Yasuyuki Kubo

STRUCTURAL ANALYSIS OF PKS1, A POLYKETIDE SYNTHASE GENE INVOLVED IN MELANIN BIOSYNTHESIS IN COLLETOTRICHUM LAGENARIUM

Albino mutants (Pks−) of Colletotrichum lagenarium form nonmelanized appressoria and possess little penetrating ability on the host plant. The defect in albino mutant 79215 (Pks−) is considered to lie in pentaketide biosynthesis and/or pentaketide cyclization during melanin biosynthesis. The cosmid pAC7, carrying the PKS1 gene, when transformed into the albino mutant restores the wild-type melanin phenotype. We have determine the DNA sequence and the transcriptional organization of the PKS1 gene. The PKS1 gene contains one open reading frame, consisting of 3 exons separated by two short introns. The predicted PKS1 polypeptide consists of 2187 amino acids and shows significant similarities with other polyketide synthases, particularly that encoded by wA in Aspergillus nidulans, involved in conidial pigmentation. The PKS1 gene contains highly conserved β-ketoacyl synthase, acetyl/malonyl transferase, and acyl carrier protein domains. We propose that the C. lagenarium PKS1 gene encodes a polyketide synthase involved in melanin biosynthesis.

Melanin biosynthesis as a prerequisite for penetration by appressoria of Colletotrichum lagenarium: Site of inhibition by melanin-inhibiting fungicides and their action on appressoria

Abstract Melanin biosynthesis by appressoria was studied in relation to their penetrating ability using tricyclazole [5-methyl-1,2,4-triazolo(3,4-b)benzothiazole], pp 389 [4,5-dihydro-4-methyltetra-zolo(1,5-a)quinazolin-5-one], and pyroquilon [1,2,5,6-tetrahydropyrrolo(3,2,1-i,j)quinolin-4-one], and color mutants of Colletotrichum lagenarium. Tricyclazole at 100 μM inhibited melanin biosynthesis by appressoria of C. lagenairum 104-T, and caused accumulation of 3,4-dihydro-4,8-dihydroxy-1(2H)naphthalenone (DDN) in the culture medium. By contrast, DDN was not detected in culture media of tricyclazole-treated mutant 8015, which is defective in the enzyme involved in the conversion of scytalone to 1,3,8-trihydroxynaphthalene (1,3,8-THN). Vermelone restored melanization of appressoria of albino mutant 79215 and of the parent strain 104-T treated with tricyclazole, pp 389, and pyroquilon; however, scytalone restored melanization only in appressoria of albino mutant 79215. These results indicate that tricyclazole, pp 389, and pyroquilon inhibit the conversion of 1,3,8-THN to vermelone in the melanin biosynthetic pathway of appressoria of C. lagenarium. Colorless appressoria formed in the presence of the three melanin-inhibiting chemicals germinated laterally on nitrocellulose membranes and rarely penetrated the membranes. On the other hand, when pigmented appressoria were restored by application of vermelone in the presence of the three chemicals, lateral germination of the appressoria was largely suppressed, and the membranes were effectively penetrated. From these results, it is concluded that the major effect of tricyclazole, pp 389, and pyroquilon on appressoria of C. lagenarium, causing failure of penetration, is the inhibition of melanization. Effects of the chemicals on other metabolic functions can be precluded as significant factors affecting the penetration process.

Genetic analysis and characterization of Cochliobolus heterostrophus colour mutants

Twenty-three colour mutants of Cochliobolus heterostrophus were obtained by mutagenesis. Mutants at six loci were identified; alb1 (type-1 white), alb3 (type-2 white), sal1 (salmon), brn1 (brown), pgr1 (pale green), and scr1 (scarlet). Crossing experiments indicated that sal1 and pgr1 were in the same linkage group 5·8% apart, and that alb1, alb3 , and brn1 were closely linked. No linkage was observed between the three closely linked genetic markers ( alb1, alb3 , and brn1 ) and sal1 or pgr1. scr1 was independent of both of these linkage groups. Furthermore, no linkage was found between these loci and the mating type ( MAT1 ) locus. Accumulation of scytalone in the sal1 mutant indicated that the melanin of this fungus is formed from 1,8-dihydroxynaphthalene, derived in turn from pentaketide, and that the genetically deficient step in this mutant was at the conversion of scytalone to 1,3,8-trihydroxynaphthalene in the melanin biosynthesis pathway. The pgr1 mutant failed to form melanin from 1,8-dihydroxynaphthalene, which suggested that the deficiency of the pgr1 mutant is involved in oxidation of 1,8-dihydroxynaphthalene. The conversion of 1,3,8-trihydroxynaphthalene to vermelone may be blocked in the brn1 mutant. The defect of the alb1 mutant seemed to be involved in some process prior to scytalone formation. The alb3 mutant, which was not coloured by scytalone, was one of the melanin-deficient mutants, but its genetically blocked point was not characterized.

Scytalone as a natural intermediate of melanin biosynthesis in appressoria ofColletotrichum lagenarium

Abstract Melanin biosynthesis of appressoria inColletotrichum lagenaru was studied using color mutants. Mutant 8015 ofC. lagenarium obtained byN-methyl-N′-nitro-N-nitroso-guanidine treatment formed a red-brown colony and secreted a substance which restored black coloration to colonies of albino mutants. This substance was identified as scytalone (3,4-dihydro-3,6,8-trihydroxy-1(2H)naphthalenone). Nineteen albino mutants formed colorless appressoria, but in the presence of 0.75 mM scytalone, the albino mutants formed darkly pigmented appressoria indistinguishable from those of the parent strain. Furthermore, the time course of appressorial pigmentation of albino mutants in the presence of scytalone was the same as that of the parent strain. On nitrocellulose membranes and host cucumber cotyledons, the colorless appressoria of albino mutants germinated laterally to form secondary appressoria, and consequently had little ability to form penetration hyphae. In the presence of 0.75 mM scytalone, however, the pigmented appressoria penetrated nitrocellulose membranes and host cucumber cell walls similarly to those of the parent strain. From these results, we conclude that scytalone is a normal precusor of melanin in appressoria, and that appressorial pigmentation is essential for the formation of penetration hyphae inC. lagenarium.

Genetic analysis of genes involved in melanin biosynthesis of Cochliobolus miyabeanus

Abstract Color mutants of Cochliobolus miyabeanus defective in melanin biosynthesis were isolated. Although the wild-type strain KU-13 formed dark green colonies, color mutants formed white, brown, and gray colonies or white colonies with red pigment secretion. From the white mutant which secreted red pigment, designated scy , a melanin precursor which restored melanization of albino mutants alm-1 was isolated and identified as scytalone. This indicated that scy mutant was defective in the conversion of scytalone to 1,3,8-trihydroxynaphthalene and that melanin of this fungus is of pentaketide origin formed from oxidation of 1,8-dihydroxynaphthalene. Albino mutants alm-1 were considered to be defective in pentaketide cyclization and brown mutants brm were considered to be defective in the conversion of 1,3,8-trihydroxynaphthalene to vermelone. Albino mutants alm-2 whose coloration was not restored by application of scytalone were also isolated. The alm-2 gene was believed to be a gene transactively regulating the pentaketide cyclization and conversion of scytalone. From crossing experiments among the color mutants, it was indicated that alm-1, alm-2 , and brm were linked and that scy segregates independently of these three mutant loci. Crossing of a methionine requiring mutant with alm and scy indicated that the three loci segregate independently of each other.

Regulation of melanin biosynthesis during appressorium formation inColletotrichum lagenarium

Regulation of melanin biosynthesis in relation to appressorium differentiation ofColletotrichum lagenarium was investigated. When spores of the parent strain 104-T were incubated at 24°C, appressorial pigmentation started at 6 h of incubation and was preceded by appressorim swelling; appressoria were darkly pigmented at 12 h of incubation. The same time course of appressorial pigmentation was observed in albino mutant 79215 when scytalone, a natural precursor of melanin biosynthesis, was applied before the swelling of appressoria. In accordance with this result, [14C]scytalone was not incorporated into germlings of albino mutant 79215 before the swelling of appressoria. Cycloheximide applied 1 h or more after incubation of spores of the parent strain 104-T, or of albino mutant 79215 treated with scytalone, inhibited neither appressorium formation nor appressorial pigmentation. These results indicate that enzymes involved in melanin biosynthesis subsequent to scytalone are preexisting enzymes or synthesized as inactive forms during 1 h of incubation, and that they are activated during appressorium differentiation. In addition, an early step(s) prior to scytalone in the melanin biosynthesis of appressoria was temperature sensitive; when colorless appressoria of the parent strain 104-T formed during 6 h of incubation at 24°C were postincubated at 32°C, the appressoria did not melanize, whereas application of scytalone to the postincubation at 32°C permitted melanization of the appressoria. Also, albino mutant 79215 formed melanized appressoria during postincubation at 32°C in the presence of scytalone. These results indicate that high temperatures inhibit melanin biosynthesis by inhibiting one or more steps prior to scytalone synthesis.

Use of mutants to indicate factors prerequisite for penetration of Colletotrichum lagenarium by appressoria

Three mutants of Colletotrichum lagenarium , with appressoria that could not penetrate nitrocellulose membranes or cell walls of the host plant, were obtained by mutagenic treatment. Appressoria of these mutants were identical in shape and colour to those of the parent strain. Scanning electron microscopy revealed that one mutant (83182) formed a penetration peg at the basal part of the appressoria and that the other mutants (82335 and 83348) did not form a penetration peg. The latter mutants are considered to have a defect in morphogenesis of the penetration peg. Application of Czapek liquid medium to mutant 83182 after appressorium formation partially restored appressorial penetration without halo formation; this mutant synthesized a protein, P-95, which was closely related to cellulose degrading enzymes. These results suggest that mutant 83182 is deficient in a secretion or translocation system for these enzymes.

Restoration of pathogenicity of a penetration-deficient mutant of Collectotrichum lagenarium by DNA complementation

Infection byColletotrichum lagenarium requires formation of an appressorium and of a penetration peg. A mutant, 83 348, defective in morphogenesis of the penetration peg was unable to penetrate into cellulose membranes or infect cucumber leaves. DNA transformation using a wild-type genomic library constructed in pKVB resulted in two transformants, Ppr1 and Ppr2, with restored penetration peg formation, from 2,000 benomylresistant transformants. However, penetration into cellulose membranes by these transformats ranged from 30 to 40% compared to greater than 90% by wild-type. Southern-blot hybridization showed that a single copy of a cosmid clone had integrated into the genome of the transformants. A 12.0-kbp fragment of the cosmid vector with the flanking region of wild-type genomic DNA was recovered by plasmid rescue from Ppr1. Using the flanking DNA sequences as a probe for colony blot hybridization, a genomic clone was identified and designated pRP46. Transformants obtained following transformation with pRP46 were able to penetrate cellulose membranes. The penetration frequency of pRP46 transformants ranged from 25 to 65%. Transformants were also pathogenic on cucumber.

Purification of a melanin biosynthetic enzyme converting scytalone to 1,3,8-trihydroxynaphthalene from Cochliobolus miyabeanus

Abstract A melanin biosynthetic enzyme, scytalone dehydratase, which converts scytalone to 1,3,8-trihydroxynaphthalene was purified from mycelia of Cochliobolus miyabeanus . Enzymatic reactions were carried out under anaerobic conditions to prevent unfavorable oxidative reactions. This enzyme had an optimum pH near 8.2 and mol w 23,000 Da. The enzymatic reaction did not require NADPH as a cofactor and was not inhibited by the melanin biosynthesis inhibitors, tricyclazole, PP 389, pyroquilon, PCBA, and phthalide

Inhibition of melanin biosynthesis by cerulenin in appressoria ofColletotrichum lagenarium

Abstract Cerulenin [(2S) (3R)2,3-epoxy-4-oxo-7,10-dodecadienoylamide], a polyketide synthesis inhibitor, inhibited appressorial pigmentation of Colletotrichum lagenarium at concentrations higher than 10 μg/ml. The inhibitory concentrations of cerulenin inhibited neither spore germination nor appressorium formation but did inhibit penetration of nitrocellulose membranes by penetrating hyphae from appressoria. The colorless appressoria germinated laterally on nitrocellulose membranes and rarely penetrated them. In the presence of cerulenin, treatment with scytalone, a melanin precursor for this fungus, restored appressorial pigmentation and also penetration by appressoria of nitrocellulose membranes. In Czapek liquid medium, mutant 8015 which is defective in the enzyme involved in the conversion of scytalone to 1,3,8-trihydroxynaphthalene in the melanin biosynthetic pathway produced scytalone after 9 days of incubation when hyphal growth reached the maximum level. Application of 10 μg/ml of cerulenin at 9 days of incubation inhibited further production of scytalone by mutant 8015. From these results, it is concluded that appressorial melanin of C. lagenarium was synthesized via the polyketide pathway.