Structure of Cutinase Gene, cDNA, and the Derived Amino Acid Sequence from Phytopathogenic Fungi

Cutinase is an extracellular fungal enzyme that allows pathogenic fungi to penetrate through the cuticular barrier into the host plant during the initial stages of the fungal infection. mRNA isolated from glucose-grown Colletotrichum capsid, induced to produce cutinase by the addition of cutin hydrolysate, was used to prepare cDNA which was cloned in the expression vector X gtl 1. The primary structure of the cutinase from C. capsid was deduced from the nucleotide sequence of the cloned cutinase cDNA. Amino acid sequences of two tryptic peptides isolated from cutinase produced by C. capsid completely matched with two segments of the amino acid sequence deduced from the nucleotide sequence, strongly suggesting that the cloned cDNA was authentic cutinase cDNA. The cDNA clone was used as a probe to screen C. capsid and Colletotrichum gloeosporioides genomic libraries constructed in Charon 35 and EMBL 3, respectively. The nucleotide sequences of the cutinase structural genes from C. capsid and C. gloeosporioides were also determined. SI mapping was used to reveal the transcriptional start sites and polyadenylation site of the primary transcript from C. capsid. The primary sequences and gene structure of the enzymes from the Colletotrichum species were compared with the primary structure and gene structure of a cutinase from Fusarium solani f. sp. pisi. A comparison of the deduced primary structures of the enzymes showed that residues involved in the catalytic triad and disulfide cross-linking of cutinase are strongly conserved. Yet, only 43% of the residues are conserved between all three enzymes. A comparison of the structure of the three genes revealed the location of the single intron has been conserved. The transcriptional start site of the C. capsid gene was centered on the sequence TCCAGACCA, the core of which (CAGAC) is found repeated after 21 nucleotides. The same core sequence, repeated after 11 nucleotides, was also identified in the 5’ nontranslated regions of the C. gloeosporioides and F. solani genes.