- Email: [email protected]
Over-expression of the cercosporin facilitator protein, CFP, in Cercospora kikuchii up-regulates production and secretion of cercosporin
FEMS Microbiology Letters 204 (2001) 89^93
www.fems-microbiology.org
Over-expression of the cercosporin facilitator protein, CFP, in Cercospora kikuchii up-regulates production and secretion of cercosporin Robert G. Upchurch a
a;b;
*, Mark S. Rose
a;b;1
, Mohamed Eweida
b;2
Agricultural Research Service, US Department of Agriculture, North Carolina State University, Raleigh, NC 27695 USA b Department of Plant Pathology, North Carolina State University, Raleigh, NC 27695 USA Received 2 August 2001; accepted 8 August 2001 First published online 18 September 2001
Abstract CFP (cercosporin facilitator protein), a light-regulated gene from the soybean fungal pathogen Cercospora kikuchii, encodes the putative major facilitator transporter of the fungal polyketide cercosporin. Gene disruption of CFP in C. kikuchii strain Gus-3 resulted in dramatically reduced cercosporin production and virulence, and increased sensitivity to the toxin. Two C. kikuchii transformant strains (10-1 and 10-11) that over-produce cercosporin were recovered from the complementation of CFP gene-disrupted strain Gus-3. Southern analysis revealed that these strains contained multiple genomic copies of CFP and over-expressed CFP transcript and protein. Although 10-1 and 10-11 produce and secrete significantly elevated levels of cercosporin, they exhibit wild-type resistance to cercosporin, and maintain a wild-type pattern of light-regulated toxin accumulation. Restoration of wild-type cercosporin resistance in 10-1 and 10-11 suggests that CFP does contribute substantially to cercosporin resistance via toxin secretion. The three-fold increase in toxin accumulation, predominately associated with the mycelium fraction of these CFP multi-copy strains, suggests that CFP may also have a significant, but unknown, role in regulating toxin production. ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Cercosporin ; CFP; Major facilitator; Over-expression ; Resistance; Cercospora kikuchii
1. Introduction CFP, a light-regulated gene encoding the ca. 65.4-kDa protein CFP (cercosporin facilitator protein) from the soybean fungal pathogen Cercospora kikuchii, was the ¢rst putative major facilitator transporter of a fungal polyketide to be described [1,2]. Members of the major facilitator superfamily [3] are integral membrane proteins that can
* Corresponding author. Tel. : +1 (919) 515-6996; Fax: +1 (919) 515-7716. E-mail address : [email protected] (R.G. Upchurch). 1 Present address: Syngenta Biotechnology Institute, 3054 Cornwallis Rd., Research Triangle Park, NC 27709-2257, USA. 2 Present address: Division of Cellular and Molecular Biology, Ontario Cancer Institute/Princess Margaret Hospital, 610 University Ave., Toronto, ON, Canada M5G 2M9.
confer resistance to drugs and other toxins through an active export mechanism energized by proton motive force. C. kikuchii and other Cercospora species produce the plant toxin cercosporin, a red, lipid-soluble, perylenequinone photosensitizer and potent generator of singlet oxygen [4,5]. Gene disruption of CFP in C. kikuchii strain Gus-3 resulted in dramatically diminished cercosporin production and virulence on the plant host, and increased sensitivity to exogenous cercosporin. Based on these ¢ndings, we proposed that CFP encodes a cercosporin transporter that contributes both to toxin resistance and to virulence by facilitated secretion [2]. When the CFP disruptant strain Gus-3 was co-transformed with the hygromycin resistance marker and a genomic fragment containing the intact CFP gene, stable, hygromycin-resistant transformants were recovered that were complemented to wild-type levels for toxin production, virulence, and cercosporin resistance [2]. Two of the restored transformant
0378-1097 / 01 / $20.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 1 ) 0 0 3 8 5 - 8
FEMSLE 10133 17-10-01
90
R.G. Upchurch et al. / FEMS Microbiology Letters 204 (2001) 89^93
strains, 10-1 and 10-11, appeared to over-accumulate cercosporin on agar medium. Here, we report that these two transformants contain multiple genomic copies of CFP and over-express CFP transcript and CFP protein. These strains produce and secrete signi¢cantly elevated levels of cercosporin, but exhibit wild-type resistance to cercosporin, and maintain a pattern of light-regulated toxin accumulation similar to the wild-type parental strain. 2. Materials and methods 2.1. C. kikuchii strains, growth conditions, and cercosporin measurements All strains described in this report were derived from wild-type C. kikuchii, isolate PR [6]. Cultures were grown for 5 days in potato dextrose broth (PDB, Difco, Detroit, MI, USA) at 23³C under either continuous white £uorescent light (approximately 80 WE s31 m32 ) or in darkness. Cultures were shaken at 180 rpm in an Innova illuminated incubator shaker (New Brunswick Scienti¢c, Edison, NJ, USA) in 125-ml Erlenmeyer £asks containing 50 ml of PDB. Dark-grown cultures were obtained by wrapping £asks with plastic-lined, black cloth and aluminum foil. For the measurement of cercosporin production, total cercosporin accumulation in mycelium plus medium was measured. Entire cultures were blended in a Waring blender for two 20-s pulses. A 10-ml aliquot of the suspension was treated with 1 vol of 5 N KOH [7] in darkness for 4 h and clari¢ed by low speed centrifugation. For the measurement of cercosporin secretion into the medium, the mycelium mass was removed by vacuum ¢ltration and a 10-ml aliquot of the mycelium-free medium was treated with KOH as described above. Cercosporin concentrations were determined spectrophotometrically [7]. The harvested mycelium was lyophilized and used for dry weight determinations. Three culture £asks were assayed for each strain in the experiment and the entire experiment was repeated. 2.2. Strain construction and molecular biological methods Strains 10-1 and 10-11 resulted from the co-transformation of C. kikuchii CFP disruptant strain Gus-3 with pgCFP [2], containing a wild-type copy of CFP on a 6.5-kb EcoRI genomic fragment, and pUCHI [8], a plasmid containing the hph gene for the selectable hygromycin B resistance marker. The GenBank accession number for CFP is AF091042. Transformants were recovered on hygromycin-containing regeneration medium as previously described [9], then patched onto PD agar for the detection of cercosporin production. C. kikuchii genomic DNA was isolated from mycelium harvested from 5-day-old PD liquid cultures as described by Garber and Yoder [10]. DNA
restriction digests (5 Wg DNA for each strain) and Southern blot analyses were performed as described by Sambrook et al. [11]. The Qiaquick gel extraction kit (Qiagen, Santa Clarita, CA, USA) was used to purify DNA fragments from agarose gels for hybridization probes. Nytran Plus membranes (Schleicher and Schuell, Keene, NH, USA) were used for nucleic acid blots using procedures supplied by the manufacturer. 32 P-radiolabeled pCFP insert DNA was made with the random priming oligolabeling kit from Pharmacia (Thousand Islands, CA, USA). For RNA isolation, lyophilized mycelium (80 mg) from each 5-day-old culture was frozen in liquid nitrogen and ground to a ¢ne powder. Total RNA was extracted from the powdered tissue using the RNeasy Plant Mini kit and protocol for isolation of total RNA from ¢lamentous fungi supplied by Qiagen. For the analysis of CFP transcript, varying amounts of fungal RNA were applied to Nytran Plus membranes with a Schleicher and Schuell Minifold II to prepare slot blots. Hybridizations were performed in a 50% formamide bu¡er at 42³C with pCFP insert DNA labeled to high speci¢c activity as described by Sambrook et al. [11]. CFP expression ratios were derived from scanning slot blot ¢lms with a laser scanning densitometer supplied by Molecular Dynamics (Sunnyvale, CA, USA). 2.3. Immunoblot analysis Soluble proteins were extracted from C. kikuchii strains as outlined by Rollins et al. [12]. 20 Wg of fungal protein was applied to a SDS^polyacrylamide gel (4% stacking and 12% separating gel) and electrophoresed for 45 min in a Bio-Rad Mini-Protean II Slab Cell (Bio-Rad Laboratories, Hercules, CA, USA). After electrophoresis, proteins were transferred to a polyvinylidene £uoride membrane (Micron Separations, Westborough, MA, USA) using a Bio-Rad transblot semi-dry transfer cell following the manufacturer's protocol. After transfer, the ¢lter was blocked by incubation in 3% (w/v) non-fat skim milk in TBST bu¡er (20 mM Tris^HCl, pH 7.5, 150 mM NaCl and 0.05% Tween 20) for 2 h at room temperature with gentle swirling. After the blocking step, the membrane was incubated for 1 h at room temperature in a 1:200 dilution of the primary rabbit anti-CFP antibody. This a¤nity-puri¢ed, polyclonal antibody is peptide-speci¢c to a hydrophilic and putatively highly antigenic region of the aminoterminus (ACREIEDPEKGQSAEIVC-amide) of CFP [2]. The antibody was prepared for us by Quality Controlled Biochemicals, Hopkinton, MA, USA. The membrane was then washed ¢ve times with TBST, 10 min each wash with gentle swirling. Following washing, the membrane was incubated for 1 h with a 1:5000 goat anti-rabbit secondary antibody conjugated to alkaline phosphatase (Promega, Madison, WI, USA). CFP protein was visualized by the color reaction of alkaline phosphatase with the western blue stabilize AP substrate (Promega).
FEMSLE 10133 17-10-01
R.G. Upchurch et al. / FEMS Microbiology Letters 204 (2001) 89^93
91
3. Results 3.1. Multiple genomic copies of CFP are present in transformants 10-1 and 10-11 Southern hybridization of EcoRI-digested PR and Gus-3 DNA probed with CFP produced patterns that were consistent with predictions for wild-type and disrupted CFP. Analysis showed that CFP was a singlecopy gene, residing on a 6.5-kb EcoRI fragment in isolate PR (Fig. 1, lane 2). Disruption of CFP in Gus-3 (lane 3), as described previously [2], was achieved by the insertion of a 7.1-kb HindIII fragment containing the L-glucuronidase UIDA expression unit from pNOM102 at a unique HindIII restriction site within CFP which introduces a unique and diagnostic EcoRI site. Co-transformation of Gus-3 with CFP/hph resulted in the recovery of 10-1 and 10-11, two stable, hygromycin-resistant, cercosporin-producing co-transformants. The CFP hybridization patterns for 10-1 and 10-11 (lanes 4 and 5) were consistent with the presence of multiple, tandem and ectopic genomic integrations of CFP in these strains. 3.2. CFP transcript is over-expressed in 10-1 and 10-11 To assess the correlation between CFP copy number and CFP transcript accumulation in PR, Gus-3, 10-1 and 10-11, we employed slot blot analysis. Cercosporin
Fig. 2. Northern slot blot analysis of CFP transcript accumulation in C. kikuchii PR and transformants 10-1 and 10-11. Cultures for RNA extraction were grown for 5 days in liquid PD medium in the light at 23³C. RNA slot blots were probed with a 1.9-kb 32 P-labeled insert of pCFP. Slot number is shown on the left and the expression of CFP transcript as a ratio of wild-type expression is shown on the right. Transcript accumulation was quanti¢ed by scanning densitometry. Slot number, strain, and RNA loading are as follows: 1, PR, 5 Wg; 2, 10-1, 5 Wg; 3, 10-11, 5 Wg; 4, PR, 1 Wg; 5, 10-1, 1 Wg; 6, 10-11, 1 Wg.
was visible in cultures of PR, 10-1 and 10-11, but not in Gus-3 when mycelium was harvested for RNA isolation. In Fig. 2, slots 1^3 (set 1) show the results of CFP hybridization with 5 Wg total RNA of each strain, while slots 4^6 (set 2) show the results with 1 Wg total RNA. Analysis of sets 1 and 2 produced essentially the same result. CFP transcript accumulation was clearly elevated in 10-1 and 10-11 to a level at least three times that of PR based on expression ratios (on the right) derived from scanning densitometry. No CFP transcript was detected in Gus-3 (data not shown).
Table 1 Cercosporin production and secretion by wild-type PR, CFP disruptant Gus-3, and transformants 10-1 and 10-11 Cercosporin (nmol mg31 fungal dry wt.)a
Strain b
Fig. 1. Southern analysis of EcoRI-digested genomic DNA of C. kikuchii strains. The hybridization probe for the fungal DNA digests was the 32 P-labeled 1.9-kb insert cDNA of clone pCFP [2]. Lane 1, V DNA molecular mass markers; lane 2, wild-type PR; lane 3, Gus-3; lanes 4 and 5, 10-1 and 10-11, CFP transformed strains of Gus-3.
PR, Lt PR, Dkc Gus-3, Lt Gus-3, Dk 10-1, Lt 10-1, Dk 10-11, Lt 10-11, Dk a
Mycelium+medium
Medium
34.1 þ 8.2 3.2 þ 1.2 1.8 þ 0.7 nd 109.7 þ 15.2 3.5 þ 0.9 116.5 þ 22.3 2.7 þ 0.8
9.5 þ 1.4 ndd nd nd 25.2 þ 3.0 nd 24.3 þ 2.5 nd
Mean þ S.E.M. for two experiments. Lt, grown in continuous light for 5 days. c Dk, grown in continuous dark for 5 days. d nd, not detected. b
FEMSLE 10133 17-10-01
92
R.G. Upchurch et al. / FEMS Microbiology Letters 204 (2001) 89^93
Fig. 3. Immunodetection of the ca. 65-kDa CFP protein in the C. kikuchii strains. Pre-stained protein molecular mass markers are to the left. Lanes are: 1, PR; 2, Gus-3; 3, 10-1; 4, 10-11.
3.3. CFP protein is over-expressed in 10-1 and 10-11 To assess the correlation between CFP transcript accumulation and CFP protein accumulation in PR, Gus-3, 101 and 10-11, we used a CFP peptide-speci¢c antibody and Western blotting to detect CFP in fungal protein extracts. An immunostained membrane transfer of a SDS^PAGE gel, equally loaded for each strain, is shown in Fig. 3. Based on visual inspection of the immunostaining intensity, 10-1 and 10-11 appear to accumulate about three-fold more of the ca. 65-kDa CFP protein than PR. No CFP protein was detected in protein extracts of Gus-3. 3.4. Over-expression of CFP in 10-1 and 10-11 is associated with the over-production and over-secretion of cercosporin Total cercosporin accumulation (mycelium+medium) and cercosporin secreted into the medium (medium) were measured for light- and dark-grown cultures of PR, Gus-3, 10-1 and 10-11 (Table 1). In the light, CFP multi-copy strains 10-1 and 10-11 produced about three times the total amount of cercosporin produced by wild-type PR, while CFP disruptant Gus-3 produced about 5% of the PR level. Total cercosporin accumulations for darkgrown PR, 10-1 and 10-11 were similar, while no cercosporin was detected in dark-grown Gus-3. In the light, CFP multi-copy strains 10-1 and 10-11 secreted about two times the wild-type PR level of cercosporin. Cercosporin (secretion) was not detected in the medium of darkgrown PR, Gus-3, 10-1 and 10-11. 4. Discussion We have shown that cercosporin production is restored to C. kikuchii CFP disruptant strain Gus-3 by co-trans-
formation with a 6.5-kb genomic fragment containing CFP and plasmid pUCHI containing the hph hygromycin resistance marker. Sequence analysis of the entire 6.5-kb EcoRI genomic fragment [2], 2.1 kb of which encodes the unspliced CFP transcript, revealed no other decipherable cercosporin metabolic gene, suggesting that the e¡ects reported here are the result of the (over-)expression of CFP. Two hygromycin-resistant transformants, 10-1 and 10-11, were recovered from the co-transformation experiment that appeared to produce elevated levels of cercosporin on PD agar medium. Southern analysis of 10-1 and 1011 revealed that these strains contained multiple genomic copies of CFP (see Fig. 1). Multi-copy CFP strains 10-1 and 10-11 exhibited a three-fold higher accumulation of CFP transcript and CFP protein compared to CFP transcript and protein accumulation in wild-type PR (see Figs. 2 and 3). Assays of cercosporin accumulation showed that 10-1 and 10-11 produced and secreted substantially greater amounts of cercosporin than the parental strain, PR. The three-fold increase in toxin accumulation, predominately in the mycelium fraction of these CFP multi-copy strains, suggests that CFP may have a signi¢cant, but unknown, role in regulating toxin production. Although cercosporin production and secretion were substantially elevated in 10-1 and 10-11, other phenotypic traits were not measurably di¡erent from wild-type PR. Experiments in which dark-grown cultures were switched to light showed that the initiation time point of cercosporin production for 10-1 and 10-11 did not di¡er from that of wild-type PR, though 10-1 and 10-11 went on to accumulate and secrete, respectively, about three-fold and twofold more cercosporin than PR by day 5. Dark-grown 10-1 and 10-11 accumulated similarly low levels of cercosporin characteristic of wild-type PR grown in the dark, suggesting that the presence of multiple copies of CFP does not alter the dark or light regulation pattern of cercosporin accumulation in 10-1 and 10-11 (Table 1). Growth yields (dry weight accumulation) for 5-day cultures of PR, Gus3, 10-1 and 10-11 grown in the light or dark do not di¡er, suggesting that the disruption of CFP in Gus-3 and the over-expression of CFP in 10-1 and 10-11 does not inhibit growth. Finally, as reported previously [2], both virulence on soybean and auto-resistance to exogenous cercosporin were restored in 10-1 and 10-11, but did not di¡er in degree from the wild-type situation. It has been hypothesized that either a change in the cell membrane structure resulting from loss of CFP interferes with wild-type cercosporin resistance or facilitated transport of cercosporin across the fungal membrane contributes substantially to resistance [5]. Gus-3 lacks wild-type resistance to cercosporin, accumulates no CFP transcript, and produces and secretes only a small fraction of the wild-type levels of cercosporin (Table 1). Restoration of wild-type cercosporin resistance and cercosporin secretion in 10-1 and 10-11 suggests that CFP does contribute substantially to cercosporin resistance. However, the three-
FEMSLE 10133 17-10-01
R.G. Upchurch et al. / FEMS Microbiology Letters 204 (2001) 89^93
fold increase in toxin accumulation in 10-1 and 10-11, which is largely associated with the fungal mycelium, is not accompanied by a hyper-resistance to cercosporin in these strains. Since the over-expression of CFP results in increased accumulation of cercosporin (predominately) in the mycelium fraction of 10-1 and 10-11 with wild-type resistance to cercosporin, crucial cytoplasmic resistance mechanisms involving toxin reduction [5] and antioxidant defense [5] must be responsible for the major portion of cercosporin resistance observed in Cercospora. On the other hand, the major role of CFP may be to rid the vulnerable cell membrane, rather than the entire cell, of this potent photosensitizer.
Acknowledgements We thank S. Gelobter for excellent technical assistance. Support for M.E. was provided by Cooperative Research and Development Agreement 58-3K95-6-449 with Pioneer Hi-Bred International, Inc.
References [1] Ehrenshaft, M. and Upchurch, R.G. (1991) Isolation of light-enhanced cDNAs of Cercospora kikuchii. Appl. Environ. Microbiol. 57, 2671^2676.
93
[2] Callahan, T.M., Rose, M.S., Meade, M.J., Ehrenshaft, M. and Upchurch, R.G. (1999) CFP, the putative cercosporin transporter of Cercospora kikuchii, is required for wild type cercosporin production, resistance, and virulence on soybean. MPMI 12, 901^910. [3] Paulsen, I.T., Brown, M.H. and Skurry, R.H. (1996) Proton-dependent multidrug e¥ux systems. Microbiol. Rev. 60, 575^608. [4] Daub, M.E. (1982) Cercosporin, a photosensitizing toxin from Cercospora spp. Phytopathology 72, 370^374. [5] Daub, M.E. and Ehrenshaft, M. (2000) The photoactivated Cercospora toxin cercosporin: contributions to plant disease and fundamental biology. Annu. Rev. Phytopathol. 38, 461^490. [6] Upchurch, R.G., Walker, D.C., Rollins, J.A., Ehrenshaft, M. and Daub, M.E. (1991) Mutants of Cercospora kikuchii altered in cercosporin synthesis and pathogenicity. Appl. Environ. Microbiol. 57, 2940^2945. [7] Jenns, A.E., Daub, M.E. and Upchurch, R.G. (1989) Regulation of cercosporin accumulation in culture by medium and temperature manipulation. Phytopathology 79, 213^219. [8] Turgeon, B.G., Garber, R.C. and Yoder, O.C. (1987) Development of a fungal transformation system based on selection of sequences with promoter activity. Mol. Cell. Biol. 7, 3297^3305. [9] Upchurch, R.G., Meade, M.J., Hightower, R.C., Thomas, R.S. and Callahan, T.M. (1994) Transformation of the fungal soybean pathogen Cercospora kikuchii with the selectable marker bar. Appl. Environ. Microbiol. 60, 4592^4595. [10] Garber, R.C. and Yoder, O.C. (1983) Isolation of DNA from ¢lamentous fungi and separation into nuclear, mitochondrial, ribosomal, and plasmid components. Anal. Biochem. 135, 416^422. [11] Sambrook, J., Fritsch, E.F. and Maniatis, T.A. (1989) Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. [12] Rollins, J.A., Ehrenshaft, M. and Upchurch, R.G. (1992) E¡ects of light- and altered-cercosporin phenotypes on gene expression in Cercospora kikuchii. Can. J. Microbiol. 39, 118^124.
FEMSLE 10133 17-10-01
www.fems-microbiology.org
Over-expression of the cercosporin facilitator protein, CFP, in Cercospora kikuchii up-regulates production and secretion of cercosporin Robert G. Upchurch a
a;b;
*, Mark S. Rose
a;b;1
, Mohamed Eweida
b;2
Agricultural Research Service, US Department of Agriculture, North Carolina State University, Raleigh, NC 27695 USA b Department of Plant Pathology, North Carolina State University, Raleigh, NC 27695 USA Received 2 August 2001; accepted 8 August 2001 First published online 18 September 2001
Abstract CFP (cercosporin facilitator protein), a light-regulated gene from the soybean fungal pathogen Cercospora kikuchii, encodes the putative major facilitator transporter of the fungal polyketide cercosporin. Gene disruption of CFP in C. kikuchii strain Gus-3 resulted in dramatically reduced cercosporin production and virulence, and increased sensitivity to the toxin. Two C. kikuchii transformant strains (10-1 and 10-11) that over-produce cercosporin were recovered from the complementation of CFP gene-disrupted strain Gus-3. Southern analysis revealed that these strains contained multiple genomic copies of CFP and over-expressed CFP transcript and protein. Although 10-1 and 10-11 produce and secrete significantly elevated levels of cercosporin, they exhibit wild-type resistance to cercosporin, and maintain a wild-type pattern of light-regulated toxin accumulation. Restoration of wild-type cercosporin resistance in 10-1 and 10-11 suggests that CFP does contribute substantially to cercosporin resistance via toxin secretion. The three-fold increase in toxin accumulation, predominately associated with the mycelium fraction of these CFP multi-copy strains, suggests that CFP may also have a significant, but unknown, role in regulating toxin production. ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Cercosporin ; CFP; Major facilitator; Over-expression ; Resistance; Cercospora kikuchii
1. Introduction CFP, a light-regulated gene encoding the ca. 65.4-kDa protein CFP (cercosporin facilitator protein) from the soybean fungal pathogen Cercospora kikuchii, was the ¢rst putative major facilitator transporter of a fungal polyketide to be described [1,2]. Members of the major facilitator superfamily [3] are integral membrane proteins that can
* Corresponding author. Tel. : +1 (919) 515-6996; Fax: +1 (919) 515-7716. E-mail address : [email protected] (R.G. Upchurch). 1 Present address: Syngenta Biotechnology Institute, 3054 Cornwallis Rd., Research Triangle Park, NC 27709-2257, USA. 2 Present address: Division of Cellular and Molecular Biology, Ontario Cancer Institute/Princess Margaret Hospital, 610 University Ave., Toronto, ON, Canada M5G 2M9.
confer resistance to drugs and other toxins through an active export mechanism energized by proton motive force. C. kikuchii and other Cercospora species produce the plant toxin cercosporin, a red, lipid-soluble, perylenequinone photosensitizer and potent generator of singlet oxygen [4,5]. Gene disruption of CFP in C. kikuchii strain Gus-3 resulted in dramatically diminished cercosporin production and virulence on the plant host, and increased sensitivity to exogenous cercosporin. Based on these ¢ndings, we proposed that CFP encodes a cercosporin transporter that contributes both to toxin resistance and to virulence by facilitated secretion [2]. When the CFP disruptant strain Gus-3 was co-transformed with the hygromycin resistance marker and a genomic fragment containing the intact CFP gene, stable, hygromycin-resistant transformants were recovered that were complemented to wild-type levels for toxin production, virulence, and cercosporin resistance [2]. Two of the restored transformant
0378-1097 / 01 / $20.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 1 ) 0 0 3 8 5 - 8
FEMSLE 10133 17-10-01
90
R.G. Upchurch et al. / FEMS Microbiology Letters 204 (2001) 89^93
strains, 10-1 and 10-11, appeared to over-accumulate cercosporin on agar medium. Here, we report that these two transformants contain multiple genomic copies of CFP and over-express CFP transcript and CFP protein. These strains produce and secrete signi¢cantly elevated levels of cercosporin, but exhibit wild-type resistance to cercosporin, and maintain a pattern of light-regulated toxin accumulation similar to the wild-type parental strain. 2. Materials and methods 2.1. C. kikuchii strains, growth conditions, and cercosporin measurements All strains described in this report were derived from wild-type C. kikuchii, isolate PR [6]. Cultures were grown for 5 days in potato dextrose broth (PDB, Difco, Detroit, MI, USA) at 23³C under either continuous white £uorescent light (approximately 80 WE s31 m32 ) or in darkness. Cultures were shaken at 180 rpm in an Innova illuminated incubator shaker (New Brunswick Scienti¢c, Edison, NJ, USA) in 125-ml Erlenmeyer £asks containing 50 ml of PDB. Dark-grown cultures were obtained by wrapping £asks with plastic-lined, black cloth and aluminum foil. For the measurement of cercosporin production, total cercosporin accumulation in mycelium plus medium was measured. Entire cultures were blended in a Waring blender for two 20-s pulses. A 10-ml aliquot of the suspension was treated with 1 vol of 5 N KOH [7] in darkness for 4 h and clari¢ed by low speed centrifugation. For the measurement of cercosporin secretion into the medium, the mycelium mass was removed by vacuum ¢ltration and a 10-ml aliquot of the mycelium-free medium was treated with KOH as described above. Cercosporin concentrations were determined spectrophotometrically [7]. The harvested mycelium was lyophilized and used for dry weight determinations. Three culture £asks were assayed for each strain in the experiment and the entire experiment was repeated. 2.2. Strain construction and molecular biological methods Strains 10-1 and 10-11 resulted from the co-transformation of C. kikuchii CFP disruptant strain Gus-3 with pgCFP [2], containing a wild-type copy of CFP on a 6.5-kb EcoRI genomic fragment, and pUCHI [8], a plasmid containing the hph gene for the selectable hygromycin B resistance marker. The GenBank accession number for CFP is AF091042. Transformants were recovered on hygromycin-containing regeneration medium as previously described [9], then patched onto PD agar for the detection of cercosporin production. C. kikuchii genomic DNA was isolated from mycelium harvested from 5-day-old PD liquid cultures as described by Garber and Yoder [10]. DNA
restriction digests (5 Wg DNA for each strain) and Southern blot analyses were performed as described by Sambrook et al. [11]. The Qiaquick gel extraction kit (Qiagen, Santa Clarita, CA, USA) was used to purify DNA fragments from agarose gels for hybridization probes. Nytran Plus membranes (Schleicher and Schuell, Keene, NH, USA) were used for nucleic acid blots using procedures supplied by the manufacturer. 32 P-radiolabeled pCFP insert DNA was made with the random priming oligolabeling kit from Pharmacia (Thousand Islands, CA, USA). For RNA isolation, lyophilized mycelium (80 mg) from each 5-day-old culture was frozen in liquid nitrogen and ground to a ¢ne powder. Total RNA was extracted from the powdered tissue using the RNeasy Plant Mini kit and protocol for isolation of total RNA from ¢lamentous fungi supplied by Qiagen. For the analysis of CFP transcript, varying amounts of fungal RNA were applied to Nytran Plus membranes with a Schleicher and Schuell Minifold II to prepare slot blots. Hybridizations were performed in a 50% formamide bu¡er at 42³C with pCFP insert DNA labeled to high speci¢c activity as described by Sambrook et al. [11]. CFP expression ratios were derived from scanning slot blot ¢lms with a laser scanning densitometer supplied by Molecular Dynamics (Sunnyvale, CA, USA). 2.3. Immunoblot analysis Soluble proteins were extracted from C. kikuchii strains as outlined by Rollins et al. [12]. 20 Wg of fungal protein was applied to a SDS^polyacrylamide gel (4% stacking and 12% separating gel) and electrophoresed for 45 min in a Bio-Rad Mini-Protean II Slab Cell (Bio-Rad Laboratories, Hercules, CA, USA). After electrophoresis, proteins were transferred to a polyvinylidene £uoride membrane (Micron Separations, Westborough, MA, USA) using a Bio-Rad transblot semi-dry transfer cell following the manufacturer's protocol. After transfer, the ¢lter was blocked by incubation in 3% (w/v) non-fat skim milk in TBST bu¡er (20 mM Tris^HCl, pH 7.5, 150 mM NaCl and 0.05% Tween 20) for 2 h at room temperature with gentle swirling. After the blocking step, the membrane was incubated for 1 h at room temperature in a 1:200 dilution of the primary rabbit anti-CFP antibody. This a¤nity-puri¢ed, polyclonal antibody is peptide-speci¢c to a hydrophilic and putatively highly antigenic region of the aminoterminus (ACREIEDPEKGQSAEIVC-amide) of CFP [2]. The antibody was prepared for us by Quality Controlled Biochemicals, Hopkinton, MA, USA. The membrane was then washed ¢ve times with TBST, 10 min each wash with gentle swirling. Following washing, the membrane was incubated for 1 h with a 1:5000 goat anti-rabbit secondary antibody conjugated to alkaline phosphatase (Promega, Madison, WI, USA). CFP protein was visualized by the color reaction of alkaline phosphatase with the western blue stabilize AP substrate (Promega).
FEMSLE 10133 17-10-01
R.G. Upchurch et al. / FEMS Microbiology Letters 204 (2001) 89^93
91
3. Results 3.1. Multiple genomic copies of CFP are present in transformants 10-1 and 10-11 Southern hybridization of EcoRI-digested PR and Gus-3 DNA probed with CFP produced patterns that were consistent with predictions for wild-type and disrupted CFP. Analysis showed that CFP was a singlecopy gene, residing on a 6.5-kb EcoRI fragment in isolate PR (Fig. 1, lane 2). Disruption of CFP in Gus-3 (lane 3), as described previously [2], was achieved by the insertion of a 7.1-kb HindIII fragment containing the L-glucuronidase UIDA expression unit from pNOM102 at a unique HindIII restriction site within CFP which introduces a unique and diagnostic EcoRI site. Co-transformation of Gus-3 with CFP/hph resulted in the recovery of 10-1 and 10-11, two stable, hygromycin-resistant, cercosporin-producing co-transformants. The CFP hybridization patterns for 10-1 and 10-11 (lanes 4 and 5) were consistent with the presence of multiple, tandem and ectopic genomic integrations of CFP in these strains. 3.2. CFP transcript is over-expressed in 10-1 and 10-11 To assess the correlation between CFP copy number and CFP transcript accumulation in PR, Gus-3, 10-1 and 10-11, we employed slot blot analysis. Cercosporin
Fig. 2. Northern slot blot analysis of CFP transcript accumulation in C. kikuchii PR and transformants 10-1 and 10-11. Cultures for RNA extraction were grown for 5 days in liquid PD medium in the light at 23³C. RNA slot blots were probed with a 1.9-kb 32 P-labeled insert of pCFP. Slot number is shown on the left and the expression of CFP transcript as a ratio of wild-type expression is shown on the right. Transcript accumulation was quanti¢ed by scanning densitometry. Slot number, strain, and RNA loading are as follows: 1, PR, 5 Wg; 2, 10-1, 5 Wg; 3, 10-11, 5 Wg; 4, PR, 1 Wg; 5, 10-1, 1 Wg; 6, 10-11, 1 Wg.
was visible in cultures of PR, 10-1 and 10-11, but not in Gus-3 when mycelium was harvested for RNA isolation. In Fig. 2, slots 1^3 (set 1) show the results of CFP hybridization with 5 Wg total RNA of each strain, while slots 4^6 (set 2) show the results with 1 Wg total RNA. Analysis of sets 1 and 2 produced essentially the same result. CFP transcript accumulation was clearly elevated in 10-1 and 10-11 to a level at least three times that of PR based on expression ratios (on the right) derived from scanning densitometry. No CFP transcript was detected in Gus-3 (data not shown).
Table 1 Cercosporin production and secretion by wild-type PR, CFP disruptant Gus-3, and transformants 10-1 and 10-11 Cercosporin (nmol mg31 fungal dry wt.)a
Strain b
Fig. 1. Southern analysis of EcoRI-digested genomic DNA of C. kikuchii strains. The hybridization probe for the fungal DNA digests was the 32 P-labeled 1.9-kb insert cDNA of clone pCFP [2]. Lane 1, V DNA molecular mass markers; lane 2, wild-type PR; lane 3, Gus-3; lanes 4 and 5, 10-1 and 10-11, CFP transformed strains of Gus-3.
PR, Lt PR, Dkc Gus-3, Lt Gus-3, Dk 10-1, Lt 10-1, Dk 10-11, Lt 10-11, Dk a
Mycelium+medium
Medium
34.1 þ 8.2 3.2 þ 1.2 1.8 þ 0.7 nd 109.7 þ 15.2 3.5 þ 0.9 116.5 þ 22.3 2.7 þ 0.8
9.5 þ 1.4 ndd nd nd 25.2 þ 3.0 nd 24.3 þ 2.5 nd
Mean þ S.E.M. for two experiments. Lt, grown in continuous light for 5 days. c Dk, grown in continuous dark for 5 days. d nd, not detected. b
FEMSLE 10133 17-10-01
92
R.G. Upchurch et al. / FEMS Microbiology Letters 204 (2001) 89^93
Fig. 3. Immunodetection of the ca. 65-kDa CFP protein in the C. kikuchii strains. Pre-stained protein molecular mass markers are to the left. Lanes are: 1, PR; 2, Gus-3; 3, 10-1; 4, 10-11.
3.3. CFP protein is over-expressed in 10-1 and 10-11 To assess the correlation between CFP transcript accumulation and CFP protein accumulation in PR, Gus-3, 101 and 10-11, we used a CFP peptide-speci¢c antibody and Western blotting to detect CFP in fungal protein extracts. An immunostained membrane transfer of a SDS^PAGE gel, equally loaded for each strain, is shown in Fig. 3. Based on visual inspection of the immunostaining intensity, 10-1 and 10-11 appear to accumulate about three-fold more of the ca. 65-kDa CFP protein than PR. No CFP protein was detected in protein extracts of Gus-3. 3.4. Over-expression of CFP in 10-1 and 10-11 is associated with the over-production and over-secretion of cercosporin Total cercosporin accumulation (mycelium+medium) and cercosporin secreted into the medium (medium) were measured for light- and dark-grown cultures of PR, Gus-3, 10-1 and 10-11 (Table 1). In the light, CFP multi-copy strains 10-1 and 10-11 produced about three times the total amount of cercosporin produced by wild-type PR, while CFP disruptant Gus-3 produced about 5% of the PR level. Total cercosporin accumulations for darkgrown PR, 10-1 and 10-11 were similar, while no cercosporin was detected in dark-grown Gus-3. In the light, CFP multi-copy strains 10-1 and 10-11 secreted about two times the wild-type PR level of cercosporin. Cercosporin (secretion) was not detected in the medium of darkgrown PR, Gus-3, 10-1 and 10-11. 4. Discussion We have shown that cercosporin production is restored to C. kikuchii CFP disruptant strain Gus-3 by co-trans-
formation with a 6.5-kb genomic fragment containing CFP and plasmid pUCHI containing the hph hygromycin resistance marker. Sequence analysis of the entire 6.5-kb EcoRI genomic fragment [2], 2.1 kb of which encodes the unspliced CFP transcript, revealed no other decipherable cercosporin metabolic gene, suggesting that the e¡ects reported here are the result of the (over-)expression of CFP. Two hygromycin-resistant transformants, 10-1 and 10-11, were recovered from the co-transformation experiment that appeared to produce elevated levels of cercosporin on PD agar medium. Southern analysis of 10-1 and 1011 revealed that these strains contained multiple genomic copies of CFP (see Fig. 1). Multi-copy CFP strains 10-1 and 10-11 exhibited a three-fold higher accumulation of CFP transcript and CFP protein compared to CFP transcript and protein accumulation in wild-type PR (see Figs. 2 and 3). Assays of cercosporin accumulation showed that 10-1 and 10-11 produced and secreted substantially greater amounts of cercosporin than the parental strain, PR. The three-fold increase in toxin accumulation, predominately in the mycelium fraction of these CFP multi-copy strains, suggests that CFP may have a signi¢cant, but unknown, role in regulating toxin production. Although cercosporin production and secretion were substantially elevated in 10-1 and 10-11, other phenotypic traits were not measurably di¡erent from wild-type PR. Experiments in which dark-grown cultures were switched to light showed that the initiation time point of cercosporin production for 10-1 and 10-11 did not di¡er from that of wild-type PR, though 10-1 and 10-11 went on to accumulate and secrete, respectively, about three-fold and twofold more cercosporin than PR by day 5. Dark-grown 10-1 and 10-11 accumulated similarly low levels of cercosporin characteristic of wild-type PR grown in the dark, suggesting that the presence of multiple copies of CFP does not alter the dark or light regulation pattern of cercosporin accumulation in 10-1 and 10-11 (Table 1). Growth yields (dry weight accumulation) for 5-day cultures of PR, Gus3, 10-1 and 10-11 grown in the light or dark do not di¡er, suggesting that the disruption of CFP in Gus-3 and the over-expression of CFP in 10-1 and 10-11 does not inhibit growth. Finally, as reported previously [2], both virulence on soybean and auto-resistance to exogenous cercosporin were restored in 10-1 and 10-11, but did not di¡er in degree from the wild-type situation. It has been hypothesized that either a change in the cell membrane structure resulting from loss of CFP interferes with wild-type cercosporin resistance or facilitated transport of cercosporin across the fungal membrane contributes substantially to resistance [5]. Gus-3 lacks wild-type resistance to cercosporin, accumulates no CFP transcript, and produces and secretes only a small fraction of the wild-type levels of cercosporin (Table 1). Restoration of wild-type cercosporin resistance and cercosporin secretion in 10-1 and 10-11 suggests that CFP does contribute substantially to cercosporin resistance. However, the three-
FEMSLE 10133 17-10-01
R.G. Upchurch et al. / FEMS Microbiology Letters 204 (2001) 89^93
fold increase in toxin accumulation in 10-1 and 10-11, which is largely associated with the fungal mycelium, is not accompanied by a hyper-resistance to cercosporin in these strains. Since the over-expression of CFP results in increased accumulation of cercosporin (predominately) in the mycelium fraction of 10-1 and 10-11 with wild-type resistance to cercosporin, crucial cytoplasmic resistance mechanisms involving toxin reduction [5] and antioxidant defense [5] must be responsible for the major portion of cercosporin resistance observed in Cercospora. On the other hand, the major role of CFP may be to rid the vulnerable cell membrane, rather than the entire cell, of this potent photosensitizer.
Acknowledgements We thank S. Gelobter for excellent technical assistance. Support for M.E. was provided by Cooperative Research and Development Agreement 58-3K95-6-449 with Pioneer Hi-Bred International, Inc.
References [1] Ehrenshaft, M. and Upchurch, R.G. (1991) Isolation of light-enhanced cDNAs of Cercospora kikuchii. Appl. Environ. Microbiol. 57, 2671^2676.
93
[2] Callahan, T.M., Rose, M.S., Meade, M.J., Ehrenshaft, M. and Upchurch, R.G. (1999) CFP, the putative cercosporin transporter of Cercospora kikuchii, is required for wild type cercosporin production, resistance, and virulence on soybean. MPMI 12, 901^910. [3] Paulsen, I.T., Brown, M.H. and Skurry, R.H. (1996) Proton-dependent multidrug e¥ux systems. Microbiol. Rev. 60, 575^608. [4] Daub, M.E. (1982) Cercosporin, a photosensitizing toxin from Cercospora spp. Phytopathology 72, 370^374. [5] Daub, M.E. and Ehrenshaft, M. (2000) The photoactivated Cercospora toxin cercosporin: contributions to plant disease and fundamental biology. Annu. Rev. Phytopathol. 38, 461^490. [6] Upchurch, R.G., Walker, D.C., Rollins, J.A., Ehrenshaft, M. and Daub, M.E. (1991) Mutants of Cercospora kikuchii altered in cercosporin synthesis and pathogenicity. Appl. Environ. Microbiol. 57, 2940^2945. [7] Jenns, A.E., Daub, M.E. and Upchurch, R.G. (1989) Regulation of cercosporin accumulation in culture by medium and temperature manipulation. Phytopathology 79, 213^219. [8] Turgeon, B.G., Garber, R.C. and Yoder, O.C. (1987) Development of a fungal transformation system based on selection of sequences with promoter activity. Mol. Cell. Biol. 7, 3297^3305. [9] Upchurch, R.G., Meade, M.J., Hightower, R.C., Thomas, R.S. and Callahan, T.M. (1994) Transformation of the fungal soybean pathogen Cercospora kikuchii with the selectable marker bar. Appl. Environ. Microbiol. 60, 4592^4595. [10] Garber, R.C. and Yoder, O.C. (1983) Isolation of DNA from ¢lamentous fungi and separation into nuclear, mitochondrial, ribosomal, and plasmid components. Anal. Biochem. 135, 416^422. [11] Sambrook, J., Fritsch, E.F. and Maniatis, T.A. (1989) Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. [12] Rollins, J.A., Ehrenshaft, M. and Upchurch, R.G. (1992) E¡ects of light- and altered-cercosporin phenotypes on gene expression in Cercospora kikuchii. Can. J. Microbiol. 39, 118^124.
FEMSLE 10133 17-10-01