Development of a Versatile Expression Plasmid Construction System

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tag and genome projects of A. oryzae3,4) would further potentiate the use of this .... pDONR™ P4-P1R (Invitrogen), generating pg50Pp,. pg50Pa, and pg50Pt ...
Biosci. Biotechnol. Biochem., 70 (8), 1882–1889, 2006

Development of a Versatile Expression Plasmid Construction System for Aspergillus oryzae and Its Application to Visualization of Mitochondria Yuka M ABASHI, Takashi K IKUMA, Jun-ichi M ARUYAMA, Manabu A RIOKA, and Katsuhiko K ITAMOTOy Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan Received January 31, 2006; Accepted April 5, 2006; Online Publication, August 1, 2006 [doi:10.1271/bbb.60052]

We report here a development of the MultiSite GatewayTM -based versatile plasmid construction system applicable for the rapid and efficient preparation of Aspergillus oryzae expression plasmids. This system allows the simultaneous connection of the three DNA fragments inserted in entry clones along with a destination vector in a defined order and orientation. We prepared a variety of entry clones and destination vectors containing promoters, genes encoding carrierproteins and fusion tags, and selectable markers, which makes it possible to generate 80 expression plasmids for each target protein. Using this system, plasmids for expression of the EGFP fused with the mitochondrialtargeting signal of citrate synthase (AoCit1) were generated. Tubular structures of mitochondria were visualized in the transformants expressing the AoCit1EGFP fusion protein. This plasmid construction system allows us to prepare a large number of expression plasmids without laborious DNA manipulations, which would facilitate molecular biological studies on A. oryzae. Key words:

Aspergillus oryzae; EGFP; expression plasmid; protein expression; mitochondria

The filamentous fungus Aspergillus oryzae has long been used in industrial fermentation processes, including sake, miso, and soy sauce production, and is therefore regarded as safe for humans.1) Due to the recent development of genetic manipulation techniques applicable in A. oryzae,2) along with its non-pathogenic nature, well-established culture methodology, and prominent ability to secrete a large amount of proteins into the culture medium, A. oryzae is widely recognized as a salient host for heterologous protein production. Moreover, accumulating data from the expressed sequence tag and genome projects of A. oryzae3,4) would further potentiate the use of this microorganism for industrial application as well as basic biological analysis. Although a variety of host-vector systems have been available in A. oryzae,5) the construction of plasmids for y

expression of target proteins in A. oryzae has often encountered difficulties, especially when selection markers or target genes are large in size and appropriate restriction sites are not available. Hence the development of an improved plasmid construction system is increasingly necessary for rapid and efficient preparation of expression plasmids for A. oryzae. The Gateway technology is a universal cloning system that provides an efficient way to transfer DNA sequences into a series of expression plasmids for functional analysis and protein expression.6–10) The sitespecific recombination reaction of bacteriophage  enzymes enables a linear DNA fragment flanked by the in vitro recombination sequences to be introduced into the specific site of donor vectors without any restriction digestion/ligation reactions. A plasmid construction system based on this technology has been developed for high-throughput protein tagging for a filamentous fungus, Aspergillus nidulans.11) With this plasmid construction system, however, it is difficult efficiently and rapidly to replace promoters, fusion tags, and selectable markers or to reverse the fusion tag site of the target gene because many donor vectors with various combinations and orders of the promoters, fusion tags, and selectable markers must be constructed by the conventional ligation technique with restriction enzymes. The MultiSite Gateway cloning system with a modified site-specific recombination reactions enables simultaneous connection of the three DNA fragments from 50 , center, and 30 entry clones along with a destination vector in a defined order and orientation to create desired expression plasmids (Fig. 1).12) Hence this system makes it possible to fuse the target gene with a large number of combinations of promoters, fusion tags, and selectable markers by only two reactions of in vitro recombination (BP and LR recombination reactions) (Fig. 1). We report here, taking advantage of this technology, the development of a novel plasmid construction system for the rapid and efficient preparation of many plasmids to be used for homologous and heterologous protein expression in A. oryzae. Individual units

To whom correspondence should be addressed. Tel: +81-3-5841-5161; Fax: +81-3-5841-8033; E-mail: [email protected]

A Novel Plasmid Construction System for A. oryzae

for construction of an expression plasmid, i.e., promoters, carrier proteins, fusion tags, and selectable markers, are provided as 50 -, center, and 30 -entry clones, and destination vectors. Subsequently, these units are assembled with the target gene cloned in either a center or 30 -entry clone through an in vitro recombination reaction into an expression plasmid (Fig. 1). By this system, a large number of expression plasmids can readily be prepared without laborious and time-consuming DNA manipulation experiments. In order to verify that the expression plasmids constructed by this system work as expected, we further employed it to construct plasmids for visualization of mitochondria in A. oryzae.

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structural genes encoding -amylase and glucoamylase,17) respectively, were amplified and used to generate pg50 PFa and pg50 PFg (Tables 1 and 2). The DNA sequences encoding enhanced green fluorescence protein (EGFP),18) DsRed2 (BD Biosciences Clontech, Palo Alto, CA), glutathione-S-transferase (GST) (Amersham Biosciences, Piscataway, NJ), and hemagglutinin epitope followed by hexahistidine sequence (HA-His6 ),19) were cloned as center and 30 entry clones. These were amplified by PCR using appropriate primers and plasmids as templates (Table 1) and inserted into pDONR 221 (Invitrogen; for generation of center entry clones) or pDONR P2R-P3 (Invitrogen; for generation of 30 entry clones) (Table 2).

Materials and Methods Strains and growth media. A. oryzae RIB40 and niaD300 (niaD )13) strains were used as a DNA donor and for transformation, respectively. DPY medium (2% dextrin, 1% polypeptone, 0.5% yeast extract, 0.5% KH2 PO4 , and 0.05% MgSO4 7H2 O, pH 5.5) and Czapek-Dox medium (CD; 0.3% NaNO3 , 0.2% KCl, 0.1% KH2 PO4 , 0.05% MgSO4 7H2 O, 0.002% FeSO4 7H2 O, 2% glucose, pH 5.5) were used for growth and as a selective medium, respectively. Escherichia coli DH5 (supE44 lacU169 (80 lacZ M15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1) was used for DNA manipulation. E. coli D.B. 3.1 (F gyrA462 endA1 (sr1-recA) mcrB mrr hsdS20 (rB mB )) was used to propagate plasmids including the ccdB cassette.

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Molecular techniques. Transformation of A. oryzae and E. coli was performed according to standard procedures.2,14) The LR and BP recombination reactions in the MultiSite Gateway system were carried out as instructed by the manufacturer (Invitrogen, Carlsbad, CA) with an exception regarding the transformation of the reaction mixture into E. coli. The larger colonies were selected because smaller colonies did not possess the expected plasmid. DNA fragments were amplified with the Pyrobest DNA polymerase (TaKaRa, Kyoto, Japan), and their nucleotide sequences were confirmed with ABI PRISM 310NT Genetic Analyzer (Applied Biosystems, Foster City, CA). Preparation of entry clones. The insert DNAs of entry clones were amplified by PCR using proper sets of primers containing additional attB sequences at the 50 end for the BP recombination reaction (Table 1). To generate 50 entry clones, DNA fragments containing the promoter regions of the pgkA (DDBJ accession no. D28484), amyB,15) and thiA16) genes (0.7, 1.3, and 1.6 kb in length, respectively) were amplified by PCR using appropriate primers and template DNAs (Table 1). The amplified DNA fragments were inserted into pDONR P4-P1R (Invitrogen), generating pg50 Pp, pg50 Pa, and pg50 Pt, respectively (Table 2). Likewise, the amyB and glaA promoters followed by their

Preparation of destination vectors. Two types of destination vectors, pgDN and pgDSN, carrying A. oryzae niaD and A. nidulans sC genes as selectable markers, respectively (Table 2), were prepared as follows: The SmaI/PvuI-fragment of pUNA,20) carrying A. oryzae niaD gene and the amyB terminator, was blunt-ended and ligated to the NdeI-digested and bluntended pDEST R4-R3, generating pgDN. A HindIII/ EcoRV-fragment containing A. oryzae niaD gene from pgDN was replaced with the XbaI/PstI-fragment of pUSC21) carrying A. nidulans sC gene, generating pgDSN. Construction of the expression plasmids for mitochondrial visualization by the high-throughput system. A DNA fragment encoding the N-terminal 76 amino acids of A. oryzae citrate synthase (AoCit1; DDBJ accession no. AB247940), which was identified as a homolog of Aspergillus niger Cit-A,22) was amplified by PCR using the primers (50 -GGGGACAAGTTTGTACAAAAAAGCAGGCTCTATGGCTTCTTCCTTGAGAATCG-30 and 50 -GGGGACCACTTTGTACAAGAAAGCTGGGTACTGGTCGAGGGTGACCTC-30 ; attB sequences are underlined) and A. oryzae genomic DNA as a template. The amplified DNA fragment was inserted into pDONR 221 by the BP recombination reaction, generating pgEAC, which was used as a center entry clone. pgEAC and three sets of other entry clones ([1] pg50 Pa and pg30 E, [2] pg50 Pa and pg30 DR, and [3] pg50 Pt and pg30 E) were mixed for the LR recombination reaction with the destination vector pgDN (Table 2), generating [1] pgACEN (for expression of the AoCit1EGFP fusion protein under the control of the amyB promoter), [2] pgACDN (for expression of the AoCit1DsRed2 fusion protein under the control of the amyB promoter), and [3] pgTCEN (for expression of the AoCit1-DsRed2 fusion protein under the control of the thiA promoter), respectively. Construction of the expression plasmid for mitochondrial visualization by the conventional ligation technique with restriction enzymes. A DNA fragment encoding the N-terminal 76 amino acids of the AoCit1

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Y. MABASHI et al. Table 1. List of Primers for Generation of the Entry Clones

Cloned DNA

Template

Primer sequence (From 50 to 30 ends)a

Primer name

50 Entry clone amyB promoter

pUNA2b

thiA promoter

pBYHI16)

pgkA promoter

pUNPc

amyB promoter and ORF

genomic DNAd attB4-amyBfs-F

attB4FM13RV attB1RniaDEF attB4-thiA-F attB1-thiA-R attB4-pgkA-F attB1-pgkA-R

GGGGacaactttgtatagaaaagttgCAGGAAACAGCTATGAC GGGGactgcttttttgtacaaacttgGGACTCACGAATAGCAAGGAATT GGGGacaactttgtatagaaaagttgTTCGGTAAATACACTATCACACAC GGGGactgcttttttgtacaaacttgGTTTCAAGTTGCAATGACTATCATC GGGGacaactttgtatagaaaagttgCCCGGGTATTGACTACTATGGTAACCAACG GGGGactgcttttttgtacaaacttgCCCGGGTGTTCTATCACACAAGGTGGG GGGGacaactttgtatagaaaagttgATGCATTTCATGGTGTTTTGATCATT

attB1-amyBfs-R GGGGactgcttttttgtacaaacttgTCGAGCTACTACAGATCTTGCTA glaA promoter and ORF

17)

pFGC32

Center entry clone EGFP pBEGFP-F18) DsRed2

pDsRed2e

gst

pGEX T4-3f

ha-his6

pMT19)

Aocit1[1–367 bp] genomic DNAd 30 Entry clone EGFP

pBEGFP-F

DsRed2

pDsRed2

gst

pGEX T4-3

ha-his6

pMT

attB4-glaAfs-F

GGGGacaactttgtatagaaaagttgGAATTCTGTAGCTGCTCTATTTC

attB1-glaAfs-R

GGGGactgcttttttgtacaaacttgAAGTCGTCGGCACCTGGCA

attB1-EGFP-F attB2-EGFP-R attB1-DsRed-F attB2-DsRed-R attB1-GST-F attB2-GST-R attB1-HA/His-F attB2-HA/His-R attB1-Aocit1-F attB2-Aocit1-R

GGGGacaagtttgtacaaaaaagcaggctAGATGGTGAGCAAGGGCGAG GGGGaccactttgtacaagaaagctgggtCCTTGTACAGCTCGTCCATGC GGGGacaagtttgtacaaaaaagcaggctAGATGGCCTCCTCCGAGAAC GGGGaccactttgtacaagaaagctgggtCCAGGAACAGGTGGTGGCG GGGGacaagtttgtacaaaaaagcaggctAGATGTCCCCTATACTAGGTTATTG GGGGaccactttgtacaagaaagctgggtCGGATCCACGCGGAACCAG GGGGacaagtttgtacaaaaaagcaggctAGCCCGGGATGTACGATGTTCCTGATTACGCTAG GGGGaccactttgtacaagaaagctgggtCCCCGGGATGGTGATGGTGATGATGGCTA GGGGacaagtttgtacaaaaaagcaggctCTATGGCTTCTTCCTTGAGAATCG GGGGaccactttgtacaagaaagctgggtACTGGTCGAGGGTGACCTC

attB2-EGFP-F attB3-EGFP-R attB2-DsRed-F attB3-DsRed-R attB2-GST-F attB3-GST-R attB2-HA/His-F attB3-HA/His-R

GGGGacagctttcttgtacaaagtggAGATGGTGAGCAAGGGCGAG GGGGacaactttgtataataaagttgATTACTTGTACAGCTCGTCCATG GGGGacagctttcttgtacaaagtggAGATGGCCTCCTCCGAGAAC GGGGacaactttgtataataaagttgACTACAGGAACAGGTGGTGG GGGGacagctttcttgtacaaagtggAGATGTCCCCTATACTAGGTTATTG GGGGacaactttgtataataaagttgATCAATCCGATTTTGGAGGATGGTC GGGGacagctttcttgtacaaagtggAGCCCGGGTACGATGTTCCTGATTACGCTAG GGGGacaactttgtataataaagttgACCCGGGCTAATGGTGATGGTGATGATGG

a

Small letters in the primer sequence represent the attB recombination sites. This plasmid was made by self-ligation of EcoRI-digested pUNA.20) c The plasmid containing the pgkA promoter and terminator, and the A. oryzae niaD marker in pUC118. d A. oryzae RIB40 strain e Invitrogen f Amersham Bioscience b

Table 2. Entry Clones and Destination Vectors for Construction of Expression Plasmids for A. oryzae 50 entry clone

Center entry clone

30 entry clone

Destination vector

amyB promoter (pg50 Pa) thiA promoter (pg50 Pt) pgkA promoter (pg50 Pp) amyB promoter + ORF (pg50 PFa) glaA promoter + ORF (pg50 PFg)

EGFP (pgEE) DsRed2 (pgEDR) gst (pgEG) ha-his6 (pgEHH) Aocit1#[1–367 bp] (pgEAC)

EGFP (pg30 E) DsRed2 (pg30 DR) gst (pg30 G) ha-his6 (pg30 HH)

A. oryzae niaD, amyB terminator (pgDN) A. nidulans sC, amyB terminator (pgDSN)

The names of entry clones and destination vectors are in parentheses.  These entry clones only were not confirmed to function for expression of proteins in A. oryzae.

protein was amplified by PCR using the primers (50 CATGCCGGCATGGCTTCTTCCTTG-30 and 50 -CATCCCGGGCTGGTCGAGGGTG-30 ; NaeI and SmaI sites are underlined) and A. oryzae genomic DNA as a template. The amplified DNA fragment was digested with NaeI and SmaI and ligated into the SmaI site of pUNA, generating pUNAC. Subsequently, the EGFP gene was amplified by PCR using the primers (50 CATCCCGGGATGGTGAGCAAGGG-30 and 50 -CATCCCGGGTTACTTGTACAGCTCGTC-30 ; SmaI sites

are underlined) and pBEGFP-F18) as a template. The amplified fragment was digested with SmaI and inserted into the SmaI site of pUNAC, generating pUNACE, which was used to express the AoCit1-EGFP fusion protein under the control of the amyB promoter. Fluorescence microscopy. Twelve transformants were obtained by introducing each expression plasmid, pgACEN, pgACDN, pgTCEN, and pUNACE, into A. oryzae niaD300 strain, from which at least two

A Novel Plasmid Construction System for A. oryzae

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Target gene

att B1

att B2

PCR BP Recombination Reaction Fusion tag

Promoter or Promoter + ORF

Gene

att L1 att L4

5’ entry clone

att R1

att R2

ccd B

CmR

att R4

att R3

amyB ter

att L3

att L2

Center entry clone

3’ entry clone Kan

Kanr Ampr

r

Kanr

Destination vector

Marker LR Recombination Reaction Gene

Tag

Promoter or Promoter + ORF

amyB ter

Expression plasmid Marker Ampr

Fig. 1. Construction of Expression Plasmids for A. oryzae by the MultiSite Gateway Technology (For C-terminal Fusion). This schematic model shows construction of the plasmid for expression of the target protein fused with a tag at the C-terminus. The target gene is amplified by adding the attB1 and attB2 recombination sequences at the 50 and 30 ends, respectively, and cloned into the center entry vector by the BP recombination reaction. The resulting center entry clone was subjected to the LR recombination reaction along with a 50 entry clone (a promoter or promoter + ORF), a 30 entry clone (a fusion tag), and a destination vector (the amyB terminator and a selectable marker). The expression plasmid is introduced into A. oryzae. For tagging at the N-terminus, the target gene amplified with addition of the attB2 and attB3 recombination sequences at the 50 and 30 ends, respectively, is cloned into 30 entry vector and connected with the center clone encoding a fusion tag.

typical strains were selected for localization studies. Approximately 103 conidia were suspended in 100 ml CD medium on a glass-bottom dish and incubated at 30  C for 10–24 h. Observations by differential interference and fluorescence microscopy were performed using Fluoview FV500 confocal laser scanning system (Olympus, Tokyo, Japan). Staining of mitochondria was performed using the mitochondrial specific dye Mitotracker Red CMXRos (Molecular Probes, Eugene, OR) according to the manufacturer’s instructions.

Results and Discussion Development of a high-throughput plasmid construction system for A. oryzae To develop a versatile plasmid construction system for homo- and heterologous protein expression in A. oryzae, we prepared a series of plasmids that served as 50 , center, or 30 entry clones and destination vectors in the MultiSite Gateway system (Table 2). To express a protein fused with the tag in A. oryzae, the target gene is amplified by adding appropriate attB sequences at both the 50 and 30 ends, and then cloned as center and 30 entry clones for C-terminal and N-terminal tagging, respectively (Fig. 1; the BP recombination reaction). For generation of the expression plasmid, the resulting entry clone and other two entry clones selected from 50 and center/30 entry clone pools are mixed for the LR

recombination reaction along with a destination vector. This reaction assembles the insert sequences from three entry clones with the destination vector in a defined order and orientation (Fig. 1). The 50 entry clones were designed to contain the promoters or the promoter plus its ORF (Fig. 1). Three different types of promoters were cloned into the 50 entry clones: the pgkA (DDBJ accession no. D28484; for constitutive expression), amyB15) (carbon source-regulated expression), and thiA16) (thiamine-regulated expression) promoters (Table 2). Expression under the control of the amyB promoter is high when A. oryzae is cultured in dextrin-containing medium, while in glucose and glycerol media expression is moderate and low, respectively.16,23) Expression under the control of the thiA promoter is repressed and up-regulated in the presence and absence, respectively, of thiamine in the medium.16,24,25) In heterologous protein production by A. oryzae higher-level production can be achieved by expressing as a fusion with a secretory protein of the host.17) The amyB and glaA promoters followed by their ORFs encoding secretory proteins of A. oryzae, amylase and glucoamylase, respectively, were chosen and prepared as 50 entry clones so that a heterologous protein could be expressed as a fusion protein (Table 2). To facilitate the purification of heterologous proteins produced by A. oryzae, and to perform localization and biochemical analyses on the target protein, the center

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and 3 -entry clones were designed to contain fusion tags. The fusion tag sequences cloned in entry clones were EGFP, DsRed2, GST, and HA-His6 (Table 2). Target genes to be expressed in A. oryzae are prepared as center or 30 -entry clones, depending on the positions of the fusion tags. For example, when a C-terminally tagged version of the target protein is expressed, a center entry clone carrying the target gene is prepared and then mixed with the 30 entry clone harboring a fusion tag sequence (Fig. 1). Two types of destination vectors containing either A. oryzae niaD13) or A. nidulans sC21) selectable markers along with the amyB terminator were prepared. A. oryzae niaD gene is often integrated homologously at the niaD locus, and thus it is a selectable marker suitable for molecular biological studies in A. oryzae.13) A. nidulans sC gene is a selectable marker favored for heterologous protein production by A. oryzae, since multiple copies of the expression plasmid can be integrated on the host genome.21) To complete the construction of an expression plasmid, an appropriate set of three entry clones is mixed in the LR recombination reaction together with a destination vector (Fig. 1). The resulting plasmids allow expression of the target gene under the control of a desirable promoter, with a suitable fusion tag and a selectable marker. Visualization of mitochondria in A. oryzae by the use of the expression plasmids constructed by the versatile system In order to demonstrate that the expression plasmids constructed by MultiSite Gateway method work as expected, we applied it to generate plasmids for visualization of mitochondria in A. oryzae. Since it has been reported that the N-terminal 76 amino acids of A. niger citrate synthase (Cit-A) are sufficient for mitochondrial targeting of green fluorescence protein (GFP) in A. nidulans,22) we used the A. oryzae homolog, AoCit1, for mitochondrial targeting of fluorescent proteins. A DNA fragment encoding the N-terminal 76 amino acids of AoCit1, found in the A. oryzae genome sequence database,4) was amplified by PCR, and its center entry clone was generated by the BP recombination reaction (Fig. 1). This clone was subjected to the LR recombination reaction along with 50 entry clones carrying the amyB or thiA promoters, 30 entry clones carrying the EGFP or DsRed2 genes, and the destination vector containing the A. oryzae niaD marker, generating the plasmids for expression of the AoCit1-EGFP or AoCit1DsRed2 fusion proteins controlled by the amyB or thiA promoters. As a control, we constructed a plasmid for expression of the AoCit1-EGFP fusion protein under the control of the amyB promoter by the conventional ligation technique with restriction enzymes. These plasmids were introduced into A. oryzae niaD300 strain (niaD ), and the cellular localizations of the AoCit1-EGFP and AoCit1-DsRed2 fusion pro-

teins were analyzed by fluorescence microscopy. In the transformants expressing the AoCit1-EGFP fusion protein under the control of the amyB promoter, tubular structures were observed in the hyphae (Fig. 2Aa and b), which resembled the mitochondrial structures visualized in other filamentous fungi, A. nidulans and Neurospora crassa.22,26) No significant differences in the localization of these structures were observed between the transformants carrying the plasmids constructed by the MultiSite Gateway-based system (Fig. 2Aa) and the conventional ligation technique (Fig. 2Ab). Hyphae expressing the AoCit1-DsRed2 fusion protein under the control of the amyB promoter fluoresced similar tubular structures (Fig. 2Ac). Furthermore, when the transformant expressing the AoCit1-EGFP fusion protein under the control of the thiA promoter was grown in the absence of thiamine (inducing condition), the fluorescent structures were similar to those observed in the strains expressing under the control of the amyB promoter (Fig. 2Ad). In contrast, no EGFP-fluorescence was detected when the transformant was grown in the presence of 10 mM thiamine (repressive condition). This indicates that expression of the AoCit1-EGFP fusion protein under the control of the thiA promoter was repressed by the addition of thiamine, as reported by Shoji et al.16) When A. oryzae wild-type strain RIB40 was stained with Mitotracker, a fluorescent dye that stains mitochondria in a membrane potential-dependent manner,27) tubular structures similar to those visualized by expressing the AoCit1-EGFP fusion protein were observed (Fig. 2Ba). To verify that the EGFP-visualized structures were mitochondria, the strain expressing the AoCit1-EGFP fusion protein was stained with Mitotracker. These two fluorescent structures co-localized (Fig. 2Bb), which indicated that the structures observed by expression of the AoCit1-EGFP fusion protein were mitochondria. Interestingly, we found that the fluorescence of Mitotracker was more intense in the hyphal tip region than in the basal region when the images were captured at a lower sensitivity (Fig. 2Bc). This is in contrast to the uniform distribution of the AoCit1-EGFP fusion protein along hyphae, resulting in yellow and green colors in the tip and basal regions, respectively, in the merged image (Fig. 2Bc, ‘‘Merge’’). This is probably because stronger activity of the membrane potential in the hyphal tip region resulted in greater fluorescence intensity of Mitotracker in the mitochondria. Since the uniform localization of the AoCit1-EGFP fusion protein in the mitochondria was not altered in any part of the hyphae, it is suggested that this visualization system might be a useful tool to investigate mitochondrial morphology throughout the whole mycelia irrespective of membrane potential activity. Considering these microscopic data together, it is evident that our plasmid construction system enables rapid and efficient preparation of plasmids for visualization of mitochondria in A. oryzae.

A Novel Plasmid Construction System for A. oryzae

A a

Aocit1

amyB pro

DIC

b

amyB pro

EGFP

EGFP

Aocit1

EGFP

ter

c

amyB pro

Merge ter

DsRed2

Aocit1

DIC

d

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thiA pro

DsRed2

EGFP

Aocit1

ter

Merge ter

(Conventional method)

DIC

EGFP

Merge

+ 10µ M thiamine

10 µm DIC

B

a

EGFP

Merge

c

b Mitotracker

DIC

EGFP

Merge

Mitotracker

Merge

DIC

Mitotracker

EGFP

Merge

10 µm

Fig. 2. Visualization of A. oryzae Mitochondria by Use of Expression Plasmids Constructed by the Versatile Construction System. A, Localization patterns of the AoCit1-EGFP and AoCit1-DsRed2 fusion proteins in A. oryzae. Transformants expressing the AoCit1-EGFP or AoCit1-DsRed2 fusion proteins were grown at 30  C for 20 h in CD medium and examined by confocal fluorescence microscopy. For expression of the AoCit1-EGFP fusion protein under the control of the amyB promoter, the expression plasmids were constructed either by the MultiSite Gateway-based system (a) or the conventional ligation technique with restriction enzymes (b). Fluorescent patterns in the transformant expressing the AoCit1-DsRed2 fusion protein under the control of the amyB promoter and the one expressing the AoCit1-EGFP fusion protein under the control of the thiA promoter are shown in c and d, respectively. Expression of the AoCit1-EGFP fusion protein by the thiA promoter was repressed in the presence of thiamine (10 mM) (d, lower). B, Co-localization of the AoCit1-EGFP fusion protein with Mitotracker-stained structures. A. oryzae wild-type strain RIB40 (a) and the transformant expressing the AoCit1-EGFP fusion protein under the control of the amyB promoter (b and c) were grown at 30  C for 24 h in CD medium. Mitochondria were stained with 50 nM Mitotracker Red CMXRos and examined by confocal fluorescence microscopy. Note that in c, the mitochondria at the hyphal tip region were stained with Mitotracker and thus fluoresced in yellow in the merged image, while those in the basal area are green, reflecting the low fluorescent intensity of Mitotracker, probably due to reduced membrane potential.

In this study, we report a versatile plasmid construction system based on the MultiSite Gateway technology, which facilitates the rapid and efficient preparation of a large number of A. oryzae expression plasmids. Theoretically, this system makes it possible to construct

80 expression plasmids for each target protein by selecting appropriate entry clones and performing the LR recombination reaction. So far, the majority of entry clones and destination vectors shown in Table 2 have been confirmed to enable expression of proteins in

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A. oryzae (this study and manuscripts in preparation), suggesting that the other entry clones will also work as expected. Although overexpression and inappropriate fusion design possibly hamper accurate localization and biochemical analyses, the promoters can be easily replaced with that of the target gene, and the fusion site is reversible by the use of the center entry clones harboring fusion tags (Table 2). In our laboratory, the plasmid construction system has been successfully used for visualization of various intracellular organelles/ structures and the production of several heterologous proteins (unpublished results). Furthermore, since our system allows faster and easier construction of DNA fragments for gene disruption by use of the center clone containing the marker gene (adeA and argB),5) we disrupted a number of genes such as protease genes by introducing DNA fragments constructed by the MultiSite Gateway technology (unpublished results). With the advent of the post-genomic era, this system will support the progress of molecular biological research on A. oryzae along with the development of applied techniques for heterologous protein production.

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Acknowledgments This study was supported in part by the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN of Japan). We thank Dr. Mamoru Ohneda and Dr. Feng Jie Jin for constructing some of the entry clones.

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References 1)

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