Analytical Biochemistry 453 (2014) 58–60
Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
Notes & Tips
Membrane-assisted culture of fungal mycelium on agar plates for RNA extraction and pharmacological analyses Mario Lange, Carolin Müller, Edgar Peiter ⇑ Plant Nutrition Laboratory, Institute of Agricultural and Nutritional Sciences (IAEW), Faculty of Natural Sciences III, Martin Luther University of Halle–Wittenberg, 06099 Halle (Saale), Germany
a r t i c l e
i n f o
Article history: Received 13 January 2014 Received in revised form 21 February 2014 Accepted 22 February 2014 Available online 4 March 2014 Keywords: Colletotrichum graminicola Fungal growth PVDF membrane RNA extraction Gene expression
a b s t r a c t Fungal mycelium grown in liquid culture is easy to harvest for RNA extraction and gene expression analyses, but liquid cultures often develop rather heterogeneously. In contrast, growth of fungal mycelium on agar plates is highly reproducible. However, this biological material cannot be harvested easily for downstream analyses. This article describes a PVDF (polyvinylidene difluoride) membrane-assisted agar plate culture method that enables the harvest of mycelium grown on agar plates. This culture method leads to a strongly reduced variation in gene expression between biological replicates and requires less growth space as compared with liquid cultures. Ó 2014 Elsevier Inc. All rights reserved.
To maximize the conclusiveness of biological experiments, it is important to minimize the variation between replicate samples. In particular, the uniformity of the organisms under investigation at the moment before sampling is crucial. Inaccuracies at this stage cannot be compensated in the downstream analysis [1]. This also holds true for the analysis of gene expression. In expression studies, the extracted RNA samples are handled and the quantification of expression is performed with great care [2,3]. However, this does not ameliorate a physiological variability of the replicate organisms. In fungal biology, transformed strains are usually cultured in liquid medium to assess the effect of knockdown or overexpression approaches on gene expression. Unfortunately, growth (and thus physiological state) of fungi propagated in liquid medium is often rather inhomogeneous [4]. For example, in liquid medium, Aspergillus niger forms highly variable microcolonies that differ in size and gene expression patterns [5]. In contrast, mycelium growth on agar plates is highly reproducible [6,7]. Agar plate-grown fungal macrocolonies show a regular formation of radial zones [5]. In those solid-state cultures, there are gradients for nutrients within a colony that are a cause for this radial zonation. These gradients reflect the situation in the natural habitats of filamentous fungi [6]. In contrast to this, in liquid cultures these gradients are absent. Physiological parameters of fungi grown on solid medium can ⇑ Corresponding author. Fax: +49 345 5527113. E-mail address: [email protected] (E. Peiter). http://dx.doi.org/10.1016/j.ab.2014.02.023 0003-2697/Ó 2014 Elsevier Inc. All rights reserved.
differ widely from those of fungi grown in liquid medium. For example, the secretion of enzymes is often enhanced in solid-state cultures [6]. In both liquid and solid-state cultures, a micro-heterogeneity between and within single neighboring hyphae has been observed [5]. To reduce the variation between replicate samples, the intrinsic micro-heterogeneity between hyphae should be normally distributed in the sampled mycelium. Due to the more homogeneous colony morphology of solid-state-grown fungi compared with liquid-grown fungi, we hypothesized that the replicateto-replicate variations in gene expression are lower in a solid-state culture. To combine an efficient harvest of mycelium or fermentation products with the growth on a solid support, a number of approaches have been developed in the past, each optimized for a certain question. Broth solidified with the block polymer pluronic polyol F-127 has been used to grow Penicillium spinulosum for extraction of fungal DNA [8]. Aqueous solutions of pluronic polyol F-127 form a gel at room temperature and liquefy when chilled. Because the time for liquefying the medium is rather long, this method is unsuitable for RNA extraction [8]. Filamentous fungi have also been grown on inert porous solids soaked with broth in order to increase the production of fermentation products, with the mycelium being trapped in the pores of the support [9–11]. Another method providing solid support for the fungus was introduced by Yasuhara and coworkers [12], who constructed an apparatus in which an inert membrane is situated above nutrient broth and grew Aspergillus oryzae on the aerial side of the
Notes & Tips / Anal. Biochem. 453 (2014) 58–60
membrane. This procedure, called membrane surface liquid culture (MSLC)1 by the authors, induced the expression of a neutral protease by more than one order of magnitude compared with standard liquid culture and simplified the enzyme extraction compared with cultivation on agar [12]. Here, we present a simplified version of MSLC by replacing the nutrient broth with an agar medium poured into commercially available standard Petri dishes. This modification drastically reduces the technical effort of a membrane-based culture and allows high-throughput applications in which filamentous fungi can easily be harvested for RNA extraction. We named this method PVDF (polyvinylidene difluoride) membrane-assisted agar plate culture, abbreviated PAAP culture. In the experiments on which this study is based, we used the PAAP culture to grow the filamentous fungus Colletotrichum graminicola, a hemi-biotrophic pathogen of corn (Zea mays). C. graminicola can infect the plant at all developmental stages and has provoked dramatic yield losses in the past [13,14]. To prepare the culture system, a PVDF membrane (Hybond-P, pore diameter = 0.45 lm, GE Healthcare, Chalfont St. Giles, UK) was prewetted in 96% ethanol (Roth, Karlsruhe, Germany) and sterilized in 70% ethanol. This procedure was followed by four sequential additions of a half-volume of liquid modified Leach’s complete medium (mLCM) containing 1% sucrose, 0.1% tryptone, 0.1% yeast extract, 0.1% Ca(NO3)2, 0.025% MgSO4 7H2O, 0.02% KH2 PO4, and 0.0054% NaCl [15,16]. Finally, the membrane was washed twice in liquid mLCM. After that, mLCM agar (liquid mLCM containing 1.5% agar) was poured into 55-mm Petri dishes (VWR International, Darmstadt, Germany), and just after solidification of the agar surface the mLCM-equilibrated PVDF membrane was placed on top. The membrane-covered agar was dried for 30 min and inoculated with a 2-ll suspension of falcate conidia containing 300 C. graminicola M2 (M1.001) wild-type spores [17]. After 3 or 4 days at 23 °C, the growing mycelium covered most of the membrane. To visualize mycelium grown on PVDF membrane, the fungus was live-stained with fluorescein diacetate (FDA) for 1 min in a solution of 0.04% FDA (Sigma–Aldrich, St. Louis, USA) and 2% acetone (Roth) in water. Pictures were captured using a stereomicroscope (SteREO Discovery V20, Carl Zeiss, Jena, Germany) equipped with a fluorescence filter set 38 (excitation = 450–490 nm, emission = 500–550 nm) and an Axiocam HRc camera (Carl Zeiss). To compare the visual homogeneity of the fungal material produced according to the PAAP culture protocol against material produced by the standard culture procedure in liquid medium, we observed the physical appearance in replicate samples. In the standard procedure, C. graminicola was grown shaking at 100 rpm in 300-ml Erlenmeyer flasks containing 100 ml of liquid mLCM for 1 week at 23 °C. As Fig. 1A shows, the culture of the filamentous fungus C. graminicola grew rather inhomogeneously in liquid medium. In contrast, a far greater homogeneity was achieved in the PAAP cultures (Fig. 1B). Growth rates of PAAP cultures were identical to those on agar plates. We next wanted to determine whether the more homogeneous visual appearance is reflected in a better reproducibility of gene expression. For this purpose, we again prepared PAAP-cultured fungal colonies but left them unstained (Fig. 1C). The harvest of fungal material from a PAAP culture was prepared by floating the Petri dish lid on liquid nitrogen. Then, the membrane carrying a whole intact fungal colony was removed from the agar and placed into the prechilled lid. The membrane and fungal biomass was lyophilized for 2 h at 1.25 mbar by using an Alpha 1-4 freeze dryer (Martin Christ, Osterrode, Germany). The margin of the colony lifted from the membrane during the drying process and both 1 Abbreviations used: MSLC, membrane surface liquid culture; PVDF, polyvinylidene difluoride; PAAP, PVDF membrane-assisted agar plate; mLCM, modified Leach’s complete medium; FDA, fluorescein diacetate; qRT–PCR, quantitative reverse transcription polymerase chain reaction; DMSO, dimethyl sulfoxide.
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could be easily separated by using a razor blade (Fig. 1D). Next, the mycelium was transferred into a 2-ml microtube containing two 3mm steel beads (Hecht, Winnenden, Germany) (Fig. 1E). The biomass of two membrane cultures was pooled to one sample. In preliminary experiments, different types of membrane were tested for this culture technique. In contrast to PVDF, nylon mesh, Miracloth, and nitrocellulose proved to be unsuitable. Nylon mesh did not prevent a close adherence of the fungus to the agar, and the fungal material remained on the plate when removing the mesh. On Miracloth, growth was strongly reduced, and agar adhered to this type of material after removal from the plate. Nitrocellulose allowed a proper removal of the mycelium-containing membrane from the supporting agar but was very brittle after freeze-drying, hence impeding the removal of the fungal material. Mycelium obtained by the standard liquid culture procedure was filtered, briefly blotted on filter paper, frozen in liquid nitrogen, and lyophilized as above. Dry biomass (10 mg) was transferred into a 2-ml microtube containing two 3-mm steel beads. The biomass obtained from the two culture methods was ground for 1 min at 30 Hz using a Tissue Lyser II (Qiagen, Hilden, Germany). RNA extraction was performed using a peqGold Plant RNA kit (Peqlab, Erlangen, Germany). The ground biomass was suspended in 450 ll of Lysis Buffer T and homogenized for 30 s at 30 Hz in the Tissue Lyser. All further steps of RNA extraction were performed according to the manufacturer’s instructions. Genomic DNA was removed using DNase I (New England Biolabs, Ipswich, MA, USA) according to the manufacturer’s instructions. RNA was LiCl precipitated (4 M final concentration), followed by two washes with 70% ethanol. Synthesis of complementary DNA (cDNA) was done with Moloney murine leukemia virus (M-MLV) reverse transcriptase (Promega, Fitchburg, WI, USA) using gene-specific reverse oligonucleotides (Eurofins MWG Operon, Ebersberg, Germany) (Table 1 in supplementary data) according to the instructions provided by Promega. Sequences for HXT and HistonH3 oligonucleotides were taken from the literature [2,18]. Quantitative reverse transcription polymerase chain reaction (qRT–PCR) was carried out on a Mastercycler ep realplex4 S (Eppendorf, Hamburg, Germany) in MicroAMP optical eight-tube strips covered with MicroAMP optical eight-cap strips using the POWER SYBR Green PCR mastermix (all from Applied Biosystems, Foster City, CA, USA). Each reaction had a volume of 12.5 ll. Reaction conditions were set as recommended by Applied Biosystems. A calibration curvebased calculation of relative expression was applied [19]. Expression levels of TRPF and HXT genes were normalized to Histone H3. Next, the mean of these normalized values of each set of four biological replicates for each gene and culture system was set to 100%. The values of the four replicates were correlated to these 100% values, resulting in relative expression values. These relative values were used to calculate standard deviations and tested for significant differences between the liquid culture and the PAAP culture using F-test statistics (Microsoft Excel 2007). The PAAP culture provided sufficient biomass (2.4 ± 0.3 mg) from two colonies of approximately 3 cm diameter to extract RNA for many qRT–PCR experiments (22.4 ± 5.4 lg) (means ± standard errors, n = 4). Most important, the variation in expression levels between the biological replicates was clearly reduced in five of six tested genes, as depicted in Fig. 1F. Taking together the data from all six tested genes, the PAAP-cultured fungi had a statistically significant lower variance, as tested by F test (P = 0.026). A further area of application of the PAAP culture protocol lies in the assessment of inhibitor or fungicide effectiveness. To discern effects on germination from effects on vegetative growth, the membrane supporting the mycelium may be transferred from a preculture agar plate to a treatment plate. To test the applicability of the PAAP culture protocol for fungicide experiments, mycelium was grown on PVDF membrane as described above for 48 h. The
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Notes & Tips / Anal. Biochem. 453 (2014) 58–60
overexpression experiments. In addition, the Petri dish-based PAAP cultures require far less space than shaking liquid vessels. Compared with agar cultures, the PAAP cultures allow a harvest of the mycelium without adhering medium or a transfer of the mycelium between solid media of different composition. Acknowledgments This work was supported by a grant from the Deutsche Forschungsgemeinschaft (DFG PE1500/2-1) within Research Unit FOR 666 (project A3) and by the Land Sachsen-Anhalt. The authors thank Holger B. Deising (Martin Luther University of Halle–Wittenberg) for providing the C. graminicola strain.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ab.2014.02.023. References
Fig.1. (A) Three individual, identically treated C. graminicola cultures grown in liquid mLCM show a rather inhomogeneous mycelium formation. (B) Three individual, identically treated colonies grown according to the newly developed PAAP culture protocol and stained with the life stain FDA exhibit a more homogeneous colony habitus and are entirely vital, as indicated by the bright fluorescence. (C) A colony of PAAP-cultured C. graminicola prior to harvest is shown. (D) The dried mycelial margin, lifted from the membrane after freeze-drying, is cut by a razor blade. (E) Harvested biomass is transferred into a 2-ml microcentrifuge tube. (F) Relative standard deviations of the genes TRPF 1 to TRPF 4, HXT 1 and HXT 5 are shown (black: conventional liquid culture; gray: PAAP culture; n = 4). (G) Application of PAAP culture for fungicide treatment is shown. Colonies were cultured for 48 h on mLCM agar to a diameter of 14 ± 1 mm. Membranes carrying whole fungal colonies were transferred under sterile conditions to mLCM containing 0.1% DMSO or 0.1% DMSO and 1 lg ml1 tebuconazol. After 32 h of growth, the colony diameter increase was measured (means ± standard deviations, n = 3).
colony diameter was marked with a needle and measured on three membranes. The membranes carrying the intact fungal colonies were transferred to agar plates containing either 0.1% dimethyl sulfoxide (DMSO; Roth) or 1 lg ml1 tebuconazol (Bayer CropScience, Monheim, Germany) and 0.1% DMSO by using a sterile forceps. The mycelium was cultured for a further 32 h, and the diameter of the colonies was measured again. The reduction of C. graminicola growth after transfer to mLCM agar containing tebuconazol (Fig. 1G) illustrates the usefulness of the PAAP culture method also for testing the effect of chemicals. In conclusion, the PAAP culture procedure has strong advantages as compared with liquid and agar cultures. Compared with liquid cultures, growth is far more homogeneous, which is also reflected in the lower variation of gene expression. This parameter is particularly important in the screening of transgenic strains for altered expression, for example, in RNA interference (RNAi) or
[1] A. Tichopad, R. Kitchen, I. Riedmaier, C. Becker, A. Ståhlberg, M. Kubista, Design and optimization of reverse-transcription quantitative PCR experiments, Clin. Chem. 55 (2009) 1816–1823. [2] U. Lingner, S. Münch, H.B. Deising, N. Sauer, Hexose transporters of a hemibiotrophic plant pathogen, Plant Physiol. 156 (2011) 1565–1576. [3] A.F. Dekairelle, S. van der Vorst, B. Tombal, J.L. Gala, Preservation of RNA for functional analysis of separated alleles in yeast: comparison of snap-frozen and RNALater solid tissue storage methods, Clin. Chem. Lab. Med. 45 (2007) 1283–1287. [4] A. Droce, J.L. Sørensena, H. Giesea, T.E. Sondergaarda, Glass bead cultivation of fungi: combining the best of liquid and agar media, J. Microbiol. Methods 94 (2013) 343–346. [5] H.A.B. Wösten, G.J. van Veluw, C. de Bekker, P. Krijgsheld, Heterogeneity in the mycelium: implications for the use of fungi as cell factories, Biotechnol. Lett. 35 (2013) 1155–1164. [6] J. Barrios-González, Solid-state fermentation: physiology of solid medium, its molecular basis, and applications, Process Biochem. 47 (2012) 175–185. [7] V. Warwar, M.B. Dickman, Effects of calcium and calmodulin on spore germination and appressorium development in Colletotrichum trifolii, Appl. Environ. Microbiol. 62 (1996) 74–79. [8] K.A. Seifert, A novel method of growing fungi for DNA extraction, Fungal Genetics Newslett. 41 (1994) 79–80. [9] C. Desgranges, C. Vergoignan, A. Lereec, G. Riba, A. Durand, Use of solid state fermentation to produce Beauveria bassiana for the biological control of European corn borer, Biotechnol. Adv. 11 (1993) 577–587. [10] A. Tomasini, C. Fajardo, J. Barrios-Gonza, Gibberellic acid production using different solid-state fermentation systems, World J. Microbiol. Biotechnol. 13 (1997) 203–206. [11] Y. Zhu, J.P. Smits, W. Knol, J. Bol, A novel solid-state fermentation system using polyurethane foam as inert carrier, Biotechnol. Lett. 16 (1994) 643–648. [12] A. Yasuhara, A. Ogawa, T. Tanaka, T. Sakiyama, K. Nakanishi, Production of neutral protease from Aspergillus oryzae by a novel cultivation method on a microporous membrane, Biotechnol. Tech. 8 (1994) 249–254. [13] G.C. Bergstrom, R.L. Nicholson, The biology of corn anthracnose, Plant Dis. 83 (1999) 596–608. [14] S. Münch, U. Lingner, D.S. Floss, N. Ludwig, N. Sauer, H.B. Deising, The hemibiotrophic lifestyle of Colletotrichum species, J. Plant Physiol. 165 (2008) 41–51. [15] J. Leach, B.R. Lang, O.C. Yoder, Methods for selection of mutants and in vitro culture of Cochliobolus heterostrophus, J. Gen. Microbiol. 128 (1982) 1719– 1729. [16] R. Horbach, Die Sfp-40 -Phosphopantetheinyltransferase CgPPT1 des Maispathogens Colletotrichum graminicola (Ces.) Wilson aktiviert Pathogenitätsfaktoren, PhD thesis, Martin Luther Universität Halle– Wittenberg, Naturwissenschaftliche Fakultät I, 2010. [17] W.M. Forgey, M.H. Blanco, W.Q. Loegering, Differences in pathological capabilities and host specificity of Colletotrichum graminicola on Zea mays, Plant Dis. Rep. 62 (1978) 573–575. [18] J.-J. Krijger, R. Horbach, M. Behr, P. Schweizer, H.B. Deising, S.G.R. Wirsel, The yeast signal sequence trap identifies secreted proteins of the hemibiotrophic corn pathogen Colletotrichum graminicola, Mol. Plant Microbe Interact. 21 (2008) 1325–1336. [19] E. Peiter, B. Montanini, A. Gobert, P. Pedas, S. Husted, F.J.M. Maathuis, D. Blaudez, M. Chalot, D. Sanders, A secretory pathway-localized cation diffusion facilitator confers plant manganese tolerance, Proc. Natl. Acad. Sci. USA 104 (2007) 8532–8537.
Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
Notes & Tips
Membrane-assisted culture of fungal mycelium on agar plates for RNA extraction and pharmacological analyses Mario Lange, Carolin Müller, Edgar Peiter ⇑ Plant Nutrition Laboratory, Institute of Agricultural and Nutritional Sciences (IAEW), Faculty of Natural Sciences III, Martin Luther University of Halle–Wittenberg, 06099 Halle (Saale), Germany
a r t i c l e
i n f o
Article history: Received 13 January 2014 Received in revised form 21 February 2014 Accepted 22 February 2014 Available online 4 March 2014 Keywords: Colletotrichum graminicola Fungal growth PVDF membrane RNA extraction Gene expression
a b s t r a c t Fungal mycelium grown in liquid culture is easy to harvest for RNA extraction and gene expression analyses, but liquid cultures often develop rather heterogeneously. In contrast, growth of fungal mycelium on agar plates is highly reproducible. However, this biological material cannot be harvested easily for downstream analyses. This article describes a PVDF (polyvinylidene difluoride) membrane-assisted agar plate culture method that enables the harvest of mycelium grown on agar plates. This culture method leads to a strongly reduced variation in gene expression between biological replicates and requires less growth space as compared with liquid cultures. Ó 2014 Elsevier Inc. All rights reserved.
To maximize the conclusiveness of biological experiments, it is important to minimize the variation between replicate samples. In particular, the uniformity of the organisms under investigation at the moment before sampling is crucial. Inaccuracies at this stage cannot be compensated in the downstream analysis [1]. This also holds true for the analysis of gene expression. In expression studies, the extracted RNA samples are handled and the quantification of expression is performed with great care [2,3]. However, this does not ameliorate a physiological variability of the replicate organisms. In fungal biology, transformed strains are usually cultured in liquid medium to assess the effect of knockdown or overexpression approaches on gene expression. Unfortunately, growth (and thus physiological state) of fungi propagated in liquid medium is often rather inhomogeneous [4]. For example, in liquid medium, Aspergillus niger forms highly variable microcolonies that differ in size and gene expression patterns [5]. In contrast, mycelium growth on agar plates is highly reproducible [6,7]. Agar plate-grown fungal macrocolonies show a regular formation of radial zones [5]. In those solid-state cultures, there are gradients for nutrients within a colony that are a cause for this radial zonation. These gradients reflect the situation in the natural habitats of filamentous fungi [6]. In contrast to this, in liquid cultures these gradients are absent. Physiological parameters of fungi grown on solid medium can ⇑ Corresponding author. Fax: +49 345 5527113. E-mail address: [email protected] (E. Peiter). http://dx.doi.org/10.1016/j.ab.2014.02.023 0003-2697/Ó 2014 Elsevier Inc. All rights reserved.
differ widely from those of fungi grown in liquid medium. For example, the secretion of enzymes is often enhanced in solid-state cultures [6]. In both liquid and solid-state cultures, a micro-heterogeneity between and within single neighboring hyphae has been observed [5]. To reduce the variation between replicate samples, the intrinsic micro-heterogeneity between hyphae should be normally distributed in the sampled mycelium. Due to the more homogeneous colony morphology of solid-state-grown fungi compared with liquid-grown fungi, we hypothesized that the replicateto-replicate variations in gene expression are lower in a solid-state culture. To combine an efficient harvest of mycelium or fermentation products with the growth on a solid support, a number of approaches have been developed in the past, each optimized for a certain question. Broth solidified with the block polymer pluronic polyol F-127 has been used to grow Penicillium spinulosum for extraction of fungal DNA [8]. Aqueous solutions of pluronic polyol F-127 form a gel at room temperature and liquefy when chilled. Because the time for liquefying the medium is rather long, this method is unsuitable for RNA extraction [8]. Filamentous fungi have also been grown on inert porous solids soaked with broth in order to increase the production of fermentation products, with the mycelium being trapped in the pores of the support [9–11]. Another method providing solid support for the fungus was introduced by Yasuhara and coworkers [12], who constructed an apparatus in which an inert membrane is situated above nutrient broth and grew Aspergillus oryzae on the aerial side of the
Notes & Tips / Anal. Biochem. 453 (2014) 58–60
membrane. This procedure, called membrane surface liquid culture (MSLC)1 by the authors, induced the expression of a neutral protease by more than one order of magnitude compared with standard liquid culture and simplified the enzyme extraction compared with cultivation on agar [12]. Here, we present a simplified version of MSLC by replacing the nutrient broth with an agar medium poured into commercially available standard Petri dishes. This modification drastically reduces the technical effort of a membrane-based culture and allows high-throughput applications in which filamentous fungi can easily be harvested for RNA extraction. We named this method PVDF (polyvinylidene difluoride) membrane-assisted agar plate culture, abbreviated PAAP culture. In the experiments on which this study is based, we used the PAAP culture to grow the filamentous fungus Colletotrichum graminicola, a hemi-biotrophic pathogen of corn (Zea mays). C. graminicola can infect the plant at all developmental stages and has provoked dramatic yield losses in the past [13,14]. To prepare the culture system, a PVDF membrane (Hybond-P, pore diameter = 0.45 lm, GE Healthcare, Chalfont St. Giles, UK) was prewetted in 96% ethanol (Roth, Karlsruhe, Germany) and sterilized in 70% ethanol. This procedure was followed by four sequential additions of a half-volume of liquid modified Leach’s complete medium (mLCM) containing 1% sucrose, 0.1% tryptone, 0.1% yeast extract, 0.1% Ca(NO3)2, 0.025% MgSO4 7H2O, 0.02% KH2 PO4, and 0.0054% NaCl [15,16]. Finally, the membrane was washed twice in liquid mLCM. After that, mLCM agar (liquid mLCM containing 1.5% agar) was poured into 55-mm Petri dishes (VWR International, Darmstadt, Germany), and just after solidification of the agar surface the mLCM-equilibrated PVDF membrane was placed on top. The membrane-covered agar was dried for 30 min and inoculated with a 2-ll suspension of falcate conidia containing 300 C. graminicola M2 (M1.001) wild-type spores [17]. After 3 or 4 days at 23 °C, the growing mycelium covered most of the membrane. To visualize mycelium grown on PVDF membrane, the fungus was live-stained with fluorescein diacetate (FDA) for 1 min in a solution of 0.04% FDA (Sigma–Aldrich, St. Louis, USA) and 2% acetone (Roth) in water. Pictures were captured using a stereomicroscope (SteREO Discovery V20, Carl Zeiss, Jena, Germany) equipped with a fluorescence filter set 38 (excitation = 450–490 nm, emission = 500–550 nm) and an Axiocam HRc camera (Carl Zeiss). To compare the visual homogeneity of the fungal material produced according to the PAAP culture protocol against material produced by the standard culture procedure in liquid medium, we observed the physical appearance in replicate samples. In the standard procedure, C. graminicola was grown shaking at 100 rpm in 300-ml Erlenmeyer flasks containing 100 ml of liquid mLCM for 1 week at 23 °C. As Fig. 1A shows, the culture of the filamentous fungus C. graminicola grew rather inhomogeneously in liquid medium. In contrast, a far greater homogeneity was achieved in the PAAP cultures (Fig. 1B). Growth rates of PAAP cultures were identical to those on agar plates. We next wanted to determine whether the more homogeneous visual appearance is reflected in a better reproducibility of gene expression. For this purpose, we again prepared PAAP-cultured fungal colonies but left them unstained (Fig. 1C). The harvest of fungal material from a PAAP culture was prepared by floating the Petri dish lid on liquid nitrogen. Then, the membrane carrying a whole intact fungal colony was removed from the agar and placed into the prechilled lid. The membrane and fungal biomass was lyophilized for 2 h at 1.25 mbar by using an Alpha 1-4 freeze dryer (Martin Christ, Osterrode, Germany). The margin of the colony lifted from the membrane during the drying process and both 1 Abbreviations used: MSLC, membrane surface liquid culture; PVDF, polyvinylidene difluoride; PAAP, PVDF membrane-assisted agar plate; mLCM, modified Leach’s complete medium; FDA, fluorescein diacetate; qRT–PCR, quantitative reverse transcription polymerase chain reaction; DMSO, dimethyl sulfoxide.
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could be easily separated by using a razor blade (Fig. 1D). Next, the mycelium was transferred into a 2-ml microtube containing two 3mm steel beads (Hecht, Winnenden, Germany) (Fig. 1E). The biomass of two membrane cultures was pooled to one sample. In preliminary experiments, different types of membrane were tested for this culture technique. In contrast to PVDF, nylon mesh, Miracloth, and nitrocellulose proved to be unsuitable. Nylon mesh did not prevent a close adherence of the fungus to the agar, and the fungal material remained on the plate when removing the mesh. On Miracloth, growth was strongly reduced, and agar adhered to this type of material after removal from the plate. Nitrocellulose allowed a proper removal of the mycelium-containing membrane from the supporting agar but was very brittle after freeze-drying, hence impeding the removal of the fungal material. Mycelium obtained by the standard liquid culture procedure was filtered, briefly blotted on filter paper, frozen in liquid nitrogen, and lyophilized as above. Dry biomass (10 mg) was transferred into a 2-ml microtube containing two 3-mm steel beads. The biomass obtained from the two culture methods was ground for 1 min at 30 Hz using a Tissue Lyser II (Qiagen, Hilden, Germany). RNA extraction was performed using a peqGold Plant RNA kit (Peqlab, Erlangen, Germany). The ground biomass was suspended in 450 ll of Lysis Buffer T and homogenized for 30 s at 30 Hz in the Tissue Lyser. All further steps of RNA extraction were performed according to the manufacturer’s instructions. Genomic DNA was removed using DNase I (New England Biolabs, Ipswich, MA, USA) according to the manufacturer’s instructions. RNA was LiCl precipitated (4 M final concentration), followed by two washes with 70% ethanol. Synthesis of complementary DNA (cDNA) was done with Moloney murine leukemia virus (M-MLV) reverse transcriptase (Promega, Fitchburg, WI, USA) using gene-specific reverse oligonucleotides (Eurofins MWG Operon, Ebersberg, Germany) (Table 1 in supplementary data) according to the instructions provided by Promega. Sequences for HXT and HistonH3 oligonucleotides were taken from the literature [2,18]. Quantitative reverse transcription polymerase chain reaction (qRT–PCR) was carried out on a Mastercycler ep realplex4 S (Eppendorf, Hamburg, Germany) in MicroAMP optical eight-tube strips covered with MicroAMP optical eight-cap strips using the POWER SYBR Green PCR mastermix (all from Applied Biosystems, Foster City, CA, USA). Each reaction had a volume of 12.5 ll. Reaction conditions were set as recommended by Applied Biosystems. A calibration curvebased calculation of relative expression was applied [19]. Expression levels of TRPF and HXT genes were normalized to Histone H3. Next, the mean of these normalized values of each set of four biological replicates for each gene and culture system was set to 100%. The values of the four replicates were correlated to these 100% values, resulting in relative expression values. These relative values were used to calculate standard deviations and tested for significant differences between the liquid culture and the PAAP culture using F-test statistics (Microsoft Excel 2007). The PAAP culture provided sufficient biomass (2.4 ± 0.3 mg) from two colonies of approximately 3 cm diameter to extract RNA for many qRT–PCR experiments (22.4 ± 5.4 lg) (means ± standard errors, n = 4). Most important, the variation in expression levels between the biological replicates was clearly reduced in five of six tested genes, as depicted in Fig. 1F. Taking together the data from all six tested genes, the PAAP-cultured fungi had a statistically significant lower variance, as tested by F test (P = 0.026). A further area of application of the PAAP culture protocol lies in the assessment of inhibitor or fungicide effectiveness. To discern effects on germination from effects on vegetative growth, the membrane supporting the mycelium may be transferred from a preculture agar plate to a treatment plate. To test the applicability of the PAAP culture protocol for fungicide experiments, mycelium was grown on PVDF membrane as described above for 48 h. The
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Notes & Tips / Anal. Biochem. 453 (2014) 58–60
overexpression experiments. In addition, the Petri dish-based PAAP cultures require far less space than shaking liquid vessels. Compared with agar cultures, the PAAP cultures allow a harvest of the mycelium without adhering medium or a transfer of the mycelium between solid media of different composition. Acknowledgments This work was supported by a grant from the Deutsche Forschungsgemeinschaft (DFG PE1500/2-1) within Research Unit FOR 666 (project A3) and by the Land Sachsen-Anhalt. The authors thank Holger B. Deising (Martin Luther University of Halle–Wittenberg) for providing the C. graminicola strain.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ab.2014.02.023. References
Fig.1. (A) Three individual, identically treated C. graminicola cultures grown in liquid mLCM show a rather inhomogeneous mycelium formation. (B) Three individual, identically treated colonies grown according to the newly developed PAAP culture protocol and stained with the life stain FDA exhibit a more homogeneous colony habitus and are entirely vital, as indicated by the bright fluorescence. (C) A colony of PAAP-cultured C. graminicola prior to harvest is shown. (D) The dried mycelial margin, lifted from the membrane after freeze-drying, is cut by a razor blade. (E) Harvested biomass is transferred into a 2-ml microcentrifuge tube. (F) Relative standard deviations of the genes TRPF 1 to TRPF 4, HXT 1 and HXT 5 are shown (black: conventional liquid culture; gray: PAAP culture; n = 4). (G) Application of PAAP culture for fungicide treatment is shown. Colonies were cultured for 48 h on mLCM agar to a diameter of 14 ± 1 mm. Membranes carrying whole fungal colonies were transferred under sterile conditions to mLCM containing 0.1% DMSO or 0.1% DMSO and 1 lg ml1 tebuconazol. After 32 h of growth, the colony diameter increase was measured (means ± standard deviations, n = 3).
colony diameter was marked with a needle and measured on three membranes. The membranes carrying the intact fungal colonies were transferred to agar plates containing either 0.1% dimethyl sulfoxide (DMSO; Roth) or 1 lg ml1 tebuconazol (Bayer CropScience, Monheim, Germany) and 0.1% DMSO by using a sterile forceps. The mycelium was cultured for a further 32 h, and the diameter of the colonies was measured again. The reduction of C. graminicola growth after transfer to mLCM agar containing tebuconazol (Fig. 1G) illustrates the usefulness of the PAAP culture method also for testing the effect of chemicals. In conclusion, the PAAP culture procedure has strong advantages as compared with liquid and agar cultures. Compared with liquid cultures, growth is far more homogeneous, which is also reflected in the lower variation of gene expression. This parameter is particularly important in the screening of transgenic strains for altered expression, for example, in RNA interference (RNAi) or
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