An efficient Agrobacterium-mediated transformation method for the edible mushroom Hypsizygus marmoreus

Microbiological Research 169 (2014) 741–748

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An efficient Agrobacterium-mediated transformation method for the edible mushroom Hypsizygus marmoreus Jin jing Zhang a,b,c,1 , Liang Shi a,1 , Hui Chen b,c , Yun qi Sun a,b,c , Ming wen Zhao a , Ang Ren a , Ming jie Chen b,c , Hong Wang b,c , Zhi yong Feng a,b,c,∗ a

College of Life Science, Nanjing Agricultural University, No. 1, Weigang Road, XuanWu District, Nanjing 210095, China National Research Center for Edible Fungi Biotechnology and Engineering, Key Laboratory of Applied Mycological Resources and Utilization, Ministry of Agriculture c Shanghai Key Laboratory of Agricultural Genetics and Breeding, Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences, No. 1000, Jinqi Road, FengXian District, Shanghai 201403, China b

a r t i c l e

i n f o

Article history: Received 16 November 2013 Received in revised form 22 January 2014 Accepted 25 January 2014 Available online 8 February 2014 Keywords: Agrobacterium-mediated transformation Hypsizygus marmoreus gpd Promoter

a b s t r a c t Hypsizygus marmoreus is one of the major edible mushrooms in East Asia. As no efficient transformation method, the molecular and genetics studies were hindered. The glyceraldehyde-3-phosphate dehydrogenase (GPD) gene of H. marmoreus was isolated and its promoter was used to drive the hygromycin B phosphotransferase (HPH) and enhanced green fluorescent protein (EGFP) in H. marmoreus. Agrobacterium tumefaciens-mediated transformation (ATMT) was successfully applied in H. marmoreus. The transformation parameters were optimized, and it was found that co-cultivation of bacteria with protoplast at a ratio of 1000:1 at a temperature of 26 ◦ C in medium containing 0.3 mM acetosyringone resulted in the highest transformation efficiency for Agrobacterium strain. Besides, three plasmids, each carrying a different promoter (from H. marmoreus, Ganoderma lucidum and Lentinula edodes) driving the expression of an antibiotic resistance marker, were also tested. The construct carrying the H. marmoreus gpd promoter produced more transformants than other constructs. Our analysis showed that over 85% of the transformants tested remained mitotically stable even after five successive rounds of subculturing. Putative transformants were analyzed for the presence of hph gene by PCR and Southern blot. Meanwhile, the expression of EGFP in H. marmoreus transformants was detected by fluorescence imaging. This ATMT system increases the transformation efficiency of H. marmoreus and may represent a useful tool for molecular genetic studies in this mushroom species. © 2014 Elsevier GmbH. All rights reserved.

Introduction Hypsizygus marmoreus is one of the major edible mushrooms in East Asia, such as in China, Japan and Korea (Lee et al., 2012). Recent research has shown that H. marmoreus is a good material for studying the developmental process of basidiomycete (Akavia et al., 2006; Jang et al., 2013). In addition, molecular genetics studies have also been conducted, resulting in cloning and char-

∗ Corresponding author at: National Research Center for Edible Fungi Biotechnology and Engineering, Key Laboratory of Applied Mycological Resources and Utilization, Ministry of Agriculture, Shanghai Key Laboratory of Agricultural Genetics and Breeding, Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences, No. 1000, Jinqi Road, FengXian District, Shanghai, China. Tel.: +86 21 62201090; fax: +86 21 62201090. E-mail address: feng [email protected] (Z.y. Feng). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.micres.2014.01.004 0944-5013/© 2014 Elsevier GmbH. All rights reserved.

acterization genes in H. marmoreus (Lee et al., 2012). However, researches about these genes functions in this mushroom are lacking. If an efficient and convenient transformation method was constructed in H. marmoreus, this will not only promote the molecular genetics studies in H. marmoreus, but also provided a good reference for other researchers of studying this mushroom. De Groot et al. (1998) was the first to apply an alternative transformation procedure based on Agrobacterium tumefaciens transfer DNA (T-DNA) (agro-transformation) for filamentous fungi. Since then, A. tumefaciens-mediated transformation (ATMT) has been used to transform various fungi, including members of the Ascomycetes, Basidiomycetes, and Zygomycetes (Mikosch et al., 2001; Michielse et al., 2004; Wang et al., 2009). The ATMT approach can generate a high number of transformants and does not require special equipment (Duarte et al., 2007). Recently, ATMT was also used to transform H. marmoreus vegetative mycelium (Hatoh et al., 2013). However, the transformation condition was not optimized

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Table 1 Oligonucleotide primers used. Name

Sequencea

gpdF gpdR 5gpdsp1 5gpdsp2 5gdpsp3 3gpdsp1 3gpdsp2 3sgpdsp3 Hm-gpd-F1 Hm-gpd-R1 Hm-gpd-F2 Hm-gpd-R2 P-gpd-F1 Hph-R P-gpd-F2 Egfp-R

5 -GGAANCAACGRTGTCCYCTG-3 5 -CGAGTRTACTNGTGTMTTCA-3 5 -CAGCTGTCAAGGAGGCTGCCAACGGCCC-3 5 -CTCGCCTTCCGTGTTCCTACTCTCGACGT-3 5 -TCCTCCACTGGTGCTGNNNNNNNNNTCGGCA-3 5 -CGCCGTGTGTGCGACCTGTTGGTGTTCG-3 5 -TGGTTGTCGTAATCGGAATAGAAAGTTGT-3 5 -AGGCTGAATATCATCTGNNNNNNNNNCGATAT-3 5 -CATG ggtaccCCTCGAGCGACATATGAATAG-3 5 -CATGggatccGGTGGATGTGTGTTGTTTTGG-3 5 -CATGctgcagCCTCGAGCGACATATGAATAG-3 5 -CATGctgcagGGTGGATGTGTGTTGTTTTGG-3 5 -GTGTGTTGTCTCCAGTAAGT-3 5 -CGGCACCTCGTGCACGCGGA-3 5 -TGACATCACCGCAAGTGCCTC-3 5 -CTCGCCCTTGCTCACCAT-3

The sequences with lowercase letters, ggtacc, ggatcc and ctgcag indicate Kpn I, BamH I, Pst I restriction sites, respectively. a DNA letter codes: M = (A, C); N = (A, T, G, C); R = (A, G); Y = (C, T).

and only several transformants were obtained. So, an efficient transformation method is needed for the functional study of H. marmoreus genes at molecular level. There are a variety of factors that can influence the transformation efficiency of higher fungi. The choice of vectors with appropriate promoters might be a key factor among them. Glyceraldehyde-3-phosphate dehydrogenase (GPD) is a crucial enzyme in glycolysis, which is reflected by the fact that it represents more than 5% of the total cellular protein in Saccharomyces cerevisiae. Homologous as well as heterologous gpd promoters have been used widely to express genes of interest. Examples include several basidiomycetous gpd promoters used in different higher fungi (Hirano et al., 2000; Van de Rhee et al., 1996; Shi et al., 2012). In this study, we cloned the gpd promoter from H. marmoreus to drive the expression of a double reporter gene fusion consisting of egfp and hph. The ATMT transformation method was optimized for H. marmoreus to achieve high efficiency. After transformation, we analyzed the expression of EGFP by fluorescence imaging and the presence of the hph by PCR and Southern blot. This method will greatly facilitate future molecular genetic studies of this fungus to gain a better understanding of the genetics of this organism. Materials and methods Strains and culture conditions H. marmoreus strain SIEF3133 was cultured at 25 ◦ C on Potato Dextrose Agar (PDA) medium and used for transformation. The Top10 strain of Escherichia coli used for plasmid amplification was grown on Luria-Bertani (LB) medium containing 100 ␮g/mL ampicillin or 50 ␮g/mL kanamycin as required. The strains of A. tumefaciens LBA4404, EHA105, GV3101 and AGL-1 were used as the T-DNA donor for fungal transformation. Minimal medium (MM) [10 mM K2 HPO4 , 10 mM KH2 PO4 , 2.5 mM NaCl, 2 mM MgSO4 ·7H2 O, 0.7 mM CaCl2 , 9 ␮M FeSO4 ·7H2 O, 4 mM (NH4)2 SO4 , 10 mM glucose, pH 7.0] was used for A. tumefaciens culture. Induction medium (IM) [MM containing 0.5% (w/v) glycerol, 0.2 mM acetosyringone (AS), 40 mM 2-(N-morpholino)ethanesulfonicacid (MES), pH 5.3] was used to co-cultivate A. tumefaciens and H. marmoreus. Cloning of the gpd gene from H. marmoreus H. marmoreus genomic DNA was isolated from two-week-old mycelium by CTAB method and total RNA was extracted using TRI Reagent® (Takara, Japan). The primers used for amplifying

the gpd gene sequence were listed in Table 1. The PCR product was purified and cloned into pGEM-T (Invitrogen, Shanghai, China,) for sequencing (Sangon Biotech, Shanghai). In order to get the gpd promoter sequence, the gDNA sequence of gpd gene was amplified by degenerate primer firstly. The promoter sequence was amplified by 5 and 3 SEFA PCR (Wang et al., 2007) using the primers which were designed dependent on the gDNA sequence of gpd gene. Then, the promoter sequence was further analyzed by Neural Network Promoter Prediction web software at http://www.fruitfly.org/seq tools/promoter.html and transcription factor binding sites searching tool at http://molsun1.cbrc. aist.go.jp/research/db/TFSEARCH.html. With the genomic DNA sequence, the transcriptional start site (TSS) and transcription terminator site (TTS) were predicted by gene structure prediction web software at http://linux1.softberry.com/berry.phtml. According to the predicted TSS and TTS, the primers were designed and the total cDNA sequence of gpd gene was amplified. Comparing the gDNA and cDNA sequence, the introns and exons were analyzed.

Plasmids and vector construction The vectors pGl-GPD/pLe-GPD used in this study were kindly provided by Professor Ming Wen Zhao (Nan Jing Agricultural University, China) (Shi et al., 2012). These vectors contain the selectable hygromycin B marker gene (hph) and the egfp reporter gene (egfp) under the control of Ganoderma lucidum and Lentinula edodes gpd promoters respectively. To construct the pHm-GPD plasmid (Fig. 4), two gpd promoters in pGl-GPD were replaced with the gpd promoter from the H. marmoreus using Pst I single restriction enzyme digestion and BamH I and Xba I double restriction enzyme digestion respectively. The primers used for PCR amplification and subcloning are listed in Table 1. The plasmids pGl-GPD and pLe-GPD also contain the kanamycin gene as a selection marker for Agrobacterium transfection. These plasmids were introduced into the four A. tumefaciens strains using 20 mM calcium chloride.

Agrobacterium-mediated transformation of H. marmoreus Protoplasts of H. marmoreus were prepared by a method previously described (Sun et al. 2001). About 105 protoplasts were used for ATMT. The transformation procedure was compared with the method described by Shi et al. (2012). A. tumefaciens strains LBA4404, EHA105, GV3101 and AGL-1 harboring pHm-GPD vector were grown at 28 ◦ C on a rotatory shaker (200 rpm) in 5 mL of LB broth supplemented with 50 ␮g/mL rifampin and 50 ␮g/mL kanamycin to an optical density (OD600) of 0.5–0.6. Bacterial cells were harvested by centrifugation at 8000 rpm, washed once with fresh IM, and resuspended in 4 ml of fresh IM to an OD600 of 0.5–0.6. Three T-DNA binary vectors with different promoters (Hm.gpd, Gm.gpd and Le.gpd) were introduced into the EHA105. The bacterial cells were then grown at 28 ◦ C on a rotatory shaker (100 rpm) for 4–6 h. Co-culture of A. tumefaciens and H. marmoreus protoplast was conducted at different bacteria-to-protoplast ratios (1:1, 10:1, 100:1, and 1000:1). The co-cultures were spread evenly on 50 mm nitrocellulose membranes laid on top of 10 mL of solidified AS-containing IM-agar plates [IM plus 1.8% agar containing various concentrations of AS (0, 0.05, 0.3 and 1 mM)] and incubated at different temperatures (22, 24, 26 and 28 ◦ C) for different time durations (12, 24, 48 and 72 h). Following co-culture, the membranes were transferred to PDA plates supplemented with 200 ␮g/mL cefotaxime and 20 ␮g/mL hygromycin B to select positive H. marmoreus transformants. Each experiment was repeated three times.

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Fig. 1. Phylogenetic tree based on GAPDH amino acid sequences. The sequence alignment and tree construction were performed with ClustalW in MegAlign, a program of the Lasergene package (DNASTAR). The GeneBank accession numbers are: Coprinopsis cinerea (BAD69793), Ganoderma lucidum (ABD64597), Pleurotus ostreatus (BAL41701), Armillariella tabescens (CAF74786), L. edodes (BAA83550), Laccaria bicolor (XP 001887370), Leucoagaricus meleagris (ABU87583), Lyophyllum shimeji (Q92243), Agaricus bisporus (EKV43342), Neurospora crassa (AAB95425), Aspergillus niger (XP 001397496), Microdochium nivale (BAE46581). The horizontal bar indicates the relative distance in the phylogenetic tree.

Molecular analysis of the transformants The presence of hph and egfp transgenes was confirmed by PCR using the primers shown in Table 1. The PCR amplification protocol started with an initial denaturation step at 94 ◦ C for 5 min, followed by 30 cycles of denaturation at 94 ◦ C for 30 s, annealing at 55 ◦ C for 30 s, and elongation at 72 ◦ C for 1 min, and a final elongation step at 72 ◦ C for 5 min. The fusion fragments containing the Hm-GPD promoter and the egfp or hph gene were amplified using primer pairs Hm-GPDF/EGF-R and Hm-GPDF/HPH-R, respectively (Table 1). The PCR amplification protocol started with an initial denaturation step at 94 ◦ C for 5 min, followed by 30 cycles of denaturation at 94 ◦ C for 30 s, annealing at 56 ◦ C for 30 s, and elongation at 72 ◦ C for 1.5 min, and a final elongation step at 72 ◦ C for 5 min. Standard procedures for restriction endonuclease digestion, agarose gel electrophoresis and Southern blotting were carried out as described by Shi et al. (2012). DNA probe labeling and hybridization were performed under conditions recommended for the digoxigenin (DIG) hybridization system by Roche (Mannheim, Germany). Mitotic stability of transformants To determine the stability of transformants, 40 transformants were randomly selected and cultured on PDA plates without hygromycin B for two weeks. Mycelia from the edge of the cultures were transferred on fresh PDA plates and grown for another two weeks. After repeating this procedure 5 times, germinating mycelia from each transformant were transferred to PDA plates containing hygromycin B (20 ␮g/mL). Results Analysis of the GPD gene and its 5 flanking region Following the degenerate PCR amplification, fragments of the same size (550 bp) were obtained from genomic DNA and total RNA of H. marmoreus. Subsequently, the entire gpd genomic sequence was obtained by 5 and 3 SEFA PCR (Wang et al. 2007), which

Fig. 2. Comparison of intron positions in gpd genes; horizontal lines represent the coding region (336–338 codons) of each gpd gene. The vertical bars mark the position of introns. The GeneBank accession numbers are: L. edodes (AB013136), H. marmoreus (JN048800), G. lucidum (DQ404344), V. volvacea (KF528323), A. niger (X99652), A. nidulans (M33539), Curvularia (X58718) and Cryphonectria (X63516).

includes an upstream region of 2707 bp and a downstream fragment of 650 bp. The coding sequence of 1017 bp corresponding to 338 amino acids is disrupted by six introns. The intron positions of gpd gene between four basidiomycetes and four ascomycetes were analyzed and the result was shown in Fig. 2. Besides, the protein was highly conserved compared to known GPD proteins with an amino acid identity of 94.0% to GPD sequences of other basidiomycetes and the phylogeny of the GPD proteins was shown in Fig. 1. In the gpd promoter, a potential transcription start site was located at 56 bp upstream of the start codon in a CT-rich region, in front of which a typical TATA-box and four CAAT-boxes were found (Fig. 3). Agrobacterium-mediated transformation In attempt to develop a simple, highly efficient transformation system for H. marmoreus, we tested the applicability of the ATMT method. The protoplast, fruit bodies and fungal mycelium were tested using the same vector pHm-GPD. This experiment was carried out in a similar way as described by Shi et al., 2012. A suspension mixture of recipient materials and bacteria was spread at a ratio of 1:1 on sterile nitrocellulose membranes on IM agar plates with 0.2 mM AS, and incubated at 25 ◦ C. After 36 h, the membranes

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Fig. 3. The upstream nucleotide sequence for H. marmoreus gpd to identify promoter elements. Possible promoter elements are framed, and the predicted transcriptional start site is underlined (GeneBank accession number JN048800).

were transferred to PDA agar plates containing 200 ␮g/mL cefotaxime and 20 ␮g/mL hygromycin B and incubated at 25 ◦ C for two weeks. A few fungal colonies were obtained on culture plates. The transformation efficiency was higher with protoplast than other materials, demonstrating that protoplast was better for H. marmoreus transformation using the ATMT method. Optimization of conditions for ATMT of H. marmoreus Based on previous studies, 22, 24, 26 and 28 ◦ C were chosen to optimize the co-cultivation temperature. The number of colonies was found to be the highest at 26 ◦ C, which is the optimal cultivation temperature for both EHA105 and H. marmoreus (Fig. 5A). When the duration of co-cultivation was elongated, the transformants were decreased (Fig. 5B). This may be due to the over-growth of the bacteria after a longer duration of co-cultivation inhibited the fungal growth. Therefore 24 h was chosen as the duration

of co-cultivation for subsequent assays. The number of colonies increased with the increase of bacteria-to-protoplast ratio and reached the peak at a ratio of 1000:1 (Fig. 5C). The number of colonies obtained at this ratio was twofold higher than the number of colonies obtained at a bacteria-to-protoplast ratio of 1:1. Four A. tumefaciens strains were evaluated and the highest transformants were obtained by using the A. tumefaciens strain EHA105, followed by LBA4404 and GV3101, and no positive colonies were obtained for AGL-1 strain (Fig. 5D). The optimal concentration of AS for cocultivation was found to be 0.3 mM (Fig. 5E). A few transformants were obtained when co-cultivation was conducted with 0.05 and 0.8 mM AS, but no positive colonies were obtained in co-cultures without AS. A comparative study was performed by co-cultivation of H. marmoreus with EHA105 carrying one of the following plasmids: pHm-gpd, pGl-GPD or pLe-GPD (Fig. 5F). These plasmids contain a cassette in which hph is under the control of different fungal

Fig. 4. Used transformation vectors based on pGl-GPE vector (Shi et al. 2012). The promoter consists of 1224 bp H. marmoreus glyceraldehyde-3-phosphate dehydrogenase (gpd) promoter (pHm-gpd) and the expression of egfp and hph as reporter genes were driven by H. marmoreus gpd promoter.

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Fig. 5. Factors affecting the efficiency of ATMT of H. marmoreus. (A) Effect of co-cultivation temperature on transformation efficiency. The growing colonies were counted as transformants. (B) Effect of length of duration of co-cultivation on transformation efficiency. (C) Effect of bacteria to protoplast ratio on transformation efficiency. (D) Effect of Agrobacterium strain on transformation efficiency. (E) Effect of AS concentration on transformation efficiency. (F) Effect of different promoters on transformation efficiency. Values reported are the mean of three independent experiments and indicate the number of transformants. Error bars indicate confidence intervals of percentages (P < 0.05).

gpd promoters. The results showed that the highest transformation efficiency was obtained using plasmid PHm-gpd, in which hph is driven by gpd promoter from H. marmoreus. A few transformants were obtained using EHA105 carrying the other two plasmids, with similar efficiencies. The hph gene in pGl-GPD and pLe-GPD is driven by promoters from G. lucidum and L. edodes, respectively.

Characterization of reporter gene integration and expression To investigate the pattern of integration of the foreign DNA fragment, three putative transformants were randomly selected for Southern blot analysis. As shown in Fig. 6A, all three transformants had the T-DNA insertion. The various sized DNA bands in the Southern blot indicated that the T-DNA into the genome of H. marmoreus randomly was obtained. The transformants obtained from H. marmoreus were analyzed for the presence of egfp/hph transgenes by PCR and EGFP expression by fluorescence microscopy. The presence of fusion fragment containing the gpd promoter-egfp/hph in the transformants was also confirmed by PCR analysis (Fig. 6B and C). The green fluorescence could only be detected in the transformants (Fig. 7), suggesting that the transformants had not only integrated egfp but also expressed this gene.

Mitotic stability of transformants Most of the transformants of H. marmoreus did not show any phenotypic difference compared to the wild-type strain under the same physiological conditions. To determine whether the transformed DNA was stably integrated in the genome of H. marmoreus, 40 transformants were grown on PDA plates without hygromycin B and replated for five generations. 34 mitotically stable transformants were obtained out of 40 colonies (85%), confirming the genetic stability of the integrated DNA in this fungus. The results suggest that this method can be used as a tool to introduce foreign DNA into H. marmoreus. Discussion In this study, we obtained the complete gpd gene sequence from H. marmoreus. The coding region was analyzed by comparing the genomic DNA sequence with the cDNA sequence. It matches perfectly with the cDNA clone, suggesting this gpd encodes a functional protein. In Mucor circinelloides and Agaricus bisporus, which harbor more than one copy of gpd sequence, only one gpd mRNA is identified, indicating that there is only one functional gpd (Harmsen et al., 1992; Wolff and Arnau, 2002). It has been reported that gpd gene in higher basidiomycetes has 5–10 introns (Kilaru and Kües, 2005). This is also true for the gpd gene in H. marmoreus. The ability of the

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Fig. 6. Identification of the foreign DNA fragments in randomly chosen H. marmoreus transformants. (A) Southern Blot analysis of three randomly selected transformants. Lane 1: pHm-GPD as positive control, Lane 2: wild-type as negative control, Lane 3–5: randomly chosen transformants. The unit of number of markers and length indicator is base pair. (B) Amplification pattern obtained with primers for the gpd promoter-egfp fusion fragment (B) and gpd promoter-hph fusion fragment (C) in genomic DNA isolated from the H. marmoreus transformants. Lane 1 untransformed H. marmoreus as negative control, Lane 2 pHm-GPD as positive control, Lane 3–6 randomly chosen transformants containing hph, Lane M, DL 2000 DNA Marker.

Fig. 7. Fluorescence microscopy analysis of two randomly selected transformants harboring egfp. (A and B) Fluorescence and bright field images of wild-type H. marmoreus mycelia respectively; (C and E) Green fluorescence of mycelia of randomly chosen transformants harboring egfp. (D and F) Bright field image of mycelia shown in C and E; Bar 100 ␮m (in A, B, C, D, E and F images).

promoter obtained in this study to drive heterologous gene expression was proved by the hygromycin resistance trait and the EGFP fluorescence of transformants. Molecular genetic studies of H. marmoreus have been limited due to the lack of an efficient gene transfer system. In the last decade, ATMT has been successfully applied to different fungal species, mainly owing to its efficiency and technical simplicity (Godio et al., 2004; Michielse et al., 2005). The main objective of this work was to establish optimal conditions of ATMT for H. marmoreus. Transformation results showed that the choices of recipient materials as well as vectors affected final efficiencies (Mikosch et al., 2001; Cho et al., 2006; Wang et al., 2008; Shi et al., 2012). In this work, the protoplast was found to produce the best results in transformation experiments as compared to fruit bodies and fungal mycelium. In A. bisporus and F. velutipes, transformation efficiencies of single gill tissues were reported to be approximately 40% and 16%, respectively (Chen et al., 2000; Cho et al., 2006). However, few transformants could be obtained from gill tissues of H. marmoreus deriving from small mushroom caps that just developed from primordium. In this study, we obtained at least 150 stable H. marmoreus transformants per 105 protoplasts, which is a relative high transformation efficiency. Various Agrobacterium strains have been tested, and it was found that the Agrobacterium strains EHA105 produced the highest transformation frequency in H. marmoreus. Transformants were also obtained by using the Agrobacterium strains LBA4404 and GV3101, but no transformants were found with AGL-1. Different Agrobacterium strains have different transformation efficiency according to different fungal strains (Michielse et al. 2005). Three studies showed that the usage of Agrobacterium strains derived from the supervirulent A281 strain (high level of vir gene expression) resulted in higher transformation frequencies in S. cerevisiae, Monascus purpureus, and the Oomycete Phytophthora infestans, compared with ATMT using Agrobacterium strain LBA1100 (Campoy et al. 2003; Piers et al. 1996; Vijn and Govers 2003). In another study, it was also found that the supervirulent Agrobacterium strain A281 and its derivative (AGL-1) were more efficient in transferring T-DNA to Cryphonectria parasitica than the Agrobacterium strain LBA4404 (Park and Kim 2004). However, systemic comparisons of these strains in relation to transformation frequencies have not been performed, making it difficult to determine which strain is the best to use (Michielse et al., 2005). From these results, we clearly know that the choice of Agrobacterium strain for ATMT of fungi can have an effect on transformation efficiency. We also found that increasing the amount of A. tumefaciens in the co-culture mixture led to an increase in the transformation frequency. However, there is an optimal ratio of A. tumefaciens to protoplast that can be used. Higher numbers of A. tumefaciens can cause bacterial over-growth during co-culture, thereby inhibiting fungal growth, probably due to nutritional or space limitations (Chen et al., 2009; Shi et al., 2012). At the same time, the addition of too many protoplasts can result in fungal over-growth, which makes the subsequent isolation of single transformants difficult (Covert et al., 2001). In most studies, the addition of AS during the Agrobacterium co-cultivation is required for transformation. In this work, transformants were obtained only when the co-culture was induced with a suitable AS concentration (0.3 mM). This is consistent with earlier reports demonstrating the essential role of AS for vir gene expression (De Groot et al. 1998; dos Reis et al., 2004; Michielse et al., 2008). Several studies have shown that each fungus has a unique combination of temperature and co-cultivation duration to obtain a maximum number of transformants (Combier et al., 2003; Meyer et al., 2003; Rolland et al., 2003; Gardiner and Howlett, 2004; Michielse et al., 2004b; Shi et al., 2012). In this study, the most suitable temperature is 26 ◦ C, which is close to the normal growth

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temperature (25 ◦ C) for H. marmoreus. At all temperatures tested, fungal transformants were obtained. This result suggests that the Agrobacterium vir gene is expressed over a wide range of temperatures, allowing for effective T-DNA transfer and integration. Similar to the co-culture temperature, co-cultivation for different time periods also resulted in positive fungal transformants. Unlike in G. lucidum, the prolonged incubation period let to irreproducible results, possibly due to the increased fungal background growth during co-cultivation (Michielse et al., 2004b). This is the first time the egfp gene is expressed in fusion with hph in the basidiomycetous mushroom H. marmoreus, under the control of H. marmoreus gpd promoter. Compared with other gpd promoters, the promoter from H. marmoreus resulted in the highest transformation efficiency. Promoters Gm-gpd and Le-gpd, which are from the basidiomycete G. lucidum and L. edodes respectively, were able to drive the expression of antibiotic resistance gene but the numbers of colonies obtained were far fewer compared to the Hm-gpd promoter. The purpose of this part is to demonstrate that promoter sequences belonging to homologous species are more active and highlight the importance of using homologous promoters to drive gene expression (Chen et al., 2000; Godio et al., 2004). Godio et al. (2004) found that a critical factor for ATMT was the nature of the promoter region used to express the antibiotic resistance marker. In G. lucidum, the construct carrying the promoter of itself resulted in the highest transformation efficiency (Shi et al., 2012). In our study, promoter from H. marmoreus had the highest transformation efficiency and promoters from two homologous species had lower transformation efficiency than H. marmoreus. Mitotic stability analysis showed that over 85% of the transformants tested remained mitotically stable even after subculture in the absence of hygromycin B. The antibiotic resistance conferred by the hph gene was still maintained. Southern blot and PCR analysis results demonstrated that T-DNA was integrated into the chromosome of H. marmoreus. In our another study, four developmental stages transcriptomes of H. marmoreus have been done and the results suggested that some genes played important roles in the fruit body formation of H. marmoreus, such as laccase genes, NADPH oxidase genes and the genes involved in MAPK signaling pathway (Cano-Dominguez et al., 2008; De Paula et al., 2008; Langfelder et al., 1998; Mu et al., 2013). As the transformation method was constructed, these genes may be as our target genes and their functions in the developmental process of H. marmoreus could be studied by this method in future study. Conclusion In this study, the promoter of gpd gene from H. marmoreus was used to drive hph and egfp gene. The ATMT transformation system of H. marmoreus was established and optimized. This ATMT transformation system increased the transformation efficiency of H. marmoreus and produced mitotically stable transformants. This transformation system can be used as a powerful tool for functional genomics research and will facilitate researches such as exogenous gene expression and protein production for biotechnological applications. References Akavia E, Wasser SP, Beharav A, Nevo E. Study of Hypsizygus marmoreus (Peck) Bigel. and Grifola frondosa (Dicks.: Fr.) S.F. Gray: Cultural-Morphological Peculiarities, Growth Characteristics. Qualitative Enzymatic Activity, and Resistance to Fungal Pest Contamination Inter J Med Mushrooms 2006;8:361–76. Campoy S, Perez F, Martin JF, Gutierrez S, Liras P. Stable transformants of the azaphilone pigment-producing Monascus purpureus obtained by protoplast transformation and Agrobacterium-mediated DNA transfer. Curr Genet 2003;43:447–52.

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