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Soybean Dwarf Virus (SbDV) is an important plant pathogen, causing economic losses in soybean. In North America, indigenous strains of SbDV mainly infect clover, with occasional outbreaks in soybean. To evaluate the risk of a US clover strain of SbDV adapting to other plant hosts, the clover isolate SbDV-MD6 was serially transmitted to pea and soybean by aphid vectors. Sequence analysis of SbDV-MD6 from pea and soybean passages identified 11 non-synonymous mutations in soybean, and six mutations in pea. Increasing virus titers with each sequential transmission indicated that SbDV-MD6 was able to adapt to the plant host. However, aphid transmission efficiency on soybean decreased until the virus was no longer transmissible. Our results clearly demonstrated that the clover strain of SbDV-MD6 is able to adapt to soybean crops. However, mutations that improve replication and/or movement may have trade-off effects resulting in decreased vector transmission., Competing Interests: The authors declare no conflict of interest. The funding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

Soybean dwarf virus (SbDV) exists as several distinct strains based on symptomatology, vector specificity, and host range. Originally characterized Japanese isolates of SbDV were specifically transmitted by Aulacorthum solani. More recently, additional Japanese isolates and endemic U.S. isolates have been shown to be transmitted by several different aphid species. The soybean aphid, Aphis glycines, the only aphid that colonizes soybean, has been shown to be a very inefficient vector of some SbDV isolates from Japan and the United States. Transmission experiments have shown that the soybean aphid can transmit certain isolates of SbDV from soybean to soybean and clover species and from clover to clover and soybean with long acquisition and inoculation access periods. Although transmission of SbDV by the soybean aphid is very inefficient, the large soybean aphid populations that develop on soybean may have epidemiological potential to produce serious SbDV-induced yield losses.

Within two decades of its discovery, Zucchini yellow mosaic virus (ZYMV) achieved a global distribution. However, whether or not seed transmission occurs in this economically significant crop pathogen is controversial, and the relative impact of seed transmission on the epidemiology of ZYMV remains unclear. Using reverse transcription-polymerase chain reaction, we observed a seed transmission rate of 1.6% in Cucurbita pepo subsp. texana and show that seed-infected C. pepo plants are capable of initiating horizontal ZYMV infections, both mechanically and via an aphid vector (Myzus persicae). We also provide evidence that ZYMV-infected seeds may act as effective viral reservoirs, partially accounting for the current geographic distribution of ZYMV. Finally, the observation that ZYMV infection of C. pepo seeds results in virtually symptomless infection, coupled with our finding that an antibody test failed to detect vertically transmitted ZYMV in infected seed, highlights the urgent need to standardize current detection methods for seed infection.

Plum pox virus (PPV) was identified in Pennsylvania in 1999. The outbreak was limited to a four-county region in southern Pennsylvania. Initial serological and molecular characterization indicated that the isolates in Pennsylvania belong to the D strain of PPV. The Pennsylvania isolates were characterized by sequence analysis, electron microscopy, host range, and vector transmission to determine how these isolates related to their previously studied European counterparts. Genetically, Pennsylvania (PPV-Penn) isolates were more closely related to each other than to any other PPV-D strains, and isolates from the United States, Canada, and Chile were more closely related to each other than to European isolates. The PPV-Penn isolates exist as two clades, suggesting the possibility of multiple introductions. Electron microscopy analysis of PPV-Penn isolates, including cytopathological studies, indicated that the virions were similar to other Potyvirus spp. PPV-Penn isolates had a herbaceous host range similar to that of European D isolates. There were distinct differences in the transmission efficiencies of the two PPV-Penn isolates using Myzus persicae and Aphis spiraecola as vectors; however, both PPV-Penn isolates were transmitted by M. persicae more efficiently than a European D isolate but less efficiently than a European M isolate.

Cucumber mosaic virus (CMV) has become a major limiting factor in snap bean production in the Great Lakes region of North America, and epidemics have occurred more frequently since the soybean aphid, Aphis glycines Matsumura, was introduced. Major aphid vectors of CMV epidemics were identified by statistically relating their temporal dispersal trends to the incidence of CMV. Alates were monitored weekly using water pan traps in 74 snap bean fields in New York and Pennsylvania from 2002 to 2006. Plants were tested for CMV by ELISA one time during late bloom in 2002 and 2003 and weekly over the season from 2004 to 2006. Principal vectors of CMV included Acyrthosiphon pisum (Harris), A. glycines, Aphis gossypii Glover, and Therioaphis trifolii (Monell). Among these, A. glycines and T. trifolii were likely responsible for severe CMV epidemics because they were among the most abundant species captured, they efficiently transmit CMV, and their dispersal activity was positively correlated with periods when CMV incidence was highest. Moreover, because high numbers of A. glycines and T. trifolii disperse during July and August, snap bean fields planted beyond late June are at risk for infection during early vegetative stages and are subsequently more at risk for yield loss. In contrast, plantings up to late June are less likely to become infected during early developmental stages and should escape yield loss because major vectors are dispersing infrequently. CMV-resistant or tolerant snap bean varieties should be planted after late June to reduce the risk of yield loss.

Cucumber mosaic virus (CMV) is a major component of the virus complex that has become more pronounced in snap bean in the midwestern and northeastern United States since 2001. Multiple-vector-transfer tests were done to estimate the CMV transmission efficiencies (p) of the main aphid species identified in commercial snap bean fields in New York and Pennsylvania. The four most efficient vectors (p > 0.05) were Aphis gossypii, A. glycines, Acyrthosiphon pisum, and Therioaphis trifolii, which were all significant species in the migratory aphid populations in snap bean. Moderately efficient vectors (0.01 < p < 0.04) were A. spiraecola, A. craccivora, Macrosiphum euphorbiae, and Rhopalosiphum maidis. Poor vectors (p < 0.01) included A. fabae, Nearctaphis bakeri, and Myzus persicae. Only one species, Sitobion avenae, failed to transmit CMV in replicated tests. Estimates of p were consistent between different clones of the same aphid species and among three different field isolates of CMV tested. Single-vector-transfer test results for a subset of the species supported those obtained via the multiple-vector-transfer approach. Our results are consistent with the notion that A. glycines is a major vector of recent CMV epidemics in snap bean, but that species is only one of several that are involved.

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 F(2) 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 F(2) 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.

Plum pox virus (PPV) populations from peaches are able to adapt consistently to herbaceous hosts, characterized by a reduction in time to symptom development, increases in inoculation efficiency and increased titres. PPV adaptation was studied by using pea (Pisum sativum) as an alternative host. Two isolates of PPV from peaches were inoculated and passaged in peas ten times using either aphid or mechanical inoculation, generating four independent passage lines. Mechanical-transmission efficiency from peach to pea improved from 3 % at passage 1 to 100 % by serial passage 4 on peas. Inoculation using aphid vectors required six to ten serial passages in pea to reach a peak of 50-60 % transmission efficiency. Sequence analyses of all four PPV population lines inoculated sequentially to pea identified a specific mutation occurring consistently in the NIb gene when compared with the same PPV isolates passaged in parallel in peach. The mutation allowed PPV to replicate up to 20 times faster in the new host. Pea-adapted strains of PPV at every passage were also tested for their ability to infect the original host, peach. Regardless of the number of previous passages, all pea-adapted PPV strains consistently infected peach at low levels using aphid inoculation.

Grapevines infected with Tomato ring spot virus (ToRSV) pose an economic risk for growers in the northeastern United States. This study describes a one-step real-time reverse-transcription polymerase chain reaction (RT-PCR) SYBR Green assay for detecting ToRSV in grapevines. Two newly designed primer pairs based on the ToRSV coat protein gene sequence were evaluated for specificity and optimized for a SYBR Green assay. The primer pair ToRSV1f/1r yielded a 130-bp product with strong primer-dimer products, whereas the primer pair ToRSV2f/2r yielded a 330-bp product with weak primer dimer products. Real-time RT-PCR detected ToRSV in more naturally infected grapevines maintained in the greenhouse than did enzyme-linked immunosorbent assay. The nucleotide sequences of the fragments amplified from grapevine growing in Pennsylvania using real-time PCR were divergent from previously published sequences.

Plum pox (Sharka) is a serious virus disease of stone fruits caused by the Plum pox virus (PPV). To determine which species could function as potential hosts and virus reservoirs, we used aphid transmission and bud or chip grafting to evaluate the susceptibility of commercial, ornamental, and wild Prunus species to isolates of PPV found in Pennsylvania, USA. Following inoculation, test trees were observed for symptoms, analyzed by enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR), back-assayed to healthy peach, and followed through at least four cold-induced dormancy (CID) cycles over 4 years. Thirty-one of 33 Prunus species and cultivars were systemically infected following aphid transmission. Systemic infection could not be detected in P. cerasus (sour cherry) and P. × 'Snofozam' (Snow Fountains) despite repeated aphid inoculation attempts. Following grafting of PPV-infected budwood, all 40 species and varieties became infected, although species differed in their susceptibility. Within most species, some individual plants remained PPV negative throughout the study despite repeated inoculations. Infection in some species could be detected only through quantitative reverse transcription (RT)-PCR. Most species displayed clear symptoms, were highly positive by ELISA and RT-PCR, and could be back-inoculated into peach seedlings following CID. Our results indicate that a wide range of native and ornamental Prunus species are susceptible to U.S. isolates of PPV-D.

ABSTRACT Sexual forms of two genotypes of the aphid Schizaphis graminum, one a vector, the other a nonvector of two viruses that cause barley yellow dwarf disease (Barley yellow dwarf virus [BYDV]-SGV, luteovirus and Cereal yellow dwarf virus-RPV, polerovirus), were mated to generate F1 and F2 populations. Segregation of the transmission phenotype for both viruses in the F1 and F2 populations indicated that the transmission phenotype is under genetic control and that the parents are heterozygous for genes involved in transmission. The ability to transmit both viruses was correlated within the F1 and F2 populations, suggesting that a major gene or linked genes regulate the transmission. However, individual hybrid genotypes differed significantly in their ability to transmit each virus, indicating that in addition to a major gene, minor genes can affect the transmission of each virus independently. Gut and salivary gland associated transmission barriers were identified in the nonvector parent and some progeny, while other progeny possessed only a gut barrier or a salivary gland barrier. Hemolymph factors do not appear to be involved in determining the transmission phenotype. These results provide direct evidence that aphid transmission of luteoviruses is genetically regulated in the insect and that the tissue-specific barriers to virus transmission are not genetically linked.

Plum pox, an invasive disease recently identified in Pennsylvania stone fruit orchards, is caused by the aphid-transmitted Plum pox virus (genus Potyvirus, family Potyviridae, PPV). To identify potential vectors, we described the aphid species communities and the seasonal dynamics of the dominant aphid species within Pennsylvania peach orchards. Aphids were trapped weekly in 2002 and 2003 from mid-April through mid-November within two central Pennsylvania orchards by using yellow and green water pan traps. In total, 42 aphid species were identified from both orchards over 2 yr. Within orchards, actual species richness ranged from 24 to 30 species. The Abundance Based Coverage Estimator predicted species richness to range from 30 to 36 species, indicating that trap catches were identifying most aphid species expected to occur in the orchard. Three species, Rhopalosiphum maidis (Fitch), Aphis spiraecola Patch, and Myzus persicae (Sulzer), were consistently dominant across locations and years. Orchard-trapped populations of these three species peaked in a similar chronological sequence each year. As expected, trap color influenced the total number and distribution of the predominate species collected. However, the same dominant species occurred in both yellow and green traps. Based on the seasonal population dynamics reported here and on published vector efficacy studies, the most probable significant PPV vector was identified as A. spiraecola. If the PPV pathogen escapes current quarantine or if subsequent reintroductions of PPV occur, these data will be useful for developing plum pox management strategies.

A Wheat streak mosaic virus (WSMV) genome lacking HC-Pro was constructed and confirmed by reverse transcription-PCR to systemically infect wheat, oat, and corn. Coupled in vitro transcription/translation reactions indicated that WSMV P1 proteinase cleaved the polyprotein at the P1/P3 junction of the HC-Pro null mutant. The WSMV HC-Pro null mutant was competent for virion formation, but the virus titer was reduced 4.5-fold relative to that of the wild type. Collectively, these results indicate that WSMV HC-Pro is dispensable for replication and movement, two essential processes that are disrupted by point and small-insertion mutations introduced into potyvirus HC-Pro.

The eriophyid mite transmitted Wheat streak mosaic virus (WSMV; genus Tritimovirus, family Potyviridae) shares a common genome organization with aphid transmitted species of the genus Potyvirus. Although both tritimoviruses and potyviruses encode helper component-proteinase (HC-Pro) homologues (required for nonpersistent aphid transmission of potyviruses), sequence conservation is low (amino acid identity, approximately 16%), and a role for HC-Pro in semipersistent transmission of WSMV by the wheat curl mite (Aceria tosichella [Keifer]) has not been investigated. Wheat curl mite transmissibility was abolished by replacement of WSMV HC-Pro with homologues of an aphid transmitted potyvirus (Turnip mosaic virus), a rymovirus (Agropyron mosaic virus) vectored by a different eriophyid mite, or a closely related tritimovirus (Oat necrotic mottle virus; ONMV) with no known vector. In contrast, both WSMV-Sidney 81 and a chimeric WSMV genome bearing HC-Pro of a divergent strain (WSMV-El Batán 3; 86% amino acid sequence identity) were efficiently transmitted by A. tosichella. Replacing portions of WSMV-Sidney 81 HC-Pro with the corresponding regions from ONMV showed that determinants of wheat curl mite transmission map to the 5'-proximal half of HC-Pro. WSMV genomes bearing HC-Pro of heterologous species retained the ability to form virions, indicating that loss of vector transmissibility was not a result of failure to encapsidate. Although titer in systemically infected leaves was reduced for all chimeric genomes relative to WSMV-Sidney 81, titer was not correlated with loss of vector transmissibility. Collectively, these results demonstrate for the first time that HC-Pro is required for virus transmission by a vector other than aphids.

Members of the Luteoviridae are transmitted by aphids in a circulative, nonpropagative manner that requires the virus to be acquired through gut tissue into the aphid hemocoel and then exit through salivary tissues. This process is aphid species-specific and involves specific recognition of the virus by unidentified components on the membranes of gut and salivary tissues. Transport through the tissues is an endocytosis/exocytosis process. Both structural proteins of the virus are involved in the transmission process, with multiple protein domains regulating the movement and survival of the virus in the aphid and plant. Here we review what is known about the genetic, cellular, and molecular mechanisms regulating these complex and specific virus-aphid interactions.

Beet western yellows virus (BWYV), family Luteoviridae, is an icosahedral plant virus which is strictly transmitted by aphids in a persistent and circulative manner. Virions cross two cellular barriers in the aphid by receptor-based mechanisms involving endocytosis and exocytosis. Particles are first transported across intestinal cells into the haemolymph and then across accessory salivary gland cells for delivery to the plant via saliva. We identified the midgut part of the digestive tract as the site of intestinal passage by BWYV virions. To analyse the role in transmission of the minor capsid component, the readthrough (RT) protein, the fate of a BWYV RT-deficient non-transmissible mutant was followed by transmission electron microscopy in the vector Myzus persicae. This mutant was observed in the gut lumen but was never found inside midgut cells. However, virion aggregates were detected in the basal lamina of midgut cells when BWYV antiserum was microinjected into the haemolymph. The presence of virions in the haemolymph was confirmed by a sensitive molecular technique for detecting viral RNA. Thus, transport of the mutant virions through intestinal cells occurred but at a low frequency. Even when microinjected into the haemolymph, the RT protein mutant was never detected near or in the accessory salivary gland cells. We conclude that the RT protein is not strictly required for the transport of virus particles through midgut cells, but is necessary for the maintenance of virions in the haemolymph and their passage through accessory salivary gland cells.

ABSTRACT Lepidopteran cells (Spodoptera frugiperda) produced isometric virus-like particles (VLP) when infected with a recombinant baculovirus Ac61 that contained the Potato leafroll virus (PLRV) coat protein gene modified with an N-terminal histidine tag (P3-6H). Cells infected with AcFL, a recombinant baculovirus that expressed cDNA copies of the PLRV genome RNA, did not produce virus-like particles (VLP). In cell lines doubly infected with Ac61 and AcFL, VLP were formed that contained PLRV-RNA packaged in P3-6H coat protein (FL). Both the P3-6H and the FL particles were morphologically indistinguishable from particles of PLRV despite the fact that they lacked the P5 readthrough protein present in wild-type PLRV. When aphids (Myzus persicae) were fed on, or injected with, purified PLRV, or VLP of either type (FL or P3-6H) and examined by electron microscopy, no differences were observed among treatments for particle endocytosis, transcellular transport, or exocytosis at the aphid midgut or accessory salivary glands. Particles were observed in the salivary canals and in the salivary duct leading out of the aphid. These results suggest that P5 readthrough protein of PLRV may not be essential for cellular transport of virus through aphid vectors.

ABSTRACT Virus isolates from forage legumes collected from eight different states were identified as luteoviruses closely related to soybean dwarf luteovirus dwarfing (SbDV-D) and yellowing (SbDV-Y) described in Japan. All isolates produced reddened leaf margins in subterranean clover and were transmitted in a persistent manner by Acrythosiphon pisum, but not by Aulacorthum solani. Specific monoclonal antibodies raised against SbDV-Y were differentially reactive with endemic isolates. Immunoblots probed with a SbDV-D polyclonal antiserum showed single 26-kDa coat protein bands, confirming close serological relatedness to SbDV. Analyses of genomic and subgenomic double-stranded RNAs and northern blot analyses confirmed genomic relatedness to SbDV. Based on our results, we conclude that the U.S. luteovirus isolates studied comprise a strain or strains of the soybean dwarf virus that have clovers as common hosts and the pea aphid as a common vector.

Barley yellow dwarf virus (BYDV) causes significant losses in yield and in overwintering ability of winter cereals. Mechanisms by which the physiology of plants is affected by the virus are not clear. To see how carbohydrates in the crown of winter cereals were affected by BYDV, fructan isomers of degree of polymerization (DP) 3-5, fructan DP>6 and the simple sugars, glucose, fructose and sucrose, were measured before and during cold hardening in three oat (Avena sativa L.) cultivars, 'Wintok', 'Coast Black' and 'Fulghum'. On a fresh weight basis fructan DP>6 decreased by 50% in infected 'Wintok' and 'Coast Black' and by 25% in 'Fulghum'. Two DP3, one DP4 and one DP5 isomer were significantly higher than non-infected controls. The percentages of simple sugars in infected crowns were significantly higher than controls in all three cultivars in every week except the first week of hardening. Crude enzyme extracts from BYDV infected plants incubated with sucrose suggested higher invertase and lower sucrose-sucrosyl transferase activity. When incubated with 1-kestose and neokestin, no significant difference was found in fructose fructosyl transferase or in hydrolase activity. The activity of unidentified enzymes catalysing the synthesis of larger (DP>5) fructan was altered by BYDV. The decrease of carbohydrates in the crown induced indirectly by BYDV may alter the plant's capacity to regenerate tillers in the spring. The ability of plants to prevent or tolerate carbohydrate fluctuations induced by BYDV infection may be an important genetically regulated characteristic for developing virus-resistant cultivars.

The potential of herbaceous weeds commonly growing in or adjacent to cucurbit crops to serve as alternate hosts and overwintering reservoirs of Erwinia tracheiphila, a causal agent of cucurbit wilt, was investigated. Methods for isolation, maintenance, long-term storage, and detection of E. tracheiphila from infected plants were developed. E. tracheiphila was consistently detected by enzyme-linked immunosorbent assay (ELISA) and reisolated from infected, susceptible, cucurbit species. When six common herbaceous weed species were inoculated, E. tracheiphila was detected in 49% (combined species) of the plants by ELISA 3 weeks after inoculation. However, we were unable to reisolate E. tracheiphila from these plants by standard techniques. Immunoaffinity isolation with a sensitivity of 2 CFU per sample also failed to recover E. tracheiphila from weed species. Comparisons of cucumber and goldenrod inoculated with live or formaldehyde-killed E. tracheiphila indicated that immunoassays could detect nonviable E. tracheiphila systemically spread in plants 3 weeks post-inoculation. In these tests, the pathogen was reisolated only from cucumber plants inoculated with live E. tracheiphila. Although we could reproduce serological evidence of E. tracheiphila antigen in the weeds investigated, our results do not support the hypothesis that E. tracheiphila can infect, survive in, or overwinter in the weed species tested.

Barley yellow dwarf luteovirus (BYDV) particles are transmitted by aphids in a species-specific manner. Transmission to plants requires that the virus particles be transported across the basal lamina and plasmalemma of the accessory salivary gland (ASG). To characterize the role of the ASG basal lamina in regulating BYDV transmission, five aphid species were microinjected with purified New York isolates BYDV-PAV or -RPV. Both viruses associated specifically only with the ASG basal lamina. The ability of virions to penetrate the basal lamina was separate from the ability to penetrate the plasmalemma. When the salivary glands of vector, Sitobion avenae, or non-vector, Rhopalosiphum maidis, aphids were incubated in vitro with New York isolate BYDV-MAV, virions only attached to the ASG basal lamina of S. avenae. When anionic and cationic ferritin were microinjected into aphids, only cationic ferritin aggregated on the surface of the ASG basal lamina and at openings of plasmalemma invaginations into the cytoplasm, suggesting that these sites had a net negative charge. In vitro studies of anionic and cationic gold penetration of ASG basal laminae indicated a macromolecular size exclusion limit of approximately 20 nm that depended on charge. Anionic gold particles did not accumulate in the basal lamina as densely as the 25 nm BYDV particles, suggesting that the virus particles have a greater affinity for the ASG basal lamina. These results indicate that both the ASG basal lamina and plasmalemma contain specific components independently involved in the recognition and transmission of luteoviruses.

DNA encoding the coat protein (P3) of a Scottish isolate of potato leafroll virus (PLRV) was inserted into the genome of Autographa californica nucleopolyhedrovirus (AcNPV) such that the coat protein was expressed either in an unmodified form or with the addition of the amino acid sequence MHHHHHHGDDDDKDAMG at the N terminus (P3-6H). Insect cells infected with these recombinant baculoviruses accumulated substantial amounts of P3 and P3-6H. P3 could not be recovered from cell extracts unless it was denatured in SDS but a proportion of the P3-6H was recoverable in a soluble form in non-denaturing conditions. Immunogold labelling of sections of infected cells showed that P3 accumulated in nuclei in large amorphous bodies. In contrast, although much of the P3-6H also accumulated in nuclei, it formed virus-like particles (VLP) which were often grouped in close-packed, almost cystalline arrays. When electron microscope grids coated with antibodies to PLRV were floated on cell extracts containing P3-6H, VLP were trapped which were indistinguishable from PLRV particles trapped from extracts of PLRV-infected plants. The VLP co- sedimented in sucrose gradients with PLRV particles which suggests that the VLP contained RNA. VLP collected from sucrose density gradient fractions contained protein which reacted with nickel chelated to nitrilotriacetic acid, a histidine-specific reagent. Cells infected with either recombinant baculovirus also synthesized a protein, with an Mr of about 17000, which was shown to be the translation product of the P4 gene which is in the +1 reading frame within the coat protein gene. This protein was also found in the nuclear fraction of infected cells but was more readily soluble than was P3.

Purified particles of barley yellow dwarf luteovirus (BYDV) contain a major 22-kDa protein and a minor protein of approximately 58 kDa. The 22-kDa capsid protein is encoded by open reading frame (ORF) 3. ORF 5 is immediately downstream and in frame with ORF 3 and a 72-kDa protein can be translated via a readthrough suppression of the ORF 3 termination codon. Antibodies were produced against two Escherichia coli expressed polypeptides that represent the amino- and carboxyl-terminal halves of a putative 50-kDa protein encoded by ORF 5. Immunological analyses indicated that the 58-kDa protein associated with purified virions contained sequences encoded by ORF 3 and ORF 5. The carboxyl terminal portion of the full-length (72 kDa) readthrough protein was absent from the 58-kDa protein. The full-length readthrough protein was detected in infected oat protoplasts and plant tissue, but was not associated with virus particles purified from plants. The carboxyl-terminal portion of the 72-kDa readthrough protein was not required for aphid transmission; however, virus was transmitted more efficiently from protoplast extracts containing virions and soluble 72-kDa readthrough protein than from mock-inoculated protoplast extracts to which plant purified virus was added. The full-length readthrough protein, although not required for transmission, may increase the transmission efficiency of BYDV by aphids.

The accumulation of fructan oligomers in cold hardened oat stems was compared to oligomer synthesis in a crude enzyme extract. Water-soluble carbohydrates were quantified at specified time intervals in three oat cultivars which had been hardened for a total of 5 wk at 2 °C. A crude enzyme extract was prepared from stems of one cultivar hardened for 6 d at 2 °C and incubated at 30 °C with 200 mM sucrose. 1-Kestose was the first oligomer to accumulate in vitro but neokestose rapidly increased after 3 h of incubation and eventually surpassed the level of 1 -kestose. Neokestose and 1-kestose accumulated at similar rates in vivo in the first week of hardening but after 1 week, 1-kestose began to decrease while neokestose remained relatively constant. The same phenomenon was observed in vitro but after only 3 h. 6-Kestose was not observed in vivo or in vitro, except for a trace before hardening began. The first tetramer to increase was 1&6 G -kestotetraose but it subsequently decreased and its concentration was surpassed by that of 6 G ,6-kestotetraose. Similarities and differences between in vivo and in vitro patterns of fructan synthesis are discussed.

Barley yellow dwarf virus (BYDV) was consistently observed in the basal lamina and in plasmalemma invaginations only of accessory salivary glands in each of 20 aphids (Sitobion avenae) reared on oats infected with the MAV or RPV isolates of BYDV. Virus particles were not found in any of 13 aphids reared in parallel on healthy oats. The MAV isolate was identified in aphids by indirect labeling with ferritin-conjugated antibody. Virus particles were also observed within cytoplasmic vesicles and intracellular canals that drain the accessory gland in all aphids reared on oats infected with MAV, which is transmitted by S. avenae, but in none of the aphids reared in parallel on oats infected with RPV, which S. avenae does not transmit. Visualization of only transmissible virus within accessory gland cytoplasmic structures indicates that the plasmalemma may be a site regulating virus uptake by the salivary gland. Presence of virus in intracellular canals and in coated pits and vesicles adjacent, to canals suggests possible transport mechanisms out of the accessory gland. The results are compatible with a virus-cell receptor mechanism for transmission specificity between luteoviruses and aphid vectors, and they suggest a route through aphids for viruses transmitted in the circulative manner.