Co-Transformation of Metarhizium Anisopliae by Electroporation Or

Home , Gene gun

ELSEiVIER FEMS Microbiology Letters 131 (1995) 289-294 Downloaded from https://academic.oup.com/femsle/article/131/3/289/512137 by guest on 28 September 2021 Co-transformation of Metarhizium anisopZiae by electroporation or using the gene gun to produce stable GUS transformants

Raymond J. St. Leger *, Susumu Shimizu, Lokesh Joshi, Michael J. Bidochka, Donald W. Roberts

Boyce Thompson Institute for Plant Research, Cornell Uniuersiry, Tower Road, Ithaca, NY 14850-1801, USA Received 17 May 1995; revised 10 July 1995; accepted 14 July 1995

Abstract

The potential of &lucuronidase as a molecular marker for studying the environmental microbiology of entomopathogenic fungi was assessed. Meturhiz&r anisopliae was stably co-transformed with plasmids (pNOM102 and pBENA3) containing the @glucuronidase and benomyl resistance ( /3-tubulin) genes, using both electroporation and biolistic delivery systems, and it was confirmed that the expressed phenotypes were not exhibited by ten randomly chosen indigenous North-American isolates. In spite of random and multiple integrations, the co-transformants showed normal growth rates and retained their pathogenicity to insects. PGlucuronidase activity in the co-transformants was used to detect histochemically the presence of fungal hyphae in infected host insects (Bombyx mori) and thus provides a practical means of marking genetically engineered pathogens for field trials.

Keywords: Metarhizium anisopliae; Fungal co-transformation; Electroporation; Biolistic gene transfer; GUS expression; ‘Tagging’ fungi

1. Introduction strain improvement through genetic manipulation and selection of fungi which are appropriate for the Fungal diseases in insects are common and control of specific target insect pests Cl]. The appli- widespread and often decimate insect populations in cation of genetic engineering to produce new strains spectacular epizootics. Consequently, much recent offers tremendous potential in pest control but also interest in mycoinsecticides has led to the marketing demands a careful and responsible approach. To of several, including Metarhizium anisopliae. The date, there is no information on the survival of possibility of utilizing a microbe for commercial specific genotypes of entomopathogenic fungi in na- purposes inevitably leads to consideration of ways of ture, nor is there experimentally derived information improving its performance. The information being on gene transfer from populations of introduced fungi gathered on the processes by which entomo- to wild-type or other fungal species. Evaluating the pathogenic fungi infect an insect host is providing a safety of recombinant organisms will require the base of knowledge for a rational approach for fungal integration of several factors including transmutabil- ity, environmental persistence, and host range to arrive at a full risk assessment. The ability to ‘tag’ ’ Corresponding author. Tel.: + 1 (607) 254 1252; Fax: + 1 populations of pathogens with a molecular marker (607) 254 1242; E-mail: [email protected]. will provide the means to follow the fate of the

Federation of European Microbiological Societies SSDI 0378-1097(95)00272-3 290 H.J. St. Lvgrr et al./ FEMS Microbiology Lerterv 131 (1995) 289-294

fungus and so assess these risks and provide funda- up to 9 h in yeast extract medium at 27°C with mental information on pathogen ecology. shaking. When germ-tubes began to emerge, crude The /I-glucuronidase (GUS) gene fusion system cellulase (1 mg ml-l; Sigma, type C-1184) and has been found to be a powerful tool for identifying /3-glucuronidase (1 mg mll’ ; Sigma, type Hl) were and localizing marked strains of saprophytic and added and the incubation resumed for up to 2 h. plant pathogenic fungi under laboratory conditions Germlings were centrifuged before they produced [2]. Here, we compare DNA delivery systems (elec- protopIasts and washed twice in electroporation Downloaded from https://academic.oup.com/femsle/article/131/3/289/512137 by guest on 28 September 2021 troporation and the gene gun) for producing, by buffer (I mM HEPES, 50 mM mannitol, pH 7.5, 1% co-transformation, strains of M. anisopliae express- (w/v) PEG 6000) containing 10 pug ml-’ aurin ing GUS, and show that GUS expression is a practi- tricarboxylic acid and resuspended in electroporation cal means of localizing the active biomass of marked buffer at approximately 5 X lo7 spores ml-‘. strains in infected insect hosts. Aliquots of 100 ~1 were mixed gently with 10 pg plasmid DNA and kept on ice for 15 min. High-voltage 5-ms pulses (25 PF capacitor and a field strength 2. Materials and methods of 12.5 kV cm- ’ ) were delivered to samples in disposable cuvettes (Bio-Rad Laboratories; inter- 2.1. Strains and growth media electrode distance of 0.2 cm) by using a gene pulse apparatus (Bio-Rad Laboratories). Immediately fol- M. anisopliae strains were described previously lowing electroporation, the spore suspension was [3]. All strains were grown in shake cultures of mixed with 1 ml potato dextrose broth plus 0.5 M Sabouraud dextrose broth or yeast extract medium as MgSO, and incubated at 25°C for 3 h. Aliquots described previously [4]. Regeneration medium (RM) containing 2 X lo5 germlings were plated onto 10 ml consists of 1% potato dextrose broth (Difco), 0.2% potato dextrose agar and incubated for 10 h before yeast extract, 0.5 M MgSO,, and 0.3% NaNO,; RM selection using an RLGA overlay containing 5 pg was solidified with 1.2% noble agar (RA) or 1.2% ml - ’ benomyl. Colonies arising on transformation sea plaque low-gelling-temperature agarose (RLGA). plates were transferred to potato dextrose agar con- X-glucuronide (5-bromo-4-chloro-3-indoyl glu- taining benomyl and X-glut (20 pg ml ’ ). curonide; X-glue; Clontech, Palo Alto, CA) was included in RA or Czapek Dox agar (Oxoid) at 20 2.4. Transformation by particle bombardment kg ml-‘. For selection of benomyl-resistant (BenR 1 High-velocity ballistic transformation of M. transformants, RLGA plates were overlaid with anisopliae germlings was performed with the DuPont molten (42°C) RLGA containing 5 pg ml-’ beno- particle delivery system PDS lOOO/HE. Briefly, 25 my1 (DuPont Co.) from a stock solution of 5 mg ~1 of a tungsten (Ml0 particles, mean diameter 1 ml-’ in dimethyl sulfoxide (DMSO). pm) suspension (60 mg ml-‘) was mixed with 2.5 pg (2 pug/p11 of plasmid DNA, 25 ~12.5 M CaCl, 2.2. Vectors and 10 ~1 0.1 M spermidine. The particle-DNA Plasmid pNOM102 harboring the GUS gene from mixture was vortexed at 4°C for 15 min and incu- E. coli under control of the Aspergillus nidulans gpd bated for 10 min on ice. The particles were collected promoter was kindly provided by R.P. Oliver and is by centrifugation and washed sequentially with wa- described elsewhere [2]. The benomyl resistance ter, 70% ethanol and absolute ethanol. The particles plasmid pBENA3 was described previously [5]. Strain were resuspended in 20 ~1 of ethanol by sonication aDH5a of E. coli was used for propagation of in a sonicator water bath (Branson 100) for 5 s. 6 ~1 plasmids. of coated particles were spread on a kapton flying- disc and used for bombardment. The particles were 2.3. Transformation by electroporation propelled toward the germinating conidia by release of helium at 1200 psi. Published protocols [6,7] were optimized for M. Before DNA was introduced into conidia, they anisopliae. lo-day-old conidia were germinated for were germinated for 9 h at 27°C with shaking (100 R.J. St. Leger et al. / FEMS Microbiology Letters 131 (1995) 289-294 291 rpm) in 0.2% yeast extract medium. For transforma- Czapek Dox agar containing benomyl (5 pg ml-’ ) tion, germlings were spread onto RA containing and X-glut. (20 pg ml-‘) and supplemented with 0.1% Igepal (Alltech Associates, Inc.). Once seeded, ampicillin, tetracycline and streptomycin at 5 pg the plates were placed into the chamber of the ml-’ each. particle delivery system at a distance of 14 cm from the launch site. The germlings were bombarded once or twice with tungsten particles coated with mixes of 3. Results and discussion Downloaded from https://academic.oup.com/femsle/article/131/3/289/512137 by guest on 28 September 2021 the plasmid DNAs. Selection for benomyl resistance was achieved with an overlay applied 10 h after Ten North American field strains of M. aniso- bombardment. pliae (ARSEF 2575, 23, 1878, 1968, 2547, 794, 1080, 3108, 3540, 3388) were inhibited by benomyl 2.5. Genomic analysis (5 wg ml-’ ) and produced negligible endogenous GUS activity as shown in quantitative spectrophoto- DNA was isolated from freeze-dried mycelium as metric assays or in qualitative plate assays using described previously [4]. Standard recombinant DNA X-glut. Consequently, transformants expressing the techniques were as described by Sambrook et al. [8]. GUS phenotype should be distinguishable from 32P-labelled DNA was produced by a primer exten- background indigenous populations. sion procedure [9]. The electrophoretic karyotypes of transformed and non-transformed strains were anal- 3.1. Transformation conditions ysed with counter-clamped homogenous electric field electrophoresis according to Simizu et al. [lo]. Transformation frequencies were previously found to be low (l-2 transformants per pg DNA) using 2.6. Protein extraction and GUS activity analysis Ca2+/PEG technology [5,13]. By contrast, transformation frequencies of ARSEF 549 utilizing electro- Mycelium was comminuted under liquid nitrogen poration were 28 + 6 (mean + S.D., n = 4) transfor- and vortexed with 50 mM potassium phosphate buffer mants per pg pBENA3 with a co-transformation (pH 7.2) containing 0.2% Triton X-100 (1 ml of frequency of 24% utilizing pBENA3 and pNOM102 buffer per g wet weight of mycelium). The super- at a molar ratio of 1:3, as measured by testing the natant was collected by centrifugation (12000 X g GUS expression of benomyl-resistant colonies on for 10 min) and assayed with p-nitrophenyl-P-D- X-glut medium plates. A co-transformation fre- glucuronide (Sigma) as described for N-acetyl-gluco- quency of 64% was obtained when pNOM102 was saminidase assays [ill. Absorbance was measured at used at 20-fold excess. In preliminary experiments it 410 nm and a unit is defined as the amount of was determined that no transformants were obtained enzyme producing 1 nmol p-nitrophenol min-’ at unless cells were pretreated with wall-digesting en- 37°C. Protein concentrations in the extracts were zymes. Pfeifer and Khachatourians [14] used electro- determined using the Bio-Rad protein assay. poration to transform protoplasts of the entomo- pathogen Beauveria bassiana to benomyl resistance 2.7. Pathogenic@ tests (l-3 transformants per pg DNA) [14], but in this study, transformation frequencies were improved ( > Groups of five Sth-instar silkworms (Bombyx 20 per pg DNA) by maintaining partial cell wall mori) were each inoculated with either one of the integrity. Few transformants ( < 5 per kg DNA) transformants or the original wild-type and infection were obtained if the enzyme pretreatment exceeded 3 assessed as described [12]. Expression of the GUS h, resulting in the formation of protoplasts. Three gene was monitored by injecting insects with X-glut GUS-positive (blue)/benomyl-resistant colonies plus in DMSO using a Hamilton 50 ~1 syringe with a a GUS-negative (benomyl-resistant) colony and an repeating dispenser. The stability of the integrated untransformed colony were selected and assayed genes after infection was tested by taking blood spectrophotometrically for GUS. Activities in the samples from infected insects [12] and plating onto blue colonies varied between 0.5 and 1.3 U (mg 292 R.J. St. Leger et al. / FEM.5 Microbiology Letters 131 (1995) 289-294

protein) l as compared with < 0.002 in the GUS- pBENA3, we selected for benomyl resistance or negative colonies. screened for blue colonies on non-selective plates Biolistic transformation protocols for M. aniso- containing X-glut. A total of 63 GUS expressing pliae were optimized by adapting methods success- colonies were obtained from three similar experi- fully used for other fungi [15,16]. Direct bombard- ments using non-selective media. These colonies ment on plates yielded transformation frequencies of were transferred to fresh X-glut plates with or with- 5, 1 and 0.2 transformants per pug DNA with 1 X 107, out added benomyl (5 /Lg ml-‘). All GUS activity Downloaded from https://academic.oup.com/femsle/article/131/3/289/512137 by guest on 28 September 2021 2 X 106, and 2 X 10’ conidia/plate, respectively. was lost during development on fresh X-glut plates, The frequency of co-transformation (1 X lo7 coni- indicating that stable gene integration had not oc- dia/plate) with pBENA3 and pNOM102 at a molar curred. Furthermore, none of these colonies were ratio of 1:3 was 12%. benomyl-resistant. Evidently, integration and full expression of the resistance gene in protoplasts of M. 3.2. Mitotic stabilio anisopliae requires much time since transformants appeared in 12-22 days on selective media as com- Transformed colonies were visible on selective (5 pared to 2-3 days for colonies to appear in non- pg benomyl ml-‘) medium 11 days after plating and selective media. This supports the conclusion of continued to appear for up to 22 days. The stability Mijnke and Schafer [17] that the limiting step in of the benomyl-resistant phenotype was determined by measuring resistance to benomyl after three serial kb WT 1 2 3 4 5 6 7 8 transfers on non-selective medium. Seventeen out of 30 transformants obtained by electroporation lost their ability to grow on Czapek Dox agar/benomyl 9.4 - medium and were considered to be non-stable or abortive transformants. Likewise, 16 out of 30 trans- 2:: : formants obtained biolistically were non-stable. The GUS phenotype was originally selected for among benomyl-resistant transformants by transfer- ring to plates containing X-glut. When ten co-trans- 2.3 - formants were transferred to plates without benomyl 2.0 - (non-selective medium), six were stable after five serial transfers while four had lost the GUS phenotype after just two transfers. Following five serial transfers, the four non-stable transformants were transferred to a plate containing benomyl. Two of the 0.56 - transformants showed normal (resistant) growth but without return of the GUS phenotype. By contrast, when single spores from the four non-stable GUS Fig. 1. Southern blot and DNA hybridization analysis of M. transformants were subject to five serial transfers anisopliae GUS transformants obtained using electroporation or through selective ( + benomyl) plates, they retained the gene gun. Genomic DNA (5 Fg) from transformants using the blue phenotype. pBENA3 and pNOM102 was digested with Hind111 (for pBENA3) or Xhol (for pNOM102) which cleave pNOM102 and pBENA3 As the GUS gene is probably selectively neutral once, respectively, and separated electrophoretically in a 1.2% as a transformation marker, we tested whether it agarose gel. Following Southern blotting, the filter was probed would provide a means for non-selective transforma- with 32P-labelled pNOM102 or pBENA3. WT is the recipient tion of M. anisopliae which like other entomo- wild-type strain probed with both plasmids. Lanes 1 and 2 are pathogens is naturally resistant to most fungicides representative transformants from electroporation probed with pBENA3; lanes 3 and 4 are abortive (non-stable) transformants currently used as selectable markers. After transfor- probed with pNOM102; lanes 5-8 are stable GUS transformants mation of 5 X lo6 protoplasts (electroporation obtained from electroporation (5-7) or biolistically (8) and probed methodology) with 25 pg each of pNOM102 and with pNOM102. R.J. St. Leger et al. /FEMS Microbiology Letters I31 (1995) 289-294 293

mb For molecular analysis of transformants, DNA was isolated from a wild-type strain and four stable 5.7 - GUS-expressing transformants, and analysed by 4.6 - Southern hybridization (Fig. 1). A labelled GUS probe hybridized only to high molecular mass DNA 3.5 - in undigested DNA from transformants (data not shown), indicating that the vector integrated into the Downloaded from https://academic.oup.com/femsle/article/131/3/289/512137 by guest on 28 September 2021 genome. Since neither pBENA3 nor pNOM102 rec- ognizes any sequence in the recipient strain DNA, it is unlikely that homologous integration will have occurred. The multiple bands in DNA from two Ben+ transformants produced by hybridization with Fig. 2. Separation of M. anisopliae intact chromosomal DNA on pBENA3 suggests multiple integration at multiple . . a counter-clamped homogenous electric field gel (a) and analysis sites in the genome. Southern blot analysis of ini- of hybridization with 32P-labelled pNOM102 (b) of transformants tially GUS-positive transformants obtained from containing a single integration event (lane 1) and multiple integration events (lane 2). The pNOM102 plasmid was introduced into M. anisopliae by co-transformation at 3-fold (lane 1) and 20-fold (lane 2) excess with pBENA3.

transformation experiments is not the uptake, but the incorporation of introduced DNA into the fungal genome.

Fig. 4. Screening of blood samples from infected Bombyx mori Fig. 3. Appearance of Bombyx mori larvae infected with trans- larvae. for distinctive blue coloration (dark patches) of hyphae formed and untransformed M. anisopliae strains. At 72 h after during growth on media containing X-glut. Blood samples (50 infection, the larvae were injected under the cuticle with 50 pg ~1) from insects 72 h after infection with a stable GUS expressing X-glut (in 25 ~1 DMSO) and incubated at 37°C for 30 min. Top, BENR transformant were plated onto CDA containing benomyl (5 uninfected B. mori; middle, stable BenR transformant; bottom, pg ml-‘) and X-glut (20 pg ml-‘). Microscopic observations stable GUS expressing Bet? transformant. Expression of the GUS were made 48 h after plating using a Wild Heerbrugg binocular gene was visible through the cuticle as blue staining regions. microscope with a camera attachment. 294 H.J. St. Leger et al. / FEMS Microbiology Let&w 131 I1 9951 289-294 non-selective transformation showed an absence of glucuronidase activity to detect infection and measure hyphal all vector DNA. biomass in infected plant tissues. Mol. Plant Microbial. Int. 6. 521-525. There is no known sexual stage for M. anisopliae Dl St. Leger, R.J., May, B., Allee, L.L., Frank, D.C. and which precludes classical genetic tests to determine Roberts, D.W. (19921 Genetic differences in allozymes and in which chromosome integration took place. Using in formation of infection structures among isolates of the counter-clamped homogenous electric field elec- entomopathogenic fungus Metarhizium anisopliae. J. Inver- trophoresis, Shimizu et al. [lo] resolved the genome tebr. Pathol. 60, 89-101. [41 St. Leger, R.J., Frank, D.C., Roberts, D.W., Staples, R.J. Downloaded from https://academic.oup.com/femsle/article/131/3/289/512137 by guest on 28 September 2021 of M. anisopliae into seven chromosomal bands. ( 19921 Molecular cloning and regulatory analysis of the Hybridization analysis using a labelled GUS probe cuticle-degrading protease structural gene from the cnto- confirmed that the transforming DNA can integrate mopathogenic fungus Meturhizium anisopliae. Eur. J. at single or multiple unlinked sites in the genome Biochem. 204, 991-1001. (Fig. 2) IS1 Goettel, M.S., St. Leger, R.J., Bhairi, S., Jung, M.K., Gak- ley, B.R. and Staples, R.C. (1989) Transformation of the entomopathogenic fungus Metarhizium anisopliae using the 3.3. Pathogenicity Aspergillus nidulans benA gene. Curr. Genet. 17, 129-132. Lb1 Chakraborty, B.W., Patterson, N.A. and Kapoor, M. (19911 Five stable co-transformants (Ben ‘---GUS + ) re- An electroporation-based system for high-efficiency transformation of a germinated conidia of filamentous fungi. Can. J. tained their pathogenicity towards Bombyx mori lar- Microbial. 37, 858-863. vae after the larvae were infected with 10’ spores [71 Goldman, G.H.. Van Montagu, M. and Herrera-Estrella, A. and incubated at 27°C for 3 days. When these insects ( 19901 Transformation of Trichoderma harzianum by high- were injected with X-glut as a histochemical sub- voltage electric probe. Curr. Genet. 17, 1169-1174. strate, the area of invasion was stained blue, marking [81 Sambrook, J., Fritsch. E.F. and Maniatis, T. (1989) Molecu- lar Cloning: A Laboratory Manual, 2nd edn. Cold Spring the presence of the transgenic fungus (Fig. 3). Fun- Harbor Laboratory, Cold Spring Harbor, NY. gal propagules in blood samples from infected in- LSI Feinberg, A.P. and Vogelstein, B. (1984) A technique for sects also retained their resistance to benomyl and radiolabelling DNA restriction endonuclease fragments to the blue phenotype (Fig. 41, indicating that the inte- high specific activity. Anal. Biochem. 137, 266-267. grated plasmid sequences are maintained during in- [lOI Shimizu, S., Arai, Y. and Matsumoto, T. (1992) Elec- trophoretic karyotype of Meturhizium anisopliae. J. Inver- fection processes. These findings indicate that GUS tebr. Pathol. 60, 185-187. expression will provide a practical means of ‘tag- [Ill St. Leger, R.J., Charnley, A.K. and Cooper, R.M. (1986) ging’ introduced fungal strains applied as mycoinsec- Cuticle-degrading enzymes of entomopathogenic fungi: syn- ticides so as to distinguish them from the wild-type thesis in culture on cuticle. J. Invertebr. Pathol. 48, 85-95. and enable ‘tracking’ of the marked organism after [121 St. Leger, R.J., Durrands, P.K., Charnley, A.K. and Cooper, R.M. (1988) Role of extracellular chymoelastase in the viru- introduction in the environment. lence of Metarhizium anisopliae for Manduca sexta. J. Invertebr. Pathol. 52, 285-293. [131 Bemier, L., Cooper, R.M., Charnley, A.K. and Clarkson, Acknowledgements D.M. (1988) Transformation of the entomopathogenic fungus Metarhizium anisopliue to benomyl resistance. FEMS Micro- biol. Lett. 60, 261-266. This work was supported in part by a grant (92- [141 Pfeifer, T.A. and Khachatourians, G.G. (1992) Beauveria 37302-7791) from the USDA Competitive Research bassiuna protoplast regeneration and transformation using Grants Office. electroporation. Appl. Microbial. Biotechnol. 38, 376-381. [lSl Armaleo, D., Ye, G.W., Klein, T.M., Shark, K.B., Sanford, J.C. and Johnston, S.A. (1990) Biolistic nuclear transformation of Saccharomyces cereuisiae and other fungi. Curr. References Genet. 12, 97-103. [I61 Bhairi, S.M. and Staples, R.C. (1992) Transient expression of Ill St. Leger, R.J. (1993) Biology and mechanisms of invasion the P-glucuronidase gene introduced into Uromyces appen- of deuteromycete fungal pathogens. In: Parasites and diculatus uredospores by particle bombardment. Phy- Pathogens of Insects, Vol. 2 (Beckage, N.C., Thompson. topathology 82, 986-989. S.N. and Federici, B.A., Eds.1, pp. 21 l-229. Academic [171 Monke, E. and Shafer, W. (19931 Transient and stable gene Press, New York, NY. expression in the fungal maize pathogen Cochliobolus het- 121Oliver, R.P., Farman, M.L., Jones, J.D.G., Hammond-Kosack, erostrophus after transformation with the P-glucuronidase K.E. (1993) Use of fungal transformants expressing p- (GUS) gene. Mol. Gen. Genet. 241, 73-80.