Analysis of Alternative Oxidase Gene in Mutants of Magnaporthe grisea Deficient in Alternative, Cyanide-resistant, Respiration.

Abstract

The fungicide SSF-126 [(E)-2-methoxyimino-N-methyl-2-(2-phenoxyphenyl) acetamide] induces cyanide-resistant, alternative respiration in the fungus Magnaporthe grisea. The activity of alternative respiration correlates with the expression level of alternative oxidase (AOX). Seven SSF-126-sensitive mutants (named MS) of M. grisea were completely deficient in alternative respiratory activity. Here, a genomic copy for the alternative oxidase gene (AOX) was isolated and analyzed. A 2661-bp AOX genomic DNA fragment containing a 613-bp of the promoter region of AOX was isolated from M. grisea wild type strain by using an inverse PCR method. The AOX was organized as three exons that are separated by short introns. Several cis-acting elements were detected in the 5'-upstream region of AOX. Northern blot analysis of AOX in MS mutants revealed that all the mutants expressed AOX mRNA in response to SSF-126 treatment, but the levels of mRNA in MS2, 5, 8, 9 and 11 were reduced several fold relative to the wild-type. All seven mutants had altered sequences in the AOX gene. These results demonstrate that sensitivity to SSF-126 and activity of alternative respiration in M. grisea are regulated by the AOX gene.