M2 is a double-stranded RNA (dsRNA) element occurring in the hypovirulent isolate Rhs 1A1 of the plant pathogenic basidiomycete Rhizoctonia solani . Rhs 1A1 originated as a sector of the virulent field isolate Rhs 1AP, which contains no detectable amount of the M2 dsRNA. The complete sequence (3,570 bp) of the M2 dsRNA has been determined. A 6.9-kbp segment of total DNA from either Rhs 1A1 or Rhs 1AP hybridizes with an M2-specific cDNA probe. The sequences of M2 dsRNA and of PCR products generated from Rhs 1A1 total DNA were found to be identical. Thus this report describes a fungal host containing full-length DNA copies of a dsRNA element. A major portion of the M2 dsRNA is located in the cytoplasm, whereas a smaller amount is found in mitochondria. Based on either the universal or the mitochondrial genetic code of filamentous fungi, one strand of M2 encodes a putative protein of 754 amino acids. The resulting polypeptide has all four motifs of a dsRNA viral RNA-dependent RNA polymerase (RDRP) and is phylogenetically related to the RDRP of a mitochondrial dsRNA associated with hypovirulence in strain NB631 of Cryphonectria parasitica , incitant of chestnut blight. This polypeptide also has significant sequence similarity with two domains of a pentafunctional polypeptide, which catalyzes the five central steps of the shikimate pathway in yeast and filamentous fungi.

A virulent field isolate of Rhizoctonia solani AG 3, Rhs 1AP, has given rise to three sectors in a period of several years. Subculturing of these sectors resulted in three hypovirulent isolates—Rhs 1A1, Rhs 1A2, and Rhs 1A3. We reported previously that five genetically different double-stranded (ds) RNAs occur in these four isolates, with each isolate containing a unique combination of two or more dsRNAs. We report here that all five dsRNA elements occur in the cytoplasm, and none in the nucleus. The mitochondria contains low to moderate concentrations of the four larger dsRNAs. The four isolates were paired in selected combinations in an attempt to transmit specific dsRNAs to cultures lacking these dsRNAs. This approach generated groups of near-isogenic lines possessing the same dsRNA elements. As many as six cultures possessing the same dsRNA genotype were found to have the same degree of pathogenicity. Consistently, acquisition of a 6.4-kb dsRNA brought about increased virulence, whereas the presence or acquisition of a 3.6-kb dsRNA resulted in diminished virulence in the recipient culture. All cultures had the same mitochondrial DNA haplotype as that of the original field isolate, Rhs 1AP. This, in conjunction with the fact that all cultures in this study had a single nuclear DNA origin (Rhs 1AP), indicates a strong correlation between certain phenotypic characters or changes thereof and dsRNA profiles or acquisition of particular dsRNAs, respectively.

The bisegmented genome of a double-stranded (ds) RNA virus from the fungusRhizoctonia solaniisolate Rhs 717 was characterized. The larger segment, dsRNA 1, is 2363 bases long whereas the smaller segment, dsRNA 2, has 2206 bases. The 5′ ends of the coding strands of dsRNA 1 and dsRNA 2 are highly conserved (100% identity over 47 bases), and contain inverted repeats capable of forming stable stem–loop structures. Analysis of the coding potential of each of the two segments showed that dsRNAs 1 and 2 could code for polypeptides of 730 aa (bases 86–2275; molecular mass 86 kDa) and 683 aa (bases 79–2130; molecular mass 76 kDa), respectively. The 86 kDa polypeptide has all the motifs of dsRNA RNA-dependent RNA polymerases (RDRP), and has significant homology with putative RDRPs of partitiviruses fromFusarium poaeandAtkinsonella hypoxylon. The 76 kDa protein shows homology with the putative capsid proteins (CP) of the same viruses. Northern blot analysis revealed no subgenomic RNA species, consistent with the fact that the long open reading frames encoding the putative RDRP and CP cover the entire length of the respective dsRNAs.