometry Identifies a Novel Interaction between a Polerovirus and a Conserved Innate Immunity Aphid Protein that Regulates Transmission Efficiency.

The vast majority of plant viruses are transmitted by insect vectors, with many crucial aspects of the transmission process being mediated by key protein-protein interactions. Still, very few vector proteins interacting with viruses have been identified and functionally characterized. Potato leafroll virus (PLRV) is transmitted most efficiently by Myzus persicae, the green peach aphid, in a circulative, non-propagative manner. Using affinity purification coupled to high-resolution mass spectrometry (AP-MS), we identified 11 proteins from M. persicaedisplaying a high probability of interaction with PLRV and an additional 23 vector proteins with medium confidence interaction scores. Three of these aphid proteins were confirmed to directly interact with the structural proteins of PLRV and other luteovirid species via yeast two-hybrid. Immunolocalization of one of these direct PLRV-interacting proteins, an orthologue of the human innate immunity protein complement component 1 Q subcomponent-binding protein (C1QBP), shows that MpC1QBP partially co-localizes with PLRV in cytoplasmic puncta and along the periphery of aphid gut epithelial cells. Artificial diet delivery to aphids of a chemical inhibitor of C1QBP leads to increased PLRV acquisition by aphids and subsequently increased titer in inoculated plants, supporting a role for C1QBP in the acquisition and transmission efficiency of PLRV by M. persicae. This study presents the first use of AP-MS for the in vivo isolation of a functionally relevant insect vector-virus protein complex. MS data are available from ProteomeXchange.org using the project identifier PXD022167.

Different potato virus Y strains frequently co-localize in single epidermal leaf cells and in the aphid stylet.

Single aphids can simultaneously or sequentially acquire and transmit multiple potato virus Y (PVY) strains. Multiple PVY strains are often found in the same field and occasionally within the same plant, but little is known about how PVY strains interact in plants or in aphid stylets. Immuno-staining and confocal microscopy were used to examine the spatial and temporal dynamics of PVY strain mixtures (PVYO and PVYNTN or PVYO and PVYN) in epidermal leaf cells of 'Samsun NN' tobacco and 'Goldrush' potato. Virus binding and localization was also examined in aphid stylets following acquisition. Both strains systemically infected tobacco and co-localized in cells of all leaves examined; however, the relative amounts of each virus changed over time. Early in the tobacco infection, when mosaic symptoms were observed, PVYO dominated the infection although PVYNTN was detected in some cells. As the infection progressed and vein necrosis developed, PVYNTN was prevalent. Co-localization of PVYO and PVYN was also observed in epidermal cells of potato leaves with most cells infected with both viruses. Furthermore, two strains could be detected binding to the distal end of aphid stylets following virus acquisition from a plant infected with a strain mixture. These data are in contrast with the traditional belief of spatial separation of two closely related potyviruses and suggest apparent non-antagonistic interaction between PVY strains that could help explain the multitude of emerging recombinant PVY strains discovered in potato in recent years.

The Recombinant Potato virus Y (PVY) Strain, PVYNTN, Identified in Potato Fields in Victoria, Southeastern Australia.

Potato virus Y (PVY) is one of the main viruses affecting potato in Australia. However, molecular characterization of PVY isolates circulating in potato in different states of Australia has not yet been thoroughly conducted. Only nonrecombinant isolates of three biological PVY strains collected from potato were reported previously from Western Australia and one from Queensland. Here, PVY isolates collected from seed potato originating in Victoria, Australia, and printed on FTA cards, were subjected to strain typing by RT-PCR, with three isolates subjected to whole genome sequencing. All the 59 PVY isolates detected during two growing seasons were identified to be recombinants based on two RT-PCR assays. No nonrecombinant PVY isolates were identified. All the RT-PCR typed isolates belonged to the PVYNTN strain. Sequence analysis of the whole genomes of three isolates suggested a single introduction of the PVYNTN strain to Australia but provided no clues as to where this introduction originated. Given the association of the PVYNTN strain with potato tuber damage, growers in Australia should implement appropriate strategies to manage PVYNTN in potato.

Evaluation of Effects of Chemical Soil Treatments and Potato Cultivars on Spongospora subterranea Soil Inoculum and Incidence of Powdery Scab and Potato Mop-Top Virus in Potato.

Spongospora subterranea is a soilborne plasmodiophorid that causes powdery scab in potato. It also transmits potato mop-top virus (PMTV), which causes necrotic arcs (spraing) in potato tubers. Three field experiments were conducted in naturally S. subterranea-infested soil to investigate the effects of two chemicals, Omega 500F (fluazinam) and FOLI-R-PLUS RIDEZ (biological extract), on powdery scab, PMTV, and changes in S. subterranea inoculum with six different potato cultivars. The efficacy of soil treatment with these two chemicals on tuber lesions, root galling, and pathogen population was also assessed in greenhouse trials. The chemical treatments did not reduce powdery scab, root gall formation, or S. subterranea inoculum in the field or greenhouse trials. Postharvest S. subterranea soil inoculum in fields varied across farms and among potato cultivars but the pathogen population consistently increased by the end of the growing season. The evaluated russet cultivars were more tolerant to powdery scab than the yellow- or red-skinned cultivars but all were susceptible to PMTV. In the field, powdery scab indices and soil inoculum changes were positively correlated, while postharvest S. subterranea inoculum was positively correlated with root galling in both greenhouse trials. Powdery scab and PMTV occurred in noninoculated potting mix, indicating that peat-based potting mix is a source for both pathogens. These results demonstrate that chemical management methods currently used by farmers are ineffective, that S. subterranea and PMTV in potting mix can cause severe epidemics in greenhouses, and that potato cultivar choices impact inoculum increases in soil.

Anthropogenic influences on emergence of vector-borne plant viruses: the persistent problem of Potato virus Y.

Potato virus Y (PVY) has reemerged as a serious impediment to seed potato production, responsible for reduced yields and tuber quality, as well as the majority of seed lot rejections by certification programs due to excessive virus incidence. This has led to seed shortages, especially in cultivars highly susceptible to infection. While seed certification programs have been effective at managing many virus diseases below economic thresholds, PVY has rapidly evolved in recent decades to become a complex of strains that evade many certification and farm management practices. The evolution of PVY strains is naturally occurring, but several human influences can be linked to the rapid change in PVY populations affecting the potato crop. Here we highlight the recent history and current status of PVY in potatoes and suggest some approaches for managing the virus moving forward.

A Stem-Loop Structure in Potato Leafroll Virus Open Reading Frame 5 (ORF5) Is Essential for Readthrough Translation of the Coat Protein ORF Stop Codon 700 Bases Upstream.

Translational readthrough of the stop codon of the capsid protein (CP) open reading frame (ORF) is used by members of the Luteoviridae to produce their minor capsid protein as a readthrough protein (RTP). The elements regulating RTP expression are not well understood, but they involve long-distance interactions between RNA domains. Using high-resolution mass spectrometry, glutamine and tyrosine were identified as the primary amino acids inserted at the stop codon of Potato leafroll virus (PLRV) CP ORF. We characterized the contributions of a cytidine-rich domain immediately downstream and a branched stem-loop structure 600 to 700 nucleotides downstream of the CP stop codon. Mutations predicted to disrupt and restore the base of the distal stem-loop structure prevented and restored stop codon readthrough. Motifs in the downstream readthrough element (DRTE) are predicted to base pair to a site within 27 nucleotides (nt) of the CP ORF stop codon. Consistent with a requirement for this base pairing, the DRTE of Cereal yellow dwarf virus was not compatible with the stop codon-proximal element of PLRV in facilitating readthrough. Moreover, deletion of the complementary tract of bases from the stop codon-proximal region or the DRTE of PLRV prevented readthrough. In contrast, the distance and sequence composition between the two domains was flexible. Mutants deficient in RTP translation moved long distances in plants, but fewer infection foci developed in systemically infected leaves. Selective 2'-hydroxyl acylation and primer extension (SHAPE) probing to determine the secondary structure of the mutant DRTEs revealed that the functional mutants were more likely to have bases accessible for long-distance base pairing than the nonfunctional mutants. This study reveals a heretofore unknown combination of RNA structure and sequence that reduces stop codon efficiency, allowing translation of a key viral protein.IMPORTANCE Programmed stop codon readthrough is used by many animal and plant viruses to produce key viral proteins. Moreover, such "leaky" stop codons are used in host mRNAs or can arise from mutations that cause genetic disease. Thus, it is important to understand the mechanism(s) of stop codon readthrough. Here, we shed light on the mechanism of readthrough of the stop codon of the coat protein ORFs of viruses in the Luteoviridae by identifying the amino acids inserted at the stop codon and RNA structures that facilitate this "leakiness" of the stop codon. Members of the Luteoviridae encode a C-terminal extension to the capsid protein known as the readthrough protein (RTP). We characterized two RNA domains in Potato leafroll virus (PLRV), located 600 to 700 nucleotides apart, that are essential for efficient RTP translation. We further determined that the PLRV readthrough process involves both local structures and long-range RNA-RNA interactions. Genetic manipulation of the RNA structure altered the ability of PLRV to translate RTP and systemically infect the plant. This demonstrates that plant virus RNA contains multiple layers of information beyond the primary sequence and extends our understanding of stop codon readthrough. Strategic targets that can be exploited to disrupt the virus life cycle and reduce its ability to move within and between plant hosts were revealed.

Sequential acquisition of Potato virus Y strains by Myzus persicae favors the transmission of the emerging recombinant strains.

In the past decade recombinant strains of Potato virus Y (PVY) have overtaken the ordinary strain, PVYO, as the predominant viruses affecting the US seed potato crop. Aphids may be a contributing factor in the emergence of the recombinant strains, but studies indicate that differences in transmission efficiency of individual PVY strains either from single or mixed infections, although variable, are not generally significant. Multiple strains of PVY are present in all potato production areas and common in many potato fields. Therefore, it is likely that individual alate aphids moving through a potato field will sequentially encounter multiple strains as they "taste test" multiple potato plants while looking for a suitable host. This study examined the transmission likelihood and efficiency of three common PVY strains when acquired sequentially by individual aphids. Green peach aphids (Myzus persicae, Sulzer) were allowed a 2-3min acquisition access period (AAP) on potato leaves infected with PVYO, PVYN:O or PVYNTN, followed by another 2-3min AAP on a second potato leaf infected with a different PVY strain before being transferred to healthy potato seedlings for a 24h inoculation access period. All possible combinations of the three strains were tested. Strain-specific infection of the recipient plants was determined by TAS-ELISA and RT-PCR 3-4wk post-inoculation. The recombinant strains, PVYN:O and PVYNTN, were transmitted more efficiently than PVYO when they were sequentially acquired regardless of the order acquired. PVYN:O and PVYNTN were transmitted with similar efficiencies when they were sequentially acquired regardless of the order. The recombinant strains appear to preferentially bind to the aphid stylet over PVYO or they may be preferentially released during inoculation. This may contribute to the increased incidence of the recombinant strains over PVYO in fields or production regions where multiple PVY strains are detected.

Phylogenetic study of recombinant strains of Potato virus Y.

Potato virus Y (PVY) exists as a complex of strains, including a growing number of recombinants. Evolution of PVY proceeds through accumulation of mutations and more rapidly through recombination. Here, the role of recombination in PVY evolution and the origin of common PVY recombinants were studied through whole genome analysis of 119 newly sequenced PVY isolates largely from U.S. potato, and subsequent combined phylogenetic and recombination analyses with an additional 166 whole PVY genomes from the GenBank database. Two novel PVYC recombinants were sequenced and identified, along with one novel PVYN:O recombinant. Sequence diversity in the parental sequences made it possible to trace the origins of all recombinant types of PVY, which also showed remarkable sequence diversity in most cases. The results suggested that the common recombinant PVY strains originated more than once, from different parental sequences.

Potato virus Y Transmission Efficiency from Potato Infected with Single or Multiple Virus Strains.

There has been a recent shift in the prevalence of Potato virus Y (PVY) strains affecting potato with the ordinary strain PVYO declining and the recombinant strains PVYNTN and PVYN:O emerging in the United States. Multiple PVY strains are commonly found in potato fields and even in individual plants. Factors contributing to the emergence of the recombinant strains are not well defined but differential aphid transmission of strains from single and mixed infections may play a role. We found that the transmission efficiencies by Myzus persicae, the green peach aphid, of PVYNTN, PVYN:O, and PVYO varied depending on the potato cultivar serving as the virus source. Overall transmission efficiency was highest from sources infected with three virus strains, whereas transmission from sources infected with one or two virus strains was not significantly different. Two strains were concomitantly transmitted by individual aphids from many of the mixed-source combinations, especially if PVYO was present. Triple-strain infections were not transmitted by any single aphid. PVYO was transmitted most efficiently from mixed-strain infection sources. The data do not support the hypothesis that differential transmission of PVY strains by M. persicae is a major contributing factor in the emergence of recombinant PVY strains in the U.S. potato crop.

Model System-Guided Protein Interaction Mapping for Virus Isolated from Phloem Tissue.

Phloem localization of plant viruses is advantageous for acquisition by sap-sucking vectors but hampers host-virus protein interaction studies. In this study, Potato leafroll virus (PLRV)-host protein complexes were isolated from systemically infected potato, a natural host of the virus. Comparing two different co-immunoprecipitation (co-IP) support matrices coupled to mass spectrometry (MS), we identified 44 potato proteins and one viral protein (P1) specifically associated with virus isolated from infected phloem. An additional 142 proteins interact in complex with virus at varying degrees of confidence. Greater than 80% of these proteins were previously found to form high confidence interactions with PLRV isolated from the model host Nicotiana benthamiana. Bioinformatics revealed that these proteins are enriched for functions related to plasmodesmata, organelle membrane transport, translation, and mRNA processing. Our results show that model system proteomics experiments are extremely valuable for understanding protein interactions regulating infection in recalcitrant pathogens such as phloem-limited viruses.

Potato leafroll virus structural proteins manipulate overlapping, yet distinct protein interaction networks during infection.

Potato leafroll virus (PLRV) produces a readthrough protein (RTP) via translational readthrough of the coat protein amber stop codon. The RTP functions as a structural component of the virion and as a nonincorporated protein in concert with numerous insect and plant proteins to regulate virus movement/transmission and tissue tropism. Affinity purification coupled to quantitative MS was used to generate protein interaction networks for a PLRV mutant that is unable to produce the read through domain (RTD) and compared to the known wild-type PLRV protein interaction network. By quantifying differences in the protein interaction networks, we identified four distinct classes of PLRV-plant interactions: those plant and nonstructural viral proteins interacting with assembled coat protein (category I); plant proteins in complex with both coat protein and RTD (category II); plant proteins in complex with the RTD (category III); and plant proteins that had higher affinity for virions lacking the RTD (category IV). Proteins identified as interacting with the RTD are potential candidates for regulating viral processes that are mediated by the RTP such as phloem retention and systemic movement and can potentially be useful targets for the development of strategies to prevent infection and/or viral transmission of Luteoviridae species that infect important crop species.

Continuous and emerging challenges of Potato virus Y in potato.

Potato virus Y (PVY) is one of the oldest known plant viruses, and yet in the past 20 years it emerged in the United States as a relatively new and very serious problem in potato. The virus exists as a complex of strains that induce a wide variety of foliar and tuber symptoms in potato, leading to yield reduction and loss of tuber quality. PVY has displayed a distinct ability to evolve through accumulation of mutations and more rapidly through recombination between different strains, adapting to new potato cultivars across different environments. Factors behind PVY emergence as a serious potato threat are not clear at the moment, and here an attempt is made to analyze various properties of the virus and its interactions with potato resistance genes and with aphid vectors to explain this recent PVY spread in potato production areas. Recent advances in PVY resistance identification and mapping of corresponding genes are described. An updated classification is proposed for PVY strains that takes into account the most current information on virus molecular genetics, serology, and host reactivity.

Cross-linking measurements of the Potato leafroll virus reveal protein interaction topologies required for virion stability, aphid transmission, and virus-plant interactions.

Protein interactions are critical determinants of insect transmission for viruses in the family Luteoviridae. Two luteovirid structural proteins, the capsid protein (CP) and the readthrough protein (RTP), contain multiple functional domains that regulate virus transmission. There is no structural information available for these economically important viruses. We used Protein Interaction Reporter (PIR) technology, a strategy that uses chemical cross-linking and high resolution mass spectrometry, to discover topological features of the Potato leafroll virus (PLRV) CP and RTP that are required for the diverse biological functions of PLRV virions. Four cross-linked sites were repeatedly detected, one linking CP monomers, two within the RTP, and one linking the RTP and CP. Virus mutants with triple amino acid deletions immediately adjacent to or encompassing the cross-linked sites were defective in virion stability, RTP incorporation into the capsid, and aphid transmission. Plants infected with a new, infectious PLRV mutant lacking 26 amino acids encompassing a cross-linked site in the RTP exhibited a delay in the appearance of systemic infection symptoms. PIR technology provided the first structural insights into luteoviruses which are crucially lacking and are involved in vector-virus and plant-virus interactions. These are the first cross-linking measurements on any infectious, insect-transmitted virus.

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.

Engineering virus resistance using a modified potato gene.

Natural mutations in translation initiation factor eIF4E confer resistance to potyviruses in many plant species. Potato is a staple food crop plagued by several potyviruses, yet to date no known eIF4E-mediated resistance genes have been identified. In this study, we demonstrate that transgenic expression of the pvr1(2) gene from pepper confers resistance to Potato virus Y (PVY) in potato. We then use this information to convert the susceptible potato ortholog of this allele into a de novo allele for resistance to PVY using site-directed mutagenesis. Potato plants overexpressing the mutated potato allele are resistant to virus infection. Resistant lines expressed high levels of eIF4E mRNA and protein. The resistant plants showed growth similar to untransformed controls and produced phenotypically similar tubers. This technique disrupts a key step in the viral infection process and may potentially be used to engineer virus resistance in a number of economically important plant-viral pathosystems. Furthermore, the general public may be more amenable to the 'intragenic' nature of this approach because the transferred coding region is modified from a gene in the target crop rather than from a distant species.

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 novel recombinant strain of Potato virus Y suggests a new viral genetic determinant of vein necrosis in tobacco.

A novel Potato virus Y (PVY) isolate, L26, recovered from a Frontier potato line was initially typed as a PVY(NTN) strain using multiplex RT-PCR and serological assays. However, L26 induced mosaic and mild vein clearing symptoms in tobacco rather than vein necrosis characteristic of the PVY (NTN) strain. The whole genome sequence was determined for L26 and two other PVY(NTN) isolates, HR1 and N4, from Idaho that did induce vein necrosis in tobacco. The sequence of all three isolates was similar to typical European PVY(NTN) isolates that contain three recombination junctions in their genome. The sequence of the L26 genome was nearly identical to the genomes HR1, N4, and to a previously characterized PVY(NTN) isolate, 423-3, differing by only five nucleotides in the entire ca. 9.7-kb genome, only one resulting in a corresponding amino acid change, D-205 to G-205 in the central region of HC-Pro. Two "signature" amino acid residues, thought involved in induction of the vein necrosis syndrome in tobacco, K-400 and E-419, were present in the C-terminal region of HC-Pro of all three isolates. Multiple alignment of the whole genome sequences of L26 and other PVY(NTN) isolates whose phenotype in tobacco has been reported, suggests that a single nucleotide change (A-1,627 to G-1,627) resulting in the single amino acid change (D-205 to G-205) in the HC-Pro cistron of L26 correlates with the loss of the vein necrosis phenotype in tobacco. Secondary structure modeling of the HC-Pro protein predicts the G-205 residue, and the previously identified residues K-400 and E-419, would all be located on the exposed surface of the protein. Taken together, these data suggest that the vein necrosis genetic determinant of PVY in tobacco is complex and includes other element(s), in addition to the C-terminal fragment of HC-Pro.

The C terminus of the polerovirus p5 readthrough domain limits virus infection to the phloem.

Poleroviruses are restricted to vascular phloem tissues from which they are transmitted by their aphid vectors and are not transmissible mechanically. Phloem limitation has been attributed to the absence of virus proteins either facilitating movement or counteracting plant defense. The polerovirus capsid is composed of two forms of coat protein, the major P3 protein and the minor P3/P5 protein, a translational readthrough of P3. P3/P5 is required for insect transmission and acts in trans to facilitate long-distance virus movement in phloem tissue. Specific potato leafroll virus mutants lacking part or all of the P5 domain moved into and infected nonvascular mesophyll tissue when the source-sink relationship of the plant (Solanum sarrachoides) was altered by pruning, with the progeny virus now being transmissible mechanically. However, in a period of months, a phloem-specific distribution of the virus was reestablished in the absence of aphid transmission. Virus from the new phloem-limited infection showed compensatory mutations that would be expected to restore the production of full-length P3/P5 as well as the loss of mechanical transmissibility. The data support our hypothesis that phloem limitation in poleroviruses presumably does not result from a deficiency in the repertoire of virus genes but rather results from P3/P5 accumulation under selection in the infected plant, with the colateral effect of facilitating transmission by phloem-feeding aphid vectors.