Benomyl resistance of Colletotrichum acutatum is caused by enhanced expression of β-tubulin 1 gene regulated by putative leucine zipper protein CaBEN1

Colletotrichum acutatum, the fungus causing anthracnose disease of various fruits and crops, has inherent resistance to benomyl. The mechanism underlying its resistance to benomyl remains unclear. We generated a benomyl-sensitive mutant CAT7-150 by transformation-mediated insertional mutagenesis. Subsequently, a CaBEN1 gene was isolated from CAT7-150 by plasmid rescue and sequenced. CaBEN1 encodes a protein of 818 amino acids containing a leucine zipper motif, and the amino acid sequence has significant similarity to hypothetical proteins found in Gibberella zeae and other fungi. Disruption and complementation of the CaBEN1 gene demonstrated that CaBEN1 was necessary for resistance to benzimidazole fungicides, but was not essential for mycelial growth. Northern analysis suggests that CaBEN1 has an important role in transcriptional activation of CaTUB1 in response to benomyl. Benomyl resistance of CaBEN1-disrupted transformant was recovered by overexpression of CaTUB1. Our results clearly demonstrate that the benomyl resistance is due to enhanced expression of CaTUB1, which is controlled by CaBEN1.