A single nucleotide substitution in the alpha a gene confers oat pathogenicity to barley stripe mosaic virus strain ND18

Abstract

A 236-nucleotide region from the alpha a gene of strain CV42 (pathogenic to oat), when substituted for the homologous region in strain ND18 (nonpathogenic to oat), was shown previously to confer a near wild-type oat pathogenicity to this strain (Weiland and Edwards, 1994, Virology 201: 116-126). The data suggested that six amino acid substitutions in the alpha a gene were responsible for the differences in oat pathogenicity, and that threonine-724, encoded by CV42, might be a critical amino acid in determining pathogenicity of barley stripe mosaic virus (BSMV) to oat. In the present work, codons specifying T-724, I-764, and N-785 (encoded by CV42 RNA alpha) were substituted individually and in combination for those coding for P-724, T-764, and K-785 (encoded by ND18 RNA alpha), respectively, by site-directed mutagenesis. The core K-733, T-734, and K-736 positions (CV42) were substituted for Q-733, S-734, and Q-736 (ND18) as a single block. The results of inoculations with these mutants indicate that the C2261-->A2261 nucleotide substitution (P-724-->T-724) by itself is sufficient to enable strain ND18 to infect oat plants, although poorly. Additional substitution of CV42 codons into ND18 RNA alpha at the remaining five positions altered symptom type, decreased the timing of the appearance of symptoms, and increased the percentage of plants infected per inoculation. Nonetheless, all mutants accumulated to similar levels in inoculated oat protoplasts after a 24-h period. Using a recombinant RNA beta from which beta-glucuronidase could be expressed, results were obtained suggesting that the multiplication of strain ND18 and the nonpathogenic variants generated in the study was restricted in the inoculated leaf. The data indicate a potential pathway by which pathogenicity to oat evolved in BSMV.