Cloning and physical mapping of DNA complementary to potato leafroll virus RNA

Abstract

Potato leafroll virus (PLRV) was aphid-transmitted from potato (Solanum tuberosum cultivar Russet Burbank) to ground cherry (Physalis floridana), where it was maintained by serial aphid transmission. Serological and plant differential tests indicated that the isolate was not contaminated with beet western yellows virus. Purified PLRV RNA was poly(A)-tailed in vitro and used as a template for reverse transcriptase, primed with oligo(dT). Alkaline gel electrophoresis of ³²P-labeled first-strand complementary DNA (cDNA) indicated a major size range of 0.1 to 3.5 kilobases (kb). A small percentage of transcripts corresponded to full length PLRV RNA (ca. 6.2 kb). Following RNase H and DNA polymerase I-mediated second strand synthesis, double-stranded cDNA was cloned into the Pst I site of the plasmid pUC9 using oligo(dC)-oligo(dG) tailing methodology. Escherichia coli JM109 transformants were screened with first-strand ³²P-cDNA in colony hybridization experiments to confirm that recombinants contained PLRV-specific sequences. Three initial clones were organized on a 3.5-kbp overlapping map based on restriction endonuclease and Southern-blot hybridization analyses. Leftward and rightward probes were derived from these initial clones and used to identify additional cloned PLRV cDNAs in a series of colony hybridization experiments. Three clones, pPLRV4-173, -228, -323, containing cDNA inserts of 3.4, 2.4, and 1.2 kbp, respectively, were identified. Restriction endonuclease and Southern-blot hybridization analyses indicated that these cDNAs formed an overlapping map representing about 5.9 kpb or 95% of the PLRV RNA genome. These clones were verified to be PLRV-specific by dot-blot hybridization to viral RNA and to total cellular RNA from PLRV-infected P. floridana plants. The 5' to 3' polarity of PLRV cDNA 228 was established by the use of M13 strand-specific hybridization probes. This information, in conjunction with the overlapping map of PLRV cDNAs, indicated that the 5' to 3' order of cDNAs 173, 228, and 323 was; 5' - cDNA 173 - cDNA 323 - cDNA 228 - 3' This conclusion was supported by partial DNA sequencing analysis which identified asymmetrically-positioned poly(dT) tracts in cloned cDNA. Northern-blot hybridization was used to detect PLRV RNA in total nucleic acid extracts of aphids. Preliminary results, described herein, indicate that procedures need to be developed for enriching total nucleic acid extracts of viruliferous aphids for PLRV RNA. The potential uses of cloned PLRV cDNA for virus replication studies, virus-vector studies, development of host plant resistance, and plant disease diagnosis are discussed.