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Apr 25, 2012 - Bo Reum Sung • Seunghoon Lee • Sung-Hwan Yun. Received: 13 January 2012 / Revised: 2 April 2012 / Accepted: 5 April 2012 / Published ...
Curr Genet (2012) 58:179–189 DOI 10.1007/s00294-012-0375-5

TECHNICAL NOTE

A split luciferase complementation assay for studying in vivo protein–protein interactions in filamentous ascomycetes Hee-Kyoung Kim • Eun Ji Cho • Seong mi Jo • Bo Reum Sung • Seunghoon Lee • Sung-Hwan Yun

Received: 13 January 2012 / Revised: 2 April 2012 / Accepted: 5 April 2012 / Published online: 25 April 2012 Ó Springer-Verlag 2012

Abstract Protein–protein interactions play important roles in controlling many cellular events. To date, several techniques have been developed for detection of protein– protein interactions in living cells, among which split luciferase complementation has been applied in animal and plant cells. Here, we examined whether the split luciferase assay could be used in filamentous ascomycetes, such as Gibberella zeae and Cochliobolus heterostrophus. The coding sequences of two strongly interacting proteins (the F-box protein, FBP1, and its partner SKP1) in G. zeae, under the control of the cryparin promoter from Cryphonectria parasitica, were translationally fused to the C- and N-terminal fragments of firefly luciferase (luc), respectively. Each fusion product inserted into a fungal transforming vector carrying the gene for resistance to either geneticin or hygromycin B, was transformed into both fungi. We detected complementation of split luciferase proteins driven by interaction of the two fungal proteins with a high luminescence intensity-to-background ratio only in the fungal transformants expressing both N-luc and C-luc fusion constructs. Using this system, we also confirmed a novel protein interaction between transcription factors, GzMCM1 and FST12 in G. zeae, which could Communicated by U. Kueck. H.-K. Kim and E. J. Cho equally contributed.

Electronic supplementary material The online version of this article (doi:10.1007/s00294-012-0375-5) contains supplementary material, which is available to authorized users. H.-K. Kim  E. J. Cho  S. Jo  B. R. Sung  S. Lee  S.-H. Yun (&) Department of Medical Biotechnology, Soonchunhyang University, Asan 336-745, South Korea e-mail: [email protected]

hardly be proven by the yeast two-hybrid method. This is the first study demonstrating that monitoring of split luciferase complementation is a sensitive and efficient method of studying in vivo protein–protein interactions in filamentous ascomycetes. Keywords Split luciferase complementation  Gibberella zeae  Cochliobolus heterostrophus  In vivo protein–protein interaction

Introduction Various types of protein–protein interaction play important roles in controlling fundamental cellular processes. To identify these interactions in living cells, several techniques have been developed (Barnard et al. 2007; Figeys 2003; Meng et al. 2005), among which the yeast twohybrid method has been used extensively due to its sensitivity and wide applicability. However, this system, which is based on the activation of a reporter gene by a transcription factor, has several limitations. It restricts protein interactions to the nucleus of a yeast cell, making identification of the interaction localization and interacting partners of membrane-bound proteins impossible. In addition, further confirmation of in vivo protein interactions (i.e., those within cells of the original organism to be studied) is necessary. Furthermore, a bait protein carrying a potential self-transactivating activity may produce false positives, which is the most common limitation of this system (Nolting and Po¨ggeler 2006a). To circumvent these drawbacks, a protein fragment-assisted complementation (PFAC) strategy (Michnick 2003) has been developed using various reporter proteins (either enzymes or fluorescent proteins), including dihydrofolate (Pelletier et al.

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1998; Remy and Michnick 2004), b-galactosidase (Rossi et al. 1997), b-lactamase (Galarneau et al. 2002), green fluorescence protein (Hu and Kerppola 2002; Wilson et al. 2004), and luciferase (Kaihara et al. 2003; Luker et al. 2004; Paulmurugan et al. 2002; Remy and Michnick 2004). Particularly, the PFAC strategy using the split luciferase from firefly (Photinus pyralis), Renilla, or Gaussia has been developed as an efficient assay for large-scale protein–protein interactions in mammalian cells and even in living small animals due to their low background signals compared with other reporters (e.g., fluorescent reporters). Recently, successful imaging of protein–protein interactions in intact plant cells has been reported (Chen et al. 2008; Fujikawa and Kato 2007). In the present study, we determined whether a split luciferase complementation assay can be used for identification of in vivo protein interactions in filamentous ascomycetous fungi, such as Gibberella zeae (anamorph: Fusarium graminearum) and Cochliobolus heterostrophus (Bipolaris maydis), which has not been attempted previously. Both fungal species cause devastating diseases in major cereal plants (e.g., head blight of wheat, barley, and rice caused by G. zeae, and Southern corn leaf blight by C. heterostrophus) (McMullen et al. 1997; Yoder 1988). In addition to the economic importance of plant diseases caused by these fungi, their genetic and molecular tractability make them useful as model fungal pathogens in molecular studies of filamentous ascomycetes. As a protein pair known to interact with each other, we chose a novel F-box protein, designated FBP1, and its interacting partner, SKP1 (Han et al. 2007). Both FBP and SKP1 are known to be components of the Skp1-Cullin-F-box protein (SCFFBP1) E3 ligase complex required for protein degradation involved in sexual development and virulence in G. zeae; the interaction of FBP1 with SKP1 was confirmed in a yeast two-hybrid assay (Han et al. 2007). As a novel protein interaction that has not been proven in most filamentous fungi, we chose the pair between G. zeae orthologues of the yeast minichromosome maintenance protein 1 (MCM1) (Bender and Sprague 1987) and homeodomain protein STE12 (Rispail and Di Pietro 2010). Both proteins, known to be transcription factors (the former is a member of MADS box transcription factor family), play important roles in regulating diverse cellular processes including sexual development in yeast and filamentous fungi (Shore and Sharroks 1995; Rispail and Di Pietro 2010). However, the interaction between these two proteins in filamentous fungi using the yeast twohybrid method was confirmed only in Sordaria macrospora (Nolting and Po¨ggeler 2006b) due to a strong self-activation activity of MCM1 (Nolting and Po¨ggeler 2006a). The objectives of the present study were as follows: (1) to construct versatile plasmid backbone carrying a gene encoding a split luciferase (either N-terminal or C-terminal

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fragment of firefly luciferase) under the control of a fungal strong promoter, and a gene for a fungal selectable marker (either hygromycin or geneticin resistance gene); (2) to insert the combinations of the FBP1 and SKP1 or the GzMCM1 and FST12 genes into the construct, resulting in fungal transforming vectors; and (3) to determine whether fungal transformants resistant to both drugs show a high luminescence intensity-to-background ratio.

Materials and methods Strains and culture conditions All fungal strains were started from 25 % glycerol stock cultures stored at -80 °C and maintained on potato dextrose agar (PDA; Difco Laboratories, Detroit, MI, USA), complete medium for G. zeae (GzCM; Leslie and Summerell 2006), or complete medium for C. heterostrophus (ChCM; Leach et al. 1982). The G. zeae Z03643 strain used in the present study is a self-fertile wild-type (WT) strain (Kim et al. 2008), and all transgenic G. zeae strains were derived from Z03643. For extraction of both total RNA and genomic DNA, strains were grown in PD broth. For vegetative growth, strains were grown in either PDA or GzCM. For perithecia formation and asexual sporulation, fungal strains were grown on carrot agar plates and CMC liquid medium, respectively, as described previously (Han et al. 2007; Leslie and Summerell 2006). C. heterostrophus WT strain C4 (MAT1-2, ATCC 48331) and its transgenic derivative strains were grown on ChCM and ChCM without salts supplemented with appropriate antibiotics, respectively. Escherichia coli strains were grown on Luria– Bertani agar or in liquid medium supplemented with appropriate antibiotics. Plasmids, primers, polymerase chain reaction (PCR), and nucleic acid manipulations The plasmids used for vector construction in the present study are listed in Table 1. The PCR primers (Tables 2, 3) used in the present study, which were obtained from the Bioneer Oligonucleotide Synthesis Facility (Bioneer Corporation, Chungwon, Korea), were resuspended at a concentration of 100 lM in sterilized water and stored at -20 °C. General PCR was performed as described previously (Kim et al. 2008). Fungal genomic DNA and total RNA were extracted as described previously (Chi et al. 2009; Kim et al. 2008). Plasmid DNA was purified from 5-ml E. coli cultures using a plasmid DNA purification kit (NucleoGen Biotech, Siheung, Korea). Standard procedures were used for restriction endonuclease digestion, ligation, agarose gel electrophoresis, and E. coli

Curr Genet (2012) 58:179–189

transformation (Sambrook and Russell 2001). Quantitative real-time PCR (qPCR) was performed with the SYBR Green Super Mix (Bio-Rad) using the first-strand cDNA synthesized from total RNA (Kim and Yun 2011). Gene expression was measured in two independent samples with three technical replicates. The G. zeae EF1A gene (FGSG_08811.3) was used as endogenous controls for data normalization. The amount of nLuc transcript from a 3-day-old mycelial sample of the GzNC strain (Table 4) grown in GzCM medium was used as a reference for comparison. Western blot hybridization For protein extraction, each G. zeae strain was cultured in GzCM broth for 4 days at 25 °C with shaking. Harvested mycelia were pulverized in liquid nitrogen and extracted by the method described previously (Bridge et al. 2004). Proteins were separated by SDS/PAGE, blotted and probed by three polyclonal primary antibodies against firefly luciferase [luciferase (251–550) (sc-32896; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-luciferase (cat# L0159; Sigma), and antibody for luciferase-aff-purified (AP21341PU-N, Acris Antibodies GmbH, Herford, Germany)]. After hybridizing with corresponding HRPconjugated secondary antibody, recombinant luciferase proteins were visualized with the ECLTM prime western blotting reagent (GE healthcare Korea).

181 Table 1 Plasmids used in this study Name

Characteristics

References

pUC19-nLUC (35S::Nluc)

The N-terminal domain (amino acids 2–416) of firefly luciferase (Luc), which was inserted between the cauliflower mosaic virus 35S promoter and Rubisco small subunit (rbs) terminator, was cloned into pUC19

Chen et al. (2008)

pUC19-cLUC (35S::Cluc)

The C-terminal domain (amino acids 398–550) of Luc, inserted between the 35S promoter and rbs terminator, was cloned into pUC19

Chen et al. (2008)

pCHPH1

The 188-bp fragment of the cryparin promoter (Crp) from Cryphonectria parasitica was fused to the hygromycin B resistance gene (hyg) in pDH25 (Cullen et al. 1987)

Kwon et al. (2009)

pSP-luc?NF Fusion vector

The full-length sequence encoding the firefly (Photinus pyralis) luciferase gene

Promega, USA

pEGFC

The full-length coding sequence of FBP1, amplified from cDNA of Z03643 was introduced into pEG202 (Golemis et al. 1996)

Han et al. (2007)

pACTgzskp

The full-length coding sequence of SKP1, amplified from cDNA of Z03643 was introduced into pACT2 (Clontech, Mountain View, CA, USA)

Han et al. (2007)

pBCATPH

The hygromycin resistance gene (hygB) under control of the Aspergillus nidulans Trp promoter and terminator (Hamer and Timberlake 1987) was introduced into pBCSK(-) (Clontech)

Yun (1998)

pBCGT

The geneticin resistance gene (gen) under control of the A. nidulans Trp promoter and terminator was introduced into pBCSK(-) (Clontech)

Kim et al., unpublished

Generation of plasmid DNAs used for split luciferase assay Versatile vector backbones pFNLucG and pFCLucH As starting materials for vector construction, we used the plasmids pUC19-nLUC and pUC19-cLUC (Table 1) that had previously been constructed for the firefly luciferase complementation assay in plant protoplasts or tissues (Chen et al. 2008), and carried a N-terminal (nLuc) and C-terminal (cLuc) fragment of the luciferase, respectively (Table 1). To replace the 35S promoter region in both plasmids with a fungal strong promoter, each was digested with EcoRI and SacI simultaneously, and then ligated with 188 bp of the cryparin gene (Crp) promoter from the ascomycetous fungus, Cryphonectria parasitica (Kwon et al. 2009), which was amplified from pCHPH1 (Table 1) using the primer pair 1 and 2, followed by EcoRI–SacI digestion, resulting in pFNLuc or pFCLuc, respectively. To introduce a fungal selectable marker into these plasmids, a gene cassette conferring resistance to either geneticin or hygromycin B, amplified from pBCGT and pBCATPH using the primers 3 and 4, was inserted into the single

HindIII site in pFNluc and pFCLuc, finally generating pFNLucG and pFCLucH, respectively (Fig. 1). These constructs can be used as a versatile vector backbone for split luciferase assay in fungi because a fungal gene pair of interest can be translationally fused with each split luciferase gene in the plasmids using a simple gene cloning procedure described below. pFNLuc-FBP1G and pFCLuc-SKP1H The coding region of the G. zeae FBP1 gene [annotated as FGSG_02095.3 in the F. graminearum genome database (http://www.broadinstitute.org/annotation/genome/fusarium_ graminearum/MultiHome.html)], which corresponds to

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182 Table 2 Primers used in this study

Curr Genet (2012) 58:179–189 Namea

1

Sequenceb (5′ 3′)

Original

Promoter/for

Amplified

Enzyme

gene

sitee

CCGAATTCCTCGAGACAGGACCAGA GA

2

Promoter-S/rev

CGAGCTCTTTGATTGAAGTTTGGAG

3

HygB-H/for

CCCAAGCTTAGGATTACCTCTAAACA

EcoRI PCrpc SacI

AGTGTACC

gend or

CCCAAGCTTAGAAGATGATATTGAAG

hygBd

4

HygB-H/rev

5

FBP1nLUC/for

AAGCTCGAGTAGTCGACATGGCCGC

6

FBP1nLUC/rev

CGTACGAGATCTGGTCGACGTTCAT

HindIII

GAGCACTT

TGCTGCCACTCCT

FBP1

SalI

SKP1

KpnI

FBP1

KpnI

SKP1

SalI

CGAGTCAGATTGAT 7

Skp1cLUC/for

CGTCCCGGGGCGGTACCTCTGAGTC TACATCTCCTCAGAA

8

Skp1cLUC/rev

TTGGATCCCCGGGTACCTTAGCGGT CCTCAGCCCACTCGTT

9

FBP1cLUC/for

TCCCGGGGCGGTACCGCCGCTGCTG CCACTCCTACT

10

FBP1cLUC/rev

GTTGGATCCCCGGGTACCTCAGTTCA TCGAGTCAGATTGATC

11

SKP1nLUC/for

GAAGCTCGAGTAGTCGACATGTCTG

12

SKP1nLUC/rev

TACGAGATCTGGTCGACGCGGTCCT

13

nLUCfor

CAGATCTCGTACGCGTCCCGGG

14

nLUCtailN

GCTCTAGTCATCCATCCTTGTCAATC

AGTCTACATCTCCT

CAGCCCACTCGTT –f nLuc



AAG 15

cLUCskp1/for

CGGAGGAGTTGTGTTTGTGGAC

16

cLUCskp1/rev

CTCGGGAGTGAAATCGTTGGTA

17

nLUC_cDNA5

ACGGTAGTGAGATGGTTGTGGATG

18

nLUC_cDNA3

CTGCATACGACGATTCTGTGATTT

19

MCM1nLUC/for

AGCTCGAGTAGTCGACATGGCCGAC ATCACAGATCAG

20

MCM1nLUC/rev

21

FST12cLUC/for

b

The region complementary to a linearized vector was underlined

TCCCGGGGCGGTACCTATTCGCAAC

22

FST12cLUC/rev

cLUCfor3

CGAAAAAGTTGCGCGGAGGAG

24

FCFST12/rev

GGATCCCCGGGTACCTTACATCATG

e

25

f

Not included

123

SalI

FST12

KpnI

CCACTAGC 23

The restriction enzyme whose recognition site was shaded in the primer sequence

GzMCM1

GGATCCCCGGGTACCTTACATCATG

A gene cassette under control of the promoter and terminator of the A. nidulans Trp

d

– –

ACTCCGCA

c

The 188 bp-promoter region of Crp

FBP1-nLuc

– –

GATGGCT

a

The primer names described in the text

TACGAGATCTGGTCGACCGACTGGT

cLuc-SKP1

– cLuc-FST12



CCACTAGC FNMCM1/for

AGCTCGAGTAGTCGACATGGCCGAC ATCACAGATCAG

26

Nluc3R

GATAGAATGGCGCCGGGCCTT

GzMCM1nLuc





Curr Genet (2012) 58:179–189 Table 3 Primers used in qPCR

Table 4 Luminescence activity in fungal cell lysates

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Name

Sequence (50 ?30 )

Amplified gene

Size (bp)

nLuc

139

cLuc

208

GzMCM1-nLuc

204

cLuc-SKP1

223

nLUC forRT

CCGCTTCCCCGACTTCCTTAGAGA

nLUC rev RT

CCGCTTCCCCGACTTCCTTAGAGA

cLUC for RT

CTTCCCGCCGCCGTTGTTGTTT

cLUC rev RT

CGATCTTTCCGCCCTTCTTG

MCM1 forRT

CAAAGCTACGTACAACGAAATCAG

nLUC rev RTf

CTCTCCAGCGGTTCCATCTTCC

cLUC forRTf

CGCGGAGGAGTTGTGTTTGTGGAC

SKP1 rev RT

TGTTTGCGCTGTCATTAGAGG

Ste12 rev RT

AGAGCTTGCTGGGCCTCAGTGC

cLuc-FST12

242

FBP forRT

ACTGGTGCAAGCACGGCGACGA

FBP1-nLuc

189

Activitya

Strain

Characteristics

Z03643

A G. zeae wild-type progenitor

8.3 ± 3.8

Z03643b

A G. zeae wild-type progenitor

7.4 ± 4.2c

C4

A C. heterostrophus progenitor

5.0 ± 2.0

GzN

A transgenic G. zeae strain carrying only pFNLucG

5.0 ± 1.0

GzC

A transgenic G. zeae strain carrying only pFCLucH

GzNC

A transgenic G. zeae strain carrying both the empty vectors pFNLucG and pFCLucH

8.0 ± 1.0 13.3 ± 5.1

GzFN

A transgenic G. zeae strain carrying only pFNLuc-FBP1G

8.0 ± 3.5

GzFNC

A transgenic G. zeae strain carrying pFNLuc-FBP1G and the empty vector pFCLucH

18.1 ± 3.2

GzCS

A transgenic G. zeae strain carrying only pFCLuc-SKP1H

10.0 ± 2.6

GzCSN

A transgenic G. zeae strain carrying pFCLuc-SKP1H and the empty vector pFNLucG

7.5 ± 1.3

GzFNCS-1

A transgenic G. zeae strain carrying both pFNLuc-FBP1G and pFCLuc-SKP1H

64,098.3 ± 14,171.7

GzFNCS-3

A transgenic G. zeae strain carrying both pFNLuc-FBP1G and pFCLuc-SKP1H

86,223.0 ± 30,147.2

GzFNCS-5

A transgenic G. zeae strain carrying both pFNLuc-FBP1G and pFCLuc-SKP1H

101,781.3 ± 20,907.2

GzSNCF-2

A transgenic G. zeae strain carrying both pFCLuc-FBP1H and pFNLuc-SKP1G

43,338.0 ± 14,662.8

GzSNCF-4

A transgenic G. zeae strain carrying both pFCLuc-FBP1H and pFNLuc-SKP1G

61,418.7 ± 4,171.8

GzFL

A transgenic G. zeae strain carrying pPcrp-FLUC (the fulllength firefly luciferase gene)

325,586.4 ± 50,528.1

ChFNCS-1

A transgenic C. heterostrophus strain carrying both pFNLuc-FBP1G and pFCLuc-SKP1H

23,207.0 ± 3,324.7

ChFNCS-7

A transgenic C. heterostrophus strain carrying both pFNLuc-FBP1G and pFCLuc-SKP1H

17,322.7 ± 3,107.3

b

GzFNCS-1b

A transgenic G. zeae strain both carrying pFNLuc-FBP1G and pFCLuc-SKP1H

3,521.3 ± 1,555.1c

c

GzFNCS-3b

A transgenic G. zeae strain both carrying pFNLuc-FBP1G and pFCLuc-SKP1H

2,667.7 ± 947.5c

Luminescence activity was obtained from three replicates a

Relative light units (RLUs) per microgram of protein per min

Fungal liquid cultures after 5-min exposure to luciferin RLUs/106 of fungal conidia/ml

amino acid position 1–703, was amplified from the plasmid pEGFC (Table 1) using the primer pair 5 and 6 (Table 2). Similarly, the SKP1 (FGSG_06922.3) region (aa 2–170) was amplified from pACTgzskp (Table 1) using the primer

pair 7 and 8 (Table 2). Both PCR products were introduced into the SalI site of pFNLucG or the KpnI site of pFCLucH, resulting in pFNLuc-FBP1G and pFCLuc-SKP1H, respectively, using In-FusionÒ HD Cloning Kit (Clontech,

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Fig. 1 Schematic representations of the versatile vector backbones constructed in the present study. Multicloning site where the gene of interest can be inserted is indicated by two thick bars between which several restriction enzyme sites are included

Mountain View, CA, USA). Since each PCR product carried 17- to 19-bp extensions at both 50 and 30 ends, which are complementary to the ends of a linearized vector (either pFNLucG digested with SalI or pFCLucH with KpnI), it could be inserted into the corresponding vector through recombination between the 17- to 19-bp overlaps of the PCR product and the vector. In addition, both genes were inserted into the same vectors in the opposite directions (i.e., FBP1 in pFCLucH and SKP1 in pFNLucG), generating pFCLuc-FBP1H and pFNLuc-SKP1G, respectively. For this, the FBP1 region (aa 2–704) and SKP1 region (aa 1–169) were amplified from pEGFC and pACTgzskp, respectively, using the primer pairs 9/10, and 11/12, respectively (Table 2), followed by recombination with pFCLucH digested with KpnI, and pFNLucG with SalI, respectively. An in-frame translational fusion of a fungal gene with a luciferase fragment was confirmed by nucleotide sequencing of the constructed vector. pFNLuc-MCM1G and pFCLuc-FST12H A putative open-reading frame (ORF) region of the Saccharomyces cerevisiae MCM1 orthologue of G. zeae (designated GzMCM1) (FGSG_08696.3), and the S. cerevisiae STE12 orthologue of G. zeae (FST12; FGSG_ 07310.3) (Lee et al. 2006) were amplified from total RNA of G. zeae Z03643 strain using the primer pairs 19/20, and 21/22, respectively (Table 2). Both PCR products were introduced into the SalI site of pFNLucG or the KpnI site of pFCLucH, resulting in pFNLuc-MCM1G and pFCLucFST12H, respectively, as described above. pPcrp-FLUC The Crp promoter from C. parasitica, which was amplified from pCHPH1 (Table 1), was fused to the full-length sequence of the firefly luciferase gene from the pSPluc?NF Fusion vector (Promega, USA), and ligated into

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the pGEMT vector (Promega, USA), resulting in pPcrpFLUC. For fungal transformation, pPcrp-FLUC was added to fungal protoplasts along with pBCGT carrying gen as a selectable marker (Table 1). Fungal transformation For transformation of G. zeae, fungal conidia harvested from 3-day-old CMC liquid cultures were inoculated into 50 ml of YPG liquid medium (Han et al. 2007) at 106 per ml, and incubated at 25 °C by shaking at 150 rpm for 24 h. Young mycelia derived from fungal conidia were harvested and incubated in 80 ml of 1 M NH4Cl containing Driselase (10 mg/ml; Desert Biologicals, Phoenix, AZ, USA) for protoplasting. For C. heterostrophus, young mycelia harvested from ChCM liquid culture of fungal conidia grown at 25 °C by shaking at 150 rpm for 18 h were digested with Driselase in 0.7 M NaCl. All other polyethylene glycolmediated transformation steps using fungal protoplasts were performed as described previously (Kim et al. 2008). Fungal transformants were selected on a regeneration medium containing either hygromycin B at 70 lg/ml or geneticin at 50 lg/ml or both. Determination of luciferase activity The fungal cell lysates, prepared from fungal mycelia grown in GzCM or ChCM liquid medium for 3 days as described previously (Lee et al. 2009), were dropped into 96-well microtiter plates (40 ll per well), and assayed by mixing with 20 ll of luciferin (Sigma, St. Louis, MO, USA). As an alternative, 96-well plates filled with l50 ll GzCM or ChCM liquid medium were inoculated with fungal conidia (106 per ml) harvested from GzCM or ChCM agar medium, and incubated for 1 day at 25 °C, as described previously (Morgan et al. 2003) before assaying. Luciferase activity was measured using GloMaxÒ 96 Microplate Luminometer (Promega) for 10 s with intervals of 1–2 s (for both cell lysate and fungal liquid culture) or

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5 min after exposure (for fungal liquid culture) to luciferin. The readings were normalized by measuring the total protein concentration in the cell lysate using the method of Bradford (1976), and represented as relative light units (RLUs) per microgram of protein per min (Paulmurugan and Gambhir 2007), or as RLU per 106 of fungal conidia per ml of fungal culture.

Results and discussion Fungal transformants carrying the constructed plasmids The plasmid DNAs constructed as described above were transformed alone or in various combinations into wildtype strains of both G. zeae and C. heterostrophus. Insertion of plasmid DNA(s) into the genome of fungal transformants was confirmed by PCR amplification of the split luciferase gene (Fig. 2a) as well as by fungal growth on drug(s) corresponding to the resistance gene present in each plasmid DNA. Expression of the transgenes in fungal transformants was confirmed by reverse transcription-PCR in fungal transformants (Figs. 2, 3). In addition, qPCR analysis determined the relative transcript amounts of the nLuc or cLuc fragments, or those fused to the fungal genes in transformants. The average PCR efficiency of the primer sets for individual genes (Table 3) ranged from 1.92 to 1.97. The transcript levels of the transgenes varied up to approximately sixfold among the fungal transformants examined, suggesting the possibility of position effects of transgenes for gene expression. However, those of both fused nLuc and cLuc genes within a single transformant (including those of nLuc and cLuc in the strain carrying an intact firefly luciferase gene) varied less than twofold changes (Fig. 4), demonstrating that two split luciferase genes were expressed at a relatively constant rate within the single cells. The accumulation of the recombinant proteins in fungal transformants was confirmed by immunoblot analysis. Specific signals for the cLuc fused to SKP1 were detected in the fungal strain expressing cLuc-SKP1 only (GzCS in Table 4) and those expressing both FBP1nLuc and cLuc-SKP1 (GzFNCS-3 and GzFNCS-5 in Table 4). The expression of cLuc-FST12 was also detected in the transformants carrying both GzMCM1-nLuc and cLuc-FST12 (GzFSM-1 and GzFSM-6 in Table 5) (Fig. 5, Fig. S1). However, none of the nLuc-fused proteins were detected although three different anti-luciferase antibodies were used, demonstrating that these antibodies were specific to the cLuc region of the firefly luciferase. There were no differences in the transformants examined compared with their wild-type progenitors in several traits, such as mycelial growth, pigmentation, and sexual development (data not shown). As negative controls, we generated

Fig. 2 Insertion and heterologous expression of the split luciferase genes in fungal transformants. a Polymerase chain reaction (PCR) amplification of the split luciferase gene from genomic DNAs of fungal transformants. The nLuc- (1.3 kb) and cLuc-SKP1-region (0.6 kb) in transformants were amplified with primers 13/14 (upper panel) and 15/16 (lower panel) (Table 2), respectively. In both panels, lane WT genomic DNA of G. zeae Z03643 strain, lane pFNLuc-FBP1G genomic DNA of the GzFN strain (Table 4) carrying only nLuc-FBP1 with primer pair 13/14, and lane pFCLuc-SKP1H genomic DNA of GzCS (Table 4) carrying cLuc-SKP1 only with primer pair 15/16. The names in the other lanes represent the transformants shown in Table 4. DNA size markers are indicated on the left side of the gels. b Transcript amplification of the fusion genes in by reverse transcription-PCR using total RNAs from fungal strain carrying both vectors, which were grown in CM (either GZCM or CHCM) liquid medium for 3 days. The cLuc-SKP1- (0.6 kb) and FBP1-nLuc region (0.58 kb) were amplified with primer pairs 15/16 and 17/18, respectively (Table 2). Lanes WT total RNA from G. zeae Z03643 strain, and other lanes total RNAs from the transformants shown in Table 4

Fig. 3 Transcript amplification of the GzMCM1 and FST12 fusion genes in by reverse transcription-PCR using total RNAs from fungal strain carrying both pFNLuc-MCM1G and pFCLuc-FST12H vectors, which were grown in GzCM for 3 days. The cLuc-FST12- (2.2 kb) and GzMCM1-nLuc-region (0.7 kb) were amplified with primer pairs 23/24 and 25/26 (Table 2), respectively. Lanes WT total RNA from G. zeae Z03643 strain, and other lanes: total RNAs from the transformants shown in Table 5

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Fig. 4 Relative expression of transgenes in the fungal transformants determined by qPCR. Both nLuc- and cLuc-specific regions (indicated by N and C, respectively, following the names of the transformants in X-axis) were amplified by the primers shown in Table 3. For example, GzNC/N and GzNC/C represent the transcript levels of nLuc and cLuc amplified by primer pairs nLUC forRT/nLUC rev RT and cLUC forRT/cLUC rev RT, respectively, from total RNA of the GzNC strain (Table 4). GzCSN/N and GzCSN/C are for those of nLuc and cLuc-SKP1 amplified by the primer pairs nLUC forRT/nLUC rev RT, and cLUC forRTf/SKP1 rev RT, respectively, from the GzCSN strain (Table 4)

Table 5 Luminescence activity driven by the interaction between GzMCM1 and FST12 in G. zeae Activitya

Strain

Characteristics

Z03643

A G. zeae wild-type progenitor

6.3 ± 2.5

GzFNCS1

A transgenic G. zeae strain carrying both pFNLuc-FBP1G and pFCLuc-SKP1H

19,178.3 ± 1,455.3

GzFM-1

A transgenic G. zeae strain carrying only pFNLuc-MCM1G

7.7 ± 1.5

GzFMC

A transgenic G. zeae strain carrying pFNLuc-MCM1G and the empty vector pFCLucH

11.8 ± 2.4

GzFS-1

A transgenic G. zeae strain carrying only pFCLuc-FST12H A transgenic G. zeae strain carrying pFCLuc-FST12H and the empty vector pFNLucG

8.0 ± 4.0

GzFSN

13.7 ± 1.6

GzFSM-1

A transgenic G. zeae strain carrying both pFNLuc-MCM1G and pFCLuc-FST12H

795.3 ± 42.3

GzFSM-6

A transgenic G. zeae strain carrying both pFNLuc-MCM1G and pFCLuc-FST12H

479.3 ± 49.2

GzFSTSKP-1

A transgenic G. zeae strain carrying both pFNLuc-MCM1G and pFCLuc-SKP1H

12.1 ± 3.5

GzFSTSKP-3

A transgenic G. zeae strain carrying both pFNLuc-MCM1G and pFCLuc-SKP1H

9.7 ± 3.4

Luminescence activity was obtained from three replicates of fungal cell lysates a

Relative light units (RLUs) per microgram of protein per min

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Fig. 5 Western blot hybridization by a polyclonal primary antibody against firefly luciferase, [luciferase (251–550): sc-32896, Santa Cruz Biotechnology]. Crude protein extracts (*50 lg) were loaded in each lane. Lane 1 G. zeae Z03643; lanes 2, 3, G. zeae strains carrying both FBP1-nLuc and cLuc-SKP1 (GzFNCS-3 and GzFNCS-5, respectively, Table 4); lane 4 a G. zeae strain carrying only FBP1-nLuc (GzN); lane 5 a G. zeae strain carrying only cLuc-SKP1 (GzCS). The arrow in the blot indicates about 38 kDa signal of cLuc-SKP1. The sizes of the standards (in kDa) are indicated on the left of blot. A nonspecific signal of *100 kDa was shown in the same gel as a loading control in the bottom panel

fungal transformants carrying an empty vector that included a split luciferase gene only (i.e., those carrying pFNLucG and/or pFCLucH) and those carrying a single fused gene only or along with the corresponding empty vector (Tables 4, 5). Luciferase activity from fungal transformants expressing the fused FBP1 and/or SKP1 protein All the cell lysates and fungal liquid cultures from the negative controls, which were transgenic strains carrying a single empty vector (GzN or GzC), a single-fused gene (GzFN or GzCS), or the single-fused gene along with the corresponding singly empty vector (GzFNC or GzCSN), exhibited only background levels of luciferase activity similar to those from the wild-type strains carrying no vector (Table 4). In addition, the fungal transformants (GzNC) carrying both empty vectors (pFNLucG and pFCLucH), which co-expressed two split luciferase fragments, showed similar levels to the other negative controls (Table 4). These observations implied not only that each split luciferase fragment is not sufficient to function, but also that no spontaneous reassembly of two split luciferase fragments occurred without interaction of proteins fused to them in filamentous fungi, as reported previously in animals (Luker et al. 2004). In contrast, all of the examined fungal transformants co-expressing two fused proteins, FBP1-nLuc and cLuc-SKP1, showed strong luciferase activities, representing increases of at least 5,000-fold in cell lysates compared with those of the negative controls (Table 4). The levels of luciferase activities in these transformants were approximately only threefold lower than those in the GzFL strain expressing the full-length firefly luciferase under the same promoter (Table 4),

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suggesting that most of the FBP1 and SKP1 proteins accumulated in fungal cells participated in protein interaction. Particularly, the fold changes in luciferase activity in cell lysates, when compared with those of b-galactosidase activity between the same protein combination in the yeast two-hybrid assay (100 to 180-fold; Han et al. 2007) suggest that the split luciferase provides an amplified signal for detecting even weak protein interactions in filamentous fungi. In addition, the protein interaction of FBP1 and SKP1, each fused to split luciferase in the opposite direction (i.e., cLuc-FBP1 and SKP1-nLuc) also led to strong luciferase activities similar to those from the interaction of FBP1-nLuc and cLuc-SKP1 in G. zeae (Table 4), demonstrating the versatility of the split luciferase fragments used in the study of protein–protein interactions in filamentous fungi. The reduced luciferase activities exhibited in C. heterostrophus transformants carrying both FBP1nLuc and cLuc-SKP1 may be attributed to the interaction of heterologous proteins in a different genetic background. Luciferase activity driven by a novel interaction between GzMCM1 and FST12 in G. zeae As in the FBP1–SKP1 interaction described above, only cell lysates of the fungal transformants co-expressing two fused proteins, GzMCM1-nLuc and cLuc-FST12, exhibited luciferase activities that were approximately 60- to 130-fold higher than background levels of the negative controls (Table 5). This clearly indicates that GzMCM1 interacts with FST12 in G. zeae as in S. macrospora (Nolting and Po¨ggeler 2006b). However, the luminescence intensities in the G. zeae strains co-expressing both proteins were much lower (with approximately 24-fold reduced level) than that of the G. zeae strain co-expressing FBP1-nLuc and cLuc-SKP1 (Table 4). Considering the levels of both transcript and protein accumulations of the fused genes between two transformants, this weak interaction signal may be attributed to possible location and/or specificity of the GzMCM1 and FST12 interaction, which are different from the FBP1 and SKP1 interaction in G. zeae. First, Son et al. (2011) identified both GzMCM1 and FST12 as transcription factors belonging to the MADS box- and homeodomain-family, respectively, and confirmed that each protein-coding gene was essential for sexual development by gene deletion analysis. This implies that the interaction between GzMCM1-nLuc and cLucFST12 proteins may be restricted for gene regulation in nuclei of fungal transformants, while the FBP1 and SKP interaction occurs in the cytoplasm since it is required for formation of the SCF (SKP1-Cullin-F-box protein) complex for proteasome-dependent degradation of various proteins in G. zeae (Han et al. 2007). Second, in contrast to an component of the SCF complex, GzMCM1 may have

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more interacting partners (other than FST12) since MCM1 is known to interact with a diverse range of transcription factors for controlling different cellular events in fungi (Messenguy and Dubois 2003; Nolting and Po¨ggeler 2006a). Similarly, FST12 may have different interacting proteins as do the yeast STE12 and its homologues of filamentous fungi, which are known to regulate mating, pseudohyphal growth, cell wall biosynthesis, pathogenicity, and sexual reproduction in the MAP kinase singling pathway (Li et al. 2005; Rispail and Di Pietro 2010; Tsuji et al. 2003; Vallim et al. 2000). In addition, the background levels of luciferase activities in the fungal strains carrying GzMCM1-nLuc and cLuc-SKP1 demonstrate that the interaction between proteins that do not naturally interact with each other can not be detected in the split luciferase assay (Table 5). Use of the split luciferase complementation assay in filamentous fungi The split luciferase assay has several advantages over the yeast two-hybrid method. First, a bait protein that is likely to have self-transactivating activity, such as a transcription factor, can be easily used to identify its binding protein(s) because this assay does not require transcriptional activation of a reporter gene induced by the bait–prey interactions within fungal nuclei. A successful complementation of split luciferase fragments driven by the interaction of GzMCM1 and FST12 in this study provides direct evidence that this assay can overcome the major limitation of the yeast-two hybrid method. In S. macrospora, a strong self-transactivating activity of the entire MCM1 protein made the confirmation of the protein interaction between MCM1 and STE12 (a homologue of FST12) difficult using the yeast two-hybrid method; it was successful when only a MCM1 N-terminus was used as bait (Nolting and Po¨ggeler 2006b). Second, the protein interaction(s) could be detected regardless of the cellular locations where they occur because a complemented luciferase in both cell lysates and within fungal cells can catalyze the reaction for bioluminescence. In this regard, this study provides positive results for protein–protein interactions that occurred in two different cellular locations such as in cytoplasm (FBP1–SKP1) and possibly in nuclei (GzMCM1–FST12). Also, the latter case suggests that the split luciferase assay provides an amplified signal for detecting even weak protein interactions in filamentous fungi. Finally, the luciferase activities measured directly from fungal cell cultures would be sufficient to detect the protein–protein interactions, which makes the analysis faster because it is not necessary to make cell-free extracts from fungal cultures. Moreover, an additional exposure of fungal culture to luciferin would not be necessary since the

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luciferase activities measured without the exposure were not different from those after 5-min exposure (Table 4). With these advantages, in addition to detection of an individual protein–protein interaction, the split luciferase assay developed here can be applied for high-throughput analysis of protein–protein interactions after constructing a fusion library with a split luciferase in filamentous fungi. A high-throughput assay for protein–protein interactions has been developed in Arabidopsis protoplasts based on this assay (Fujikawa and Kato 2007). Moreover, a native temporal and/or spatial interaction between two proteins could be investigated if expression of an nLuc- or cLuc-fusion protein is under the control of its native promoter instead of the constitutive promoter (PCrp) used in the present study. Recently, we found that a fungal native promoter (that of TRI6, which encodes a transcription factor controlling production of trichothecene mycotoxin) generated luciferase activity in G. zeae, which was clearly detected with a luminometer, but approximately 200-fold lower than those in the GzFL strain expressing the full-length firefly luciferase under PCrp (Table 4) (Kim et al., unpublished data). This implies that it may be sufficiently sensitive to detect protein–protein interactions with such a relatively weak native promoters in fungi. In addition, the luminescence could be visualized at the cellular level using a bioluminescence microscope imaging system (e.g., LV200; Olympus, Tokyo, Japan). Furthermore, no requirement for external light stimulation and the high signal-to-noise ratio (S/N) would make the split luciferase assay more efficient and sensitive than BiFC using fluorescent reporters. Despite the positive results provided in the present study, further studies are required to determine the applicability of the split luciferase assay. The strong protein interaction demonstrated in the current study is based on formation of the SCF complex for protein degradation in G. zeae (Han et al. 2007). As this interaction occurs in the cytoplasm, it would be relatively easy to detect even using the yeast two-hybrid method. Therefore, the versatility of split luciferase complementation must be tested for more diverse protein interactions in different cellular location. Moreover, the fusion between a protein of interest and split luciferase may influence the success of protein interactions, including luciferase complementation, activity of luciferase, and/or function of the original protein in fungal cells. Thus, it will be necessary to determine whether the fused protein is as functional as the native molecule by genetic complementation of fungal strains lacking the original protein with the gene encoding the fused protein. In addition, it will be necessary to test other types of protein interaction, such as those involving proteins that do not naturally interact with each other (tested as the interaction between GzMCM1-nLuc and cLuc-SKP1 in this study) or those that can dissociate under some conditions.

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Nevertheless, successful complementation of split luciferase fragments in both G. zeae and C. heterostrophus makes this method a very useful tool for the study of protein–protein interactions in filamentous fungi. Acknowledgments This research was supported by the Agricultural Research Center program of the Ministry for Food, Agriculture, Forestry and Fisheries, Korea, and by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MEST) (2009-0075256). We thank Ae Ran Park, Seoul National University for technical assistance in the western blot analysis.

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