Combined gas chromatography-mass spectrometry showed that C21, C23, and C25 n-alkanes accumulated in the suberized layers during wound healing of cores of potato tuber tissue. Treatment (10 min) of freshly-cut tissue with trichloroacetate (TCA), an inhibitor of fatty-acid chain elongation, severely inhibited accumulation of hydrocarbons and fatty alcohols associated with the suberized layer in the wound healing tissue (maximum inhibition at 4 mM) but had very little effect on the deposition of the major aliphatic components of the suberin polymer. This preferential inhibition of wax synthesis resulted in severe inhibition of the development of diffusion resistance of the tissue to water vapor. These results strongly indicate that the waxes associated with the suberin polymer, rather than the polymer itself, consitute the major diffusion barrier formed during wound healing. Electron-microscopic examination showed that inhibition of wax synthesis by TCA disrupted the formation of the lamellar structure of suberin specifically by preventing the formation of the light bands. This evidence strongly suggests that the light bands in the suberin complex are composed of waxes.

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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...

Rabbit antibody to cutinase-I, isolated from Fusarium solani f. pisi, was conjugated to ferritin. With this fermtin-conjugated antibody it was shown that germinating spores of this fungus excreted cutinase during the penetration of the host pisum sativum. This result constitutes the most specific and strongest evidence for an enzymic penetration of a plant cuticle by a pathogen during infection.Cuticle constitutes the first barrier between a higher plant and its environment. The mode of penetration of this barrier by phytopathogenic fungi has been a controversial subject for a long time (4). Penetration is either by the mere physical force of fungal growth or by enzymic dissolution of the cuticle. The nature of the opening created by the penetrating fungus has been used as a supporting evidence for the enzymic penetration theory (5). However, in the absence of convincing evidence to support either side, the arguments remained unsettled. The structural component of plant cuticle is a biopolyester, cutin, derived from hydroxy and epoxy fatty acids (3) and therefore this polymer constitutes the major physical barrier between the invading fungus and the plant. Recently we have isolated in homogeneous form cutinases from the extracellular fluid of several fungal pathogens grown on cutin as the sole source of carbon (6, 7). We were able to prepare antibodies against these pure enzymes and using ferritin-labeled antibody a specific test was performed to determine whether a fungal pathogen excretes cutinase during the cuticular penetration phase of the infection process. The results presented in this communication provide convincing evidence that Fusarium solani f. pisi excretes cutinase during infection of the host tissue. MATERIALS AND METHODSCutinase-I was isolated in homogeneous form from the extracellular fluid of F. solani f. pisi grown on apple cutin as the sole source of carbon (6). Rabbit antiserum was prepared against the pure cutinase (7) and the immunoglobulin fraction was isolated from this antiserum by repeated ammonium sulfate fractionation (2). Ferritin was conjugated to the immunoglobulin using xylylene metadiisocyanate as the coupling agent (1 Epicotyls from 5-day-old etiolated Pisum sativum seedlings were excised and rinsed in distilled H20 several times. The cuticular surface of these epicotyl segments, 0.5 cm long, was inoculated with small (about 40-,ul) droplets of a thick suspension of micro-and macroconidia of F. solani f. pisi from 15-to 20-day-old cultures grown on 2% potato dextrose agar. The inoculated segments of epicotyl were placed in a Petri dish in humid atmosphere. After varying periods of incubation at room temperature the infected segments were immersed in the ferritin-conjugated anticutinase-I preparation for 30 min. Controls were similarly treated with nonconjugated ferritin or ferritinconjugated immunoglobulin fraction prepared from a rabbit which was not immunized with cutinase-I. The incubated specimens were rinsed in 0.1 M Na-phosphate buffer (pH 7.2) for 30 min and fix...

The cutinase gene from Fusarium solani f. sp. pisi (Nectria hematococa) was cloned and sequenced. Sau3A fragments of genomic DNA from the fungus were cloned in a k Charon 35 vector. When restriction fragments generated from the inserts were screened with 5' and 3' probes from cutinase cDNA, a 5.5-kilobase SstI fragment hybridized with both probes, suggesting the presence of the entire cutinase gene. A 2,818-base pair segment was sequenced, revealing a 690-nucleotide open reading frame that was identical to that found in the cutinase cDNA with a single 51-base pair intron. Transformation vectors were constructed containing a promoterless gene for hygromycin resistance, Which was translationally fused to flanking sequences of the cutinase gene. When protoplasts and mycelia were transformed with these vectors, hygromycin-resistant transformants were obtained. Successful transformation was assessed by Southern blot analysis by using radiolabeled probes for the hygromycin resistance gene and the putative promoter. The results of'Southern blot analysis indicated that the plasmid had integrated into the Fusarium genome and that the antibiotic resistance was a manifestation of the promoter activity of the cutinase flanking sequences. Transformation of Colletotrichum capsici with the same construct confirmed the promoter activity of the flanking region and the integration of the foreign DNA. Transformation and deletion analysis showed that promoter activity resided within the 360 nucleotides immediately 5' to the cutinase initiation codon.Several lines of evidence strongly suggested that the ability to produce cutinase might determine whether a fungal pathogen can penetrate intact plant organs and infect them (22). In vitro translation of mRNA from cultures induced to produce cutinase (25) revealed that the primary translation product of cutinase was 2,100 daltons larger than the mature enzyme (13). By using the induced mRNA, a cDNA library was constructed, cutinase cDNA was isolated, and the nucleotide sequence was determined, revealing an open reading frame for a 23,951-dalton protein (44). The availability of the cloned cDNA provided a probe for the cutinase gene.Recently, strong evidence was presented that fungal spores sense cotitact with a plant surface by using the small amount of cutinase carried by the spore. Upon contact with the cuticle, the enzyme generates small amounts of cutin monomers and the most unique monomers trigger cutinase synthesis (47). Dot blot analysis with cloned cutinase cDNA as the probe demonstrated that cutinase gene transcripts became measurable within 15 min after the spores contacted cutin. To determine how the cutin monomers trigger cutinase gene expression, a knowledge of the cutinase gene structure is essential. In this paper we report the cloning of the cutinase gene from Fusarium solani f. sp. pisi and its nucleotide sequence, including the sequences of the flanking regions. We also report the transformation of F. solani f. sp. pisi and Colletotrichum capsici using the hygromycin r...

ci-Hydroxylation of fatty acids, which is a key reaction in the biosynthesis of cutin and suberin, has been demonstrated for the first time in a cell-free preparation from a higher plant. A crude microsomal fraction (105,000g pellet) from germinating embryonic shoots of Vicia faba catalyzed the conversion of palmitic acid to ci-hydroxypaimitic acid. As the crude cell-free preparation also catalyzes the formation of other hydroxy acids such as a-and l8-hydroxy acids, the o-hydroxylation product was identified by gas chromatography on a polyester column and reverse phase, high performance liquid chromatography, two techniques which were shown to resolve the positional isomers. Gas chromatographic analysis of the dicarboxylic acid obtained by CrO3 oxidation of the enzymic product also confirmed the identity of the enzymic whydroxylation product. This enzymic hydroxylation required 02 and NADPH, but substitution of NADH resulted in nearly half the reaction rate obtained with NADPH. Maximal rates of w-hydroxylation occurred at pH 8 and the rate increased in a sigmoidal manner with increasing concentrations of palmitic acid. This w-hydroxylation was inhibited by the cassicad mixed function oxidase inhibitors such as metal chelators (ophenanthroline, 8-hydroxyquinoline, and a,a-dipyridyl), NaN3 and thiol reagents (N-ethylmaleimide andp-chloromercuribenzoate). As expected of a hydroxylase, involving cytochrome P450, the present c-hydroxylase was inhibited by CO and this enzyme system showed unusually high sensitivity to this inhibition; 10% CO caused inhibition and 30% CO completely inhibited the reaction. Another unusual feature was that the inhibition caused by any level of CO could not be reversed by light (420-460 nm).Palmitic acid, 16-hydroxypalmitic acid, and 10,16-dihydroxypalmitic acid constitute the most common components of cutin (10,15). Exogenous labeled palmitic acid was incorporated into both hydroxy acids, whereas labeled 16-hydroxypalmitic acid was incorporated specifically into the dihydroxy acid in Vicia faba leaf discs (12). From such results we concluded that palmitic acid was hydroxylated first at the co-carbon followed by another hydroxylation at C-10. Incorporation of specifically labeled precursors into cutin and suberin in tissues slices showed that w-hydroxylation of linoleic acid and/or oleic acid was also involved in the biosynthesis of the major monomers of cutin and suberin (5,13 (soluble PVP, Mr 400,000) was provided by GAF Corp. Carbon monoxide (research grade, 99.99%) was purchased from Matheson Gas Products, while the other gases were obtained from Liquid Air, Inc. Hexadecane-1,16-dioic acid was purchased from K & K Lab., 16-hydroxyhexadecanoic acid from Aldrich Chemical Co., and 2-hydroxyhexadecanoic acid from Analabs, Inc. Hexadecane-1,16-diol and hexadecane-1,2-diol were prepared by LiAlH4 reduction of hexadecane-1,16-dioic acid and 2-hydroxyhexadecanoic acid, respectively. Both of these diol standards were purified by TLC on Silica Gel G with diethyl ether-hexane-methanol (...

A long open reading frame (ORF) closely linked to the Cephalosporium acremonium gene cefEF was identified by DNA sequencing. The cefEF gene encodes the enzyme involved in cephalosporin C (CPC) biosynthesis known as expandase/hydroxylase. Complementation of a C. acremonium cefG mutant, as well as expression of the gene in Aspergillus niger, showed this ORF to be the cefG gene, encoding cephalosporin C acetyltransferase, which catalyzes the last step in CPC biosynthesis. Analysis of transformants containing additional copies of this gene showed that a direct relationship exists between cefG copy number, cefG message levels, and CPC titers. This gene encodes an enzyme for what may be a rate-limiting step in CPC production.

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