Characterization of double-stranded RNA genetic elements associated with biological control of chestnut blight: organization of terminal domains and identification of gene products.

We have determined the organization within the terminal domains of the major large double-stranded RNA genetic elements associated with the hypovirulent strain EP713 of the chestnut blight pathogen Cryphonectria (Endothia) parasitica. Only the polyadenylated strand contained long open reading frames. Furthermore, only RNA of the same polarity as the polyadenylated strand was detectable in a single-stranded form, indicating that the polyadenylated strand is the coding or plus strand. The organization of the 5'-proximal portion of the plus strand consisted of a 495 nucleotide non-coding leader sequence followed by two overlapping open reading frames. The first, ORF1, extended 957 nucleotides while the second, ORF2, began 68 nucleotides upstream of the ORF1 termination codon and extended at least 1412 nucleotides. No open reading frames of significant size were detected within 0.8 kb of the poly(A) tail. In vitro translation of synthetic transcripts containing ORF1 yielded a polypeptide of Mr 29 kd. The ORF1 product was also detected in lysates of the hypovirulent strain but was absent in lysates of the isogenic virulent strain. It represents the first protein to be identified as a gene product encoded by a hypovirulence-associated double-stranded RNA genetic element.