Transformation of Epichloë typhina by electroporation of conidia
© Dombrowski et al; licensee BioMed Central Ltd. 2011
Received: 21 September 2010
Accepted: 5 March 2011
Published: 5 March 2011
Choke, caused by the endophytic fungus Epichloë typhina, is an important disease affecting orchardgrass (Dactylis glomerata L.) seed production in the Willamette Valley. Little is known concerning the conditions necessary for successful infection of orchardgrass by E. typhina. Detection of E. typhina in plants early in the disease cycle can be difficult due to the sparse distribution of hyphae in the plant. Therefore, a sensitive method to detect fungal infection in plants would provide an invaluable tool for elucidating the conditions for establishment of infection in orchardgrass. Utilization of a marker gene, such as the green fluorescent protein (GFP), transformed into Epichloë will facilitate characterization of the initial stages of infection and establishment of the fungus in plants.
We have developed a rapid, efficient, and reproducible transformation method using electroporation of germinating Epichloë conidia isolated from infected plants.
The GFP labelled E. typhina provides a valuable molecular tool to researchers studying conditions and mechanisms involved in the establishment of choke disease in orchardgrass.
Orchardgrass (Dactylis glomerata L.) is an important forage grass species. About 97% of orchardgrass seed used for pastures and hay in North America is produced in the Willamette Valley in western Oregon. Choke disease, caused by the endophytic fungus Epichloë typhina, was first reported in the Willamette Valley during the mid 1990s . Infected plants remain asymptomatic during most of the year. In the spring, E. typhina proliferates within reproductive tillers, encasing the developing seed head in a dense mycelial mat (stroma). However, the stem continues to grow, revealing an elongated, white stromal mass that resembles a small cattail. Conidia of one or two mating types are produced on the surface of each stroma. Fertilization requires the transfer of conidia of one mating type to stroma of the opposite mating type. This is typically accomplished by flies in the genus Botanophila. Following fertilization, perithecia develop within the stroma surface [3, 4]. The perithecia produce ascospores, which are dispersed by wind and cause new plant infections. The pathogen has not been shown to be transmitted through the seed in orchardgrass, but it is seed transmitted in other grasses [3–6].
The recent introduction and rapid spread of choke disease is a serious problem for orchardgrass seed producers in the Willamette Valley [1, 7, 8]. In England, where the disease has occurred for many years, the number of fields with choke disease increased from zero to a few during the first year of production to 33-81% by the second to fifth year of seed production . In France, choke was reported to affect up to 30% of the tillers in a field by the fourth year of seed production [10, 11]. Since the first incidence of choke reported in Oregon in the mid 1990s , the fungus has spread to ~90% of orchardgrass seed production fields and has caused yield losses of up to 65% in individual fields .
Very little is known about the conditions necessary for successful infection of orchardgrass by E. typhina. Fertilization of the fungal stroma is facilitated by flies [2, 12], but fly density was not correlated with reproductive success of the fungus in western Oregon [2, 13]. Fertilization of E. typhina by ascospores has been reported recently and may contribute to the rapid spread of choke in orchardgrass . Attempts to infect orchardgrass foliage or flowers with conidia or ascospores were not successful . Germination of E. typhina ascospores and conidia on the cut ends of seed stalks of orchardgrass and growth of hyphae down the pith has been reported [4–6], although it was not established whether these plants ultimately became infected through the stalks. Infection of young tillers by ascospores or conidia produced by ascospores under very favorable experimental conditions was recently reported . It is not known when or how infections occur in the field or how long the latent period is between infection and manifestation of symptoms in the field.
Detection of E. typhina early in the disease cycle can be difficult due to sparse distribution of hyphae in the plant. Therefore a sensitive method to detect the fungus in plants would provide an invaluable tool for elucidating the conditions and establishment of the infection in orchardgrass. Green Fluorescent Protein (GFP) has been utilized extensively as a marker to aid in the development of fungal transformation systems and to examine early stages in plant/fungal interactions [reviewed in ,[17–22]]. Translation fusions between GFP and fungal genes of interest have also been utilized to determine gene expression patterns and to investigate the role of specific genes in the infection process [16, 23]. Transformation of E. typhina with the GFP marker gene will facilitate the characterization of the initial stages of infection and progression of the disease in plants . There are several different methods available for fungal transformation, such as chemical induction, Agrobacterium-mediated transformation, particle bombardment and electroporation [[16, 24–27], reviews and references within, [28, 29]]. Chemical transformation of protoplasts of Epichloë festucae has been reported and was used to determine the expression patterns of the lolitrem biosynthetic genes  and to look at the infection process of E. festucae in Lolium perenne. Electroporation of germinating conidia has been used successfully to transform a range of fungal species [32–34]. We report here a simple and rapid method using electroporation to transform germinating Epichloë conidia isolated from infected plants. The GFP labelled E. typhina provides a valuable molecular tool for researchers studying the infection process in orchardgrass. The transformation protocol will also be valuable for gene function studies in Epichloë-plant interactions.
Materials and methods
The pCT74 GFP expression vector  was used as an intact circular plasmid or as a linearized plasmid for transformation experiments. The plasmid was digested overnight at 37°C with Xho I to linearize the plasmid. The digested DNA was precipitated with 1/10 volume of 3 M NaOAc, pH 5.2, and 2 volumes of EtOH. One μL of blue dextran (10 mg/mL) was added to aid in visualization of the pellet. The precipitated DNA was resuspended in sterile distilled water at a final concentration of 0.5 μg/μL.
Collection and preparation of conidia for transformation
Sensitivity of untransformed conidia to hygromycin B
The plasmid used for transformation contains the hygromycin B phosphotransferase gene which confers resistance to hygromycin and enables the selection of transformed colonies. To determine the concentration of hygromycin to be used for selection, conidia (~5000 conidia/plate) were placed on increasing concentrations of hygromycin B, from 0-400 mg/L, on Corn Meal Malt Agar (CMMA - 19 g corn meal agar, 2 g yeast extract, 20 g malt extract and 5 g agar per liter). Since conidia were collected from plants grown in the greenhouse, 150 μg/mL of ampicillin or ticarcillin was added to the selection medium to inhibit bacterial growth.
Determination of optimal electrical field strength for conidia transformation
Electroporation was performed using the Bio-Rad Gene Pulser II on 100 μL of suspended conidia (~5000 conidia) in a 0.2 cm cuvette. The percent survival of germinating conidia at various combinations of voltage settings between 1 and 2 kV, resistances between 400-800 ohms, and 25 μF capacitance was tested. Percent survival at each resistance level (400, 600, 800 ohms) at the three voltages was determined by calculating the number of colonies from each sample (based on colony counts obtained from plating out serial dilutions from each sample) and comparing that to the number of colonies obtained from non-electroporated germinating conidia.
Transformation of conidia
Approximately 105 — 106 conidia in 100 μL of sterile electroporation buffer were combined with 0.1-2.0 μg of circular or linearized DNA in sterile 500 μL microfuge tubes and incubated on ice for 20 min. The DNA/conidial mixture was then transferred into a cold 0.2 cm electroporation cuvette and subjected to a specific electrical field using Bio-Rad Gene Pulser II. Immediately following the pulse, the conidia were transferred to a sterile 15 mL snap-cap tube containing 900 μL of cold sterile Regeneration Medium (RM - 14.5 g mannitol, 0.4 g yeast extract and 1.5 g potato dextrose broth per 100 mL distilled water). The conidia were incubated on ice for 20-30 min and were then placed on a rotating shaker (150-180 rpm) at room temperature for 5 hours prior to plating on selective solid medium (200 μL/plate). Since conidia were collected from plants grown in the greenhouse, 150 μg/mL of ampicillin or ticarcillin was added to the selection medium to inhibit bacterial growth. Hygromycin B-resistant colonies started to develop in 3-4 weeks. Colonies were examined with a LEICA Fluorescent Dissecting Microscope MZFLIII using a PLANAPO 1.0X Lens to confirm GFP expression in E. typhina transformants. In order to visualize the untransformed control fungus, low intensity white light was used in addition to the UV light.
Southern analysis to determine copy number
DNA was isolated using a modified CTAB (hexadecyltrimethylammonium bromide) protocol  including treatment with RNase. The DNA was treated with Nucleon PhytoPure™resin, a component found in the Illustra Nucleon Phytopure™Genomic DNA extraction kit (GE Healthcare UK Limited, Buckinghamshire, UK) which covalently binds polysaccharides. Two μL of the resin was added per 100 μL of solution, mixed gently, and then centrifuged to pellet the resin. The DNA remained in the supernatant. Approximately 5 μg of DNA was digested with EcoRI, using the manufacturer's supplied buffer, and separated in a 1.2% agarose gel. DNA was transferred to a Hybond-N+ membrane (Amersham; GE Healthcare) by downward capillary transfer following standard protocols . PCR was used to synthesize a digoxigenin (DIG; Roche, Cat. No. 11573152910) labelled probe for a portion of the GFP gene following the recommendations of the company (https://www.roche-applied-science.com). Primers were designed using Primer3 software . Primers for probe synthesis were GFPF64 5'-GACGTAAACGGCCACAAGTTC and GFPR671 5'-GAACTCCAGCAGGACCATGTG producing an amplicon of ~ 600 bp (between nucleotide 64 and 671 of the GFP open reading frame). Hybridization and detection were performed following the protocol of Engler-Bloom et al.  with modifications described by Krueger and Williams .
Results and discussion
Sensitivity of untransformed conidia to hygromycin B
Determination of optimal electrical field strength for conidia transformation
Most reports indicate that the best electroporation conditions to use for transformation are those that result in 40-60% survival of the cells. With increasing levels of resistance, the 40-60% survival rate occurred at lower voltages. Approximately 50% of the conidia survived using 1 kV at 800 Ω, 1.2 kV at 600 Ω, and between 1.2 and 1.5 kV at 400 Ω (Additional file 2).
Transformation of conidia
Number of hygromycin-resistant colonies expressing GFP at designated electroporation conditions.
# GFP/total # HygR colonies
Southern analysis to determine copy number
An efficient method for the transformation of germinating conidia of E. typhina by electroporation was established. Two critical parameters for successful transformation were the use of a linearized plasmid and conidia that were collected from newly formed stromata. Transformed fungi were selected on 200 and 300 μg/mL hygromycin B. Over 80% of transformed lines continued to express GFP after successive transfers and storage. Fifteen lines were chosen and have been maintained for over 5 years and are still expressing GFP. We observed variable levels of GFP expression and some different colony morphologies. The selected isolates were verified as E. typhina by sequencing the PCR products obtained using species specific primers for the actin 1 locus. Southern analysis revealed the presence of a single insert in 70% of the lines tested. The GFP labelled E. typhina lines provide a valuable molecular tool to researchers studying conditions and mechanisms involved in the establishment of choke in orchardgrass. While the mode of infection for E. typhina in orchardgrass is uncertain, several researchers have successfully introduced E. typhina into perennial ryegrass and orchardgrass [5, 41] however, it was not determined if the inoculated plants would ultimately manifest choke symptoms. It will be interesting to see if these methods will work to introduce the GFP expressing E. typhina into orchardgrass, and more importantly, if that will lead to the manifestation of choke disease in subsequent years. This would allow monitoring of the infection process under natural mechanisms of infection which would be quite different from artificial methods of inoculation.
Green Fluorescent Protein
Corn Meal Malt Agar
Special thanks are extended to Dr. Jennifer M. Lorang and Dr. Lynda M. Ciuffetti, Department of Botany and Plant Pathology, Oregon State University, for providing the pCT74 plasmid. Experimental methods performed in this research complied with current laws and regulations of the U.S.A. The use of trade, firm, or corporation names in this publication is for the information and convenience of the reader. Such use does not constitute an official endorsement or approval by the United States Department of Agriculture or the Agricultural Research Service of any product or service to the exclusion of others that may be suitable. Relevance to ARS National Programs. NP205: Rangeland, Pasture and Forages: Component "Plant Resources". NP302: Plant Biological and Molecular Processes: Component "Biological Processes that Determine Plant Productivity and Quality". and NP303 Plant Diseases: Component "Pathogen Biology, Genetics, Population Dynamics, Spread, and Relationship with Hosts and Vectors".
- Alderman SC, Pfender WF, Welty RE, Mellbye ME, Cook RL, Spatafora JW, Putnam M: First report of choke caused by Epichloë typhina on orchardgrass in Oregon. Plant Dis. 1997, 81: 1335-1339. 10.1094/PDIS.19184.108.40.2065A.View ArticleGoogle Scholar
- Rao S, Baumann D: The interaction of a Botanophila fly species with an exotic Epichloë fungus in a cultivated grass: Fungivore or mutualist?. Entomol Exp Appl. 2004, 112: 99-105. 10.1111/j.0013-8703.2004.00189.x.View ArticleGoogle Scholar
- Sampson K: The systemic infection of grasses by Epichloë typhina (Pers.) Tul. Trans Brit Mycol Soc. 1933, 18: 30-47. 10.1016/S0007-1536(33)80025-8.View ArticleGoogle Scholar
- Western JH, Cavett JJ: The choke disease of cocksfoot (Dactylis glomerata) caused by Epichloë typhina (Fr.) Tul. Trans Brit Mycol Soc. 1959, 42: 298-307. 10.1016/S0007-1536(56)80037-5.View ArticleGoogle Scholar
- Chung KR, Hollin W, Siegel MR, Schardl CL: Genetics of host specificity in Epichloë typhina. Phytopathology. 1997, 87: 599-605. 10.1094/PHYTO.19220.127.116.119.PubMedView ArticleGoogle Scholar
- Chung KR, Schardl CL: Sexual cycle and horizontal transmission of the grass symbiont, Epichloë typhina. Mycol Res. 1997, 101: 295-301. 10.1017/S0953756296002602.View ArticleGoogle Scholar
- Pfender WF, Alderman SC: Geographic distribution and incidence of orchardgrass choke caused by Epichloë typhina in Oregon. Plant Dis. 1999, 83: 754-758. 10.1094/PDIS.1918.104.22.1684.View ArticleGoogle Scholar
- Pfender WF, Alderman SC: Regional development of orchardgrass choke disease and estimation of seed yield loss. Plant Dis. 2006, 90: 240-244. 10.1094/PD-90-0240.View ArticleGoogle Scholar
- Large EC: Surveys for choke (Epichloë typhina) in cocksfoot seed crops 1951-1953. Plant Pathol. 1954, 3: 6-11. 10.1111/j.1365-3059.1954.tb00674.x.View ArticleGoogle Scholar
- Raynal GE: Libération des ascospores d'Epichloë typhina, agent de la quenouille du dactyle: Conséquences pour l'épidémiologie et la lutte. Fourrages. 1991, 127: 345-358.Google Scholar
- Fermaud M: Epidémiologie de la quenouille du dactyle porte-graine due à Epichloë typhina (Pers. ex Fr.) Tulasne. PhD Thesis, Ecole Nationale Supérieure d'Agronomie, Montpellier, France. 1986Google Scholar
- Bultman TL, White JF, Bowdish TI, Welch AM, Johnston JJ: Mutualistic transfer of Epichloë spermatia by Phorbia flies. Mycologia. 1995, 87: 182-189. 10.2307/3760903.View ArticleGoogle Scholar
- Kaser JM, Rao S: Mapping the choke pathogen in cultivated orchardgrass fields in the Willamette Valley. Seed production research at Oregon State University USDA-ARS cooperating. Edited by: Young W III. 2009, Dept. of Crop and Soil Science Ext/CrS, 129: 1-5.Google Scholar
- Alderman SC, Rao S: Ascosporic fertilization of Epichloë typhina in Dactylis glomerata seed production fields in Oregon and implications for choke management. Plant Health Prog. 2008Google Scholar
- Leyronas C, Raynal G: Role of fungal ascospores in the infection of orchardgrass (Dactylis glomerata) by Epichloë typhina agent of choke disease. J Plant Pathol. 2008, 90: 15-21.Google Scholar
- Lorang JM, Tuori RP, Martinez JP, Sawyer TL, Redman RS, Rollins JA, Wolpert TJ, Johnson KB, Rodriguez RJ, Dickman MB, Ciuffetti LM: Green fluorescent protein is lighting up fungal biology. Appl Environ Microbiol. 2001, 67: 1987-1994. 10.1128/AEM.67.5.1987-1994.2001.PubMedPubMed CentralView ArticleGoogle Scholar
- Liu T, Liu L, Hou J, Li G, Gao S, Chen J: Expression of green fluorescent protein in Curvularia lunata causing maize leaf spot. Can J Plant Pathol. 2010, 32: 225-228. 10.1080/07060661.2010.484213.View ArticleGoogle Scholar
- Mansouri S, van Wijk R, Rep M, Fakhoury AM: Transformation of Fusarium virguliforme, the causal agent of sudden death syndrome of soybean. J Phytopathol. 2009, 157: 319-320. 10.1111/j.1439-0434.2008.01485.x.View ArticleGoogle Scholar
- Pliego C, Kanematsu S, Ruano-Rosa D, de Vicente A, López-Herrera C, Cazorla FM, Ramos C: GFP sheds light on the infection process of avocado roots by Rosellinia necatrix. Fungal Genet Biol. 2009, 46: 137-145. 10.1016/j.fgb.2008.11.009.PubMedView ArticleGoogle Scholar
- de Silva AP, Bolton MD, Nelson BD: Transformation of Sclerotinia sclerotiorum with the green fluorescent protein gene and fluorescence of hyphae in four inoculated hosts. Plant Pathol. 2009, 58: 487-492. 10.1111/j.1365-3059.2009.02022.x.View ArticleGoogle Scholar
- Maruthachalam K, Nair V, Rho HS, Choi J, Kim S, Lee YH: Agrobacterium tumefaciens-mediated transformation in Colletotrichum falcatum and C. acutatum. J Microbiol Biotechnol. 2008, 18: 234-241.PubMedGoogle Scholar
- Sukno SA, Garcia VM, Shaw BD, Thon MR: Root infection and systemic colonization of maize by Colletotrichum graminicola. Appl Environ Microbiol. 2008, 74: 823-832. 10.1128/AEM.01165-07.PubMedPubMed CentralView ArticleGoogle Scholar
- Heneghan MN, Porta C, Zhang C, Burton KS, Challen MP, Bailey AM, Foster GD: Characterization of serine proteinase expression in Agaricus bisporus and Coprinopsis cinerea by using green fluorescent protein and the A. bisporus SPR1 promoter. Appl Environ Microbiol. 2009, 75: 792-801. 10.1128/AEM.01897-08.PubMedPubMed CentralView ArticleGoogle Scholar
- Richey MG, Marek ET, Schardl CL, Smith DA: Transformation of filamentous fungi with plasmid DNA by electroporation. Phytopathology. 1989, 79: 844-847. 10.1094/Phyto-79-844.View ArticleGoogle Scholar
- Gold SE, Duick JW, Redman RS, Rodriguez RJ: Molecular transformation, gene cloning, and gene expression for filamentous fungi. Applied Mycology and Biotechnology. Edited by: Khachatourians GG, Aroua DP. 2001, Oxford, UK: Elsevier Science, 1: 199-238. 10.1016/S1874-5334(01)80010-1.Google Scholar
- Michielse CB, Hooykaas PJJ, van den Hondel CAMJJ, Ram AFJ: Agrobacterium-mediated transformation as a tool for functional genomics in fungi. Curr Genet. 2005, 48: 1-17. 10.1007/s00294-005-0578-0.PubMedView ArticleGoogle Scholar
- Meyer V: Genetic engineering of filamentous fungi - Progress, obstacles and future trends. Biotechnol Adv. 2008, 26: 177-185. 10.1016/j.biotechadv.2007.12.001.PubMedView ArticleGoogle Scholar
- Lianhui J, Jiang ZD, Liu Y, Koh CMJ, Zhang LH: A simplified and efficient method for transformation and gene tagging of Ustilago maydis using frozen cells. Fungal Genet Biol. 2010, 47: 279-287. 10.1016/j.fgb.2010.01.002.View ArticleGoogle Scholar
- Betts MF, Tucker SL, Galadima N, Meng Y, Patel G, Li L, Donofrio N, Floyd A, Nolin S, Brown D, Mandel MA, Mitchel TK, Xu JR, Dean RA, Farman ML, Orback MJ: Development of high throughput transformation system for insertional mutagenesis in Magnaporthe oryzae. Fungal Genet Biol. 2007, 44: 1035-1049. 10.1016/j.fgb.2007.05.001.PubMedView ArticleGoogle Scholar
- May KJ, Bryant MK, Zhang X, Ambrose B, Scott B: Patterns of expression of a lolitrem biosynthetic gene in Epichloë festucae-perennial ryegrass symbiosis. Mol Plant-Microbe Interact. 2008, 21: 188-197. 10.1094/MPMI-21-2-0188.PubMedView ArticleGoogle Scholar
- Christensen MJ, Bennett RJ, Ansari HA, Koga H, Johnson RD, Bryan GT, Simpson WR, Koolaard JP, Nickless EM, Voisey CR: Epichloë endophytes grow by intercalary hyphal extension in elongating grass leaves. Fungal Genet Biol. 2008, 45: 84-93. 10.1016/j.fgb.2007.07.013.PubMedView ArticleGoogle Scholar
- Marchand G, Fortier E, Neveu B, Bolduc S, Belzile F, Bélanger RR: Alternative methods for genetic transformation of Pseudozyma antarctica, a basidiomycetous yeast-like fungus. J Microbiol Methods. 2007, 70: 519-529. 10.1016/j.mimet.2007.06.014.PubMedView ArticleGoogle Scholar
- Lakshmi Prabha V, Punekar NS: Genetic transformation of Aspergilli: Tools of the trade. Indian J Biochem Biophys. 2004, 41: 205-215.PubMedGoogle Scholar
- Dobrowolska A, Staczek P: Development of transformation system for Trichophyton rubrum by electroporation of germinated conidia. Curr Genet. 2009, 55: 537-542. 10.1007/s00294-009-0264-8.PubMedView ArticleGoogle Scholar
- Doyle JJ, Doyle JL: Isolation of plant DNA from fresh tissue. Focus. 1990, 12: 13-15.Google Scholar
- Sambrook J, Russell DW: Molecular Cloning, A Laboratory Manual. 2001, Cold Spring Harbor, Cold Spring Harbor Press, New YorkGoogle Scholar
- Rozen S, Skaletsky HJ: Primer3 on the WWW for general users and for biologist programmers. Bioinformatics Methods and Protocols: Methods in Molecular Biology. Edited by: Misener S, Krawetz SA. 2000, Totowa, New Jersey: Humana Press, 365-386.Google Scholar
- Engler-Blum G, Meier M, Frank J, Müeller GA: Reduction of background problems in nonradioactive Northern and Southern blot analyses enables higher sensitivity than 32P-based hybridizations. Anal Biochem. 1993, 210: 235-244. 10.1006/abio.1993.1189.PubMedView ArticleGoogle Scholar
- Krueger SK, Williams DE: Quantification of digoxigenin-labeled DNA hybridized to DNA and RNA slot blots. Anal Biochem. 1995, 229: 162-169. 10.1006/abio.1995.1398.PubMedView ArticleGoogle Scholar
- Baldwin JC, Dombrowski JE, Alderman SC: A DNA assay for the detection of choke in orchardgrass. Seed production research at Oregon State University USDA-ARS cooperating. Edited by: Young W III. 2004, Dept. of Crop and Soil Science Ext/CrS, 124: 19-20.Google Scholar
- Latch GCM, Christensen MJ: Artificial infections of grasses with endophytes. Ann Appl Biol. 1985, 107: 17-24. 10.1111/j.1744-7348.1985.tb01543.x.View ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.