Molecular characterization of clones of the Myzus persicae complex (Hemiptera: Aphididae) differing in their ability to transmit the potato leafroll luteovirus (PLRV)

A prerequisite to studying the specific interactions involved in the persistent transmission of luteoviruses such as the potato leafroll virus (PLRV) is the characterization of both the virus and its vectors. A range of techniques was used to assess genetic differentiation among 27 clones belonging to the Myzus persicae complex (M. persicae (Sulzer), M. antirrhinii (Macchiati) and M. nicotianaeBlackman) and showing different efficiencies in transmitting PLRV isolates. All M. persicae/M. nicotianae clones belonged to one of two karyotypes, both 2n = 12, either normal or carrying an autosomal translocation (A1,3), and all M. antirrhinii clones had 13 or 14 chromosomes. Amplified esterase 4 genes were detected by PCR–REN assay in M. persicae/M. nicotianae taxa, with gene expression being modified by methylation. Similarly, amplified E4 genes were revealed in M. antirrhinii but they all showed unmethylated. Two allozyme and 11 microsatellite loci discriminated 10 different genotypic classes among the 27 clones. Analysis of genetic relatedness between these genotypic classes revealed that M. nicotianae clones were very closely related to M. persicaeclones, whereas the genetic differentiation between M. antirrhinii and M. persicae was greater. The implications of these results for the taxonomic status of these genotypes within the complex, and the transmission of PLRV, are discussed.

Infectious transcripts from cloned cDNA of potato leafroll luteovirus.

Infectious transcripts play a key role in the research on plant viruses at the molecular level. A number of cDNA clones covering the whole genome of the Polish isolate of potato leafroll virus were constructed. Four overlapping clones were selected and assembled using restriction sites. The full copy was positioned between T7 RNA polymerase promoter and unique ScaI site. The full-length capped transcripts of the sequence of the viral genome synthesised in vitro were able to replicate in protoplasts and to produce the viral coat protein.

Improved Detection of Potato Leafroll Luteovirus in Leaves and Tubers with a Digoxigenin-Labeled cRNA Probe

A digoxigenin-labeled cRNA probe of approximately 2,100 bp was more than 2,000 times more sensitive in detecting potato leafroll virus (PLRV) in leaf extracts of Datura stramonium, Physalis floridana, and potatoes than enzyme-linked immunosorbent assay (ELISA). The limit of detecting PLRV with the probe was 1 pg/ml compared with 2 ng/ml by ELISA. The probe detected PLRV easily in dormant tuber tissues at dilutions of up to 1:100. There was no background reaction with healthy extracts. No reactions were observed between the probe and potato X potexvirus or potato Y potyvirus.

Expression of the Potato Leafroll Virus ORF0 Induces Viral-Disease-like Symptoms in Transgenic Potato Plants

The role of the open reading frame 0 (ORF0) of luteoviruses in the viral infection cycle has not been resolved, although the translation product (p28) of this ORF has been suggested to play a role in host recognition. To investigate the function of the potato leafroll luteovirus (PLRV) p28 protein, transgenic potato plants were produced containing the ORF0. In the lines in which the ORF0 transcripts could be detected by Northern (RNA) analysis, the plants displayed an altered phenotype resembling virus-infected plants. A positive correlation was observed between levels of accumulation of the transgenic transcripts and severity of the phenotypic aberrations observed. In contrast, potato plants transformed with a modified, untranslatable ORF0 sequence were phenotypically indistinguishable from wild-type control plants. These results suggest that the p28 protein is involved in viral symptom expression. Southern blot analysis showed that the transgenic plants that accumulated low levels of ORF0 transcripts detectable only by reverse transcription-polymerase chain reaction, contained methylated ORF0 DNA sequences, indicating down-regulation of the transgene provoked by the putatively unfavorable effects p28 causes in the plant cell.