Identification of genes expressed during cercosporin biosynthesisin Cercospora zeae-maydis

Physiological and Molecular Plant Pathology (2002) 61, 237±248 doi:10.1006/pmpp.2002.0437, available online at http://www.idealibrary.com on

Identi¢cation of genes expressed during cercosporin biosynthesis in Cercospora zeae-maydis WO N - B O S H I M and L A R RY D . D U N K L E * Crop Production and Pest Control Research, U.S. Department of Agriculture-Agricultural Research Service, Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907-1155, U.S.A. (Accepted for publication 2 September 2002) Gray leaf spot, caused by the fungus Cercospora zeae-maydis, is the most destructive foliar disease of maize in the United States. However, little is known about the biochemical and molecular events of pathogenesis. C. zeae-maydis produces cercosporin, a phytotoxin shown to be a virulence factor in diseases caused by other Cercospora species. To identify genes involved in cercosporin biosynthesis and ultimately determine the role of cercosporin in disease development, we constructed a cDNA subtraction library of C. zeae-maydis by suppression subtractive hybridization. Poly (A) ‡ RNA isolated from cultures grown in a cercosporinsuppressing medium was subtracted from poly (A) ‡ RNA isolated from cultures grown in a cercosporininducing medium, resulting in transcripts that are speci®c to the cercosporin-producing culture. Analyses of 768 sequences in this cDNA subtraction library revealed 197 cDNAs with high similarity to genes in the GenBank and Saccharomyces Genome Database, and these genes were grouped into nine categories based on predicted functions of the encoded proteins. Northern analysis of seven selected clones with predicted functions in fatty acid metabolism ( fatty acid synthase, oleate D-12 desaturase, and linoleate diol synthase) and secondary metabolism [cytochrome P450 oxidoreductase, cytochrome P450 monooxygenase, dihydrogeodin ( phenol) oxidase, and coproporphyrinogen oxidase] indicated that those genes were expressed in cercosporin-inducing conditions. Analysis of expression kinetics con®rmed that those genes are expressed concomitantly with cercosporin accumulation. Published by Elsevier Science Ltd. Keywords: Cercospora zeae-maydis; cercosporin biosynthesis; suppression subtractive hybridization; gene expression; secondary metabolite.

INTRODUCTION Gray leaf spot, caused by the fungus Cercospora zeae-maydis Tehon & E.Y. Daniels, has emerged as the most common and destructive foliar disease of maize throughout the major maize-growing regions of the United States [30, 42]. The disease has also been reported worldwide, particularly in Africa, causing substantial reductions in grain yield and quality [42]. Gray leaf spot incidence and severity have increased substantially in the United States concomitant with an increase in no-till or other conservation tillage practices [42]. Although the disease cycle, from initial infection to dissemination of secondary conidial inoculum from newly formed lesions, requires considerable time * To whom all correspondence should be addressed. E-mail: [email protected] Names are necessary to report factually on available data. However, the USDA neither guarantees nor warrants the standard of the product, and the use of the names implies no approval of the product to the exclusion of others that may also be suitable.

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[6, 7], the abundance of primary inoculum in overwintered diseased maize tissue apparently is sucient to initiate an epidemic and compensate for the lengthy stages of pathogenesis and symptom development [17, 37]. Like many members of the genus Cercospora [4, 22, 23, 32], C. zeae-maydis produces cercosporin [18, 24, 29, 41], a phytotoxin suggested to be a virulence factor in other plant diseases caused by Cercospora species [11]. Cercosporin, a photosensitizing perylenequinone [14, 16, 43], generates toxic reactive oxygen species, i.e. hydroxyl radicals and singlet oxygen, which damage the plant cell membrane [14±16]. In the light, cercosporin is converted to an electronically excited state that reacts with molecular oxygen to yield singlet oxygen. These singlet oxygen species can catalyse membrane lipid peroxidation, leading to the loss of membrane integrity, leakage of cytoplasmic contents, and cell death [14, 16]. For several cercosporin-producing fungi, it is suggested that high light intensities and cercosporin production are required for disease development [16, 40]. Published by Elsevier Science Ltd.

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Despite the physiological signi®cance of cercosporin, the molecular mechanisms of cercosporin biosynthesis and the role of cercosporin in disease development are not well characterized [16]. In an e€ort to identify genes involved in cercosporin-related plant pathogenesis, Ehrenshaft et al. [20] and Chung et al. [13] isolated singlet oxygen resistance (SOR1) and cercosporin resistance gene (CRG1), respectively, from. C. nicotianae. SOR1 was identi®ed as a gene required for resistance to singlet oxygen-generating photosensitizers in a diverse array of organisms [20] and CRG1 as a gene speci®cally required for cercosporin resistance in C. nicotianae [13]. Signi®cantly, SOR1 transcripts also were observed in the fungus grown in cercosporin non-inducing environments, indicating that the SOR1 gene product is not directly involved in cercosporin biosynthesis. The precise function of this protein was later established as an essential component in the biosynthesis of pyridoxine (vitamin B6), which is capable of quenching singlet oxygen [19]. Consequently, the name of the SOR1 gene was changed to PDX1. Callahan et al. [11] screened cDNAs obtained from lightenhanced transcripts of C. kikuchii and isolated the cercosporin facilitator protein (CFP) gene. Transcript levels of CFP increased 20-fold in cultures grown in the light, and the temporal expression pattern paralleled cercosporin accumulation. After further characterization, CFP was identi®ed as a membrane-bound transporter that is speci®c for exporting cercosporin from fungal cells [11]. Although the chemical structure of cercosporin [14, 16, 43] suggests that fatty acids and polyketides are logical precursors, no genes involved in the cercosporin biosynthetic pathway have been identi®ed. Because C. zeae-maydis lacks a sexual stage, genetic analysis has been dicult. To overcome this barrier and identify genes involved in cercosporin biosynthesis, we constructed a cDNA subtraction library of C. zeae-maydis. Poly (A) ‡ RNA from cultures grown in cercosporinsuppressing medium was subtracted from poly (A) ‡ RNA obtained from cultures grown in cercosporin-inducing medium, resulting in transcripts that are highly upregulated in the cercosporin-induced culture. Here we describe seven genes that were identi®ed from the cDNA subtraction library. Sequence data and northern analysis demonstrated that these genes are expressed concurrently with cercosporin production, suggesting they are involved in cercosporin biosynthesis in. C zeae-maydis.

MATERIALS AND METHODS

Fungal strain, media, and cercosporin assay The C. zeae-maydis strain (SCOH1) used in this study was isolated in September 1999, from infected maize in a ®eld near South Charleston, OH, U.S.A. A single-spore isolate

was recovered from conidia produced from a gray leaf spot lesion and maintained as previously described [37]. C. zeae-maydis was grown on 0.2 potato dextrose agar (PDA) (Difco, Detroit, MI, U.S.A.) to evaluate cercosporin production and on V8 juice agar to obtain conidia [29, 41]. For determination of cercosporin production, the fungus was grown on 0.2 PDA at 258C under a 14 h light (25 mE m 2 s 1)/10 h dark cycle for 7 days, and agar blocks containing the fungal mycelium and underlying medium were removed with a cork borer (8 mm diameter). Cercosporin was detected by spectrophotometric analysis of 5 N KOH extracts of the mycelium and agar blocks [18, 29] and quanti®ed by application of the molar extinction coecient in alkali (e ˆ 23 300) [43]. Conidia produced on 3 day old V8 agar plates were dislodged with a camel-hair brush, collected in deionized water, and adjusted to 5  105 conidia per ml. The conidial suspension (200 ml) was inoculated into 100 ml of 0.2 potato dextrose broth (PDB) (Difco, Detroit, MI, U.S.A.) or complete medium [CM; 10 g glucose, 1 g yeast extract, 1 g casein hydrolysate, 1 g Ca(NO3)2.4H2O, and 10 ml of mineral solution (2 g KH2 PO4, 2.5 g MgSO4.7H2O, 1.5 g NaCl in 100 ml water, pH 5.3) in 1 l of water] and incubated on a rotary shaker (150 rpm) at 258C under constant light (15 mE m 2 s 1). For preparation of nucleic acids, fungal mycelia were harvested after 7 days of incubation. To detect and quantify the production of cercosporin in these liquid cultures, a 1 ml sample of fungal culture was extracted with an equal volume of 5 N KOH and analysed by spectrophotometry [18, 29].

Extraction and analysis of total RNA To obtain total RNA, a conidial suspension was inoculated into 0.2 PDB or CM and grown for 7 days as described earlier. Fungal cultures (1 g fresh wt) were harvested by ®ltration through Miracloth (Calbiochem, La Jolla, CA, U.S.A.) and immediately frozen in liquid nitrogen. The samples were ground to a ®ne powder and transferred to sterile tubes. Total RNA was isolated with Trizol reagent (Gibco BRL, Grand Island, NY, U.S.A.) according to the manufacturer's instructions. The quality and concentration of RNA samples were determined by spectrophotometric methods as well as by visual assessment of u.v.-induced ¯uorescence emitted by ethidium bromide following electrophoresis on a 1.2 % denaturing agarose gel [36]. Northern analysis of PDX1 was performed to con®rm the quality of the RNA preparations used for construction of the cDNA subtraction library. Standard methods for northern analysis were used [38, 39]. The PDX1 DNA used as the 32P-labelled probe was prepared by PCR ampli®cation with primers Sor1fs (50 -ACAAAGGGTGAAGCAGGAAT-30 ) and Sor1ra

Cercosporin biosynthesis genes (50 -GGCAGCTTGTCGCAGTTGAT-30 ) [20]. PCR ampli®cation was performed in a 50 ml reaction mixture with AmpliTaq DNA polymerase (Roche, Branchburg, NJ, U.S.A.) in a DNA thermal cycler 480 (Perkin Elmer Cetus, Norwalk, CT, U.S.A.). The reactions were carried out for 30 cycles of 45 s of denaturation at 948C, 45 s of annealing at 588C, and 45 s of extension at 728C. To obtain total RNA samples for assessing the kinetics of gene expression, C. zeae-maydis conidial suspension was inoculated into CM broth (100 ml) and grown as described earlier. After 3 days of incubation, the culture was harvested by ®ltration through Miracloth, and 1 g of mycelium ( fresh wt) was suspended in fresh 0.2 PDB (100 ml), CM broth (100 ml) or 0.2 PDB with 10 mM ammonium phosphate (AP; 100 ml). The fungal cultures were incubated on a rotary shaker (150 rpm) at 258C under constant light (15 mE m 2 s 1) for 7 days. Mycelial samples (100 mg fresh wt) were collected daily from each fungal culture, and cercosporin production was assayed. Total RNA from each sample was extracted with Trizol reagent and stored at 808C.

Construction of the cDNA subtraction library Poly (A) ‡ RNA from C. zeae-maydis cultures grown in cercosporin-inducing medium (0.2 PDB) and poly (A) ‡ RNA from cultures grown in cercosporin-suppressing medium (CM), which were incubated on a rotary shaker (150 rpm) at 258C under constant light (15 mE m 2 s 1), were puri®ed from corresponding total RNA samples with Oligotex mRNA spin-columns (Qiagen, Valencia, CA, U.S.A.). The PCR-Select cDNA Subtraction Kit (Clontech, Palo Alto, CA, U.S.A.) was used to construct a Czm-P subtraction library (genes expressed only in cercosporin-inducing conditions) and a Czm-C subtraction library (genes expressed only in cercosporin-suppressing conditions). Ampli®ed PCR products were cloned into the pGEM-T Easy Vector (Promega, Madison, WI, U.S.A.), and Library Eciency DH5a competent cells (Gibco BRL, Grand Island, NY, U.S.A.) were used for library construction. A total of 768 clones from the Czm-P cDNA subtraction library and 384 clones from the Czm-C cDNA subtraction library were sequenced and analysed at the Agricultural Genome Center, Purdue University. Similarity searches were done via BLAST algorithm [3] against the genes in GenBank and the Saccharomyces genome database (SGD).

Northern analysis Genes identi®ed from analysis of the Czm-P cDNA subtraction library were used as probes for northern analysis. Total RNA samples (15 mg) obtained from C. zeae-maydis grown in 0.2 PDB or CM were subjected to electrophoresis on 1.2 % denaturing agarose gels and

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transferred by capillary action to Nytran SPC Membrane (Schleicher & Schuell, Keene, NH, U.S.A.). Probes were 32 P-labelled with a Prime It II Random Primer Labelling Kit (Stratagene, La Jolla, CA, U.S.A.). Northern hybridizations were performed as previously described [38, 39]. In all analyses, gels were stained with ethidium bromide to con®rm the uniformity of RNA loading.

Nucleotide sequence accession numbers The nucleotide sequences and the deduced polypeptide sequences of the genes identi®ed in this study were submitted to GenBank, and the accession numbers are listed in Table 1.

RESULTS

Fungal growth and cercosporin production in 0.2 PDB or CM Fungal growth of C. zeae-maydis was more vigorous in CM than in 0.2 PDB. After 7 days, an average of 1.7 g ( fresh wt) of mycelia was obtained from CM, whereas an average of 1.0 g ( fresh wt) of mycelia was obtained from 0.2 PDB. Despite the di€erence in growth rates, cercosporin production was visually detectable only in 0.2 PDB cultures [Fig. 1(A) and (B)]. Spectrophotometric analysis con®rmed the production of the toxin (17.8±20.2 ng ml 1) in 0.2 PDB cultures [Fig. 1(C)] and its absence in CM cultures [Fig. 1(D)]. Fig. 1(C) shows the characteristic and diagnostic absorption spectrum of cercosporin in alkali with Amax at 480, 595, and 640 nm. Although light is indicated as a critical factor for induction of cercosporin synthesis in other species [5, 11, 21, 32], in our study with C. zeae-maydis, dark-grown cultures produced low, but detectable levels of cercosporin (data not shown).

Subtraction cDNA library analysis Northern analysis of PDX1, a constitutively expressed gene [20], con®rmed the quality of the poly (A) ‡ RNA used for construction of the cDNA subtraction library (Fig. 2). PDX1 transcripts (1.3 kb) were intact in both total RNA samples and were expressed at comparable levels, indicating that these RNA samples were of suitable quality for library construction. A total of 768 clones from the Czm-P subtraction library, which includes cDNAs of genes expressed in cercosporin-inducing medium, were sequenced and analysed. In addition, 384 sequences from the Czm-C subtraction library were analysed to eliminate false positives from the Czm-P library and to con®rm the absence from both libraries of constitutively expressed

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T A B L E 1. Putative cercosporin biosynthesis genes identi®ed from the Czm-P cDNA subtraction library from C. zeae-maydis Clone

GenBank

Insert size

P01C22

AF448825

550 bp

P02D03

AF448826

470 bp

P02F09

AF448827

250 bp

P01J14

AF448828

500 bp

P02I19

AF448829

290 bp

P01F17

AF448830

500 bp

P02G06

AF448831

300 bp

P02E07

AF448832

560 bp

Putative function1 FAS: Fatty acid synthase, a subunit, Aspergillus nidulans (GenBank U75347) ODEA: Oleate D-12 desaturase, Aspergillus nidulans (GenBank AF262955) LDS: Linoleate diol synthase precursor, Gaeumannomyces graminis var. graminis (GenBank AF124979) CPRA: NADPH cytochrome P450 oxidoreductase, Aspergillus niger (GenBank S38427) STC: Sterigmatocystin biosynthesis P450 monooxygenase, Aspergillus nidulans (GenBank Q00707) DHGO: Dihydrogeodin oxidase, Aspergillus terreus (GenBank D49538) COX: Coproporphyrinogen oxidase, Mus musculus (GenBank A48049) CFP: Cercosporin transporter, Cercospora kikuchii (GenBank AF091042)

E value2

Identity3

Predicted size4

1E-83

89 %

6.5 kb

1E-47

70 %

1.4 kb

1E-13

47 %

4.4 kb

9E-37

54 %

2.3 kb

5E-12

49 %

1.8 kb

6E-29

58 %

2.9 kb

2E-27

62 %

1.2 kb

5E-72

76 %

2.4 kb

1

Putative function was designated based on BLAST analyses. Gene function with the highest score is presented in the table. Expectation value. 3 The extent to which two amino acid sequences are invariant. 4 Predicted transcript size based on northern analysis of corresponding genes. 2

genes, e.g. PDX1. From the Czm-P library, 197 clones containing cDNA sequences with high similarity (P 5 10 5) to genes in the databases (GenBank and SGD) were analysed for the size of the cDNA insert, which ranged from 136 to 754 bp in length with an average length of 510 bp (Fig. 3). Gene functions were designated based on information obtained from the databases, and these 197 clones were grouped into nine categories: carbohydrate metabolism, amino acid/protein metabolism, fatty acid metabolism, cell di€erentiation/development, membrane transporter, stress response, signal transduction, secondary metabolism, and hypothetical proteins with unknown functions (Fig. 4). The two categories that we considered to be potentially most informative are fatty acid metabolism and secondary metabolism, since the enzymes encoded by the genes in these categories perform functions that are pertinent to biosynthesis of secondary metabolites in ®lamentous fungi, such as toxin precursor synthesis and various structural modi®cations [1, 8±10].

Identi®cation of genes expressed during cercosporin biosynthesis We selected eight clones from the Czm-P cDNA subtraction library for further analysis. Three clones (P01C22, P02D03, and P02F09) were from the fatty acid metabolism category, and four clones (P01F17, P02I19, P02G06, and P01J14) were from the secondary metabolism

category. In addition, one clone (P02E07), which contains cDNA highly similar to CFP, was selected from the membrane transporter category. Table 1 summarizes the detailed information of these eight cDNA clones and lists their putative functions. Among the genes in the fatty acid metabolism category, clone P01C22 had interesting characteristics relevant to cercosporin biosynthesis. It was one of seven clones in the Czm-P cDNA subtraction library with high similarity to a fatty acid synthase (FAS) gene, including two with similarity to the b subunit. This clone contained a 550 bp insert that is highly similar to FAS (a subunit) of Aspergillus nidulans, particularly at the polypeptide level in the b-ketoacyl reductase domain (89 % identity) [Fig. 5(A)]. Northern blot analysis showed a transcript (ca. 6.5 kb) expressed only in the cercosporin-producing culture [Fig. 5(D)]. Published reports [1, 8, 9] suggest that FAS may play an important role in biosynthesis of fungal secondary metabolites, particularly in the early steps of the pathways. Two other clones, P02D03 and P02F09, were highly similar to oleate D-12 desaturase (ODEA) of A. nidulans and linoleate diol synthase (LDS) of Gaeumannomyces graminis var. graminis, respectively [Fig. 5(B) and (C)]. Northern analysis of these two genes con®rmed that they are uniquely expressed by cercosporin-producing cultures [Fig. 5(E) and (F)] and revealed the presence of transcripts of the predicted size (Table 1).

Cercosporin biosynthesis genes

B

A

0.5

0.5

Absorbance

D

Absorbance

C

241

400

700

Wavelength (nm)

400

700

Wavelength (nm)

F I G . 1. C. zeae-maydis cultures. (A) Cercosporin-inducing environment: 0.2 PDB. (B) Cercosporin non-inducing environment: complete medium. (C) Absorption spectrum ( from 400 to 700 nm) of culture ®ltrate of 0.2 PDB culture. (D) Absorption spectrum ( from 400 to 700 nm) of culture ®ltrate of CM culture.

Two clones from the secondary metabolism category, P01J14 and P02I19, contained sequences highly similar to cytochrome P450 monooxygenase (STC) and cytochrome P450 oxidoreductase (CPRA), respectively. These genes are members of a cytochrome P450 family that is well characterized and involved in secondary metabolite biosynthesis, particularly in sterigmatocystin biosynthesis by Aspergillus species [10]. The polypeptide sequences deduced from these two clones were nearly 50 % identical to their corresponding proteins in the database [Table 1, Fig. 6(A) and (B)], and the transcripts were expressed only in 0.2 PDB cultures [Fig. 6(E) and (F)]. Two other clones, P01F17 and P02G06, contained gene fragments similar to dihydrogeodin oxidase (DHGO), a phenol oxidase, of A. terreus and coproporphyrinogen oxidase (COX) of Mus musculus, respectively [Fig. 6(C) and (D)]. Deduced amino acid sequences of these two genes were 58 and 62 % identical, respectively, to the corresponding proteins in the database. In addition, transcript levels of these genes were distinctly higher in cercosporin-producing cultures [Fig. 6(G) and (H)] than other genes identi®ed in this

study. However, the roles of DHGO and COX in fungal secondary metabolism are not clearly understood. Clearly, C. zeae-maydis does not produce geodin, but the presence of this phenol oxidase suggests that an enzyme with a similar mechanism functions in cercosporin biosynthesis. Signi®cantly, a clone containing a nucleotide sequence with high similarity to the CFP gene of C. kikuchii was identi®ed from the Czm-P subtraction library. This gene (CzmCFP) was 76 % identical to CFP at the polypeptide level [Fig. 7(A)] and was highly expressed only in 0.2 PDB cultures [Fig. 7(B)]. The presence of this clone only in the Czm-P cDNA subtraction library and the absence of PDX1 from both the Czm-P and Czm-C libraries suggested that suppressive hybridization during library construction was performed e€ectively.

Kinetics of gene expression We performed northern analyses over a 7 day time period on four of the genes identi®ed in this study, two from fatty acid metabolism (FAS and LDS) and two from secondary

242

A

W.-B. Shim and L. D. Dunkle cDNA insert size (bp)

PDB CM

> 700 > 601 - 700 > 501 - 600 > 401 - 500 > 301 - 400

PDX1 (1.3 kb)

> 201 - 300 < 200

0

10

20

30

40

50

60

70

Number of clones

B

F I G . 2. (A) Northern blot analysis of PDX1. Total RNA samples (15 mg) were obtained from C. zeae-maydis mycelia grown in cercosporin-inducing (PDB) and non-inducing (CM) media. C. zeae-maydis PDX1 used for the hybridizations was prepared by PCR ampli®cation (see Materials and Methods). (B) RNA samples were stained with ethidium bromide after electrophoresis in a 1.2 % denaturing agarose gel.

metabolism (STC and DHGO), to show that expression patterns of these genes are coincident with cercosporin biosynthesis. Expression of CFP and PDX1 was analysed as induced and constitutively expressed genes, respectively. Three media were used to grow C. zeae-maydis and to follow gene expression during cercosporin production: 0.2 PDB, CM broth, and 0.2 PDB supplemented with 10 mM AP, which completely suppresses cercosporin production (Fig. 8). We selected 0.2 PDB ‡ AP as an additional cercosporin-suppressing medium in this experiment to test the hypothesis that di€erential gene expression observed in the subtraction library was a consequence of di€erences in media composition. Fig. 8(A) shows cercosporin production by C. zeaemaydis in the three media. Cercosporin production was ®rst detected on day 3 after transfer of fungal mycelium and reached maximal levels on day 5 in 0.2 PDB. Cercosporin production was not detected in CM or 0.2 PDB ‡ AP.

F I G . 3. Analysis of cDNA insert size of the clones identi®ed in the C. zeae-maydis cDNA subtraction library. A total of 768 clones from the Czm-P library (genes expressed only by the cercosporin-producing culture) were sequenced. Of those, 197 sequences with high similarity (P 5 10 5) to genes in the databases (GenBank and SGD) were analysed for the size of the cDNA insert. Inserts ranged from 136 to 754 bp in length with an average length of 510 bp.

Northern analysis showed that all four of the selected genes were expressed during cercosporin biosynthesis [Fig. 8(B)]. Transcripts of these genes were detected in C. zeae-maydis grown in 0.2 PDB, while very low levels or no transcripts were detected in the fungus grown in cercosporin-suppressing media (CM or 0.2 PDB ‡ AP). Signi®cantly, the expression pattern for three genes (FAS, STC and DHGO) was very similar to that of CzmCFP. In studies with C. kikuchii, transcript levels of CFP increased

Predicted gene functions HP SM ST SR MT CD FAM AAPM CM

0

10

20

30

40

50

60

Number of clones

F I G . 4. Predicted gene functions of the clones identi®ed in the C. zeae-maydis cDNA subtraction library. Of the 768 clones obtained from Czm-P library (genes expressed only by the cercosporin-producing culture), 197 clones contained cDNA sequences with high similarity (P 5 10 5) to genes in the databases (GenBank and SGD). These cDNAs were analysed, and the putative functions were assigned based on information obtained from the database. We grouped these cDNAs into nine categories: CM, carbohydrate metabolism; AAPM, amino acid/ protein metabolism; FAM, fatty acid metabolism; CD, cell di€erentiation/development; MT, membrane transporter; SR, stress response; ST, signal transduction; SM, secondary metabolism; and HP, hypothetical protein.

Cercosporin biosynthesis genes

A

P01C22: 534

AnFAS:

635

P01C22: 354

AnFAS:

695

P01C22: 174

AnFAS:

B

755

P02D03: 470

ODEA:

303

P02D03: 290

ODEA:

C

363

P02F09: 21

GggLDS: 442 P02F09: 201

GggLDS: 502

D

FAS

PDB CM

243

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

PHYTGDAWNFTRGAAATIDREFGFIGRTLMHGIVETHVLHHYVSTIPFYHADEATEAIKP 291 PHY ++W F RGAAATIDREFGFIGR ++HGI+ETHVLHHYVSTIPFYHADEA+EAIK PHYQPESWTFARGAAATIDREFGFIGRHILHGIIETHVLHHYVSTIPFYHADEASEAIKK 362 IMGRHYRANTEGGSVGFVKSLWSSARWCQWVEPNEGATGENSKVLFFRNRNGLGLPP 120 +MG HYR+ G +GF+K+LW+SAR C WVEP EG GEN+ VLFFRN NG+G+PP VMGSHYRSEAHTGPLGFLKALWTSARVCHWVEPTEGTKGENAGVLFFRNTNGIGVPP 419

YEDDDLANLLISSIEDCANAMGPRQVPTVMKAIEILGIQQARTWRCATLNEMRKHFDLQP 200 ++D +L +L SIED A A GP VP M+AIEILGI+Q+RTW ATLNE R+ L P FDDTELVKILQESIEDVAGAFGPNHVPACMRAIEILGIKQSRTWNVATLNEFRQFIGLTP 501 HETFESITDNKEV 239 H++F + + ++ HDSFYHMNPDPKI 514

E

ODEA

PDB CM

F

LDS

PDB CM

F I G . 5. BLAST results (A±C) and northern analysis (D±F) of C. zeae-maydis genes selected from the fatty acid metabolism category of the Czm-P cDNA subtraction library: FAS (A and D), oleate D-12 desaturase (B and E) and LDS (C and F). Amino acid sequences included for comparison are FAS from A. nidulans (A), ODEA from A. nidulans (B), and LDS from G. graminis var. graminis (C). Similar amino acids are indicated as ‡. For Northern blots, total RNA samples (15 mg) obtained from C. zeae-maydis mycelia grown in cercosporin-inducing (PDB) and non-inducing (CM) media were subjected to electrophoresis in a 1.2 % denaturing agarose gel. Standard methods for northern analysis were used, and gels were stained with ethidium bromide to con®rm uniformity of loading in each lane (not shown). FAS (D), ODEA (E), and LDS (F) cDNA fragments obtained from corresponding clones were used for the hybridizations.

20-fold in cultures grown in the light, and the temporal expression pattern paralleled cercosporin accumulation [11]. Expression of LDS was di€erent from the other genes. In 0.2 PDB cultures, the LDS transcript level increased with time, whereas in CM cultures the transcript decreased from constitutive levels to an undetectable level. Elevated levels of LDS gene expression were not detected in 0.2 PDB ‡ AP. As expected [16, 19, 20], PDX1 was expressed constitutively in all media.

DISCUSSION The biosynthetic pathway of cercosporin is poorly understood despite e€orts to understand the pathway by labelling with 14 C-acetate [36, 43], by isolating and analysing light-enhanced cDNAs from C. kikuchii [11, 21], and by isolating C. kikuchii mutants a€ected in cercosporin production [40]. Genes required for resistance to cercosporin, such as PDX1 and CRG1, and a gene speci®cally

244

W.-B. Shim and L. D. Dunkle

A

P02G06: 279 QVLIKSEGDTNAAGVAQGMSTAEMINNASVLVLAGAETSATTLSGATYLLLKHPNTMKLL 100 +V++KS D N +G GMS EMINNA+V+V+AG+ET+++ L G TYLL K + M STC: 271 RVIVKS-ADGNQSG--DGMSYGEMINNAAVMVVAGSETTSSALCGCTYLLCKF-DKMDKA 326 P02G06: 99

STC:

B

IDEIRAAFKCDEEIDARSVSRLSYL 25 + E+R AF ++ID SVSRL YL 327 VAEVRGAFAAADQIDLVSVSRLPYL 351

P01J14: 478 RYQLEICAPISRQFVSTLAQFAPNDEIKEKATKIGNDKDVFAEEVAKKNFNIGQLLEHLS 299 RY +E+CAP+SRQFV+TLA FAP + +++ + + +F E + Q L+ ++ AnCPRA: 369 RYYMEVCAPVSRQFVATLAAFAPMRKARQRLC-VWVAQGLFPREGHQPMLQHAQALQSIT 427 P01J14: 298 GGQVWDKIPFSLFIEGITKIQPRYYSISSSSLVQKHKVAITAVVESVEVPGAPHVVKGVT 119 + + +PFSL IEGITK+QPRYYSISSSSLVQK K++ITAVVESV +PGA H+VKGVT AnCPRA: 428 S-KPFSAVPFSLLIEGITKLQPRYYSISSSSLVQKDKISITAVVESVRLPGASHMVKGVT 486 P01J14: 118 TNYLLALKLKQHGDPNPDPHGLNYALNGPRNKYLG 14 TNYLLALK KQ+G P L+ +GPRNKY G AnCPRA: 487 TNYLLALKQKQNGRSLSRPSRLDLLHHGPRNKYDG 521

C

P01F17: 213 CTNGPNSRRCWSGGFDIDTDF-DQDWPETGRTVSYDFTITNTTMSPDGFERLVYAINGQY 389 CTNGP+SRRCW GFDI +D+ D G+ V YD T+T T+SPDG+ERL NGQY DHGO: 28 CTNGPSSRRCWQDGFDIWSDYTDPKVAPPGKLVEYDLTVTQVTISPDGYERLGTVFNGQY 87 P01F17: 390 PGPTIYANWGDTIHVTVHNELEH-NGTSIHWHGLRMW 497 PGP I A+WGDT+ +TVHN L + NGT++HWHG+R++ DHGO: 88 PGPLIEADWGDTLRITVHNNLTNGNGTAVHWHGIRLF 124

D

P02I19: 23

COX:

PRYLFDEDAIHFHRTIKEACDAHDKNYYSRFKKWCDEYFNVKHRGESRGVGGIFFDDLDE 202 PRYL EDA+HFHRT+KEACD H + Y +FKKWCD+YF + HRGE RG+GGIFFDDLD 185 PRYLNQEDAVHFHRTLKEACDQHGPDIYPKFKKWCDDYFFIVHRGERRGIGGIFFDDLDS 244

P02I19: 203 TEKDQESLFAFCQTCLNAFLPSYLP 277 K E F F +TC A +PSY+P COX: 245 PSK--EEAFRFVKTCAEAVVPSYVP 267

E

STC

PDB CM

F

CPRA

PDB CM

G

DHGO

PDB CM

H

COX

PDB CM

F I G . 6. BLAST results (A±D) and northern analysis (E±H) of C. zeae-maydis genes selected from the secondary metabolism category of the Czm-P cDNA subtraction library: STC (A and E), CPRA (B and F), a phenol oxidase with high similarity to DHGO (C and G) and COX (D and H). Amino acid sequences included for comparison are STC from A. nidulans (A), CPRA from A. niger (B), DHGO from A. terreus (C) and COX from M. musculus (D). Similar amino acids are indicated as ‡. For Northern blots, total RNA samples (15 mg) obtained from C. zeae-maydis mycelia grown in cercosporin-inducing (PDB) and non-inducing (CM) media were subjected to electrophoresis in a 1.2 % denaturing agarose gel. Standard methods for northern analysis were used, and gels were stained with ethidium bromide to con®rm uniformity of loading in each lane (not shown). STC (E), CPRA (F), DHGO (G) and COX (H) cDNA fragments obtained from corresponding clones were used for the hybridizations.

required for cercosporin transport, CFP, have been identi®ed in C. nicotianae and C. kikuchii, respectively [11, 13, 20]. However, no pathway intermediates, enzymes, or genes that are directly involved in biosynthesis of cercosporin have been identi®ed [16].

In an e€ort to identify the genes in the cercosporin biosynthetic pathway, we constructed a cDNA subtraction library of C. zeae-maydis by suppression subtractive hybridization (SSH). We analysed this library to identify genes whose expression corresponds with cercosporin

Cercosporin biosynthesis genes

A

245

P02E07: 29 CFP:

YTSGEALAATAFQQPFGRAYLLTDLKWTF------------ICGVANSSVLLIIGR---- 208 Y SGEALAATAFQ PFGRAYLL DLKWTF ICGVANSS LLI GR 112 YNSGEALAATAFQLPFGRAYLLMDLKWTFLVSLALYLIGSLICGVANSSELLIFGRSIAG 171

P02E07: 209 -----------IISRNVPLRKRALYAGLVGATFAIAAVLGPVLGGIFTDRVSWRWCFYI 388 II+RNVPLRKRALYAGLVGATFAIAAVLGPVLGGIFTDR+SWRWC YI CFP: 172 VGNAGVFAGVFIIIARNVPLRKRALYAGLVGATFAIAAVLGPVLGGIFTDRISWRWCLYI 231 P02E07: 389 NLPIGAVAVAIIVFLLPPRPGEKAAEVKDLSWWKFFLKLNPFGAALLLGSLTCLFLALQ 565 NLPIGAV VAII+FLLP RPGEKAAEVKDLSWW+FFLKLNPFG+ALLLGSLTC FLALQ CFP: 232 NLPIGAVRVAIIIFLLPSRPGEKAAEVKDLSWWQFFLKLNPFGSALLLGSLTCFFLALQ 290

B

CFP

PDB CM F I G . 7. BLAST results and northern analysis of putative cercosporin transporter (CFP) gene of C. zeae-maydis. This gene was selected from the membrane transporter category of the Czm-P cDNA subtraction library and served as a positive control during SSH. (A) Amino acid sequence included for comparison is CFP from C. kikuchii (A). Similar amino acids are indicated as ‡. (B) For Northern blots, total RNA samples (15 mg) obtained from C. zeae-maydis mycelia grown in cercosporin-inducing (PDB) and noninducing (CM) media were subjected to electrophoresis in a 1.2 % denaturing agarose gel. Standard methods for northern analysis were used, and gels were stained with ethidium bromide to con®rm uniformity of loading in each lane (not shown). The CFP cDNA fragment obtained from the corresponding clone was used for hybridization (B).

accumulation in culture media. SSH is a powerful technique to identify di€erentially expressed genes when exploring the genetic basis of biological processes. However, one of the concerns of SSH is the high frequency of false positive clones that can arise during the experimental procedure. These false positive clones are background clones representing commonly expressed genes that escape the SSH. In an e€ort to overcome this drawback, we analysed the quality and quantity of poly (A) ‡ RNA samples that were used in this study by spectrophotometry and performed Northern blot analyses prior to SSH. In addition, we analysed the SSH library enriched in transcripts expressed in the absence of cercosporin production (the Czm-C library). Importantly, Northern blot analysis of the PDX1 gene, which is constitutively expressed in Cercospora species [20], indicated that the transcripts were intact and were present at comparable levels in both 0.2 PDB and CM fungal cultures (Fig. 2). The absence of a clone containing PDX1 cDNA from both libraries and the presence of a clone containing CFP cDNA in the Czm-P library support the conclusion that subtractive hybridization was performed eciently during library construction. We designated putative gene functions of these 197 clones based on information obtained from the database

via BLAST and grouped these genes into nine categories with respect to their biochemical functions (Fig. 4). Genes predicted to be involved in carbohydrate metabolism (52 clones), amino acid/protein metabolism (35 clones), and hypothetical proteins with no known functions (28 clones) comprised over 58 % of the unique clones in this library. However, no evidence implicated genes in those categories in cercosporin biosynthesis. On the other hand, the ®nding of numerous genes with functional roles in signal transduction (9 clones) and membrane transport (33 clones) in this cDNA subtraction library is potentially signi®cant. Proteins encoded by these genes may not have a direct enzymatic function in the cercosporin biosynthetic pathway, but they may provide valuable information about the activation of toxin biosynthesis and transport of substrates, intermediates, and ®nal products into or out of the fungal cell. Based on the chemical structure of cercosporin [14, 16, 43], we anticipated that genes in the fatty acid metabolism and secondary metabolism categories would be most informative in deducing the cercosporin biosynthetic pathway. Our data indicate that these genes are expressed uniquely in cercosporin-inducing medium and concurrently with cercosporin accumulation. Enzymes similar to FAS, ODEA, and LDS may be responsible for

246

W.-B. Shim and L. D. Dunkle cercosporin production assay

A cercosporin (ng/ml)

40 35

PDB

30

CM

25

PDB+AP

20 15 10 5 0 0 day

B

1 day

2 day

PDB 01 2 3 4 5 6 7

3 day

4 day

5 day

CM 01 2 3 4 5 6 7

6 day

7 day

PDB + AP 01 2 3 4 5 6 7

FAS LDS STC DHGO CFP PDX1 rRNA

F I G . 8. Cercosporin accumulation and kinetics of gene expression. (A) A 1 ml sample of C. zeae-maydis grown in 0.2 PDB, CM or 0.2 PDB ‡ AP was removed daily and extracted with an equal volume of 5 N KOH, and cercosporin production was analysed by spectrophotometry. No cercosporin was detected in cultures grown in CM or 0.2 PDB supplemented with AP. (B) For northern analysis, total RNA samples (15 mg) obtained daily from C. zeae-maydis mycelia grown in 0.2 PDB, CM and 0.2 PDB ‡ AP media were subjected to electrophoresis in a 1.2 % denaturing agarose gel. Standard methods for northern analysis were used, and gels were stained with ethidium bromide to con®rm uniformity of loading in each lane (rRNA). For hybridization, FAS, LDS, STC, DHO, and CFP cDNA fragments were obtained from the corresponding clones, and PDX1 was prepared by PCR ampli®cation.

synthesis and modi®cation of the cercosporin backbone as they are in the synthesis of sterigmatocystin and a¯atoxin [9, 10, 12, 26]. Furthermore, it is well documented that genes such as CPRA, STC, phenol oxidase (DHGO), and COX are required for modi®cation and bioconversion of various secondary metabolites [2, 9, 27, 33]. In addition to the identi®cation of a FAS gene, another revealing discovery from this study was the absence of a polyketide synthase (PKS) gene in the Czm-P cDNA subtraction library, since cercosporin has been suggested to be of polyketide origin [11, 16, 40, 43]. Polyketides and fatty acids are structurally disparate molecules that are synthesized by PKS and FAS, respectively, which share

evolutionary origin [25, 28]. Long-chain fatty acids synthesized by FAS generally function as structural components of cell membranes and lipid bodies, whereas polyketides produced by PKS have obscure and unknown functions and are often categorized as secondary metabolites. FAS can play critical roles in the synthesis of moieties that are linked to polyketides and other secondary metabolites [1, 8, 9], as well as components of membranes and precursors of hormones and signaling molecules. PKS and FAS carry out some of the same enzymatic functions due to the conservation of certain domains. Also, PKS has been shown to be involved in synthesis of fatty acids [34]. Thus, we hypothesize that

Cercosporin biosynthesis genes the PKS predicted to be involved in cercosporin biosynthesis is not unique to this pathway and was expressed in both poly (A) ‡ RNA populations or that additional sequences must be analysed to detect the PKS that is speci®cally involved. The multiplicity of PKS genes in fungi and the lack of a cercosporin-speci®c PKS probe precluded attempts to detect such a gene by northern analysis [31, 35]. The possibility that cercosporin biosynthesis proceeds without PKS activity seems remote but cannot be eliminated with available data. Interestingly, Brobst and Townsend [8] suggested that a specialized FAS initiates the production of precursors, such as hexanoyl CoA, in secondary metabolite biosynthesis, and that PKS are responsible for subsequent condensations. In addition, it has been shown recently that FAS involved in fungal secondary metabolism, e.g. HC-toxin biosynthesis in Cochliobolus carbonum [1] and sterigmatocystin biosynthesis in A. nidulans [9, 10], are distinct from FAS required for primary metabolism and fungal growth. Similarly, the FAS homolog identi®ed in the Czm-P subtraction library may play a similar role in C. zeae-maydis for the synthesis of initial precursors that are unique to the cercosporin biosynthetic pathway. Further study, such as targeted gene disruption, is necessary to characterize the functional role of this FAS homolog in cercosporin biosynthesis. In this study, we have developed a means to e€ectively induce or suppress cercosporin production in cultures of C. zeae-maydis by manipulation of the nutritional status of the culture medium. By SSH and analysis of a cDNA subtraction library, we discovered genes expressed during cercosporin biosynthesis by C. zeae-maydis, a fungal pathogen with no previous molecular information. With the exception of genes for self-defence against cercosporin (PDX1, CFP, and CRG1), no genes encoding proteins associated with or directly participating in cercosporin synthesis had been identi®ed in Cercospora. Analysis of the expression and regulation of genes identi®ed in this study will help us understand their role in cercosporin biosynthesis. Gene disruption and mutant phenotype determination will facilitate a critical evaluation of the role of cercosporin in gray leaf spot of maize. These ®ndings will, in turn, impact the potential of innovative disease management strategies that target the action or synthesis of cercosporin.

Research reported is a cooperative investigation of the U. S. Department of Agriculture±Agricultural Research Service and the Purdue University Agricultural Experiment Station. Published as paper no. 16680, Purdue University Agricultural Experiment Station. The authors thank Mark McClenning and Dina Huntington for excellent technical assistance.

247 REFERENCES

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