F. E. Gildow

Role of accessory salivary glands in aphid transmission of barley yellow dwarf virus.

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.

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.

Studies on the role of the minor capsid protein in transport of Beet western yellows virus through Myzus persicae

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.

Complete Deletion of Wheat Streak Mosaic Virus HC-Pro: a Null Mutant Is Viable for Systemic Infection

ABSTRACT 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.

Plum Pox in North America: Identification of Aphid Vectors and a Potential Role for Fruit in Virus Spread

ABSTRACT Thirteen aphid species were tested for their ability to transmit Pennsylvania isolates of Plum pox virus (PPV) collected in Columbia (PENN-3), Franklin (PENN-4), and York (PENN-7) Counties, PA. Four species, Aphis fabae, A. spiraecola, Brachycaudus persicae, and Myzus persicae, consistently transmitted PPV in preliminary transmission tests. Two species, Metopolophium dirhodum and Rhopalosiphum padi, were occasional inefficient vectors. Toxoptera citricida, from Florida, also was an effective vector but it does not occur in major stone-fruit-growing states. Species not transmitting PPV in parallel tests included Acyrthosiphon pisum, Aphis glycines, Aulacorthum solani, Macrosiphum euphorbiae, Rhopalosiphum maidis, and Sitobion avenae. When given a 3-day probing access period simultaneously on PPV-infected peach seedlings and healthy peach seedlings, Myzus persicae, Aphis spiraecola, A. fabae, and B. persicae transmitted PPV to 63, 31, 38, and 32% of the healthy peach seedlings, respectively. When given a similar probing period on PPV-infected peach fruit and healthy peach seedlings, the same aphid species transmitted PPV to 50, 35, 0, and 0% of seedlings, respectively. Results support the hypothesis of secondary PPV spread by indigenous aphids in Pennsylvania, and suggest that PPV-infected fruit has the potential to function as a virus source for long-distance dispersal.

Plant Virus HC-Pro Is a Determinant of Eriophyid Mite Transmission

ABSTRACT 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, ∼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.

Aphid Acquisition and Cellular Transport of Potato leafroll virus-like Particles Lacking P5 Readthrough Protein

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.

Transmission Efficiency of Cucumber mosaic virus by Aphids Associated with Virus Epidemics in Snap Bean

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.

Genetic regulation of polerovirus and luteovirus transmission in the aphid Schizaphis graminum

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.