Stewart M. Gray

Discussion paper: The naming of Potato virus Y strains infecting potato.

SummaryPotato virus Y (PVY) strain groups are based on host response and resistance gene interactions. The strain groups PVYO, PVYC and PVYN are well established for the isolates infecting potato in the field. A switch in the emphasis from host response to nucleotide sequence differences in the virus genomes, detection of isolates recombining sequences of different strains, and the need to recognize isolates that cause necrotic symptoms in potato tubers have led to the assignment of new acronyms, especially to isolates of the PVYN strain group. This discussion paper proposes that any newly found isolates should be described within the context of the original strain groups based on the original methods of distinguishing strains (i.e., tobacco and potato assays involving use of ‘differential’ potato cultivars). Additionally, sequence characterization of the complete genomes of isolates is highly recommended. However, it is acceptable to amend the names of PVY isolates with additional, specific codes to show that the isolate differs at the molecular, serological or phenotypic level from the typical strains within a strain group. The new isolates should preferably not be named using geographical, cultivar, or place-association designations. Since many new variants of PVY are being discovered, any new static classification system will be meaningless for the time being. A more systematic investigation and characterization of PVY from potato at the biological and molecular levels should eventually result in a biologically meaningful genetic strain concept.

Use of group-specific primers and the polymerase chain reaction for the detection and identification of luteoviruses

A general diagnostic assay for a number of distinct luteoviruses was developed using the polymerase chain reaction (PCR) and restriction enzyme analysis. Two minimally degenerate, group-specific primers were derived from previously published RNA sequences of three luteoviruses. This primer pair generated specific PCR fragments of about 530 bp from extracts of plants infected with potato leafroll virus, beet western yellows virus, or New York barley yellow dwarf virus (BYDV) serotypes MAV, PAV, RMV, RPV and SGV, which span much of the respective viral coat protein gene. Each virus was easily distinguished from the others by restriction enzyme analysis of the amplified DNA products. Samples from BYDV-infected oat and wheat collected in Nebraska were identified as containing PAV-like serotypes; micro-heterogeneity was detected in several samples. This method provides a rapid, sensitive and relatively inexpensive means of luteovirus detection and identification. It is the first test capable of simultaneously detecting all five BYDV serotypes.

Potato virus Y: An Evolving Concern for Potato Crops in the United States and Canada

North American potato production differs from other geographical regions such as Europe in that it is essentially a closed system, i.e., seed potatoes are not imported and production is dominated by only a few cultivars. The lack of significant seed imports provides a mechanism for seed certification to be extremely effective at minimizing virus levels in seed lots, especially if the changes in seed laws, postharvest testing, and tolerance limits discussed above are adopted. This is an opportunity to effectively manage PVY at levels that are at or below detection and well below economic significance. Aiding the seed certification programs in the adoption of the Canada/US-Management Plan for Potato Viruses that Cause Tuber Necrosis has and continues to build consensus and cooperation within the industry to reform and modernize seed certification practices and, as importantly, modernize best management practices that growers can implement so that their production meets or exceeds virus tolerances set within the seed certification standards. Seed inspectors could also benefit from continually updated information from the research community to help them better recognize the spectrum of symptoms caused by the various strains and variants of PVY in all the different cultivars now being grown in their states and provinces. They could also benefit from improved field diagnostics that will assist them and the growers in identifying problem plants that should be rogued. If PVY levels in seed can be minimized and on-farm management strategies can be optimized, then PVY incidence in the potato crop will be marginalized. The restricted distribution of the tuber necrotic strains also offers an opportunity to prevent these strains from becoming economically significant if appropriate testing of seed lots in those areas could prevent them from being planted. Shipping point inspections of tubers will also help in identifying and eliminating tuber necrotic viruses. The dominance of a few cultivars has been eroding in recent years. Russet Burbank, a cultivar introduced over 100 years ago, still accounts for 40 to 50% of the U.S. acreage, but acreage in the Northwestern United States has been declining steadily as other russet cultivars come on the market and gain acceptance. Potato cultivar has had a significant impact on the PVY problem, as with the release and widespread acceptance of Shepody, Russet Norkotah, and other asymptomatic carriers of PVY (http://oregonstate. edu/potatoes/latenttoPVYlist.htm), which in 2008 comprised more than 15 and 12% of the total U.S. and Canadian seed acreage, respectively. These cultivars have certainly contributed to the overall increase in PVY in the seed potato crop and by extension the commercial potato crop. The increased diversity of potato cultivars grown in both countries has also introduced a wider spectrum of PVY symptoms, most notably the milder symptoms that are characteristic of the PVYN/NTN and PVYN-Wi strains on many cultivars. Since the success of seed certification is dependent upon visual assessment of the crop, mild or absent symptoms means that many more infected plants go unnoticed. The more symptomatic PVYO strains are observed and removed, but the other strains remain in the crop and are passed along in the seed, contributing to an overall increase in PVY incidence and more importantly to a shift in PVY strain composition. The U.S. and Canadian potato industry stakeholders are increasingly aware of the PVY-associated challenges and have been moving rapidly to work with researchers and all aspects of the industry to implement plans to suppress PVY incidence. Continued education of growers, seed certification officials, and researchers alike, coupled with the development and adoption of new or revised best management practices and diagnostic tools, and the renewed inter est of breeders to develop virus resistant cultivars, will be the keys to success in bringing PVY incidence under control and in minimizing tuber necrotic strains.

Analysis of Genetic Bottlenecks during Horizontal Transmission of Cucumber Mosaic Virus

ABSTRACT Genetic bottlenecks may occur in virus populations when only a few individuals are transferred horizontally from one host to another, or when a viral population moves systemically from the infection site. Genetic bottlenecks during the systemic movement of an RNA plant virus population were reported previously (H. Li and M. J. Roossinck, J. Virol. 78:10582-10587, 2004). In this study we mechanically inoculated an artificial population consisting of 12 restriction enzyme marker mutants of Cucumber mosaic virus (CMV) onto young leaves of squash plants and used two aphid species, Aphis gossypii and Myzus persicae, to transmit the virus populations from infected source plants to healthy squash plants. Horizontal transmission by aphids constituted a significant bottleneck, as the population in the aphid-inoculated plants contained far fewer mutants than the original inoculum source. Additional experiments demonstrated that genetic variation in the artificial population of CMV is not reduced during the acquisition of the virus but is significantly reduced during the inoculation period.

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.

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.

The C Terminus of the Polerovirus P5 Readthrough Domain Limits Virus Infection to the Phloem

ABSTRACT 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.

Coupling Genetics and Proteomics To Identify Aphid Proteins Associated with Vector-Specific Transmission of Polerovirus (Luteoviridae)

ABSTRACT Cereal yellow dwarf virus-RPV (CYDV-RPV) is transmitted specifically by the aphids Rhopalosiphum padi and Schizaphis graminum in a circulative nonpropagative manner. The high level of vector specificity results from the vector aphids having the functional components of the receptor-mediated endocytotic pathways to allow virus to transverse the gut and salivary tissues. Studies of F2 progeny from crosses of vector and nonvector genotypes of S. graminum showed that virus transmission efficiency is a heritable trait regulated by multiple genes acting in an additive fashion and that gut- and salivary gland-associated factors are not genetically linked. Utilizing two-dimensional difference gel electrophoresis to compare the proteomes of vector and nonvector parental and F2 genotypes, four aphid proteins (S4, S8, S29, and S405) were specifically associated with the ability of S. graminum to transmit CYDV-RPV. The four proteins were coimmunoprecipitated with purified RPV, indicating that the aphid proteins are capable of binding to virus. Analysis by mass spectrometry identified S4 as a luciferase and S29 as a cyclophilin, both of which have been implicated in macromolecular transport. Proteins S8 and S405 were not identified from available databases. Study of this unique genetic system coupled with proteomic analysis indicated that these four virus-binding aphid proteins were specifically inherited and conserved in different generations of vector genotypes and suggests that they play a major role in regulating polerovirus transmission.

Serological properties of ordinary and necrotic isolates of Potato virus Y: a case study of PVYN misidentification.

In the course of a multi-year survey of Potato virus Y (PVY) incidence and diversity in the U.S. seed potato crop, an unusual PVY variant was identified in low but significant levels in multiple states. This variant, PVYO-O5, was initially detected by a commercially available PVYN-specific monoclonal antibody, 1F5. This antibody is widely used by U.S. Seed Certification programs to test for PVYN and is one of two antibodies designated by the North American Plant Protection Organization (NAPPO) for pre-shipment testing of tuber lots that are to be transported between countries. Consequently, PVYN positives identified by the 1F5 antibody have triggered quarantine actions, prevented cross-border shipments and impacted trade. Here, we demonstrate by a variety of methods that the PVYO-O5 is a variant within the ordinary PVY strain (PVYO). Specifically, the PVYO-O5 variant likely arose due to a single amino acid substitution within the capsid protein. This variant does not induce vein necrosis in tobacco or tuber necrosis in susceptible varieties of potato. Furthermore, it is identified by RT-PCR based diagnostics as PVYO and it has a typical PVYO genome sequence. We demonstrate that another PVYN specific monoclonal antibody, SASA-N, recognizes an epitope distinct from that recognized by 1F5, and correctly identifies the PVYO-O5 variants as belonging to the PVYO serotype. Since the PVYO-O5 variant is present in many seed producing states and misidentification of PVYO-O5 as PVYN/NTN has clear quarantine implications for export shipments of potato, the limitations of the commercially available monoclonal antibodies should be considered in any certification or phytosanitary testing program.ResumenA lo largo del estudio de varios años sobre la incidencia y diversidad del virus Y de la papa (PVY) en los cultivos de papa para semilla en los Estados Unidos (EU), se identificó a una variante inusual a niveles bajos pero significativos en múltiples estados. Esta variante, PVYO-O5, se detectó inicialmente con un anticuerpo monoclonal comercialmente disponible específico para PVYN, el 1F5. Este anticuerpo es ampliamente usado por los Programas de Certificación de Semilla en los EU para PVYN, y es uno de los dos anticuerpos designados por la Organización Norteamericana de Protección de Plantas (NAPPO) para pruebas de pre-envío de lotes de tubérculos que serán transportados entre países. Consecuentemente, los PVYN positivos identificados con el anticuerpo 1F5 han disparado acciones cuarentenarias, evitando envíos trans-fronteras y han impactado al comercio. Aquí, nosotros demostramos con diversos métodos que PVYO-O5 es una variante del PVY ordinario (PVYO). Específicamente, la variante PVYO-O5 es probable que haya surgido debido a una substitución de un aminoácido dentro de la proteína de la cápside. Esta variante no induce necrosis de las venas en tabaco o necrosis del tubérculo en variedades susceptibles de papa. Aún mas, se le identifica como PVYO mediante RT-PCR y tiene la típica secuencia genómica del PVYO. Demostramos que otro anticuerpo monoclonal específico para PVYN, el SASA-N, reconoce un epítope distinto al reconocido por 1F5, e identifica correctamente a las variantes PVYO-O5 como pertenecientes al serotipo PVYO. Tomando en cuenta que la variante PVYO-O5 esta presente en muchos estados que producen semilla, y que la identificación equivocada de PVYO-O5 como PVYN/NTN tiene claras implicaciones cuarentenarias para envíos de exportación de papa, se deberían de considerar las limitaciones de los anticuerpos monoclonales disponibles comercialmente en cualquier programa de pruebas para certificación o fitosanidad.

Plant virus proteins involved in natural vector transmission

Abstract Plant viruses transmitted by invertebrate vectors either reversibly bind to vector mouthparts or are internalized by the vector and later secreted. Viral proteins mediate the binding of plant viruses to vector mouthparts and the transport of virus across vector-cell membranes. Both mechanisms probably involve conformational changes of virus proteins during their association with the vector.