Molecular Cloning of Sugarcane Mosaic Virus Complementary-Dna: Use as a Probe for the Detection of Virus Infection and Viral-Rnas.

DNA complementary (cDNA) to the RNA genome of sugarcane mosaic virus strain H (SCMV-H) was synthesized using avian myeloblastosis virus reverse transcriptase and oligo(dT) as primer. Second strand synthesis used the same enzyme and oligo(dG) primer after tailing the first strand with oligo(dC). Double-stranded cDNA was inserted into the PstI site of plasmid pBR322 by the G-C tailing method and cloned in Esherichia coli HB101. Twenty recombinant clones containing SCMV-H sequences were obtained, but most had inserts less than 500 base pairs (0.5 kbp). Two plasmids, S47-6 and S47-20, however, had larger inserts of 1.2 kbp and 2.7 kbp, respectively and were used for further study. These two plasmids had some SCMV-H sequences in common, but did not share any BamHI, EcoRI, HindIII, PstI, or SalI restriction sites. Dot blot hybridization, which involves spotting crude plant extracts on nitrocellulose filters and hybridizing with (\u2732)P-labeled recombinant S47-6 or S47-20 plasmid DNA, proved to be a rapid and sensitive method for detecting SCMV-H infection. Sap from SCMV-infected plants diluted 1/1000 to 1/3000 gave detectable hybridization signals as did 15 to 40 pg purified viral RNA. Dot blot hybridization also revealed that SCMV strain I, but not SCMV strains A, B, D, M, and J, has sequences in common with the strain H-derived clones. Northern hybridization of single-stranded RNA from SCMV-infected tissue showed that, besides genomic RNA, a series of smaller RNAs with sizes of 7.9, 6.6, 4.7, 2.8, 1.5, 1.2, 0.9, and 0.7 kb hybridized to the probe. No discrete viral double-stranded RNA species were found. Serologically specific electron microscopy of plant extracts was used to demonstrate a series of discrete less-than-full-length virus particles, two of which correlate with the 7.9 and 6.6 kb RNAs