Amino Acid Alterations in the β-Tubulin of Metarhizium anisopliae That Confer Benomyl Resistance

We cloned the β-tubulin genes from the wild type strain and two benomyl-resistant mutants of Metahizium anisopliae and determined their nucleotide sequences. A β-tubulin encoding 448-residue protein from wild type M. anisopliae shows strong homology to other β-tubulins. The coding region is interrupted by four introns. Comparisons of intron position between the M. anisopliae gene and other fungal β-tubulin genes show considerable positional conservation. The mutations responsible for benomyl resistance were determined in two spontaneous mutants, S-18 and S-19. One mutant S-18 substituted glutamate for asparagine at position 33 and lysine for glutamine at position 134. The other mutant S-19 showed alterations at three positions of β-tubulin; arginine for tryptophan at position 21, lysine for asparagine at position 33, and phenylalanine for leucine at position 240. These data suggest that regions of β-tubulin containing amino acids 21, 33,134, and 240 interact to form the binding site of benomyl.