Cloning and structure determination of cDNA for cutinase, an enzyme involved in fungal penetration of plants

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Abstract
The primary structure of cutinase, an extracellular fungal enzyme involved in the penetration of plants by pathogenic fungi, was determined from the nucleotide sequence of cloned cDNA [of Fusarium solani f. sp. pisi]. Clones containing cDNA made from poly(A)+ RNA isolated from fungal cultures induced to synthesize cutinase was screened for their ability to hybridize with the [32P]cDNA for mRNA unique to the induced culture. The 75 cDNA clones thus identified were screened for the cutinase genetic code by hybrid-selected translation and examination of products with anti-cutinase IgG. This method yielded 15 clones containing cDNA for cutinase, and Southern blots showed that the size of the cDNA inserts ranged 279-950 nucleotides. Blot analysis showed that cutinase mRNA contained 1050 nucleotides, indicating that the clone containing 950 nucleotides represented nearly the entire mRNA. This near-full-length cDNA and the restriction fragments subcloned from it were sequenced by a combination of the Maxam-Gilbert and the phage M13-dideoxy techniques. cDNAs from 2 other clones, containing the bulk of the coding region for cutinase, were also completely sequenced, and the results confirmed the sequence obtained with the 1st cloned. A peptide isolated from a trypsin digest of cutinase was sequenced and the amino acid sequence as well as the initiation and termination codons were used to identify the coding region of the cDNA. The primary structure of the enzyme so fat determined by amino acid sequencing (.apprxeq. 40% of the total) agreed completely with the nucleotide sequencing results. Thus, the complete primary structure of the mature enzyme and that of the signal peptide region were ascertained.