Diversity of cutinases from plant pathogenic fungi: cloning and characterization of a cutinase gene from Alternaria brassicicola

Abstract

Alternaria brassicicola produced two cutinase isozymes in the presence of cutin monomers as specific inducers. A cDNA library was constructed from poly(A)+RNA isolated from mycelium incubated with cutin monomers. Specific cDNA clones were selected from the library according to their abilities to hybridize with first strand cDNA prepared from induced cultures, but not from glucose-grown cultures. Cutinase-specific cDNA clones were identified by Southern analysis of plasmid DNA, employing a mixture of two heterologous cutinase cDNAs and one cutinase gene as probes. The largest 984 bp insert found among positive clones contained the entire cutinase coding region composed of 209 amino acids. The amino acid sequence predicted from the cDNA nucleotide sequence contained amino acids and sequences highly conserved among fungal cutinases. The sequences of four additional positive cDNAs were identical and thus gave no indication for the presence of a second gene of origin. Southern analysis of genomic DNA of A. brassicicola yielded a similar result. The structural gene of cutinase (CUTAB1) was contained within a 1545 bp genomic DNA fragment. Nucleotide sequences of the cDNA and the gene were identical, with the exception of one intron of 56 bp. The location of the intron was identical with introns identified in other fungal cutinase genes. The potential role of CUTAB1 in pathogenicity will be determined by gene disruption.