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

Abstract

The primary structure of cutinase, an extracellular fungal enzyme involved in the penetration of plants by pathogenic fungi, has been determined from the nucleotide sequence of cloned cDNA. Clones containing cDNA made from poly(A)+ RNA isolated from fungal cultures induced to synthesize cutinase were 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 from 279 to 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 two other clones, containing the bulk of the coding region for cutinase, were also completely sequenced, and the results confirmed the sequence obtained with the first clone. 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 far determined by amino acid sequencing (=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.Cutinase, a glycoprotein excreted by phytopathogenic fungi, catalyzes the hydrolysis of cutin, the structural polyester of the plant cuticle (1, 2). Cutinase was shown to be present at the site of fungal penetration of the host plant cuticle and specific inhibition of cutinase was shown to protect plants against fungal penetration and consequently infection (1, 2). The active site of cutinase is composed of a catalytic triad involving serine, histidine, and a carboxyl group (3). The enzyme contains one disulfide bridge, which is essential for the activity of the enzyme (2). Recently the amino acid sequence of the active-serine-containing tryptic peptide of cutinase was determined (4). An understanding of the structure and mechanism of action of cutinase could help in the development of effective inhibitors for use as antipenetrants to protect plants against fungal attack.Recent evidence suggested that the infecting capacity of certain pathogenic fungi can be determined by the ability of the pathogen to produce cutinase (5, 6). Thus the regulation of expression of cutinase gene could be highly relevant to pathogenesis. To investigate these molecular aspects of host-pathogen interaction, labeled cDNA probes for cutinase would be highly beneficial. In this paper we report the cloning of cutinase cDNA and the complete nucleotide sequence of the c...

Regulation of Constitutively Expressed and Induced Cutinase Genes by Different Zinc Finger Transcription Factors inFusarium solani f. sp. pisi(Nectria haematococca)

“…Amino acid sequence of some peptides obtained from one of these enzymes (cutinase 1) matched with that predicted from the cDNA of the induced cutinase transcript (10). The present results reveal the occurrence of other cutinase genes that could encode enzymes that are similar but not identical to cutinase 1.…”

“…Samples of each cutinase were reduced, and SH groups were derivatized with 14 C-labeled iodoacetamide (2.8 Ci/mol, PerkinElmer Life Sciences) and digested with trypsin as described before (10). The digests were fractionated by high pressure liquid chromatography on a Nova-PacC 18 Radial-Pac column (Waters Associates) with a linear gradient of 5-65% acetonitrile in water containing 0.1% trifluoroacetic acid.…”

“…The presence of the multiple cutinase genes was indicated by Southern analysis of the genome of F. solani pisi (11) and by the production of multiple cutinases (8). However, only the highly inducible cutinase gene had been cloned (10). Therefore, we first examined a genomic DNA library for cutinase genes.…”

“…Whether the constitutively expressed cutinase and the inducible cutinase are encoded by the same or different genes is not known, and the nature of the transcription factors that are involved in the constitutive expression of cutinase is unknown. Two different but very similar cutinases had been isolated from F. solani pisi (8,9), and the gene previously cloned matched the amino acid sequence of one of these proteins (10), suggesting that another gene encodes the other. Such a gene had not been previously cloned, although Southern analysis had indicated the presence of multiple cutinase genes in the F. solani pisi genome (11).…”

Infection structures of fungal plant pathogens – a cytological and physiological evaluation

“…Subsequently, the liberated fragments are able to induce de novo synthesis of cutinase (Wolosbuk & Kolattukudy, 1986). The gene encoding cutinase of F. solani has been cloned (Soliday et al, 1984), and it has been shown that plant cutin monomers, together with a protein factor of approximately 100 kDa, are necessary to initiate transcription of the cutinase gene in isolated nuclei (Podila, Dickman & Kolattukudy, 1988). Insertion of the cutinase gene from E. solani into the genome of a wound pathogen of papaya fruits, the ascomycete Mycosphaerella sp., enabled this fungus to infect papaya fruits through the intact cuticle (Dickman, Podila & Kolattukudy, 1989).…”