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Molecular Cloning of Hypovirulence-associated Double-stranded RNA in Endothia parasitica and Detection of Sequence-related Single-stranded RNA

Shivanand Hiremath^1,

¹ Department of Biochemistry
² Department of Plant Pathology, University of Kentucky, Lexington, Kentucky 40536
and³ Department of Cell and Developmental Biology, Roche Institute of Molecular Biology, Nutley, New Jersey 07110, U.S.A.

Recombinant plasmids containing cDNA copies of the dsRNAs present in one hypovirulent strain of Endothia (Cryphonectria) parasitica, EP713, were constructed and analysed by restriction endonuclease mapping and Southern hybridization. Overlapping inserts of four plasmids were found to represent most of the large dsRNA (L-dsRNA) sequences. Inserts which represented the two termini of the L-dsRNAs, designated ‘homopolymer’ and ‘heteropolymer’, were identified. These plasmids were used as probes in Northern hybridization experiments in an attempt to detect other RNAs having sequences related to those of the L-dsRNAs. No additional RNAs were detected that hybridized to plasmids representing the middle region of L-dsRNAs. However, plasmids representing the termini of L-dsRNAs hybridized to several RNAs in EP713 ranging in size from 300 to 1300 nucleotides. These RNAs were absent from EP155, the isogenic virulent strain of E. parasitica. RNAs related to the homopolymer terminus were more abundant than those related to the heteropolymer terminus. All were sensitive to digestion by S1 nuclease but resistant to RNase III, indicating that they were single-stranded. Only those ssRNAs related to the homopolymer terminus of L-dsRNAs were retained on oligo(dT)-cellulose. Single-stranded M13 phage DNAs containing the insert of one of the plasmids, in each orientation, were used as probes in Northern hybridization experiments. Hybridization to the ssRNAs was observed with only one of these probes, indicating that the transcripts are derived from only one strand of the L-dsRNAs. These results establish the existence of a set of poly(A)-containing ssRNAs that are 3'-coterminal with the homopolymer terminus of L-dsRNAs and have the same polarity as the poly(A)-containing strand of L-dsRNAs.

Keywords: chestnut blight, Endothia parasitica, cloning

Present address: Department of Entomology, University of California, Riverside, California 92521-1037, U.S.A.

Received 8 February 1988; accepted 20 June 1988.