Keith L. Perry

Transmission of plant viruses by aphid vectors.

SUMMARY Aphids are the most common vector of plant viruses. Mechanisms of transmission are best understood by considering the routes of virus movement in the aphid (circulative versus non-circulative) and the sites of retention or target tissues (e.g. stylets, salivary glands). Capsid proteins are a primary, but not necessarily sole, viral determinant of transmission. A summary is presented of the taxonomic affiliations of the aphid transmitted viruses, including 8 families, 18 genera, and taxonomically unassigned viruses.

Transmissibility of field isolates of Soybean viruses by Aphis glycines

During the 2001 growing season, 191 symptomatic soybean (Glycine max (L.) Merr.) plants were dug from production plots in Indiana, Wisconsin, and Kentucky. Alfalfa mosaic virus (AMV), Bean pod mottle virus (BPMV), Bean yellow mosaic virus (BYMV), Peanut stunt virus (PSV), Tobacco ringspot virus (TRSV), and Soybean mosaic virus (SMV) were identified. No mixed infections were observed. The ability of the soybean aphid (Aphis glycines Matsamura) to transmit field isolates of these viruses was tested. Using naturally infected field- or greenhouse-grown soybean plants as sources, six isolates of SMV and two isolates of AMV were transmitted using a short feeding assay. One of two isolates of TRSV was transmitted by A. glycines in one of four experiments using an extended feeding transmission assay. BPMV was not transmitted by A. glycines in assays involving 11 field isolates and over 840 aphids. One field isolate each of BYMV and PSV were tested and no transmission by A. glycines was observed.

ARGONAUTE2 mediates RNA-silencing antiviral defenses against Potato virus X in Arabidopsis.

RNA-silencing mechanisms control many aspects of gene regulation including the detection and degradation of viral RNA through the action of, among others, Dicer-like and Argonaute (AGO) proteins. However, the extent to which RNA silencing restricts virus host range has been difficult to separate from other factors that can affect virus-plant compatibility. Here we show that Potato virus X (PVX) can infect Arabidopsis (Arabidopsis thaliana), which is normally a nonhost for PVX, if coinfected with a second virus, Pepper ringspot virus. Here we show that the pepper ringspot virus 12K protein functions as a suppressor of silencing that appears to enable PVX to infect Arabidopsis. We also show that PVX is able to infect Arabidopsis Dicer-like mutants, indicating that RNA silencing is responsible for Arabidopsis nonhost resistance to PVX. Furthermore, we find that restriction of PVX on Arabidopsis also depends on AGO2, suggesting that this AGO protein has evolved to specialize in antiviral defenses.

The structure of cucumber mosaic virus and comparison to cowpea chlorotic mottle virus.

ABSTRACT The structure of cucumber mosaic virus (CMV; strain Fny) has been determined to a 3.2-Å resolution using X-ray crystallography. Despite the fact that CMV has only 19% capsid protein sequence identity (34% similarity) to cowpea chlorotic mottle virus (CCMV), the core structures of these two members of the Bromoviridaefamily are highly homologous. As suggested by a previous low-resolution structural study, the 305-Å diameter (maximum) of CMV is ∼12 Å larger than that of CCMV. In CCMV, the structures of the A, B, and C subunits are nearly identical except in their N termini. In contrast, the structures of two loops in subunit A of CMV differ from those in B and C. These loops are 6 and 7 residues longer than the analogous regions in CCMV. Unlike that of CCMV, the capsid of CMV does not undergo swelling at pH 7.0 and is stable at pH 9.0. This may be partly due to the fact that the N termini of the B and C subunits form a unique bundle of six amphipathic helices oriented down into the virion core at the threefold axes. In addition, while CCMV has a cluster of aspartic acid residues at the quasi-threefold axis that are proposed to bind metal in a pH-dependent manner, this cluster is replaced by complementing acids and bases in CMV. Finally, this structure clearly demonstrates that the residues important for aphid transmission lie at the outermost portion of the βH-βI loop and yields details of the portions of the virus that are hypothesized to mediate binding to aphid mouthparts.

A Conserved Capsid Protein Surface Domain of Cucumber Mosaic Virus Is Essential for Efficient Aphid Vector Transmission

ABSTRACT A prominent feature on the surfaces of virions of Cucumber mosaic virus (CMV) is a negatively charged loop structure (the βH-βI loop). Six of 8 amino acids in this capsid protein loop are highly conserved among strains of CMV and other cucumoviruses. Five of these amino acids were individually changed to alanine or lysine (an amino acid of opposite charge) to create nine mutants (the D191A, D191K, D192A, D192K, L194A, E195A, E195K, D197A, and D197K mutants). Transcripts of cDNA clones were infectious when they were mechanically inoculated onto tobacco, giving rise to symptoms of a mottle-mosaic typical of the wild-type virus (the D191A, D191K, D192A, E195A, E195K, and D197A mutants), a systemic necrosis (the D192K mutant), or an atypical chlorosis with necrotic flecking (the L194A mutant). The mutants formed virions and accumulated to wild-type levels, but eight of the nine mutants were defective in aphid vector transmission. The aspartate-to-lysine mutation at position 197 interfered with infection; the only recovered progeny (the D197K∗ mutant) harbored a second-site mutation (denoted by the asterisk) of alanine to glutamate at position 193, a proximal site in the βH-βI loop. Since the disruption of charged amino acid residues in the βH-βI loop reduces or eliminates vector transmissibility without grossly affecting infectivity or virion formation, we hypothesize that this sequence or structure has been conserved to facilitate aphid vector transmission.

Complete Genome Sequence of a New Circular DNA Virus from Grapevine

ABSTRACT A novel circular DNA virus sequence is reported from grapevine. The corresponding genomic organization, coding potential, and conserved origin of replication are similar to those of members of the family Geminiviridae, but the genome of 3,206 nucleotides is 4% larger than the largest reported geminiviral genome and shares only 50% overall sequence identity.

Transcription of tomato ribosomal DNA and the organization of the intergenic spacer.

SummaryThe organization of the intergenic spacer of a 9.04 kb tomato ribosomal RNA gene (rDNA) was determined. The 3258 by spacer contains two major repeat elements enclosing a region which includes 351 by of an 81.8% A–T rich sequence. A block of nine 53 by repeats begins 388 by downstream from the 3′ end of the 25S rRNA. The A–T rich domain is followed by a block of six 141 by repeats terminating 818 by upstream from the 5′ end of the 18S rRNA. Major pre-rRNAs of 7.6 and 6.5 kb were observed by Northern hybridization analysis. The 5′ termini of these RNAs were identified through combined S1 nuclease and primer extension analyses. The 7.6 kb RNA is likely to be the primary transcript; its 5′ terminus lies within a sequence motif, TATA(R)TA(N)GGG, conserved at the termini of transcripts mapped in three other plant species. The 6.5 kb RNA is interpreted as a 5′ end processed transcript derived from the 7.6 kb RNA. Comparative analysis of transcribed sequences revealed a 25 by domain of the intergenic spacer which is relatively conserved among five plant species. The conservation of spacer sequences in plants is in contrast to the extensive sequence divergence of the intergenic spacer in other non-plant systems and suggests a conserved function directed by these sequences.

A natural M RNA reassortant arising from two species of plant- and insect-infecting bunyaviruses and comparison of its sequence and biological properties to parental species

Reassortment allows multicomponent viruses to exchange genome segments, a process well-documented in the vertebrate- and arthropod-infecting members of the family Bunyaviridae but not between distinct species of the plant- and insect-infecting members of the genus Tospovirus. Genome sequence comparisons of a virus causing severe tospovirus-like symptoms in Florida tomato with Groundnut ringspot virus (GRSV) and Tomato chlorotic spot virus (TCSV) demonstrated that reassortment has occurred, with the large (L) and small (S) RNAs coming from GRSV and the medium (M) RNA coming from TCSV (i.e. L(G)M(T)S(G)). Neither parental genotype is known to occur in the U.S. suggesting that L(G)M(T)S(G) was introduced as a reassortant. L(G)M(T)S(G) was transmitted by western flower thrips (Frankliniella occidentalis [Pergande]), and was not able to overcome the Sw5 resistance gene of tomato. Our demonstration of reassortment between GRSV and TCSV suggests caution in defining species within the family Bunyaviridae based on their ability to reassort.

Transmissibility of Cucumber mosaic virus by Aphis gossypii Correlates with Viral Accumulation and Is Affected by the Presence of Its Satellite RNA

ABSTRACT Satellite RNAs (satRNAs) are associated with Cucumber mosaic virus (CMV) in tomato, most often causing severe epidemics of necrotic plants, and not associated with specific host symptoms. Laboratory studies on virus transmission by the aphid vector Aphis gossypii were performed to better understand the dynamics of field populations of CMV. The presence of satRNAs correlated with lower concentrations of virus in infected plants and with a decrease in the efficiency of transmission from satRNA-infected plants. Both the concentration of virus in CMV-infected tomato and the efficiency of transmission varied more extensively with nonnecrogenic satRNAs than with necrogenic satRNAs. A negative effect of satRNAs on virus accumulation can account, in part, for a decrease in the field transmission and recovery of CMV + satRNAs. Aphids behaved differently and probed less readily on plants infected with CMV + necrogenic satRNAs compared with plants containing non-necrogenic satRNAs. Aphid-mediated satRNA-free CMV infections were observed in test plants when aphids were fed on source plants containing CMV + nonnecrogenic satRNA; no comparable satRNA-free test plants occurred when aphids were fed on source plants containing necrogenic satRNAs. These results indicate that factors associated with transmission can be a determinant in the evolution of natural populations of CMV and its satRNA.

Insect-mediated transmission of mixed and reassorted cucumovirus genomic RNAs

Transmissions of virus using the aphid Myzus persicae were performed using plants co-infected with two cucumoviruses, tomato aspermy virus (V-TAV) and cucumber mosaic virus (M-CMV). Five of the aphid-transmitted progeny viruses (3.7%) induced symptoms distinct from those induced by either parental virus. Northern blot hybridization analysis of encapsidated RNAs from these novel progeny demonstrated that all of the RNA profiles were characteristic of pseudo-recombinants, i.e. viruses with reassorted genomic RNAs. The two larger RNAs, 1 and 2, originated from V-TAV, whereas RNA 3 was derived from M-CMV. A more sensitive RNase protection assay analysis of both unencapsidated and encapsidated RNAs revealed the presence of minor populations of V-TAV-derived RNA 3 in all of these novel progeny, and of M-CMV-derived RNA 1 (and presumably RNA 2) in one of the progeny. A bias against the encapsidation of the minor populations of RNAs by the M-CMV coat protein was observed, suggesting that there is specificity or competition with regard to the encapsidation of cucumoviral RNAs in vivo. This study demonstrates that insect vectors can mediate the establishment of pseudorecombinants with mixed populations of RNA 3.