The Effects of Bean Leafroll Virus on Life History Traits and Host Selection Behavior of Specialized Pea Aphid (Acyrthosiphon pisum, Hemiptera: Aphididae) Genotypes.

Intraspecific specialization by insect herbivores on different host plant species contributes to the formation of genetically distinct "host races," but the effects of plant virus infection on interactions between specialized herbivores and their host plants have barely been investigated. Using three genetically and phenotypically divergent pea aphid clones (Acyrthosiphon pisum L.) adapted to either pea (Pisum sativum L.) or alfalfa (Medicago sativa L.), we tested how infection of these hosts by an insect-borne phytovirus (Bean leafroll virus; BLRV) affects aphid performance and preference. Four important findings emerged: 1) mean aphid survival rate and intrinsic rate of population growth (Rm) were increased by 15% and 14%, respectively, for aphids feeding on plants infected with BLRV; 2) 34% of variance in survival rate was attributable to clone × host plant interactions; 3) a three-way aphid clone × host plant species × virus treatment significantly affected intrinsic rates of population growth; and 4) each clone exhibited a preference for either pea or alfalfa when choosing between noninfected host plants, but for two of the three clones tested these preferences were modestly reduced when selecting among virus-infected host plants. Our studies show that colonizing BLRV-infected hosts increased A. pisum survival and rates of population growth, confirming that the virus benefits A. pisum. BLRV transmission affected aphid discrimination of host plant species in a genotype-specific fashion, and we detected three unique "virus-association phenotypes," with potential consequences for patterns of host plant use by aphid populations and crop virus epidemiology.

Modeling the competition between viruses in a complex plant-pathogen system.

In this article, we propose a mathematical model that describes the competition between two plant virus strains (MAV and PAV) for both the host plant (oat) and their aphid vectors. We found that although PAV is transmitted by two aphids and MAV by only one, this fact, by itself, does not explain the complete replacement of MAV by PAV in New York State during the period from 1961 through 1976; an interpretation that is in agreement with the theories of A. G. Power. Also, although MAV wins the competition within aphids, we assumed that, in 1961, PAV mutated into a new variant such that this new variant was able to overcome MAV within the plants during a latent period. As shown below, this is sufficient to explain the swap of strains; that is, the dominant MAV was replaced by PAV, also in agreement with Power's expectations.

Presence of rose spring dwarf-associated virus in Chile: partial genome sequence and detection in roses and their colonizing aphids.

Rose is one of the most important cut flowers produced in the world. It is also grown in landscape plantings and public gardens for ornamental purposes. However, there is no detailed information available about viruses infecting roses in Chile. In order to gain insight about the viruses that could be present, a plant showing yellow vein chlorosis in its leaves was collected from a garden in Santiago. Double-stranded RNA (dsRNA) was isolated and after a random primed RT-PCR amplification procedure followed by sequencing, Rose spring dwarf-associated virus (RSDaV) presence was established. In order to widen the survey, several additional symptomatic and asymptomatic plants as well as aphids were screened by RT-PCR using two different pairs of virus-specific primers. RSDaV was detected in 24% of the analyzed samples. To our knowledge, this is the first report of RSDaV in Chilean rose plants and Rhodobium porosum (Sanderson) aphids.

Genetic characterization and mapping of major gene resistance to potato leafroll virus in Solanumtuberosum ssp. andigena.

Major gene inheritance of resistance to Potato leafroll virus (PLRV) was demonstrated in a parthenogenic population derived from the highly resistant tetraploid andigena landrace, LOP-868. This major gene or chromosome region seems to control a single mechanism for resistance to infection and virus accumulation in this source. About 149 dihaploid lines segregated in a ratio of 107 resistant to 32 susceptible, fitting the expected ratio for inheritance of a duplex gene under random chromatid segregation. A tetraploid AFLP map was constructed using as reference the ultra high density (UHD) map. All AFLP markers associated with PLRV resistance mapped to the same linkage group. Map position was confirmed by analysis of previously-mapped SSR markers. Rl (adg) is located on the upper arm of chromosome V, at 1 cM from its most closely linked AFLP marker, E35M48.192. This marker will be used to develop allele-specific primers or a pair of flanking PCR-based markers for their use in marker assisted selection.

Molecular characterization of a virus from the family Luteoviridae associated with cotton blue disease.

Cotton blue disease is an aphid-transmitted cotton disease described in Brazil in 1962 as Vein Mosaic "var. Ribeirão Bonito". At present it causes economically important losses in cotton crops if control measures are not implemented. The observed symptoms and mode of transmission have prompted researchers to speculate that cotton blue disease could be attributed to a member of the family Luteoviridae, but there was no molecular evidence supporting this hypothesis. We have amplified part of the genome of a virus associated with this disease using degenerate primers for members of the family Luteoviridae. Sequence analysis of the entire capsid and a partial RdRp revealed a virus probably belonging to the genus Polerovirus. Based on our results we propose that cotton blue disease is associated with a virus with the putative name Cotton leafroll dwarf virus (CLRDV).

Analysis of differential selective forces acting on the coat protein (P3) of the plant virus family Luteoviridae.

The coat protein (CP) of the family Luteoviridae is directly associated with the success of infection. It participates in various steps of the virus life cycle, such as virion assembly, stability, systemic infection, and transmission. Despite its importance, extensive studies on the molecular evolution of this protein are lacking. In the present study, we investigate the action of differential selective forces on the CP coding region using maximum likelihood methods. We found that the protein is subjected to heterogeneous selective pressures and some sites may be evolving near neutrality. Based on the proposed 3-D model of the CP S-domain, we showed that nearly neutral sites are predominantly located in the region of the protein that faces the interior of the capsid, in close contact with the viral RNA, while highly conserved sites are mainly part of beta-strands, in the protein's major framework.

Analysis of differential selective forces acting on the coat protein (P3) of the plant virus family Luteoviridae

The coat protein (CP) of the family Luteoviridae is directly associated with the success of infection. It participates in various steps of the virus life cycle, such as virion assembly, stability, systemic infection, and transmission. Despite its importance, extensive studies on the molecular evolution of this protein are lacking. In the present study, we investigate the action of differential selective forces on the CP coding region using maximum likelihood methods. We found that the protein is subjected to heterogeneous selective pressures and some sites may be evolving near neutrality. Based on the proposed 3-D model of the CP S-domain, we showed that nearly neutral sites are predominantly located in the region of the protein that faces the interior of the capsid, in close contact with the viral RNA, while highly conserved sites are mainly part of beta-strands, in the protein's major framework.

Replicase mediated resistance against potato leafroll virus in potato Desirée plants.

Potato leafroll virus (PLRV) is a major menace for the potato production all over the world. PLRV is transmitted by aphids, and until now, the only strategy available to control this pest has been to use large amounts of insecticides. Transgenic approaches involving the expression of viral replicases are being developed to provide protection for plants against viral diseases. The purpose of this study was to compare the protection afforded by the differential expression of PLRV replicate transgene in potato plants cv. Desirée, Plants were genetically modified to express the complete sense PLRV replicase gene. Two constructions were used, one containing the constitutive 35SCaMV promoter and the other the phloem-specific RolA promoter from Agrobacterium rhizogenes. Transgenic plants were infected with PLRV in vitro, using infested aphids. In plants in which 35SCaMV controlled the expression of the PLRV replicase gene, signs of infection were initially detected, although most plants later developed a recovery phenotype showing undetectable virus levels 40 days after infection. In turn, those plants with the RolA promoter displayed an initial resistance that was later overcome. Different molecular mechanisms are likely to participate in the response to PLRV infection of these two types of transgenic plants.

Replicase mediated resistance against Potato Leafroll Virus in potato Desiree plants

Potato leafroll virus (PLRV) is a major menace for the potato production all over the world. PLRV is transmitted by aphids, and until now, the only strategy available to control this pest has been to use large amounts of insecticides. Transgenic approaches involving the expression of viral replicases are being developed to provide protection for plants against viral diseases. The purpose of this study was to compare the protection afforded by the differential expression of PLRV replicate transgene in potato plants cv. Desirée, Plants were genetically modified to express the complete sense PLRV replicase gene. Two constructions were used, one containing the constitutive 35SCaMV promoter and the other the phloem-specific RolA promoter from Agrobacterium rhizogenes. Transgenic plants were infected with PLRV in vitro, using infested aphids. In plants in which 35SCaMV controlled the expression of the PLRV replicase gene, signs of infection were initially detected, although most plants later developed a recovery phenotype showing undetectable virus levels 40 days after infection.

Analyses of genotypic diversity among North, South, and Central American isolates of sugarcane yellow leaf virus: evidence for Colombian origins and for intraspecific spatial phylogenetic variation.

We have analyzed the genotypic diversity of sugarcane yellow leaf virus (SCYLV) collected from North, South, and Central America by fingerprinting assays and selective cDNA cloning and sequencing. One group of isolates from Colombia, designated the C-population, has been identified as residing at the root node between a separable superpopulation structure of SCYLV and other members of the family Luteoviridae, indicating that the progenitor viruses of the North, South, and Central American isolates of the SCYLV superpopulation most likely arose from a C-population structure. From a model of intrafamilial evolution (F. Moonan et al., Virology 269:156-171, 2000), a prediction could be made that within the SCYLV species, the capacity of genomic sequence divergence would range from lowest in the capsid protein open reading frame 3 (ORF 3) to highest in a region spanning across the carboxy-terminal end of the RNA-dependent RNA polymerase ORF. We have demonstrated the validity and applicability of this intrafamilial model for the prediction of intraspecies SCYLV diversity. Analysis of spatial phylogenetic variation (SPV) within the SCYLV isolates could not be assessed by application of a "partial likelihoods assessed through optimization" (PLATO)-derived intraspecies model alone. However, application of a PLATO-derived intrafamilial model with the intraspecies-derived model allowed distinction of three forms of SPV. Two of the SPV forms identified correspond to the extremes in a continuum of sequence evolution displayed in a SCYLV superpopulation structure, and the third form was diagnostic of a C-population structure. The application of these types of models has value in terms of predicting the types of SCYLV intraspecies diversity that may exist worldwide, and in general, may be useful in application for more informed design of transgenes for use in the elicitation of homology-dependent virus resistance mechanisms in transgenic plants.

Transgenic resistance in potato plants expressing potato leaf roll virus (PLRV) replicase gene sequences is RNA-mediated and suggests the involvement of post-transcriptional gene silencing.

Genetically engineered expression of replicase encoding sequences has been proposed as an efficient system to confer protection against virus diseases by eliciting protection mechanisms in the plant. Potato leaf-roll was one of the first diseases for which this kind of protection was engineered in potato plants. However, details of the protecting mechanism were not reported, so far. The ORF2b of an Argentinean strain of PLRV was cloned and sequenced finding 94% and 97% of homology with Australian and Dutch strains, respectively. To elucidate the mechanism of protection against PLRV infection, three versions of ORF2b (non-translatable sense, translatable sense with an engineered ATG and antisense) were constructed under the control of the 35S CaMV promoter and the nos terminator and introduced in potato plants (cv. Kennebec) by Agrobacterium tumefaciens-mediated transformation. Grafting infection experiments showed that resistant transgenic plants could be obtained with any of the constructs, suggesting that the mechanism of protection is independent of the expression of protein and is RNA mediated. Field trial infection confirmed that resistant transgenic events were obtained. Biolistic transient transformation experiments of leaves derived from transgenic plants using a gene coding for the fusion protein GUS-ORF2b, followed by scoring of the number of GUS expressing leaf spots, supported that the protection is mediated by a post-transcriptional gene silencing mechanism.

Molecular evidence that sugarcane yellow leaf virus (ScYLV) is a member of the Luteoviridae family.

A previously uncharacterized virus was reported in southeast Brazil causing a yellowing leaf disease in sugarcane. The virus, termed sugarcane yellow leaf virus (ScYLV), shares features typical of the luteoviruses. To start the molecular characterization of ScYLV, the nucleotide sequence of the coat protein (CP), 17 kDa protein and C-terminus of the RNA-dependent RNA polymerase coding regions was determined from an RT-PCR amplification product. Comparisons showed that the deduced amino acid sequences share a considerable degree of identity and similarity with corresponding sequences of known luteoviruses, thus clearly establishing ScYLV as a member of the family Luteoviridae. The authenticity of the CP open reading frame was confirmed by its expression in Escherichia coli. The recombinant CP positively reacted in immunoblot assays with polyclonal antibodies raised against native ScYLV. Furthermore, phylogenetic analyses also suggest that the 5' and 3' coding blocks of the ScYLV genome possess different taxonomic affinities within the Luteoviridae family, as does also the genome of soybean dwarf virus.

Molecular cloning and nucleotide sequence of the coat protein gene of a Cuban isolate of potato leafroll virus and its expression in Escherichia coli.

Total RNA from infected Physalis floridana was isolated to generate complementary DNA corresponding to the coat protein (GP) gene of a Cuban isolate of potato leaf roll virus (PLRV). This cDNA was amplified by the polymerase chain reaction (PCR) and cloned into the bacterial expression vectors pEX(1-3) for fusion protein expression in E. coli. The product was detected by antibodies specific for the PLRV CP. The coding sequence of the CP gene was determined, and the predicted length of the CP was 208 amino acids (23 kD). The nucleotide sequences and deduced amino acid sequences were compared with the other PLRV isolates and found to be 97-99.5% identical at both the nucleotide and amino acid sequence level of other isolates. Comparison of the deduced amino acid sequences of the PLRVcub CP revealed considerable homology to other luteoviruses. We believe that the protocol described could be applicable to other plant viruses of low abundance or of cumbersome isolation, since this method is less time consuming than the traditional methods of cloning coat protein genes of plant viruses with known sequences.