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Regulation of melanin biosynthesis during appressorium formation inColletotrichum lagenarium
EXPERIMENTAL MYCOLOGY8, 364-369 (1984)
Regulation
of Melanin Biosynthesis during Appressorium in Colleto trichum lagenarium
Formation
YASUYUKI KUBO, IWAO FURUSAWA, AND MASAKI YAMAMOTO Laboratory
of Plant Pathology, Faculty of Agriculture,
Kyoto University, Kyoto 606, Japan
Accepted for publication August 22, 1984 KUBO, Y., FLJRUSAWA,I., AND YAMAMOTO, M. 1984. Regulation of melanin biosynthesis during appressorium formation in Colletotrichum lagenarium. Experimental Mycology 8, 364-369. Regulation of melanin biosynthesis in relation to appressorium differentiation of Colletotrichum 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, [i4C]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. 0 1984 Academic press, Inc. INDEX DESCRIPTORS:Colletotrichum lagenarium; appressoria; melanin; differentiation; albino; pigment; scytalone; [i4C]scytalone; enzymes; anthracnose.
In some plant pathogenic fungi, a prerequisite to invasion of the host plant is a differentiation of the nonpathogenic germinated spore into a pathogenic stage with an appressorium that functions as an infection structure. We have already reported that melanization of appressoria in Colletotrichum lagenarium is essential for expression of the penetration ability of the appressoria; colorless appressoria of albino mutants, or those of the parent strain 104-T in the presence of tricyclazole (5 methyl[l,2,4]triazolo[3,4 - blbenzothiazole), pp 389 (4,5 - dihydro - 4 - methyltetrazolo[1,5 - alquinazolin - 5 - one), or pyroquilon (1,2,5,6
- tetrahydropyrro]o[3,2,1
lin - 4 - one), inhibitors
- iJlquh@
of melanin
biosyn364
0147-5975184 $3.00 Copyright AU rights
0 1984 by Academic Press, Inc. of reproduction in any form reserved.
thesis, germinated laterally to form secondary appressoria, and consequently had little ability to penetrate the host plant (Kubo et al., 1982a,b, 1983, 1984; Suzuki et al., 1982). The melanin of appressoria in C. lagenarium was indicated to be allomelanin derived from 1,8-dihydroxynaphthalene (1,8DHN)‘; appressoria of albino mutant 79215 converted both scytalone and vermelone to melanin, and the parent strain 104-T in the presence of tricyclazole, pp 389, or pyroquilon converted vermelone but not scyta’ Abbreviations used: 1,8-DHN, 1 ,S-dihydroxynaphthalene; PSA, potato sucrose agar; 1,3,6,8-THN, 1,3,6,8-tetrahydroxynaphthalene; 1,3,8-THN, 1,3,8trihydroxynaphthalene.
MELANIN
REGULATION
lone (Kubo et al., 1983, 1984). The melanin biosynthetic pathway was partially elucidated (Kubo et al., 1984). Our previous observation of the penetration process with the albino mutant 79215 revealed that melanized cells were confined to the appressoria; spores, germ tubes, and penetration hyphae were not colored by application of scytalone, indicating that melanin biosynthesis was a developmentally regulated system (Kubo et al., 1983). Although the relationships between cell differentiation and regulation of melanin biosynthesis mediated by polyphenoloxidases have been investigated in several fungi such as Schizophyllum commune (Leonard, 1971; Leonard and Phillips, 1973; Phillips and Leonard, 89X), Physarum polycephalum (Chet and Htittermann, 1977), Podosporn anserina (Esser, 1968), and Scleuotium rofiii (Chet and Hiittermann, 1982), the regulation of allomelanin synthesis is poorly understood. In this study, the relation of appressorium differentiation of C. lagenarium to the metabolic development of melanin biosynthesis was investigated using the albino mutant and a melanin precursor, scytalone. MATERIALS
AND
METHODS
Fungus. Colletotrichum lagenarium (Pass.) Ellis and Halsted strain 104-T (stock c&u-e in Laboratory of Plant Pathology, Kyoto University) was used as a parent strain. An albino mutant 79215 was isolated from the parent strain previously by ultraviolet n-radiation (Kubo et al., 1982a). Cultural condition and spores. The two strains of 6. lagenarium were cultured on potato sucrose agar (LISA) medium. Spores on mycehal mats cultured on PSA medium at 24°C for 7 days were collected with a brush and washed with ice-cold distilled water three times. spore germination. Spores suspended in sterile deionized water (1 ml) to give about 1 x lo5 spores/ml were poured into a Petri dish (diameter 2 cm) and incubated at 24°C.
IN
Colletot~ichum
365
Scytalone was applied as solution in the spore suspension or by exchanging the water for 1 mB of solution containing scytalone at various time after the onset of incubation. Scytalone was obtained from either mutant 8015 of C. Eagenarium (Kubc~ et al., 1983) or Verticillium dahliae brm-I (ATCC 44571) (Bell et &., B976a). [14C]Scytalone. V. dahliae brm-1 was cultured at 24°C in two 3QO-ml flasks each containing 100 ml of sucrose nitrate medium (Brandt, 1964). After incubation for 8 days, 100 bCi [U-14C]acetic acid (sodium salt; 689 @/mg) was added to each tiask. which was incubated in a sealed glass case containing NaOH for an additional 7 days. The whole 25-day-old culture solution was adjusted to pH 5.0 with H,P0,, saturated with NaCl, and extracted with ethyl acetate three times (100 ml each). The ethyl acetate extract was combined and concentrated by evaporation under reduced pressure. The concentrate was applied to a thin-layer plate (Kieselgel 60 F,,,, 0.25 mm thick, Merck) and developed with benzene-acetone (l/l, v/v). The developed thin-layer piate was observed under 365 nm hght for fluorescence to indicate t e location of scytalone as judged from comparison with a standard. The spot was scraped off with a razor blade and ehtte with ethyl acetate. The ethyl acetate eluate was concentrate to dryness to give white crystals (scytalone; 0.5 mg, sp act 3.12 x lo7 cpmlmg). Incorporation of [14Clscytalone into appressorial melanin. To investigate the activity of enzymes involved in melanin biosynthesis subsequent to scytalone synthesis during appressorium formation, 2 mH of spore suspension (5 X PO4 spores/ml) was poured into a Petri dish (diame cm) and the germlings were labele [14C]scytalone (7.2 x IO5 cpm) for successive 2-h periods through 12 h of incubation. At the end of each period, the Petri dish was then placed on ice and mefanin infermediates were removed by exchanging the incubation solution successively with ace-
366
KUBO,
FURUSAWA,
tone, ethyl acetate, acetone, and water (2 ml each for 30 min). After the extraction of the intermediates, the germlings were collected on Whatman 3MM filter paper (diameter, 24 mm). Each filter paper was washed three times with 30 ml of cold distilled water. The radioactivity was measured in a liquid scintilation spectrophotometer (Packard Model 2425) as described previously (Furusawa et al., 1977). RESULTS
Time Course of Appressorium Formation and Pigmentation When spores of the parent strain 104-T were incubated at 24°C germination occurred after 3 h of incubation. The tips of germ tubes swelled until 6 h of incubation and structurally mature, colorless appressoria were observed. As the appressorium swelled, protoplasm migrated from the spore into the appressorium. Visible pigmentation of appressoria started after they reached full size and the intensity of pigmentation gradually increased to form completely pigmented appressoria at 12 h of incubation. More than 95% of spores differentiated to form appressoria almost synchronously following the time course described above. Melanin Biosynthetic Activity Subsequent to Scytalone Synthesis The melanin biosynthetic activity subsequent to scytalone synthesis during appressorium formation of C. Zagenarium was also investigated. First, the time course of appressorial pigmentation was determined by applying scytalone to albino mutant 79215 at various times after the onset of spore incubation. When 1 n-&! scytalone was applied to albino mutant 79215 after 0, 2, or 4 h of incubation, visible pigmentation of appressoria started 6 h after incubation; at this time the migration of protoplasm from spores into appressoria was complete and appressoria were swollen to full size. The
AND YAMAMOTO
intensity of appressorial pigmentation gradually increased during further incubation until 12 h. In these case, the time course of appressorial pigmentation for albino mutant 79215 was the same as that of the parent strain 104-T (Fig. 1). When scytalone was applied after 6 h of incubation, visible pigmentation of appressoria was observed from within 30 min after the treatment and completely pigmented appressoria were observed by 12 h (Fig. 1). Addition of scytalone after the formation of structurally mature appressoria permitted visible pigmentation of appressoria from within 30 min and the duration needed for completion of pigmentation of appressoria depended on the time of scytalone addition; 6, 8, and 10 h incubation after the addition of scytalone were required for complete pigmentation of appressoria when scytalone was applied 8, 12, and 16 h after the onset of spore incubation, respectively (Fig. 1). Germinating spores were labeled with [t4C]scytalone at 24°C for 2-h periods at O-2, 2-4, 4-6, 6-8, 8-10, and lo-12 h after the onset of incubation. Incorporation of [14C]scytalone into the melanin fraction was first detected at 4-6 h and the highest incorporation was observed at 8- 10 h (Fig. 2). Incorporation of [14C]scytalone was coincident with the period of appressorial swelling and the increase in the intensity was in accordance with maximal visual pigmentation of appressoria. The results
0
4
8
12 Incubation
I 16 20 time (: h)
24
28
1
FIG. 1. Relationship between appressorial pigmentation and the time of addition of I mM scytalone in albino mutant 79215 of Colletotrichum lagenarium. Strain 104-T is the parent to albino mutant 79215. Arrows indicate the time of scytalone application. I-I, Time needed for completion of appressorial pigmentation after first visual pigmentation was observed.
MELANIN
REGULATION
IN
Colletotrichum
367
Effect of Temperature
MLmbtii Time
after
is incubation
(h)
FIG. 2. Incorporation of [‘4C]scytalone into ethyl acetate-insoluble fraction during appressorium formation by albino mutant 79215 of Colletotrichwn iagenarium. The [14C]scytalone was applied during the indicated 2-h periods after start of spore incubation.
clearly indicate that the enzymes involved in melanin biosynthesis subsequent to scytalone synthesis were not active before appressorium differentiation occurred. Effect of Protein
Synthesis Inhibitor
We used the inhibitor cycloheximide to determine if de nova protein synthesis was required for development of the enzyme activities involved in melanin biosynthesis. Treatment with cycloheximide at 3.55 pJ4 [a minimum concentration sufficient to suppress protein synthesis of C. lagenarium; Furusawa et al., 1977; Suzuki et al., 1981) from the onset of spore incubation, inhibited spore germination of both the parent strain 104-T and albino mutant 79215 (Table 1). When 3.55 $I4 cycloheximide was applied 1 h after the start of spore incubation with the parent strain 104-T, darkly pigmented appressoria indistinguishable from those without cycloheximide were observed at 12 h of incubation (Table 1). Also, in the case of albino mutant 79215, applica.tion of cycloheximide at 1 h after the start of spore incubation permitted formation of structurally mature colorless appressoria, and application of 1 mM scytalone in the presence of 3.55 $k! cycloheximide restored the pigmentation of the appressoria (Table 1).
Temperature sensitivity of the enzymes involved in melanin biosynthesis was investigated. When spores of the parent strain 104-T and albino mutant 79215 were incubated at 32°C from the onset of spore incubation, germination tubes elongated continuously and appressoria were not formed (Tani et al., 1977; Kubo et al., 1982a). When spores of the parent strain 104-T were preincubated at 24°C for 5.5 h so that structurally mature colorless appressoria were formed, and then incubated at 32°C for an additional 6.5 h: appressoria did not become pigmented (Table 2)* On t other hand, when the postincubation of the parent strain 104-T at 32°C was ~erf~~~~d in the presence of 1 mM scytalone, appressoria became pigmented (Table 2). Also, in the case of albino mutant 79215, postincubation at 32°C in the presence of 1 m talone permitted appressorial p~grn~~tat~Q~ (Table 2). DISCUSSION
We have reported previously that appressorial melanin of C. lagenarium is dlomelanin derived from 1 ,8-DHN, and the melanin biosynthetic pathway was ~art~a~~y elucidated (Kubo et al., 1983, 1984). The melanin biosynthetic pathway is similar if not identical to that proposed for V. dahliae (Bell et al., 1976b; Stipanovic and Bell, 1976, 1977; Tokousbahdes and Sisler, 1979), Thielaviopsis basic& (Wheeler and Stipanovic, 1979; Stipanovic and Wheeler, 1980), and Pyricdaria oryzae (Wolos~~~~, et al., 1980). Wheeler (1982) reported that a reducfase acts to convert 1,3,6,8-tetrahydroxynaphthalene (1,3,6&THN) to scytalone 1,3&trihydroxynaphthalene (1,3,8-T to vermelone and that a dehydratase acts to convert scytalone to 1,3,8-TEN and vermelone to 1,8-DHN in melanin biosy~tbes~s by microsclerotia of V. dahliae. Ib is COPPceivable that the two enzymes also are involved in melanin biosynthesis ~~r~~~ appressorium formation of C. lagerzarium.
KUBO,
368
FURUSAWA,
AND YAMAMOTO
TABLE I Effect of 3.55 pk? Cycloheximide and 1 mM Scytalone on Appressorial Colletotrichum legenarium” Time of cycloheximide addition 04
Strain 104-T
Presence of scytalone
Controlb 0 1 Controlb Controlb 0 1 1
79215
+ +
% Appressorium formation 90 1.4 81 95 96 1.1 87 98
Pigmentation of
Appressorial pigmentation + + + + +
’ Spores were incubated at 24°C for 12 h. b In the absence of cycloheximide.
The results obtained in the present experiments clearly show that the activity of the putative reductase and dehydratase involved in melanin biosynthesis were expressed soon after the onset of appressorium differentiation. Also, the dark pigment found in appressoria of albino mutant 79215 treated with scytalone was not found in spores, germ tubes, or penetration hyphae (Kubo et al., 1983). Thus the enzymes involved in melanin biosynthesis are considered to be developmentally regulated enzymes. The study with cycloheximide indicated that enzymes involved in the melanin biosynthesis remained active even TABLE 2 Effect of Temperature and 1 mM Scytalone on Appressorial Pigmentation following 5.5 h Preincubation at 24°C in Colletotrichum lagenariumO
Strain 104-T 79215
Temperature (“C) to which spores transferred after 5.5 h
Presence of scytalone
Appressorial pigmentation
32 32 24 32 32 24 24
+ + +
+ + + +
’ Spores were incubated for a total of 12 h.
when de nova protein synthesis was blocked from 1 h after the start of spore incubation. Therefore, the enzymes, including the putative reductase and dehydratase, are considered either to be preexisting enzymes in spores or to be synthesized as inactive forms during the first hour of incubation. These enzymes appeared to be activated in some manner during appressorium differentiation. The results of the experiment to assess the temperature sensitivity of melanin biosynthesis indicate that the melanin biosynthetic pathway in appressoria of C. lagenavium is divided into two steps; certain processes leading to pentaketide cyclization are temperature sensitive and later processes, subsequent to the cyclization mediated by the putative reductase and dehydratase, are not. Postincubation of structurally mature, colorless appressoria of the parent strain 104-T at 32°C without scytalone did not permit melanization of appressoria. Therefore, the amount of scytaIone required for visual pigmentation of appressoria appears not to be synthesized before appressorium differentiation. It is possible that early processes leading to pentaketide cyclization are also activated at the time of appressorium differentiation. The relation of appressorium differentiation to the post-translational regulation that
MELANIN
REGULATION
leads to the metabolic development of melanin biosynthesis is intriguing. A study of this regulation is now in progress through which the earlier processes of melanin biosynthesis leading to pentaketide cyclization should be elucidated. Since synchronous differentiation of a single cell accompanies melanin biosynthesis and is easily regulated, we consider that appressorium formation by C. lagenariwn is a good experimental system for the study of regulation of melanin biosynthesis in relation to morphogenesis. REFERENCES BELL, A. A., STIPANOVIC, R. D., AND PUHALLA, J. E. 1976a. Pentaketide metabolites of Verticillium dahliae. Identification of (+ )-scytalone as a natural precursor to melanin. Tetrahedron 32: 1353-1356. BELI,. A. A., PUHALLA, J. E., TOLMSOFF, W. J., AND STIPANOVIC, R. D. 1976b. Use of mutants to establish (+)-scytalone as an intermediate in melanin biosynthesis by Verticillium dahliae. Canad. .I. Microbiol. 22: 781-799. ERANDT, W. II. 1964. Morphogenesis in Verticillium: Effects of light and ultraviolet radiation on microsclerotia and melanin. Canad. J. Bot. 42: 10171023. CHET, I., AND HOTTERMANN, A. 1977. Melanin biosynthesis during differentiation of Physurum polycephalum. Biochim. Biophys. Acta 499: 148-155. CHET. I.. AND HOTTERMANN, A. 1982. De nova synthesis of polyphenol oxidase (Lactase) during formation of sclerotia in Sclerotiurn rolfsii. FEMS Microbiol. Lett. 14: 211-21.5. ESSER, K. 1968. Phenol oxidases and morphogenesis in Podospora anserina. Genetics 60: 281-288. FURUSAWA, I., NI~HICXJCZHI, M., TANI, M., AND ISHIDA, N. 1977. Evidence of early protein synthesis essential to the spore germination of Colletotrichum lagenarium. J. Gen. Microbial. 101: 307-310. Kuso, Y., SUZUKI, K., FURUSAWA, I., ISHIDA. N., AND YAMAMOTO, M. 1,982a. Relation of appressorium pigmentation and penetration of nitrocellulose membranes by Colletotrichum lagenarium. Phytoparhology 72: 498-501. KUBO, Y., SUZUKI, K., FURUSAWA, I., AND YAMAMOTO, M. 198?b. Effect of tricyclazole on appressorial pigmentation and penetration from appressoria of Colletotrichum lagenarium. Pkytopathology 72: 1198-1200. KUBO, Y.: SUZUKI, K.. FURUSAWA, I., AND YAMAMOTO, M. 1983. Scytalone as a natural intermediate of melanin biosynthesis in appressoria of Colletotrickum lagenarium. Exp. Mycol. 7: 208-215.
IN Coiletorriclurn
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KUBO, Y., SUZUKI, K., FURUSAWA, I., AND YAMAMOTO, M. 1984. Melanin biosynthesis as a prerequisite for penetration by appressoria of Co&tot+ chum lagenarium: Site of inhibition by melanin inhibitory fungicides and their action on appressoria. Pesric. Biochem. Physiol., in press. LEONARD, T. J. 1971. Phenoloxidase activity and fruiting body formation in Schitopkyikan commune. J. Bacterial. 106: 162-167. LEONARD, T. J., AND PHILLIPS, L. E. 1913. Study of polyphenoloxidase activity during the reproductive cycle in Sckizopkyllum commune. J. Bacterial 114: 7- 10. PHILLIPS, L. E., AND LEONARD, T. J. 1976. Extracelluiar and intracellular phenoloxidase activity during growth and development in Sckizopkyllum. Mycologia 68: 268-276. STJPANOVIC, R. D., AND BELL, A. A. 1976. Pentaketide metabolites of Verticillium dahliae. 3. Ident% cation of (- )-3,4-dihydro-3,8-dihydroxy-I(ZFI)naphthalenone [( -)-Vermelone] as a precursor to melanin. J. Org. Chem. 41: 2468-2469. STIPANOVIC, R. D., AND BELL. A. A. 1977. Pen?aketide metabolites of Verticillium dakliae. II. Accamulation of naphthol derivatives by the aberrant melanin mutant brm-2. Mycologia 69: 164-172. STIPANOVIC, R. D., AND WHEELER, M. H. 1980. Accumulation of 3,3’-biflaviolin, a melanin shunt product, by tricyclazole-treated Thiebaviopsis basicola. Pestic. Biockem. Physiol. 13: 198-201. SUZUKI, K.; FURUSAWA, I., ISHIDA, N.: AND YAMAMOTO, M. 1981. Protein synthesis during germination and appressorium formation of Colletotrichum lagenarium spores. J. Gen. Microbioi. 124: 61-69. SUZUKI, K., KUBO, Y., FURLJSAWA, I., ISHIDA, N., AND YAMAMQTO, M. 1982. Behavior of colorless appressoria in an albino mutant of Colletotrickum iagenarium. Canad. J. Microbial. 28: 121O-i2i3. TANI, M., ISHIDA, N., AND F~RUSAWA, I. 1977. ,Effects of temperature and antibiotics on appressorium formation in spores of Colietotrichum lageflniizim. &mad. J. Microbioi. 23: 626-629. TOKOUSBALIDES, M. C.. AND SISLER, H. D. 1979. Site of inhibition by tricyclazole in the melanin biosycthetic pathway of Verticiliium dukliae. Pestic. Biochem. Pkysiol. 11: 64-73. WHEELER, M. II. 1982. Melanin biosynthesis in Verticillium dahliae: Dehydration and reduction reactions in cell-free homogenates. Exp. Mycol. 6: 171179. WHEELER,
M.
H.,
AND
STIPANOVIC,
R. 10. 1979.
Mel-
anin biosynthesis in Thielaviopsis basicoia. Exp. Mycol. 3: 340-350. WOLOSHUK, C. P.; SISLER, M. D.; TOKOUSBALIDES. M. C., AND DUTKU, S. R. !980. Melanin biosynthesis in Pyricularia oryzae: Site of tricyclazole inhibition and pathogenic&y of melanin-deficient mntants. Pestic. Biochem. Physiol. 14: 256-264.
Regulation
of Melanin Biosynthesis during Appressorium in Colleto trichum lagenarium
Formation
YASUYUKI KUBO, IWAO FURUSAWA, AND MASAKI YAMAMOTO Laboratory
of Plant Pathology, Faculty of Agriculture,
Kyoto University, Kyoto 606, Japan
Accepted for publication August 22, 1984 KUBO, Y., FLJRUSAWA,I., AND YAMAMOTO, M. 1984. Regulation of melanin biosynthesis during appressorium formation in Colletotrichum lagenarium. Experimental Mycology 8, 364-369. Regulation of melanin biosynthesis in relation to appressorium differentiation of Colletotrichum 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, [i4C]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. 0 1984 Academic press, Inc. INDEX DESCRIPTORS:Colletotrichum lagenarium; appressoria; melanin; differentiation; albino; pigment; scytalone; [i4C]scytalone; enzymes; anthracnose.
In some plant pathogenic fungi, a prerequisite to invasion of the host plant is a differentiation of the nonpathogenic germinated spore into a pathogenic stage with an appressorium that functions as an infection structure. We have already reported that melanization of appressoria in Colletotrichum lagenarium is essential for expression of the penetration ability of the appressoria; colorless appressoria of albino mutants, or those of the parent strain 104-T in the presence of tricyclazole (5 methyl[l,2,4]triazolo[3,4 - blbenzothiazole), pp 389 (4,5 - dihydro - 4 - methyltetrazolo[1,5 - alquinazolin - 5 - one), or pyroquilon (1,2,5,6
- tetrahydropyrro]o[3,2,1
lin - 4 - one), inhibitors
- iJlquh@
of melanin
biosyn364
0147-5975184 $3.00 Copyright AU rights
0 1984 by Academic Press, Inc. of reproduction in any form reserved.
thesis, germinated laterally to form secondary appressoria, and consequently had little ability to penetrate the host plant (Kubo et al., 1982a,b, 1983, 1984; Suzuki et al., 1982). The melanin of appressoria in C. lagenarium was indicated to be allomelanin derived from 1,8-dihydroxynaphthalene (1,8DHN)‘; appressoria of albino mutant 79215 converted both scytalone and vermelone to melanin, and the parent strain 104-T in the presence of tricyclazole, pp 389, or pyroquilon converted vermelone but not scyta’ Abbreviations used: 1,8-DHN, 1 ,S-dihydroxynaphthalene; PSA, potato sucrose agar; 1,3,6,8-THN, 1,3,6,8-tetrahydroxynaphthalene; 1,3,8-THN, 1,3,8trihydroxynaphthalene.
MELANIN
REGULATION
lone (Kubo et al., 1983, 1984). The melanin biosynthetic pathway was partially elucidated (Kubo et al., 1984). Our previous observation of the penetration process with the albino mutant 79215 revealed that melanized cells were confined to the appressoria; spores, germ tubes, and penetration hyphae were not colored by application of scytalone, indicating that melanin biosynthesis was a developmentally regulated system (Kubo et al., 1983). Although the relationships between cell differentiation and regulation of melanin biosynthesis mediated by polyphenoloxidases have been investigated in several fungi such as Schizophyllum commune (Leonard, 1971; Leonard and Phillips, 1973; Phillips and Leonard, 89X), Physarum polycephalum (Chet and Htittermann, 1977), Podosporn anserina (Esser, 1968), and Scleuotium rofiii (Chet and Hiittermann, 1982), the regulation of allomelanin synthesis is poorly understood. In this study, the relation of appressorium differentiation of C. lagenarium to the metabolic development of melanin biosynthesis was investigated using the albino mutant and a melanin precursor, scytalone. MATERIALS
AND
METHODS
Fungus. Colletotrichum lagenarium (Pass.) Ellis and Halsted strain 104-T (stock c&u-e in Laboratory of Plant Pathology, Kyoto University) was used as a parent strain. An albino mutant 79215 was isolated from the parent strain previously by ultraviolet n-radiation (Kubo et al., 1982a). Cultural condition and spores. The two strains of 6. lagenarium were cultured on potato sucrose agar (LISA) medium. Spores on mycehal mats cultured on PSA medium at 24°C for 7 days were collected with a brush and washed with ice-cold distilled water three times. spore germination. Spores suspended in sterile deionized water (1 ml) to give about 1 x lo5 spores/ml were poured into a Petri dish (diameter 2 cm) and incubated at 24°C.
IN
Colletot~ichum
365
Scytalone was applied as solution in the spore suspension or by exchanging the water for 1 mB of solution containing scytalone at various time after the onset of incubation. Scytalone was obtained from either mutant 8015 of C. Eagenarium (Kubc~ et al., 1983) or Verticillium dahliae brm-I (ATCC 44571) (Bell et &., B976a). [14C]Scytalone. V. dahliae brm-1 was cultured at 24°C in two 3QO-ml flasks each containing 100 ml of sucrose nitrate medium (Brandt, 1964). After incubation for 8 days, 100 bCi [U-14C]acetic acid (sodium salt; 689 @/mg) was added to each tiask. which was incubated in a sealed glass case containing NaOH for an additional 7 days. The whole 25-day-old culture solution was adjusted to pH 5.0 with H,P0,, saturated with NaCl, and extracted with ethyl acetate three times (100 ml each). The ethyl acetate extract was combined and concentrated by evaporation under reduced pressure. The concentrate was applied to a thin-layer plate (Kieselgel 60 F,,,, 0.25 mm thick, Merck) and developed with benzene-acetone (l/l, v/v). The developed thin-layer piate was observed under 365 nm hght for fluorescence to indicate t e location of scytalone as judged from comparison with a standard. The spot was scraped off with a razor blade and ehtte with ethyl acetate. The ethyl acetate eluate was concentrate to dryness to give white crystals (scytalone; 0.5 mg, sp act 3.12 x lo7 cpmlmg). Incorporation of [14Clscytalone into appressorial melanin. To investigate the activity of enzymes involved in melanin biosynthesis subsequent to scytalone synthesis during appressorium formation, 2 mH of spore suspension (5 X PO4 spores/ml) was poured into a Petri dish (diame cm) and the germlings were labele [14C]scytalone (7.2 x IO5 cpm) for successive 2-h periods through 12 h of incubation. At the end of each period, the Petri dish was then placed on ice and mefanin infermediates were removed by exchanging the incubation solution successively with ace-
366
KUBO,
FURUSAWA,
tone, ethyl acetate, acetone, and water (2 ml each for 30 min). After the extraction of the intermediates, the germlings were collected on Whatman 3MM filter paper (diameter, 24 mm). Each filter paper was washed three times with 30 ml of cold distilled water. The radioactivity was measured in a liquid scintilation spectrophotometer (Packard Model 2425) as described previously (Furusawa et al., 1977). RESULTS
Time Course of Appressorium Formation and Pigmentation When spores of the parent strain 104-T were incubated at 24°C germination occurred after 3 h of incubation. The tips of germ tubes swelled until 6 h of incubation and structurally mature, colorless appressoria were observed. As the appressorium swelled, protoplasm migrated from the spore into the appressorium. Visible pigmentation of appressoria started after they reached full size and the intensity of pigmentation gradually increased to form completely pigmented appressoria at 12 h of incubation. More than 95% of spores differentiated to form appressoria almost synchronously following the time course described above. Melanin Biosynthetic Activity Subsequent to Scytalone Synthesis The melanin biosynthetic activity subsequent to scytalone synthesis during appressorium formation of C. Zagenarium was also investigated. First, the time course of appressorial pigmentation was determined by applying scytalone to albino mutant 79215 at various times after the onset of spore incubation. When 1 n-&! scytalone was applied to albino mutant 79215 after 0, 2, or 4 h of incubation, visible pigmentation of appressoria started 6 h after incubation; at this time the migration of protoplasm from spores into appressoria was complete and appressoria were swollen to full size. The
AND YAMAMOTO
intensity of appressorial pigmentation gradually increased during further incubation until 12 h. In these case, the time course of appressorial pigmentation for albino mutant 79215 was the same as that of the parent strain 104-T (Fig. 1). When scytalone was applied after 6 h of incubation, visible pigmentation of appressoria was observed from within 30 min after the treatment and completely pigmented appressoria were observed by 12 h (Fig. 1). Addition of scytalone after the formation of structurally mature appressoria permitted visible pigmentation of appressoria from within 30 min and the duration needed for completion of pigmentation of appressoria depended on the time of scytalone addition; 6, 8, and 10 h incubation after the addition of scytalone were required for complete pigmentation of appressoria when scytalone was applied 8, 12, and 16 h after the onset of spore incubation, respectively (Fig. 1). Germinating spores were labeled with [t4C]scytalone at 24°C for 2-h periods at O-2, 2-4, 4-6, 6-8, 8-10, and lo-12 h after the onset of incubation. Incorporation of [14C]scytalone into the melanin fraction was first detected at 4-6 h and the highest incorporation was observed at 8- 10 h (Fig. 2). Incorporation of [14C]scytalone was coincident with the period of appressorial swelling and the increase in the intensity was in accordance with maximal visual pigmentation of appressoria. The results
0
4
8
12 Incubation
I 16 20 time (: h)
24
28
1
FIG. 1. Relationship between appressorial pigmentation and the time of addition of I mM scytalone in albino mutant 79215 of Colletotrichum lagenarium. Strain 104-T is the parent to albino mutant 79215. Arrows indicate the time of scytalone application. I-I, Time needed for completion of appressorial pigmentation after first visual pigmentation was observed.
MELANIN
REGULATION
IN
Colletotrichum
367
Effect of Temperature
MLmbtii Time
after
is incubation
(h)
FIG. 2. Incorporation of [‘4C]scytalone into ethyl acetate-insoluble fraction during appressorium formation by albino mutant 79215 of Colletotrichwn iagenarium. The [14C]scytalone was applied during the indicated 2-h periods after start of spore incubation.
clearly indicate that the enzymes involved in melanin biosynthesis subsequent to scytalone synthesis were not active before appressorium differentiation occurred. Effect of Protein
Synthesis Inhibitor
We used the inhibitor cycloheximide to determine if de nova protein synthesis was required for development of the enzyme activities involved in melanin biosynthesis. Treatment with cycloheximide at 3.55 pJ4 [a minimum concentration sufficient to suppress protein synthesis of C. lagenarium; Furusawa et al., 1977; Suzuki et al., 1981) from the onset of spore incubation, inhibited spore germination of both the parent strain 104-T and albino mutant 79215 (Table 1). When 3.55 $I4 cycloheximide was applied 1 h after the start of spore incubation with the parent strain 104-T, darkly pigmented appressoria indistinguishable from those without cycloheximide were observed at 12 h of incubation (Table 1). Also, in the case of albino mutant 79215, applica.tion of cycloheximide at 1 h after the start of spore incubation permitted formation of structurally mature colorless appressoria, and application of 1 mM scytalone in the presence of 3.55 $k! cycloheximide restored the pigmentation of the appressoria (Table 1).
Temperature sensitivity of the enzymes involved in melanin biosynthesis was investigated. When spores of the parent strain 104-T and albino mutant 79215 were incubated at 32°C from the onset of spore incubation, germination tubes elongated continuously and appressoria were not formed (Tani et al., 1977; Kubo et al., 1982a). When spores of the parent strain 104-T were preincubated at 24°C for 5.5 h so that structurally mature colorless appressoria were formed, and then incubated at 32°C for an additional 6.5 h: appressoria did not become pigmented (Table 2)* On t other hand, when the postincubation of the parent strain 104-T at 32°C was ~erf~~~~d in the presence of 1 mM scytalone, appressoria became pigmented (Table 2). Also, in the case of albino mutant 79215, postincubation at 32°C in the presence of 1 m talone permitted appressorial p~grn~~tat~Q~ (Table 2). DISCUSSION
We have reported previously that appressorial melanin of C. lagenarium is dlomelanin derived from 1 ,8-DHN, and the melanin biosynthetic pathway was ~art~a~~y elucidated (Kubo et al., 1983, 1984). The melanin biosynthetic pathway is similar if not identical to that proposed for V. dahliae (Bell et al., 1976b; Stipanovic and Bell, 1976, 1977; Tokousbahdes and Sisler, 1979), Thielaviopsis basic& (Wheeler and Stipanovic, 1979; Stipanovic and Wheeler, 1980), and Pyricdaria oryzae (Wolos~~~~, et al., 1980). Wheeler (1982) reported that a reducfase acts to convert 1,3,6,8-tetrahydroxynaphthalene (1,3,6&THN) to scytalone 1,3&trihydroxynaphthalene (1,3,8-T to vermelone and that a dehydratase acts to convert scytalone to 1,3,8-TEN and vermelone to 1,8-DHN in melanin biosy~tbes~s by microsclerotia of V. dahliae. Ib is COPPceivable that the two enzymes also are involved in melanin biosynthesis ~~r~~~ appressorium formation of C. lagerzarium.
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FURUSAWA,
AND YAMAMOTO
TABLE I Effect of 3.55 pk? Cycloheximide and 1 mM Scytalone on Appressorial Colletotrichum legenarium” Time of cycloheximide addition 04
Strain 104-T
Presence of scytalone
Controlb 0 1 Controlb Controlb 0 1 1
79215
+ +
% Appressorium formation 90 1.4 81 95 96 1.1 87 98
Pigmentation of
Appressorial pigmentation + + + + +
’ Spores were incubated at 24°C for 12 h. b In the absence of cycloheximide.
The results obtained in the present experiments clearly show that the activity of the putative reductase and dehydratase involved in melanin biosynthesis were expressed soon after the onset of appressorium differentiation. Also, the dark pigment found in appressoria of albino mutant 79215 treated with scytalone was not found in spores, germ tubes, or penetration hyphae (Kubo et al., 1983). Thus the enzymes involved in melanin biosynthesis are considered to be developmentally regulated enzymes. The study with cycloheximide indicated that enzymes involved in the melanin biosynthesis remained active even TABLE 2 Effect of Temperature and 1 mM Scytalone on Appressorial Pigmentation following 5.5 h Preincubation at 24°C in Colletotrichum lagenariumO
Strain 104-T 79215
Temperature (“C) to which spores transferred after 5.5 h
Presence of scytalone
Appressorial pigmentation
32 32 24 32 32 24 24
+ + +
+ + + +
’ Spores were incubated for a total of 12 h.
when de nova protein synthesis was blocked from 1 h after the start of spore incubation. Therefore, the enzymes, including the putative reductase and dehydratase, are considered either to be preexisting enzymes in spores or to be synthesized as inactive forms during the first hour of incubation. These enzymes appeared to be activated in some manner during appressorium differentiation. The results of the experiment to assess the temperature sensitivity of melanin biosynthesis indicate that the melanin biosynthetic pathway in appressoria of C. lagenavium is divided into two steps; certain processes leading to pentaketide cyclization are temperature sensitive and later processes, subsequent to the cyclization mediated by the putative reductase and dehydratase, are not. Postincubation of structurally mature, colorless appressoria of the parent strain 104-T at 32°C without scytalone did not permit melanization of appressoria. Therefore, the amount of scytaIone required for visual pigmentation of appressoria appears not to be synthesized before appressorium differentiation. It is possible that early processes leading to pentaketide cyclization are also activated at the time of appressorium differentiation. The relation of appressorium differentiation to the post-translational regulation that
MELANIN
REGULATION
leads to the metabolic development of melanin biosynthesis is intriguing. A study of this regulation is now in progress through which the earlier processes of melanin biosynthesis leading to pentaketide cyclization should be elucidated. Since synchronous differentiation of a single cell accompanies melanin biosynthesis and is easily regulated, we consider that appressorium formation by C. lagenariwn is a good experimental system for the study of regulation of melanin biosynthesis in relation to morphogenesis. REFERENCES BELL, A. A., STIPANOVIC, R. D., AND PUHALLA, J. E. 1976a. Pentaketide metabolites of Verticillium dahliae. Identification of (+ )-scytalone as a natural precursor to melanin. Tetrahedron 32: 1353-1356. BELI,. A. A., PUHALLA, J. E., TOLMSOFF, W. J., AND STIPANOVIC, R. D. 1976b. Use of mutants to establish (+)-scytalone as an intermediate in melanin biosynthesis by Verticillium dahliae. Canad. .I. Microbiol. 22: 781-799. ERANDT, W. II. 1964. Morphogenesis in Verticillium: Effects of light and ultraviolet radiation on microsclerotia and melanin. Canad. J. Bot. 42: 10171023. CHET, I., AND HOTTERMANN, A. 1977. Melanin biosynthesis during differentiation of Physurum polycephalum. Biochim. Biophys. Acta 499: 148-155. CHET. I.. AND HOTTERMANN, A. 1982. De nova synthesis of polyphenol oxidase (Lactase) during formation of sclerotia in Sclerotiurn rolfsii. FEMS Microbiol. Lett. 14: 211-21.5. ESSER, K. 1968. Phenol oxidases and morphogenesis in Podospora anserina. Genetics 60: 281-288. FURUSAWA, I., NI~HICXJCZHI, M., TANI, M., AND ISHIDA, N. 1977. Evidence of early protein synthesis essential to the spore germination of Colletotrichum lagenarium. J. Gen. Microbial. 101: 307-310. Kuso, Y., SUZUKI, K., FURUSAWA, I., ISHIDA. N., AND YAMAMOTO, M. 1,982a. Relation of appressorium pigmentation and penetration of nitrocellulose membranes by Colletotrichum lagenarium. Phytoparhology 72: 498-501. KUBO, Y., SUZUKI, K., FURUSAWA, I., AND YAMAMOTO, M. 198?b. Effect of tricyclazole on appressorial pigmentation and penetration from appressoria of Colletotrichum lagenarium. Pkytopathology 72: 1198-1200. KUBO, Y.: SUZUKI, K.. FURUSAWA, I., AND YAMAMOTO, M. 1983. Scytalone as a natural intermediate of melanin biosynthesis in appressoria of Colletotrickum lagenarium. Exp. Mycol. 7: 208-215.
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KUBO, Y., SUZUKI, K., FURUSAWA, I., AND YAMAMOTO, M. 1984. Melanin biosynthesis as a prerequisite for penetration by appressoria of Co&tot+ chum lagenarium: Site of inhibition by melanin inhibitory fungicides and their action on appressoria. Pesric. Biochem. Physiol., in press. LEONARD, T. J. 1971. Phenoloxidase activity and fruiting body formation in Schitopkyikan commune. J. Bacterial. 106: 162-167. LEONARD, T. J., AND PHILLIPS, L. E. 1913. Study of polyphenoloxidase activity during the reproductive cycle in Sckizopkyllum commune. J. Bacterial 114: 7- 10. PHILLIPS, L. E., AND LEONARD, T. J. 1976. Extracelluiar and intracellular phenoloxidase activity during growth and development in Sckizopkyllum. Mycologia 68: 268-276. STJPANOVIC, R. D., AND BELL, A. A. 1976. Pentaketide metabolites of Verticillium dahliae. 3. Ident% cation of (- )-3,4-dihydro-3,8-dihydroxy-I(ZFI)naphthalenone [( -)-Vermelone] as a precursor to melanin. J. Org. Chem. 41: 2468-2469. STIPANOVIC, R. D., AND BELL. A. A. 1977. Pen?aketide metabolites of Verticillium dakliae. II. Accamulation of naphthol derivatives by the aberrant melanin mutant brm-2. Mycologia 69: 164-172. STIPANOVIC, R. D., AND WHEELER, M. H. 1980. Accumulation of 3,3’-biflaviolin, a melanin shunt product, by tricyclazole-treated Thiebaviopsis basicola. Pestic. Biockem. Physiol. 13: 198-201. SUZUKI, K.; FURUSAWA, I., ISHIDA, N.: AND YAMAMOTO, M. 1981. Protein synthesis during germination and appressorium formation of Colletotrichum lagenarium spores. J. Gen. Microbioi. 124: 61-69. SUZUKI, K., KUBO, Y., FURLJSAWA, I., ISHIDA, N., AND YAMAMQTO, M. 1982. Behavior of colorless appressoria in an albino mutant of Colletotrickum iagenarium. Canad. J. Microbial. 28: 121O-i2i3. TANI, M., ISHIDA, N., AND F~RUSAWA, I. 1977. ,Effects of temperature and antibiotics on appressorium formation in spores of Colietotrichum lageflniizim. &mad. J. Microbioi. 23: 626-629. TOKOUSBALIDES, M. C.. AND SISLER, H. D. 1979. Site of inhibition by tricyclazole in the melanin biosycthetic pathway of Verticiliium dukliae. Pestic. Biochem. Pkysiol. 11: 64-73. WHEELER, M. II. 1982. Melanin biosynthesis in Verticillium dahliae: Dehydration and reduction reactions in cell-free homogenates. Exp. Mycol. 6: 171179. WHEELER,
M.
H.,
AND
STIPANOVIC,
R. 10. 1979.
Mel-
anin biosynthesis in Thielaviopsis basicoia. Exp. Mycol. 3: 340-350. WOLOSHUK, C. P.; SISLER, M. D.; TOKOUSBALIDES. M. C., AND DUTKU, S. R. !980. Melanin biosynthesis in Pyricularia oryzae: Site of tricyclazole inhibition and pathogenic&y of melanin-deficient mntants. Pestic. Biochem. Physiol. 14: 256-264.