Department of Applied Biological Chemistry, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan1
Faculty of Agriculture, Kyoto Prefectural University, Shimogamo, Kyoto 606-8522, Japan2
Graduate School of Pharmaceutical Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan3
Morinaga and Co., Ltd, Tsurumi-ku, Yokohama 230-8504, Japan4
Author for correspondence: Shohei Sakuda. Tel: +81 3 5841 5133. Fax: +81 3 5841 8022. e-mail: asakuda{at}mail.ecc.u-tokyo.ac.jp
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ABSTRACT |
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Keywords: aflastatin A, Colletotrichum lagenarium, melanin, PKS1, RT-PCR
Abbreviations: AsA, aflastatin A; 1,8-DHN, 1,8-dihydroxynaphthalene; 1,3,6,8-THN, 1,3,6,8-tetrahydroxynaphthalene; 1,3,8-THN, 1,3,8-trihydroxynaphthalene
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INTRODUCTION |
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The melanin of this fungus is a polymer formed from 1,8-dihydroxynaphthalene (1,8-DHN). 1,8-DHN is biosynthesized from five molecules of malonyl-CoA (Fujii et al., 2000 ) via four biosynthetic intermediates, 1,3,6,8-tetrahydroxynaphthalene (1,3,6,8-THN), scytalone, 1,3,8-trihydroxynaphthalene (1,3,8-THN) and vermelone (Fig. 1
; Bell & Wheeler, 1986
). Several key enzymes and genes involved in the biosynthetic pathway have been clarified. In some fungal pathogens such as C. lagenarium or Magnaporthe grisea, melanization of appressoria is essential for penetration into the host plant (Kubo & Furusawa, 1991
). Fungal conidia germinate and the tips of germ tubes differentiate into appressoria, which synthesize melanin. The melanin mediates the build-up of pressure in the appressorium and this high pressure provides the essential driving force for mechanical penetration into the host plant (Howard & Ferrari, 1989
). Since blocking of melanin biosynthesis renders the fungus unable to generate the high pressure required for host leaf penetration by appressoria, melanin is an ideal target for the development of an effective drug to protect host plants from infection by pathogens (Kubo & Furusawa, 1991
). Inhibitors of enzymes involved in the melanin biosynthetic pathway are actually applied to control rice blast disease (Bell & Wheeler, 1986
; Yamaguchi & Kubo, 1992
).
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METHODS |
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Chemicals.
AsA was extracted from the mycelium of Streptomyces sp. MRI 142 and purified as described by Ono et al. (1997) . Scytalone was prepared by the method of Kubo et al. (1983)
with some modifications. Briefly, a culture filtrate (potato-sucrose medium) of strain 9201Y was adjusted to pH 5·0 with H3PO4 and saturated with NaCl, and extracted with ethyl acetate. The ethyl acetate extract was evaporated in vacuo and the oily residue was subjected to silica gel chromatography. The column was washed with n-hexane/ethyl acetate (2:1, v/v), and scytalone was eluted with n-hexane/ethyl acetate (1:1, v/v).
Induction of melanin synthesis.
Melanin biosynthesis at the vegetative mycelial growth stage was induced as described previously (Takano et al., 1995 ). Conidia of C. lagenarium 104-T were inoculated into 100 ml of the potato-sucrose liquid medium containing 0·2% of yeast extract and grown at 26·5 °C in a rotary shaker (120 r.p.m.) for 40 h. For rapid induction of melanin synthesis, the mycelia were separated from the medium by filtration through two layers of sterile cheesecloth, and transferred into 50 ml 1·2 M sucrose solution. The mycelial suspension (2 ml) was added to 2 ml 1·2 M sucrose solution containing AsA (02 µg ml-1, final concentration) in a sterile test tube [1·8 cm (i.d.)x20 cm (length)] and incubated at 26·5 °C in a test tube shaker (250 r.p.m.) for 20 h. AsA was dissolved in DMSO at appropriate concentrations and added to the sucrose solution. Melanin pigment in the culture filtrate was determined by measuring the A585 on a spectrophotometer (model FP777; JASCO).
Induction of scytalone synthesis.
Scytalone biosynthesis was induced as described above except that strain 9201Y instead of wild-type strain 104-T was used. After induction by incubation in 1·2 M sucrose, the culture filtrate was adjusted to pH 5·0 with H3PO4 and scytalone was extracted with ethyl acetate. The amount of scytalone in the ethyl acetate extract was determined semi-quantitatively by TLC (silica gel 60 F254; Merck). TLC plates were developed with chloroform/methanol (9:1, v/v) and inspected by short wavelength UV illumination.
Restoration of melanin production by exogenous scytalone in the presence of AsA.
Restoration of melanin production by addition of exogenous scytalone was examined in the presence of AsA (1 µg ml-1) using strain 104-T. Scytalone (0·5 or 1·0 mM, final concentration) was added to 1·2 M sucrose, and melanin production was inspected at 20 h after the induction in the same way as described above.
Measurement of polyketide synthase activity.
A. oryzae M-2-3(pTAPSG) was grown at 30 °C in CzapekDox medium containing starch, and a cell lysate was prepared by grinding in liquid nitrogen with a mortar and pestle. The mycelial powder (8 g) was then suspended in 40 ml extraction buffer (50 mM potassium phosphate, pH 7·5, 30% glycerol, 2 mM ß-mercaptoethanol, 1 mM EDTA and 0·1 mM benzamidine). Polyclar VT (0·8 g; Gokyou Sangyou) was added to the mixture, and the mixture was stirred on ice for 1 h and filtered through four-layered gauze. The obtained filtrate was centrifuged at 15000 g for 20 min to remove residual cell debris. Bio-Beads S-X1 (4 g; Bio-Rad) were added to the supernatant, and the mixture was stirred on ice for 30 min. After removing the beads by filtration, the filtrate was centrifuged at 210000 g for 2 h. The obtained pellet was dissolved in 4 ml of the extraction buffer mentioned above and used as a crude enzyme solution.
Enzyme reaction mixture, containing 175 µl 1 mM potassium phosphate buffer, pH 7·2, 25 µl 1 µM malonyl-CoA, 50 µl enzyme solution and 5 µl DMSO with or without AsA (5·0 or 10·0 µg ml-1), was incubated for 1 h at 24 °C. The reaction mixture was heated at 100 °C for 20 min to convert 1,3,6,8-THN to flaviolin and acidified with addition of 50 µl 6 M HCl. Products were extracted with 300 µl ethyl acetate, dried by flushing of nitrogen gas, and dissolved in 100 µl acetonitrile for HPLC analysis. HPLC conditions were as follows: SenshuPak ODS-H-1151 (4·6x150 mm; Senshu Kagaku), maintained at 40 °C; mobile phase, linear gradient from 5% CH3CN in H2O to 35% CH3CN in H2O (each contained 2% acetic acid) over 30 min with detection at 305 nm; flow rate, 0·8 ml min-1; retention time of flaviolin, 21·2 min (Fujii et al., 2000 ; Funa et al., 1999
).
RT-PCR.
Melanin biosynthesis was induced as described above in the presence or absence of 1 µg AsA ml-1. Samples were taken periodically and cells were collected by filtration. Cells (5070 mg) were ground under liquid nitrogen to a fine powder using a mortar and pestle and then transferred to an Eppendorf tube. Total cellular RNA was prepared using the RNeasy Plant Mini Kit (Qiagen) as recommended by the manufacturer. Total RNA (1 µg) was treated with 1 unit DNase I (Gibco-BRL) to remove residual genomic DNA and used for RT-PCR. First-strand cDNA was prepared using random hexamer primers and ReverTra Ace (Toyobo) reverse transcriptase in a final volume of 20 µl. cDNA was used as template in PCR reactions with the following primer sets: PKS1 5'-CCGAGCATCCTTGCGAAGACGTTCC-3' and 5'-TGAGTCTCTCCGCCAGCGAACCGAC-3' (amplified fragment size, 398 bp), SCD1 5'-TTGCGGTACCAGTGCATGTTGTAGC-3' and 5'-CTCCTACGACTCCAAGGACTGGGAC-3' (324 bp), THR1 5'-CCTTTGGCCACGTCAAGGACGTCAC-3' and 5'-TCAACACCCTCGTCGTCGAGCTCAC-3' (348 bp) and CMR1 5'-GCATGGAGTGGCGACAAATGGTTG-3' and 5'-TGAAGGTCGGTGATGGCCAGTTGA-3' (260 bp). A housekeeping gene, G3PDH 5'-ATCGGTCGTATCGTCTTCCGCAAC-3' and 5'-CGTTGACACCCATCACGTACATGG-3' (369 bp), was used as a control to certify that the same amount of RNA was used in all reactions. The PCR reactions were performed with AmpliTaq Gold DNA polymerase (PE Applied Biosystems) and a buffer provided by the manufacturer in the presence of 200 µM deoxynucleoside triphosphate, 0·5 µM each primer, 1 µl cDNA and 2·5 U enzyme in a final volume of 50 µl. The reactions consisted of 3240 cycles. The first cycle was for 9 min at 95 °C (for denaturation and enzyme activation) and 30 s at 68 °C (for annealing and extension); the denaturation step was shortened to 30 s in the subsequent cycles. Control reactions without reverse transcriptase gave no signal.
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RESULTS |
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Effects of AsA on the expression of genes responsible for melanin biosynthesis
The effect of AsA on the expression of three genes encoding melanin biosynthetic enzymes, PKS1, SCD1 and THR1 (Takano et al., 1995 ; Kubo et al., 1996
; Perpetua et al., 1996
), and a regulatory gene (CMR1) was examined. Expression of G3PDH (glyceraldehyde-3-phosphate dehydrogenase) was used as a control. C. lagenarium was cultured in the induction medium with or without AsA (1·0 µg ml-1) and samples were taken periodically. Total RNA was prepared and used for RT-PCR analysis. At 0 time, transcription of these melanin-related genes was not strongly activated (Fig. 5
). After induction without AsA, expression of these genes was activated and maintained at high levels during the incubation for 820 h. When AsA was added to the culture, the expression of PKS1 was severely impaired at all incubation times. Expression of all other genes was unaffected by this treatment.
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DISCUSSION |
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In the present experiments, AsA inhibited scytalone production, and addition of exogenous scytalone was able to restore melanin production under conditions where scytalone production was completely inhibited by AsA. This suggests that AsA targets a step before the synthesis of scytalone in the melanin biosynthetic pathway. This conclusion is supported by the fact that expression of SCD1, THR1 and CMR1 was not impaired by AsA. SCD1 catalyses the biosynthetic steps from scytalone to 1,3,8-THN and vermelone to 1,8-DHN, and THR1 catalyses the step from 1,3,8-THN to vermelone (Fig. 1). CMR1 encodes a regulatory protein which positively regulates the transcription of SCD1 and THR1 in the melanin induction system in C. lagenarium (Tsuji et al., 2000
). There are two biosynthetic enzymes before the synthesis of scytalone, namely PKS1 and a reductase catalysing the step from 1,3,6,8-THN to scytalone (Fig. 1
). AsA did not inhibit PKS1 activity and may not inhibit the reductase since no accumulation of 1,3,6,8-THN, which was analysed as flaviolin, was observed in the culture filtrate cultured with AsA (data not shown). However, expression of PKS1 was severely impaired by AsA. This reduction of PKS1 expression may be very important for AsAs inhibitory effect on melanin production. It is not clear if the expression of PKS1 is strongly regulated by CMR1 similarly to the case of SCD1 and THR1 (Tsuji et al., 2000
). The expression pattern of the four genes caused by the addition of AsA may suggest that the regulatory system of the expression of PKS1 is different from that of SCD1 and THR1. Our results may suggest that AsA inhibits an early regulatory step prior to the expression of PKS1 in melanin production or alters PKS1 expression directly. Expression of PKS1 was drastically inhibited by AsA, but it was not completely inhibited (Fig. 5
). It is not clear if this effect of AsA on PKS1 expression is enough for complete inhibition of melanin production. It may possibly be presumed that AsA also impairs other parts in the pathway of melanin production, for example, expression of a gene encoding the reductase or production of malonyl-CoA.
Since it has not been clarified how expression of PKS1 is regulated, AsA may be useful as a probe to investigate the molecular mechanism of the regulatory system. Knowledge of this mechanism could be very important for developing new effective inhibitors of melanin production. It is not clear whether AsA has a common target in melanin production by C. lagenarium and aflatoxin production by A. parasiticus. We are now examining the effects of AsA on the biosynthetic pathways of aflatoxin and other fungal polyketides.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 18 January 2001;
revised 19 April 2001;
accepted 1 June 2001.