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Development of a transformation system in the swainsonine producing, slow growing endophytic fungus, Undifilum oxytropis
Journal of Microbiological Methods 81 (2010) 160–165
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Journal of Microbiological Methods j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j m i c m e t h
Development of a transformation system in the swainsonine producing, slow growing endophytic fungus, Undifilum oxytropis Suman Mukherjee a,⁎, Angus L. Dawe a,c, Rebecca Creamer a,b a b c
Molecular Biology Program, New Mexico State University, Las Cruces, New Mexico, 88003, USA Department of Entomology, Plant Pathology, and Weed Science, New Mexico State University, Las Cruces, New Mexico, 88003, USA Biology Department, New Mexico State University, Las Cruces, New Mexico, 88003, USA
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Article history: Received 25 November 2009 Received in revised form 28 February 2010 Accepted 28 February 2010 Available online 6 March 2010 Keywords: Endophytes Undifilum oxytropis Swainsonine Green Florescent Protein (GFP) Transformation system
a b s t r a c t Undifilum oxytropis (Phylum: Ascomycota; Family: Pleosporaceae) is a slow growing endophytic fungus that produces a toxic alkaloid, swainsonine. This endophyte resides in locoweeds, which are perennial flowering legumes. Consumption of this fungus by grazing animals induces a neurological disorder called locoism. The alkaloid swainsonine, an α-mannosidase inhibitor, is responsible for the field toxicity related to locoism. Little is known about the biosynthetic pathway of swainsonine in endophytic fungi. Genetic manipulation of endophytic fungi is important to better understand biochemical pathways involved in alkaloid synthesis, but no transformation system has been available for studying such enzymes in Undifilum. In this study we report the development of protoplast and transformation system for U. oxytropis. Fungal mycelia required for generating protoplasts were grown in liquid culture, then harvested and processed with various enzymes. Protoplasts were transformed with a fungal specific vector driving the expression of Enhanced Green Florescent Protein (EGFP). The quality of transformed protoplasts and transformation efficiency were monitored during the process. In all cases, resistance to antibiotic hygromycin B was maintained. Such manipulation will open avenues for future research to decipher fungal metabolic pathways. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Locoweeds commonly refer to a group of perennial flowering legumes that are found in the dry and arid regions of United States. Common genera of locoweed found in the western USA are Oxytropis sericia, Oxytropis lambertii and Astragalus mollissimus (Allred, 1991). Aerial parts of locoweeds can be poisonous when consumed by grazing animals (James and Panter, 1989). Consumption of locoweed by cattle causes a neurological disorder called locoism, which is characterized by loss of muscular coordination and staggered gait (Broquist, 1985). In contrast, other locoweed species, such as Astragalus crassiparus or some populations of O. lambertii are not toxic to grazing animals (Ralphs et al., 2002). Toxic locoweeds are found to be associated with a slow growing endophytic fungus, Undifilum oxytropis (Pryor et al., 2009). U. oxytropis is an Embellisia-like fungus that does not sporulate or produce external mycelia on the host plant. The fungus is found to be in a symbiotic relationship with the locoweeds and found in associated with seed coat, stem and leaves (Ralphs et al., 2002). When cultured in vitro, the fungus grows 0.03–0.34 mm/day with thin
⁎ Corresponding author. Tel.: + 1 575 646 6160; fax: + 1 575 646 8087. E-mail address: [email protected] (S. Mukherjee). 0167-7012/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2010.02.015
septate hyphae (Braun et al., 2003) on potato dextrose agar (PDA, Difco, Detroit, MI) plates at room temperature. Incubation of cultures for more than 30 days is often required to attain a colony diameter of at least 5 mm. After colonies reached 5–6 mm in diameter, continued radial growth is extremely slow or arrested. These endophytic fungi also produce a polyhydroxy alkaloid, swainsonine, (1, 2, 8-trihydroxyindolizidine), naturally and in culture (Braun et al., 2003; Harris et al., 1988). Levels of swainsonine in locoweeds correlate well with presence or absence of endophytic fungi in plants (Braun et al., 2003). Production of swainsonine is dependent on various factors such as environmental conditions and the specific fungal isolate (Braun, 1999; Oldrup, 2005). Swainsonine is a very stable compound that can induce toxic reactions even when the locoweed plants have been dried for many years. Swainsonine inhibits lysosomal α-mannosidase (involved in the biosynthesis and catabolism of glycoproteins) and golgi α-mannosidase II (Elbein, 1991; Stegelmeier et al., 1999). The metabolic pathway of swainsonine has been partially determined in Rhizoctonia leguminicola and Metarhizium anisopliae (Sim and Perry, 1997; Wickwire et al., 1990). Biochemical details of the swainsonine pathway in alkaloid producing fungi such as U. oxytropis have not been deciphered, principally due to the absence of methods for genetic manipulation. The primary step to such manipulation would be to establish methods for generation of protoplasts and develop a transformation
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system. In related Ascomycetes like Alternaria alternata and Penicillium chrysogenum, preparation and regeneration of protoplasts and transformation system have been established. (Akamatsu et al., 1997; Fierro et al., 1993). There are reports of protoplast isolation from slow growing grass endophytes (Panaccione et al., 2001, Wang et al., 2004). However, there is no established protocol for generating protoplasts from swainsonine producing slow growing endophytic fungi like U. oxytropis. The aim of this study was to establish a protocol for generation of protoplasts and develop a transformation system for U. oxytropis. In the current study, protoplasts of U. oxytropis were prepared and transformed with a fungus specific vector, pPd-EGFP driving the expression of Enhanced Green Florescent Protein (EGFP) (Suzuki et al., 2000). The quality of transformed protoplasts and transformation efficiency were monitored during the process by visualizing GFP in the transformed protoplasts. Screening of the regenerated fungal mycelia for stable expression of GFP was performed.
on an orbital shaker for 3 h. Digestion buffer consisted of 1 ml of β-glucuronidase (Sigma Aldrich, St. Louis, MO), 75 mg lysing enzyme (Sigma Aldrich, St. Louis, MO), 800 mg β-D-Glucanase G (Sigma Aldrich, St. Louis, MO), 600 mg Bovine Serum Albumin (Sigma Aldrich, St. Louis, MO) dissolved in 100 ml osmotic medium (1.2 M MgSO4 with NaH2PO4, pH 5.8). After incubation, 8 ml of the digested mycelial mass was collected in 30 ml corex tubes and combined with 10 ml of Trapping Buffer (0.4 M Sorbitol in 100 mM Tris–HCl, pH 7.0). Thereafter, the solution was centrifuged at 6000 rpm at 4 °C for 15 min. Protoplasts were collected at the interface of the two layers and 2 volumes of 1 M sorbitol were added. The trapped protoplasts were centrifuged at 6000 rpm at 4 °C for 5 min. Pellets were collected after decanting the supernatant and collected protoplasts were suspended in STC buffer (1 M sorbitol in 100 mM Tris–HCl, pH 8.0, 100 mM CaCl2). Protoplasts suspended in STC buffer were kept on ice for transformation experiments. Protoplasts generated were counted using a hemocytometer.
2. Materials and methods
2.4. Transformation and regeneration of protoplasts
2.1. Fungal strains and culture conditions
Freshly prepared protoplasts in a volume of 100 μl were transferred to pre-cooled 50 ml tubes on ice. Starting protoplasts concentration was 109/ml. Five μg of pPd-EGFP vector diluted in 10 μl of Tris–EDTA (TE) buffer was added to the protoplast solution and incubated on ice for 30 min. For the control reaction, 10 μl of the pPdEGFP vector was replaced by TE buffer. Thereafter, 1 ml of PTC buffer (PTC buffer: 40% Poly Ethylene Glycol, 4000 Molecular Weight, 100 mM Tris–HCl, pH 8.0 and 100 mM CaCl2) was added to each tube, and incubated for an additional 25 min at room temperature. The transformation mixture was evenly distributed as 2, 20, and 200 μl droplets onto petri dishes. 12.5 ml of regeneration medium composed of 1 M of sucrose, 0.001 w/v of yeast extract, 0.001 w/v of casein hydrolysate and 0.016% of Bacto Agar was added to each plate. Further 12.5 ml of regeneration media containing 60 μg/ml HygB was overlaid on each plate. The concentration of HygB was more than double the initial lethal concentration (20 μg/ml) for U. oxytropis. High concentration of HygB was added to each plate in order to avoid dilution of the antibiotic. Further experiments were supplemented with 20 μg/ml HygB. Concentration of protoplasts was maintained constant throughout the process. Plates were incubated for four days at room temperature to observe hyphal growth. Fungal cultures produced after regeneration were transferred to fresh PDA–HygB containing plates. Transfer of fungal cultures was performed after three weeks of growth, which correlates to one fungal passage. Control transformation was also transferred to PDA plates and overlaid with media without HygB to compare phenotype of putative transformants. U. oxytropis protoplasts were also regenerated on hygB- negative PDA plates as an additional control. Transformation experiments were repeated four times.
U. oxytropis isolate 25-1 was isolated from leaves of Oxytropis sericea (white locoweed), which were collected from sites around Green River, WY, USA. Samples were pressed to dry the intact plant for subsequent isolation and culturing of the endophyte. The greenest tissues were selected and surface sterilized for 30 s in 70% ethanol, followed by 3 min in 20% bleach, and then 30 s in sterile water. Tissues were dried on sterile paper towels and plated on water agar media. Plates were stored at room temperature (25 °C). Fungal hyphae were transferred to potato dextrose agar (PDA) plates and grown at room temperature (Ralphs et al., 2008) for at least 14 days. Hyphal mass of the recovered endophytes were transferred onto PDA plates and maintained at 18 °C. The 25-1 isolate described above has been preserved as desiccated mycelia and stored at both 4 °C and − 80 °C at the New Mexico State University Center for Natural History Collections (NMSU-CNHC). 2.2. HygromycinB selection In this study, transformation of U. oxytropis was performed using pPd-EGFP vector. pPd-EGFP, is a fungal specific expression vector that confers resistance to antibiotic hygromycin B (Hyg B) because the vector expresses the HygB phosphotransferase gene driven by the Aspergillus nidulans trpC-promoter (Suzuki et al., 2000). Once transformed, the vector expresses EGFP driven by the Cryphonectria parasitica glyceraldehydes-3-phosphate dehydrogenase (gpd) promoter. To evaluate the sensitivity of U. oxytropis to HygB, the fungi was grown in presence of varying concentrations of HygB (MP Biomedicals, OH), 0 μg/ml to 40 μg/ml. The cultures were grown for 14 days at room temperature. Measurement of hyphal growth was performed by counting the length of radial growth in mm beyond a point of reference point (start of culture on day zero). Hygromycin selection was performed on three independent fungal cultures grown on PDA plates for each concentration. 2.3. Preparation of protoplasts Fungal protoplasts were prepared based on methods described by Churchill et al. (1990) with modifications as described below. Liquid suspension cultures of U. oxytropis were started in 100 ml of Potato Dextrose Broth (PDB) and allowed to shake for two weeks on a platform shaker at 200 rpm at room temperature. Hyphal suspensions were harvested by filtration using miracloth (EMD Biosciences, San Diego, CA) in a Buchner funnel. Mycelial mass was resuspended in 100 ml of digestion buffer and incubated at room temperature
2.5. Sporulation and hyphal tipping To generate mononucleate cultures, mycelia were transferred to water agar plates for sporulation. Single spores and single hyphal tips were collected from water agar plates to obtain a pure mononucleate culture. Single spore and single hypha were grown separately in different PDA plates again for three weeks to visualize EGFP expression microscopically. 2.6. Microscopy Zeiss (Thornwood, NY) inverted florescence microscope, Axiovert 200 M was used to capture florescence images at a combined objective and eyepiece magnification of 40× and 10×, respectively. Nuclear DNA was stained using 4′, 6-diamidino-2-phenylindole
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(DAPI, Molecular Probes, Carlsbad, CA) in the generated protoplasts. Axiovision software was used to merge the images. 2.7. Immunoblot Fungal tissues from transformed and untransformed U. oxytropis were ground using liquid nitrogen, homogenized in extraction buffer (50 mM Tris–HCl, pH 7.5, 100 mM NaCl, 50 mM NaF, 2 mM phenylmethylsulphonyl floride, 5 mM EDTA, 1% Triton-X 100, 10% glycerol) and centrifuged at 4 °C for 10 min at 13,000 rpm (Bruno et al., 2004). Protein concentration of lysates was quantified using Bio-Rad protein assay kit (Bio-Rad, Herculus, CA). Total protein (20 μg) was separated on a 12% SDS-polyacrylamide gel and transferred to Immobilon polyvinylidene fluoride (PVDF) transfer membranes (Millipore, Billerica, MA). Membranes were blocked with Bovine Serum Albumin and incubated with 1:1000 EGFP antibody (Abcam, Cambridge, MA). Antirabbit Horse Radish Peroxidase conjugated IgG was used as the secondary antibody at a 1:2000 dilution (Kirkegaard and Perry Laboratories, Gaithersburg, MD). Blots were visualized using the ECL system (GE Healthcare, Piscataway, NJ). Loading control for the gel was immunoblotted in a similar protocol but was incubated with 1:5000 α-tubulin antibody (Santa Cruz Biotechnologies, Santa Cruz, CA). 2.8. Swainsonine assay Fungal suspension cultures were grown in Potato Dextrose Broth (PDB, Difco Laboratories Franklin lakes, NJ), media for two weeks. The fungal mass was filtered and dried at 60 °C overnight. The dried tissue was homogenized in liquid nitrogen and 100 mg of dried tissue was treated with 5 ml of 2% acetic acid and 4 ml chloroform overnight using culture tube shaker (40 rpm). The extract was then centrifuged at 2000 rpm for 15 min and passed through a cation exchange column that was prepared from Dowex 50WX8-100 mesh (Sigma Aldrich, St. Louis, MO) resin. Swainsonine was eluted from the column by displacement with ammonium hydroxide. Ammonium hydroxide was evaporated from the extracted samples using a flow of nitrogen at 60 °C. Dehydrated samples were then hydrated in 100 μl of water and stored for LC-MS measurement (Gardner et al., 2001). LC-MS system consisted of an HP1100 binary solvent pump, autosampler, a Betasil C18 reversed phase HPLC column (Acquity LC system, Waters Corporation, MA) and a Micromass, Q-Tof Micro mass spectrometer (Waters Corporation, MA). The assay was replicated four times with the transformed samples being taken from a single PDA plate.
Table 1 Average growth in mm of Undifilum oxytropis in presence of Hygromycin B antibiotic for 14 days (SE = standard error). Hygromycin conc. (μg/ml)
Average growth ± 1 SE (over 14 days)a
0 5 8 10 20 40
6.16 ± 0.2 mm 5.72 ± 0.1 mm 5.01 ± 0.3 mm 4.70 ± 0.4 mm 0.33 ± 0.12 mm (no growth) 0.33 ± 0.1 mm (no growth)
a N = Average growth was calculated from readings of three independent fungal culture grown on PDA plates for each concentration of hygromycin.
After protoplast purification, five μg of pPD-EGFP vector was used to transform 109 per ml protoplasts in a pre-cooled microcentrifuge tube. One-hour post transformation the protoplasts were observed using 488 nm filter on a Axiovert 200 M microscope. Microscopic examination of protoplasts demonstrated expression of EGFP in selected protoplasts (Fig. 2). The untransformed protoplasts were also imaged to check for auto-florescence. Protoplasts after transformation was diluted in 1 ml of PTC buffer, plated in PDA plates and overlaid with regeneration media. Serial dilution of transformant solution was plated to ensure separation of individual transformants. Growth in the form of mycelia was observed for seven days after plating the transformants. Previous studies on other Ascomycetes fungi (Gorfer et al., 2007) required 24 to 48 h for protoplasts to regenerate as mycelia but due to the slow-growing characteristic of U. oxytropis fungal transformants were visible four
3. Results In order to use the pPd-EGFP vector, which confers resistance to HygB, the sensitivity of U. oxytropis to HygB was tested using increasing concentrations of HygB antibiotic compared to a control plate without HygB. U. oxytropis showed high sensitivity to HygB antibiotic. Concentration of 10 μg/ml HygB showed little impediment of hyphal growth on PDA plates and concentrations of b10 μg/ml were not inhibitory. Particularly, 20 μg/ml and higher HygB concentrations showed complete inhibition of hyphal growth (Table 1). Preparation of quality protoplasts from slow growing, swainsonine producing U. oxytropis was the rate-limiting step for establishment of the transformation system. In the current study the combinations of enzymes used for generation of maximum and efficient protoplasts along with the incubation was varied from 2–3 h. The 3 h incubation time was observed to be optimum for almost complete digestion of fungal mass and production of sufficient protoplasts for U. oxytropis. Protoplast yields were approximately 109 per ml using the above-mentioned protocol. Staining of protoplasts with DAPI was used to confirm nucleated protoplast production (Fig. 1).
Fig. 1. Protoplasts generated from Undifilum oxytropis mycelial culture. A. Light microscope images of protoplasts; B. Florescent images of protoplast after nucleic acid staining with DAPI stain.
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that allowed comparison of the phenotype of putative transformants (control 2). Growth pattern of regenerated fungal mycelia on HygBcontaining PDA plates was observed but no morphological differences were found between untransformed and putatively transformed colonies. Microscopic examination of selected transformants was performed to evaluate the quality of transformants, after three weeks of growth under HygB selection. Mycelium demonstrated strong expression of the EGFP gene, as observed under at 488 nm using an Axiovert 200 M microscope (Fig 3A). Transformed fungi were transferred to fresh HygB plates, which are thereafter referred to as second passage of transformants. The fungal cultures were allowed to grow again for three weeks before transferring to fresh HygB plates. Microscopic observation showed EGFP expression in the transformed mycelium at week six. EGFP expression was also detected in the mycelial mass at 18 weeks post transformation (Fig. 3B and C). Control untransformed mycelia was also observed (Fig. 3D). Single spore and single hypha from eighteen-week old transformed cultures were transferred to HygB containing water agar plates to ensure complete separation of clonal hyphae and to verify maximum GFP expression in the transformed cultures. Sporulation of transformed fungi was observed after three weeks of transfer. Single hyphal tips and single spores from the clonally separated fungal cultures were transferred back to fresh PDA plates and allowed to grow for three weeks. Emerged mycelia on PDA plates containing HygB were screened for expression of EGFP. Expression was close to 100% in mycelia as observed (data not shown). U. oxytropis isolates produce colonies composed of two hyphal types: a basal layer of densely packed, short, torulose, and interwined hyphal segments, and a surface layer of more typical elongated filamentous aerial hyphae (Pryor et al., 2009). Both types of fungal hyphae retained EGFP expression from hyphal tips grown on PDA plates containing HygB (data not shown). Three, six and eighteen-week old transformed and untransformed mycelia were collected and analyzed using immunoblot assay. Fig. 4 indicates that EGFP expression is present in all cultures which were transformed. No expression of GFP was observed in the untransformed U. oxytropis (Fig. 4). Screening of transformants indicated stable expression of EGFP throughout several passages post transformation. Naïve U. oxytropis produces the toxic alkaloid swainsonine. In order to evaluate whether transformed U. oxytropis retained regular swainsonine production characteristic, the swainsonine concentration from fungal mass was measured both before and after EGFP expression. Swainsonine levels detected in unmanipulated U. oxytropis (2.02% total swainsonine in 0.1 g of starting fungal mass) before transformation correlated with the levels of swainsonine found in transformed cultures (1.8% total swainsonine in 0.1 g of starting fungal mass). Fig. 2. A. Florescence microscopic image of DAPI stained protoplasts generated from Undifilum oxytropis mycelial culture; B. Expression of GFP in protoplasts in panel A; C. Overlap of panels A and B.
days post transformation. Although serial dilutions were performed on PDA plates, individual colonies were not observed after seven days because the transformants merged with each other. On an average three individual fungal colonies were observed after regeneration on PDA plates. However, those may have been the result of merged transformants before penetration of the overlay and therefore an exact transformation efficiency cannot be calculated. Fourteen days after transformation, complete mycelia formation observed on HygBcontaining PDA plates. Fungal cultures were transferred between passages. No hyphae were observed in untransformed protoplasts plated on HygB containing PDA plates (control 1). Untransformed protoplasts were regenerated on PDA plates without HygB selection
4. Discussion Previously, various transformation systems have been developed for ascomycete fungi including endophytes (Abello et al., 2008), but this study describes the first successful transformation and GFP expression in an isolate of very slow growing, swainsonine-producing endophytic fungus, U. oxytropis. The expression of both hygromycin-B phosphotransferase driven by a promoter from A. nidulans (class Eurotiomycetes) and EGFP controlled by an element from C. parasitica (class Sordariomycetes) also indicates that U. oxytropis (class Dothidiomycetes) is tolerant of non-native promoter systems. This system may allow for the use of controlled expression options, such as the recently identified copper-responsive element from C. parasitica (Willyerd et al., 2009). Assay of swainsonine levels show that transformed U. oxytropis fungal mycelia retains the ability to produce of toxic alkaloid, swainsonine after stable expression of a foreign heterologous gene. This system can therefore be used to perform
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Fig. 3. Mycelial growth of Undifilum oxytropis. A: Florescence microscope images of mycelia three weeks post transformation on hygromycin selection plates; B. Florescent images of mycelia expressing EGFP, six weeks post transformation on hygromycin selection plates; C. Florescent images of mycelia expressing EGFP, eighteen weeks post transformation on hygromycin selection plates; D. Untransformed mycelia of Undifilum oxytropis on hygromycin negative plates.
genetic manipulation on U. oxytropis. These procedures for generating stable transformants will be used to study metabolically important genes in the lysine biosynthetic pathway or swainsonine metabolic pathway in U. oxytropis. Such manipulation will open avenues for
future research to decipher the role of enzymes in fungal metabolic pathways including the swainsonine degradation pathway. Acknowledgements The authors would like to thank Deana Baucom, Jorge Achata, and Dr. Soum Sanogo (EPPWS) for technical assistance and constructive discussion. Funding for this project was provided by USDA Special grant 59-5428-1-327 and New Mexico State University Agricultural Experiment Station. References
Fig. 4. Immunoblot of hyphal samples after three weeks, six weeks, eighteen weeks of Undifilum oxytropis growth using anti-EGFP antibody. Lane 1: Three weeks post transformation U. oxytropis hyphae; Lane 2: Untransformed U. oxytropis hyphae; Lane 3: Six weeks post transformation U. oxytropis hyphae; Lane 4: Eighteen weeks post transformation U. oxytropis hyphae. Bottom panel: Total protein loading control of the samples from Lane 1–4 blotted with anti-tubulin antibody.
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Journal of Microbiological Methods j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j m i c m e t h
Development of a transformation system in the swainsonine producing, slow growing endophytic fungus, Undifilum oxytropis Suman Mukherjee a,⁎, Angus L. Dawe a,c, Rebecca Creamer a,b a b c
Molecular Biology Program, New Mexico State University, Las Cruces, New Mexico, 88003, USA Department of Entomology, Plant Pathology, and Weed Science, New Mexico State University, Las Cruces, New Mexico, 88003, USA Biology Department, New Mexico State University, Las Cruces, New Mexico, 88003, USA
a r t i c l e
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Article history: Received 25 November 2009 Received in revised form 28 February 2010 Accepted 28 February 2010 Available online 6 March 2010 Keywords: Endophytes Undifilum oxytropis Swainsonine Green Florescent Protein (GFP) Transformation system
a b s t r a c t Undifilum oxytropis (Phylum: Ascomycota; Family: Pleosporaceae) is a slow growing endophytic fungus that produces a toxic alkaloid, swainsonine. This endophyte resides in locoweeds, which are perennial flowering legumes. Consumption of this fungus by grazing animals induces a neurological disorder called locoism. The alkaloid swainsonine, an α-mannosidase inhibitor, is responsible for the field toxicity related to locoism. Little is known about the biosynthetic pathway of swainsonine in endophytic fungi. Genetic manipulation of endophytic fungi is important to better understand biochemical pathways involved in alkaloid synthesis, but no transformation system has been available for studying such enzymes in Undifilum. In this study we report the development of protoplast and transformation system for U. oxytropis. Fungal mycelia required for generating protoplasts were grown in liquid culture, then harvested and processed with various enzymes. Protoplasts were transformed with a fungal specific vector driving the expression of Enhanced Green Florescent Protein (EGFP). The quality of transformed protoplasts and transformation efficiency were monitored during the process. In all cases, resistance to antibiotic hygromycin B was maintained. Such manipulation will open avenues for future research to decipher fungal metabolic pathways. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Locoweeds commonly refer to a group of perennial flowering legumes that are found in the dry and arid regions of United States. Common genera of locoweed found in the western USA are Oxytropis sericia, Oxytropis lambertii and Astragalus mollissimus (Allred, 1991). Aerial parts of locoweeds can be poisonous when consumed by grazing animals (James and Panter, 1989). Consumption of locoweed by cattle causes a neurological disorder called locoism, which is characterized by loss of muscular coordination and staggered gait (Broquist, 1985). In contrast, other locoweed species, such as Astragalus crassiparus or some populations of O. lambertii are not toxic to grazing animals (Ralphs et al., 2002). Toxic locoweeds are found to be associated with a slow growing endophytic fungus, Undifilum oxytropis (Pryor et al., 2009). U. oxytropis is an Embellisia-like fungus that does not sporulate or produce external mycelia on the host plant. The fungus is found to be in a symbiotic relationship with the locoweeds and found in associated with seed coat, stem and leaves (Ralphs et al., 2002). When cultured in vitro, the fungus grows 0.03–0.34 mm/day with thin
⁎ Corresponding author. Tel.: + 1 575 646 6160; fax: + 1 575 646 8087. E-mail address: [email protected] (S. Mukherjee). 0167-7012/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2010.02.015
septate hyphae (Braun et al., 2003) on potato dextrose agar (PDA, Difco, Detroit, MI) plates at room temperature. Incubation of cultures for more than 30 days is often required to attain a colony diameter of at least 5 mm. After colonies reached 5–6 mm in diameter, continued radial growth is extremely slow or arrested. These endophytic fungi also produce a polyhydroxy alkaloid, swainsonine, (1, 2, 8-trihydroxyindolizidine), naturally and in culture (Braun et al., 2003; Harris et al., 1988). Levels of swainsonine in locoweeds correlate well with presence or absence of endophytic fungi in plants (Braun et al., 2003). Production of swainsonine is dependent on various factors such as environmental conditions and the specific fungal isolate (Braun, 1999; Oldrup, 2005). Swainsonine is a very stable compound that can induce toxic reactions even when the locoweed plants have been dried for many years. Swainsonine inhibits lysosomal α-mannosidase (involved in the biosynthesis and catabolism of glycoproteins) and golgi α-mannosidase II (Elbein, 1991; Stegelmeier et al., 1999). The metabolic pathway of swainsonine has been partially determined in Rhizoctonia leguminicola and Metarhizium anisopliae (Sim and Perry, 1997; Wickwire et al., 1990). Biochemical details of the swainsonine pathway in alkaloid producing fungi such as U. oxytropis have not been deciphered, principally due to the absence of methods for genetic manipulation. The primary step to such manipulation would be to establish methods for generation of protoplasts and develop a transformation
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system. In related Ascomycetes like Alternaria alternata and Penicillium chrysogenum, preparation and regeneration of protoplasts and transformation system have been established. (Akamatsu et al., 1997; Fierro et al., 1993). There are reports of protoplast isolation from slow growing grass endophytes (Panaccione et al., 2001, Wang et al., 2004). However, there is no established protocol for generating protoplasts from swainsonine producing slow growing endophytic fungi like U. oxytropis. The aim of this study was to establish a protocol for generation of protoplasts and develop a transformation system for U. oxytropis. In the current study, protoplasts of U. oxytropis were prepared and transformed with a fungus specific vector, pPd-EGFP driving the expression of Enhanced Green Florescent Protein (EGFP) (Suzuki et al., 2000). The quality of transformed protoplasts and transformation efficiency were monitored during the process by visualizing GFP in the transformed protoplasts. Screening of the regenerated fungal mycelia for stable expression of GFP was performed.
on an orbital shaker for 3 h. Digestion buffer consisted of 1 ml of β-glucuronidase (Sigma Aldrich, St. Louis, MO), 75 mg lysing enzyme (Sigma Aldrich, St. Louis, MO), 800 mg β-D-Glucanase G (Sigma Aldrich, St. Louis, MO), 600 mg Bovine Serum Albumin (Sigma Aldrich, St. Louis, MO) dissolved in 100 ml osmotic medium (1.2 M MgSO4 with NaH2PO4, pH 5.8). After incubation, 8 ml of the digested mycelial mass was collected in 30 ml corex tubes and combined with 10 ml of Trapping Buffer (0.4 M Sorbitol in 100 mM Tris–HCl, pH 7.0). Thereafter, the solution was centrifuged at 6000 rpm at 4 °C for 15 min. Protoplasts were collected at the interface of the two layers and 2 volumes of 1 M sorbitol were added. The trapped protoplasts were centrifuged at 6000 rpm at 4 °C for 5 min. Pellets were collected after decanting the supernatant and collected protoplasts were suspended in STC buffer (1 M sorbitol in 100 mM Tris–HCl, pH 8.0, 100 mM CaCl2). Protoplasts suspended in STC buffer were kept on ice for transformation experiments. Protoplasts generated were counted using a hemocytometer.
2. Materials and methods
2.4. Transformation and regeneration of protoplasts
2.1. Fungal strains and culture conditions
Freshly prepared protoplasts in a volume of 100 μl were transferred to pre-cooled 50 ml tubes on ice. Starting protoplasts concentration was 109/ml. Five μg of pPd-EGFP vector diluted in 10 μl of Tris–EDTA (TE) buffer was added to the protoplast solution and incubated on ice for 30 min. For the control reaction, 10 μl of the pPdEGFP vector was replaced by TE buffer. Thereafter, 1 ml of PTC buffer (PTC buffer: 40% Poly Ethylene Glycol, 4000 Molecular Weight, 100 mM Tris–HCl, pH 8.0 and 100 mM CaCl2) was added to each tube, and incubated for an additional 25 min at room temperature. The transformation mixture was evenly distributed as 2, 20, and 200 μl droplets onto petri dishes. 12.5 ml of regeneration medium composed of 1 M of sucrose, 0.001 w/v of yeast extract, 0.001 w/v of casein hydrolysate and 0.016% of Bacto Agar was added to each plate. Further 12.5 ml of regeneration media containing 60 μg/ml HygB was overlaid on each plate. The concentration of HygB was more than double the initial lethal concentration (20 μg/ml) for U. oxytropis. High concentration of HygB was added to each plate in order to avoid dilution of the antibiotic. Further experiments were supplemented with 20 μg/ml HygB. Concentration of protoplasts was maintained constant throughout the process. Plates were incubated for four days at room temperature to observe hyphal growth. Fungal cultures produced after regeneration were transferred to fresh PDA–HygB containing plates. Transfer of fungal cultures was performed after three weeks of growth, which correlates to one fungal passage. Control transformation was also transferred to PDA plates and overlaid with media without HygB to compare phenotype of putative transformants. U. oxytropis protoplasts were also regenerated on hygB- negative PDA plates as an additional control. Transformation experiments were repeated four times.
U. oxytropis isolate 25-1 was isolated from leaves of Oxytropis sericea (white locoweed), which were collected from sites around Green River, WY, USA. Samples were pressed to dry the intact plant for subsequent isolation and culturing of the endophyte. The greenest tissues were selected and surface sterilized for 30 s in 70% ethanol, followed by 3 min in 20% bleach, and then 30 s in sterile water. Tissues were dried on sterile paper towels and plated on water agar media. Plates were stored at room temperature (25 °C). Fungal hyphae were transferred to potato dextrose agar (PDA) plates and grown at room temperature (Ralphs et al., 2008) for at least 14 days. Hyphal mass of the recovered endophytes were transferred onto PDA plates and maintained at 18 °C. The 25-1 isolate described above has been preserved as desiccated mycelia and stored at both 4 °C and − 80 °C at the New Mexico State University Center for Natural History Collections (NMSU-CNHC). 2.2. HygromycinB selection In this study, transformation of U. oxytropis was performed using pPd-EGFP vector. pPd-EGFP, is a fungal specific expression vector that confers resistance to antibiotic hygromycin B (Hyg B) because the vector expresses the HygB phosphotransferase gene driven by the Aspergillus nidulans trpC-promoter (Suzuki et al., 2000). Once transformed, the vector expresses EGFP driven by the Cryphonectria parasitica glyceraldehydes-3-phosphate dehydrogenase (gpd) promoter. To evaluate the sensitivity of U. oxytropis to HygB, the fungi was grown in presence of varying concentrations of HygB (MP Biomedicals, OH), 0 μg/ml to 40 μg/ml. The cultures were grown for 14 days at room temperature. Measurement of hyphal growth was performed by counting the length of radial growth in mm beyond a point of reference point (start of culture on day zero). Hygromycin selection was performed on three independent fungal cultures grown on PDA plates for each concentration. 2.3. Preparation of protoplasts Fungal protoplasts were prepared based on methods described by Churchill et al. (1990) with modifications as described below. Liquid suspension cultures of U. oxytropis were started in 100 ml of Potato Dextrose Broth (PDB) and allowed to shake for two weeks on a platform shaker at 200 rpm at room temperature. Hyphal suspensions were harvested by filtration using miracloth (EMD Biosciences, San Diego, CA) in a Buchner funnel. Mycelial mass was resuspended in 100 ml of digestion buffer and incubated at room temperature
2.5. Sporulation and hyphal tipping To generate mononucleate cultures, mycelia were transferred to water agar plates for sporulation. Single spores and single hyphal tips were collected from water agar plates to obtain a pure mononucleate culture. Single spore and single hypha were grown separately in different PDA plates again for three weeks to visualize EGFP expression microscopically. 2.6. Microscopy Zeiss (Thornwood, NY) inverted florescence microscope, Axiovert 200 M was used to capture florescence images at a combined objective and eyepiece magnification of 40× and 10×, respectively. Nuclear DNA was stained using 4′, 6-diamidino-2-phenylindole
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(DAPI, Molecular Probes, Carlsbad, CA) in the generated protoplasts. Axiovision software was used to merge the images. 2.7. Immunoblot Fungal tissues from transformed and untransformed U. oxytropis were ground using liquid nitrogen, homogenized in extraction buffer (50 mM Tris–HCl, pH 7.5, 100 mM NaCl, 50 mM NaF, 2 mM phenylmethylsulphonyl floride, 5 mM EDTA, 1% Triton-X 100, 10% glycerol) and centrifuged at 4 °C for 10 min at 13,000 rpm (Bruno et al., 2004). Protein concentration of lysates was quantified using Bio-Rad protein assay kit (Bio-Rad, Herculus, CA). Total protein (20 μg) was separated on a 12% SDS-polyacrylamide gel and transferred to Immobilon polyvinylidene fluoride (PVDF) transfer membranes (Millipore, Billerica, MA). Membranes were blocked with Bovine Serum Albumin and incubated with 1:1000 EGFP antibody (Abcam, Cambridge, MA). Antirabbit Horse Radish Peroxidase conjugated IgG was used as the secondary antibody at a 1:2000 dilution (Kirkegaard and Perry Laboratories, Gaithersburg, MD). Blots were visualized using the ECL system (GE Healthcare, Piscataway, NJ). Loading control for the gel was immunoblotted in a similar protocol but was incubated with 1:5000 α-tubulin antibody (Santa Cruz Biotechnologies, Santa Cruz, CA). 2.8. Swainsonine assay Fungal suspension cultures were grown in Potato Dextrose Broth (PDB, Difco Laboratories Franklin lakes, NJ), media for two weeks. The fungal mass was filtered and dried at 60 °C overnight. The dried tissue was homogenized in liquid nitrogen and 100 mg of dried tissue was treated with 5 ml of 2% acetic acid and 4 ml chloroform overnight using culture tube shaker (40 rpm). The extract was then centrifuged at 2000 rpm for 15 min and passed through a cation exchange column that was prepared from Dowex 50WX8-100 mesh (Sigma Aldrich, St. Louis, MO) resin. Swainsonine was eluted from the column by displacement with ammonium hydroxide. Ammonium hydroxide was evaporated from the extracted samples using a flow of nitrogen at 60 °C. Dehydrated samples were then hydrated in 100 μl of water and stored for LC-MS measurement (Gardner et al., 2001). LC-MS system consisted of an HP1100 binary solvent pump, autosampler, a Betasil C18 reversed phase HPLC column (Acquity LC system, Waters Corporation, MA) and a Micromass, Q-Tof Micro mass spectrometer (Waters Corporation, MA). The assay was replicated four times with the transformed samples being taken from a single PDA plate.
Table 1 Average growth in mm of Undifilum oxytropis in presence of Hygromycin B antibiotic for 14 days (SE = standard error). Hygromycin conc. (μg/ml)
Average growth ± 1 SE (over 14 days)a
0 5 8 10 20 40
6.16 ± 0.2 mm 5.72 ± 0.1 mm 5.01 ± 0.3 mm 4.70 ± 0.4 mm 0.33 ± 0.12 mm (no growth) 0.33 ± 0.1 mm (no growth)
a N = Average growth was calculated from readings of three independent fungal culture grown on PDA plates for each concentration of hygromycin.
After protoplast purification, five μg of pPD-EGFP vector was used to transform 109 per ml protoplasts in a pre-cooled microcentrifuge tube. One-hour post transformation the protoplasts were observed using 488 nm filter on a Axiovert 200 M microscope. Microscopic examination of protoplasts demonstrated expression of EGFP in selected protoplasts (Fig. 2). The untransformed protoplasts were also imaged to check for auto-florescence. Protoplasts after transformation was diluted in 1 ml of PTC buffer, plated in PDA plates and overlaid with regeneration media. Serial dilution of transformant solution was plated to ensure separation of individual transformants. Growth in the form of mycelia was observed for seven days after plating the transformants. Previous studies on other Ascomycetes fungi (Gorfer et al., 2007) required 24 to 48 h for protoplasts to regenerate as mycelia but due to the slow-growing characteristic of U. oxytropis fungal transformants were visible four
3. Results In order to use the pPd-EGFP vector, which confers resistance to HygB, the sensitivity of U. oxytropis to HygB was tested using increasing concentrations of HygB antibiotic compared to a control plate without HygB. U. oxytropis showed high sensitivity to HygB antibiotic. Concentration of 10 μg/ml HygB showed little impediment of hyphal growth on PDA plates and concentrations of b10 μg/ml were not inhibitory. Particularly, 20 μg/ml and higher HygB concentrations showed complete inhibition of hyphal growth (Table 1). Preparation of quality protoplasts from slow growing, swainsonine producing U. oxytropis was the rate-limiting step for establishment of the transformation system. In the current study the combinations of enzymes used for generation of maximum and efficient protoplasts along with the incubation was varied from 2–3 h. The 3 h incubation time was observed to be optimum for almost complete digestion of fungal mass and production of sufficient protoplasts for U. oxytropis. Protoplast yields were approximately 109 per ml using the above-mentioned protocol. Staining of protoplasts with DAPI was used to confirm nucleated protoplast production (Fig. 1).
Fig. 1. Protoplasts generated from Undifilum oxytropis mycelial culture. A. Light microscope images of protoplasts; B. Florescent images of protoplast after nucleic acid staining with DAPI stain.
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that allowed comparison of the phenotype of putative transformants (control 2). Growth pattern of regenerated fungal mycelia on HygBcontaining PDA plates was observed but no morphological differences were found between untransformed and putatively transformed colonies. Microscopic examination of selected transformants was performed to evaluate the quality of transformants, after three weeks of growth under HygB selection. Mycelium demonstrated strong expression of the EGFP gene, as observed under at 488 nm using an Axiovert 200 M microscope (Fig 3A). Transformed fungi were transferred to fresh HygB plates, which are thereafter referred to as second passage of transformants. The fungal cultures were allowed to grow again for three weeks before transferring to fresh HygB plates. Microscopic observation showed EGFP expression in the transformed mycelium at week six. EGFP expression was also detected in the mycelial mass at 18 weeks post transformation (Fig. 3B and C). Control untransformed mycelia was also observed (Fig. 3D). Single spore and single hypha from eighteen-week old transformed cultures were transferred to HygB containing water agar plates to ensure complete separation of clonal hyphae and to verify maximum GFP expression in the transformed cultures. Sporulation of transformed fungi was observed after three weeks of transfer. Single hyphal tips and single spores from the clonally separated fungal cultures were transferred back to fresh PDA plates and allowed to grow for three weeks. Emerged mycelia on PDA plates containing HygB were screened for expression of EGFP. Expression was close to 100% in mycelia as observed (data not shown). U. oxytropis isolates produce colonies composed of two hyphal types: a basal layer of densely packed, short, torulose, and interwined hyphal segments, and a surface layer of more typical elongated filamentous aerial hyphae (Pryor et al., 2009). Both types of fungal hyphae retained EGFP expression from hyphal tips grown on PDA plates containing HygB (data not shown). Three, six and eighteen-week old transformed and untransformed mycelia were collected and analyzed using immunoblot assay. Fig. 4 indicates that EGFP expression is present in all cultures which were transformed. No expression of GFP was observed in the untransformed U. oxytropis (Fig. 4). Screening of transformants indicated stable expression of EGFP throughout several passages post transformation. Naïve U. oxytropis produces the toxic alkaloid swainsonine. In order to evaluate whether transformed U. oxytropis retained regular swainsonine production characteristic, the swainsonine concentration from fungal mass was measured both before and after EGFP expression. Swainsonine levels detected in unmanipulated U. oxytropis (2.02% total swainsonine in 0.1 g of starting fungal mass) before transformation correlated with the levels of swainsonine found in transformed cultures (1.8% total swainsonine in 0.1 g of starting fungal mass). Fig. 2. A. Florescence microscopic image of DAPI stained protoplasts generated from Undifilum oxytropis mycelial culture; B. Expression of GFP in protoplasts in panel A; C. Overlap of panels A and B.
days post transformation. Although serial dilutions were performed on PDA plates, individual colonies were not observed after seven days because the transformants merged with each other. On an average three individual fungal colonies were observed after regeneration on PDA plates. However, those may have been the result of merged transformants before penetration of the overlay and therefore an exact transformation efficiency cannot be calculated. Fourteen days after transformation, complete mycelia formation observed on HygBcontaining PDA plates. Fungal cultures were transferred between passages. No hyphae were observed in untransformed protoplasts plated on HygB containing PDA plates (control 1). Untransformed protoplasts were regenerated on PDA plates without HygB selection
4. Discussion Previously, various transformation systems have been developed for ascomycete fungi including endophytes (Abello et al., 2008), but this study describes the first successful transformation and GFP expression in an isolate of very slow growing, swainsonine-producing endophytic fungus, U. oxytropis. The expression of both hygromycin-B phosphotransferase driven by a promoter from A. nidulans (class Eurotiomycetes) and EGFP controlled by an element from C. parasitica (class Sordariomycetes) also indicates that U. oxytropis (class Dothidiomycetes) is tolerant of non-native promoter systems. This system may allow for the use of controlled expression options, such as the recently identified copper-responsive element from C. parasitica (Willyerd et al., 2009). Assay of swainsonine levels show that transformed U. oxytropis fungal mycelia retains the ability to produce of toxic alkaloid, swainsonine after stable expression of a foreign heterologous gene. This system can therefore be used to perform
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Fig. 3. Mycelial growth of Undifilum oxytropis. A: Florescence microscope images of mycelia three weeks post transformation on hygromycin selection plates; B. Florescent images of mycelia expressing EGFP, six weeks post transformation on hygromycin selection plates; C. Florescent images of mycelia expressing EGFP, eighteen weeks post transformation on hygromycin selection plates; D. Untransformed mycelia of Undifilum oxytropis on hygromycin negative plates.
genetic manipulation on U. oxytropis. These procedures for generating stable transformants will be used to study metabolically important genes in the lysine biosynthetic pathway or swainsonine metabolic pathway in U. oxytropis. Such manipulation will open avenues for
future research to decipher the role of enzymes in fungal metabolic pathways including the swainsonine degradation pathway. Acknowledgements The authors would like to thank Deana Baucom, Jorge Achata, and Dr. Soum Sanogo (EPPWS) for technical assistance and constructive discussion. Funding for this project was provided by USDA Special grant 59-5428-1-327 and New Mexico State University Agricultural Experiment Station. References
Fig. 4. Immunoblot of hyphal samples after three weeks, six weeks, eighteen weeks of Undifilum oxytropis growth using anti-EGFP antibody. Lane 1: Three weeks post transformation U. oxytropis hyphae; Lane 2: Untransformed U. oxytropis hyphae; Lane 3: Six weeks post transformation U. oxytropis hyphae; Lane 4: Eighteen weeks post transformation U. oxytropis hyphae. Bottom panel: Total protein loading control of the samples from Lane 1–4 blotted with anti-tubulin antibody.
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