Genetic Transformation and Mutagenesis of the Entomopathogenic Fungus Paecilomyces fumosoroseus

Journal of Invertebrate Pathology 74, 281–288 (1999) Article ID jipa.1999.4885, available online at http://www.idealibrary.com on

Genetic Transformation and Mutagenesis of the Entomopathogenic Fungus Paecilomyces fumosoroseus Frank A. Cantone and John D. Vandenberg1 USDA Agricultural Research Service, U. S. Plant, Soil and Nutrition Laboratory, Tower Road, Ithaca, New York 14853 Received March 22, 1999; accepted June 8, 1999

We have developed a DNA-mediated transformation system for the entomopathogenic fungus Paecilomyces fumosoroseus based on resistance to the herbicide glufosinate ammonium. The resistance is provided by the bar gene from Streptomyces hygroscopicus and is under the control of the Aspergillus nidulans trpC promoter and terminator sequences. Frequencies of up to 110 transformants/␮g of linearized plasmid DNA were obtained aided by the addition of the nuclease inhibitor, aurintricarboxylic acid (ATA). The transformants were stable for drug resistance for five consecutive vegetative transfers under selective and nonselective conditions. Southern analyses revealed that the transforming DNA integrated into the P. fumosoroseus genome in single and multiple copies. Single-copy integration events were generated with higher efficiency by restriction enzyme-mediated integration (REMI), although rates of transformation were generally not as high as those treatments containing ATA. Two mutant strains altered in sporulation capacity and virulence toward the Russian wheat aphid were recovered using this approach. This transformationbased manipulation of P. fumosoroseus will facilitate insertional mutagenesis and the functional analysis of various genes. Key Words: Paecilomyces fumosoroseus; genetic transformation; REMI mutagenesis; Streptomyces hygroscopicus bar gene; glufosinate ammonium; Russian wheat aphid; Diuraphis noxia.

INTRODUCTION

The filamentous fungus Paecilomyces fumosoroseus is a common insect pathogen and soilborne organism that has been isolated from a wide variety of insects from different orders located throughout the world (Humber, 1992). Because of its potential to cause epizootics naturally, P. fumosoroseus is considered to be

1 To whom correspondence should be addressed. Fax: (607) 2551132. E-mail: [email protected].

a good candidate for microbial control of insect pests (Jackson et al., 1997). The fungus has recently been genetically characterized by vegetative compatibility groupings and RAPD-PCR fingerprint patterns, which revealed broad variability (Cantone and Vandenberg, 1998; Tigano-Milani et al., 1995). However, limited information on the processes and genes responsible for pathogenicity and virulence restricts our capacity to manipulate and make targeted improvements of this fungus for the purpose of biological control. Because the fungus has no known sexual stage, functional analyses of genes will require a transformation-based technique. Recently, Barreto et al. (1997) described the transformation of P. fumosoroseus to hygromycin resistance using a PEG-based protoplast method and microprojectile bombardment of intact conidia. Transformation systems using the bar gene from Streptomyces hygroscopicus, which confers resistance to the nonselective herbicides bialaphos and glufosinate ammonium, have been developed for fungi such as Cercospora kikuchii (Upchurch et al., 1994), Neurospora crassa (Avalos et al., 1989), and Pleurotus ostreatus (Yanai et al., 1996). These herbicides contain the active ingredient phosphinothricin, an analogue of glutamic acid, and inhibit the activity of glutamine synthetase, killing the organism by ammonia poisoning (Avalos et al., 1989). The bar gene encodes the enzyme phosphinothricin acetyltransferase, which inactivates phosphinothricin by acetylation. In this paper, we report the transformation of P. fumosoroseus to glufosinate ammonium resistance. Sites of DNA integration were examined and mitotic stability of the transformants was assessed. We also tested the addition of restriction enzymes to the reaction mixture to increase the rate of transformation and introduce randomly tagged mutations in the fungal genome, a procedure known as Restriction Enzyme-Mediated Integration or REMI (Kupsa and Loomis, 1992; Schiestl and Petes, 1991). Finally, we evaluated selected transformants for changes in sporulation capacity and virulence.

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Protoplast Isolation Single-spore isolate 5540 (United States Department of Agriculture, ARS Collection of Entomopathogenic Fungal Cultures, Ithaca, NY) of P. fumosoroseus was grown on Saboraud’s dextrose agar medium supplemented with 1% yeast extract (SDAY; pH adjusted to 7.2 with NaOH) for 14 days at 25°C with a 15:9 h light:dark cycle. Conidial suspensions were prepared in sterile 0.1% Tween 80 and used to inoculate submerged cultures at a final concentration of 5 ⫻ 104 conidia ml⫺1. Liquid complete medium was composed of casamino acids, mineral salts, vitamins, and glucose prepared according to Jackson et al. (1997). Liquid cultures (100 ml of medium in 250-ml Erlenmeyer flasks) were grown for 4 days in a shaker-incubator at 28°C and 300 rpm. Blastospores (submerged conidia) were harvested by centrifugation (1500g for 5 min at 4°C) and resuspended in 100 ml of sterile 25 mM ␤-mercaptoethanol and 5 mM EDTA and incubated for 20 min at 25°C. Protoplasts were isolated using the procedures of Churchill et al. (1990) with the following modifications. Blastospores were pelleted at 1500 g for 10 min at 4°C and washed with 100–150 ml of 0.6 M MgSO4. This step was repeated one additional time. Spore walls were digested for 2 to 3 h with 10 mg ml⫺1 Mureinase (United States Biochemical, Cleveland, OH) in 100 ml of osmotic medium (1.2 M MgSO4 in 10 mM NaH2PO4 ). Ten-milliliter aliquots of the protoplast suspension were mixed with 10 ml of osmotic medium in 50-ml centrifuge tubes. The suspension was very gently overlaid with 20 ml of trapping buffer (0.4 M sorbitol, 100 mM Tris, pH 8) followed by centrifugation at 1750g for 10 min at 4°C. Protoplasts were removed from the interface with a sterile Pasteur pipette and transferred to a clean 50-ml tube. Trapping buffer was removed and protoplast suspensions were overlaid with fresh trapping buffer and pelleted as before. Protoplasts were pooled, mixed with two volumes of 1 M sorbitol, and pelleted as before. The supernatant was removed to clean tubes and protoplasts were pelleted again. The protoplasts were combined, resuspended in 30–50 ml of 1 M sorbitol, and pelleted as before. The pellet was resuspended in 30–50 ml of STC (1 M sorbitol, 50 mM CaCl2, 100 mM Tris, pH 8), subjected to centrifugation as before, and resuspended in 10–20 ml of STC, and the concentration of protoplasts was determined. The protoplasts were diluted to a final concentration of 1 ⫻ 109 ml⫺1 in 80% STC/20% PTC (60% polyethylene glycol 3350, 100 mM Tris, pH 8, 50 mM CaCl2 ). Dimethylsulfoxide was added to a concentration of 1%, and aliquots of protoplasts were transferred to cryovials. The cryovials were placed in isopropanol baths at 4°C for at least 1 h and then the baths containing the vials were

transferred to ⫺80°C for storage. Vials containing protoplasts were thawed on ice just prior to use. Transformation The following plasmids containing the bar gene from S. hygroscopicus were screened for their ability to transform protoplasts of P. fumosoroseus to glufosinate ammonium resistance: pBARGEM7-2 (Fungal Genetics Stock Center, University of Kansas Medical Center, Kansas City, KS), pGPDH-Bar.1 (Dr. Olen Yoder, Department of Plant Pathology, Cornell University, Ithaca, NY), and pJA4 (Dr. Mary Case, Department of Genetics, University of Georgia, Athens, GA). Plasmid DNA was digested with restriction enzyme (which did not cut within any functional sequences), subjected to phenol/ chloroform extraction, precipitated with ethanol, dried, and resuspended in TE buffer (10 mM Tris, pH 8, and 1 mM EDTA). Initial transformation conditions included 10 µg of linear plasmid, 1 mM spermidine, 125 ng heparin, and 50 µg salmon sperm DNA in 25 µl combined with 100 µl of protoplasts (1 ⫻ 108 ) in STC and incubated on ice for 15 min. Next, 125 µl of PTC was added, and protoplasts were incubated at room temperature (22–25°C) for about 20 min. Ten volumes of liquid minimal medium (Correll et al., 1987) supplemented to a final concentration of 0.6 M sucrose were added to the protoplasts. Aliquots of this suspension were then added to cooled, molten minimal medium with 0.6 M sucrose, and protoplasts were allowed to regenerate for about 1 h. Plates were overlaid with minimal medium with 0.6 M sucrose containing 25 µg/ml of glufosinate ammonium (Ignite [20% active ingredient]; AgrEvo USA, Wilmington, DE) and incubated at 25°C with a 15:9 h light:dark cycle. Optimized conditions included 25 µg heparin and 1 mM aurintricarboxylic acid (ATA) (Churchill et al., 1990) along with 1 mM spermidine and 50 µg salmon sperm DNA. Protoplasts were allowed to regenerate for 15 h in minimal medium before they were overlaid with 50 µg/ml of glufosinate ammonium. Individual transformants were transferred to fresh selective medium before further analysis. For restriction enzyme-mediated integration (REMI), individual restriction enzymes (BamHI, EcoRI, and XhoI at 10, 25, 50, or 200 units per 125-µl reaction) were added to the transformation mixture just prior to the addition of the protoplasts. Extraction of Genomic DNA Blastospores of P. fumosoroseus were grown for 4 days in Saboraud’s dextrose broth medium, harvested by centrifugation at 1750g for 5 min, frozen in a dry ice/ethanol bath, and lyophilized. Genomic DNA was extracted from the blastospores according to the proce-

TRANSFORMATION AND MUTAGENESIS OF P. fumosoroseus

dure of Kim et al. (1990) with some modifications. Proteinase K was eliminated from the lysis buffer, and ␤-mercaptoethanol was added at a final concentration of 0.2% along with the addition of SDS. Following the addition of CTAB, samples were extracted three times with chloroform/isoamyl alcohol (CHCl3/IAA) (24:1) and layers separated by centrifugation at 5000 g for 5 min. The DNA was precipitated with 2 vol of 100% ethanol at ⫺20°C. The DNA was centrifuged at 1750g for 5 min, the supernatant was discarded, and the pellet was rinsed with 70% ethanol and dried under vacuum. The pellet was resuspended in 500 µl of TE and the RNA in the preparation was digested with RNase-Plus (58 to 38, Inc., Boulder, CO) at a dilution of 1:125 and incubated at 37°C for at least 2 h. The DNA was transferred to 1.5-ml tubes and extracted with an equal vol of phenol/ CHCl3. The layers were separated by centrifugation at 13,000 rpm for 10 min and the upper aqueous layer was removed to a 2.2-ml tube. Two vol of 100% ethanol and 5 M NaCl (to a final concentration of 1.5 M) were added and the DNA was precipitated at ⫺20°C. The DNA was pelleted at 13,000 rpm for 10 min, the supernatant was discarded, and the pellet was washed with 70% ethanol. The pellet was dried under vacuum for several min and resuspended in 500 µl of TE buffer. Southern Analyses Genomic DNA (7–10 µg) of individual transformants was digested with one of three restriction enzymes (BamHI, EcoRI, or XhoI) according to supplier’s directions (New England Biolabs, Beverly, MA), and electrophoretically separated on 0.8% agarose (FMC BioProducts, Rockland, ME) gels in 1⫻ TBE buffer (89 mM Tris–borate, 2 mM EDTA). The DNA was transferred onto Nytran nylon membranes (Schleicher & Schuell, Keene, NH) with an S&S TurboBlotter transfer system using 3 M NaCl and 8 mM NaOH. Hybridizations included 25 ng of either the entire linearized pBARGEM7-2 plasmid or a 1.1-kb XbaI–NsiI fragment of pBARGEM7-2 that contains the Aspergillus nidulans trp C promoter and bar gene from S. hygroscopicus. The probes were labeled with 32P-dCTP (3000 Ci per mmol; DuPont Biomedical, Boston, MA) by random priming with the RadPrime DNA labeling system (GIBCO BRL, Gaithersburg, MD) for 30–60 min at 37°C. Hybridizations were carried out in 10–15 ml of buffer (50% formamide, 7% SDS, 0.25 M sodium phosphate (pH 7.2), 1 mM EDTA) in a Micro Hybridization Oven (Bellco Glass, Inc., Vineland, NJ) for 16–18 h at 42°C. Membranes were washed one time with 2⫻ SSPE/0.1% SDS at 42°C, one wash with 0.1⫻ SSPE/0.5% SDS at 42°C, and a final wash with 0.1⫻ SSPE/0.5% SDS at 65°C, each for 20–30 min. Membranes were exposed to Kodak BioMax MS film (Eastman Kodak Co., Rochester, NY) at ⫺80°C.

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Analysis of Selected Transformants Putative transformants were transferred to 12-well culture plates containing fresh minimal medium with 50 µg/ml glufosinate ammonium. Two strains with distinct alterations in culture appearance and sporulation capacity, 20-A5 and 55-A3, were selected for further analyses. Small agar plugs (2–3 mm) from cultures grown on minimal medium were transferred to 90-mm plates containing 20 ml of SDAY medium in triplicate. The cultures were grown for 21 days at 25°C with a 15:9 h light:dark cycle. Colony characteristics were noted and diameters were measured, and the entire contents of the plates were blended in a Waring stainless steel blender jar with 100 ml of 0.1% Tween 80 for 30 s (two 15-s bursts). The number of conidia per plate was estimated using hemacytometer counts. Insect bioassays of transformants 20-A5 and 55-A3 were done against the Russian wheat aphid, Diuraphis noxia, according to the method of Vandenberg (1996). Insects were inoculated 15 per dish with a computercontrolled sprayer (Burkard Ltd., Rickamsworth, England) at a dosage of approximately 100 spores per square centimeter. Aphids were maintained on excised barley leaves and monitored daily for survival. Upon death, cadavers were removed and incubated on water agar until sporulation to verify diagnosis. Reisolation from selected cadavers was used to verify recovery of the same strain used for inoculation. Each assay was done twice. Eight cadavers, resulting from inoculation with either wild type 5540 or transformants 20-A5 and 55-A3, were placed individually in 1.5-ml tubes and vortexed in 0.2 ml (transformants) or 0.75 ml (wild type) of 0.1% Tween 80. Conidia were then counted in a hemacytometer to estimate the number of conidia per cadaver. Analysis of variance was used to test for differences among strains in sporulation, both in vitro and in vivo, and aphid survival times. RESULTS AND DISCUSSION

P. fumosoroseus was sensitive to glufosinate ammonium in a preliminary test. Agar plugs from isolate 5540 were inoculated onto minimal medium or minimal medium supplemented with 50 µg/ml glufosinate ammonium. After 1 week no growth was observed on medium supplemented with glufosinate ammonium, whereas colonies on unsupplemented control plates were 20 mm in diameter. Initial tests to identify a suitable selection agent revealed that our strain of P. fumosoroseus was not inhibited when inoculated on minimal agar medium supplemented with up to 500 µg/ml of the antibiotic hygromycin B (data not shown). In contrast, Barreto et al. (1997) added 150 µg/ml of hygromycin to their selection media to obtain transformants of P. fumosoroseus. These conflicting results are not surprising since

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FIG. 1. Southern blot analysis of glufosinate ammonium-resistant isolates of Paecilomyces fumosoroseus transformed with linearized pBARGEM7-2. DNA (7 µg/lane) was separated by electrophoresis on a 0.8% agarose gel, transferred to Nytran membranes, and hybridized with a 32P-labeled 1.1-kb XbaI–NsiI fragment of pBARGEM7-2 that contains the Aspergillus nidulans trp C promoter and bar gene from Streptomyces hygroscopicus. Lanes 1–16, DNA from eight transformants; odd-numbered lanes contain uncut DNA, whereas even-numbered lanes contain DNA digested with EcoRI. Lane 17 contains uncut DNA from the untransformed wild-type strain 5540. Lane 18 contains pBARGEM7-2 digested with EcoRI. DNA molecular size markers (in kb) are indicated on the right.

individual strains can vary greatly in their response to various inhibitory agents. Transformants resistant to glufosinate ammonium were obtained with each plasmid tested and emerged from the selective minimal medium within 7–10 days. Frequencies under nonoptimized conditions were 15, 10, and 3 transformants/µg of linear plasmid DNA, respectively, for pBARGEM7-2, pGPDH-Bar.1, and pJA4. Transformations under optimized conditions yielded 69 and 46 transformants/µg of DNA for pBARGEM7-2 and pGPDH-Bar.1, respectively. Furthermore, the timing of overlay with selective medium was important to achieve maximum numbers of transformants. Initially, protoplasts were allowed to regenerate for 1 h before overlaying with selective medium. However, the number of transformants increased to 110/µg of EcoRIlinearized pBARGEM7-2 if protoplasts were allowed to regenerate for about 15 h before overlaying with glufosinate ammonium. This along with the addition of the endonuclease inhibitor, ATA, are probably responsible for the rise in transformation frequency. The inhibitor prevents the action of restriction enzymes in vitro (data not shown) and presumably inhibits the action of nucleases carried over during protoplast formation. The plasmid pBARGEM7-2, which has the bar gene under the control of the A. nidulans trpC promoter and terminator sequences, was used for all subsequent transformation reactions. We selected Paecilomyces transformants resistant to glufosinate ammonium for Southern analysis to confirm integration of EcoRI-linearized pBARGEM7-2. Genomic DNA from eight transformants hybridized to a 32P-labeled 1.1-kb XbaI–NsiI fragment that contains the trp C promoter and bar gene (Fig. 1). Hybridization to only undigested high-molecular-weight DNA (odd number lanes in Fig. 1) verified that the plasmid integrated into the fungal genome. DNA digested with EcoRI displayed one to several hybridizing bands suggesting single (e.g., Fig. 1, lane 4) and multiple (e.g., Fig. 1, lane 10) integrations of the plasmid. The bar gene/trpC promoter probe did not hybridize to genomic

DNA from untransformed wild-type isolate 5540 (lane 17 in Fig. 1). We cultured eight different transformants through five consecutive vegetative transfers on selective minimal medium and nonselective SDAY and probed genomic DNA with the entire linearized pBARGEM7-2. Most of the transformants were mitotically stable, showing no apparent DNA rearrangements, although changes in band intensity were observed (lanes 3 and 7, Figs. 2A and 2B), perhaps due to changes in plasmid copy number. One transformant displayed an altered hybridization pattern (lane 1, Figs. 2A and 2B), whereas another transformant (lane 6, Figs. 2A and 2B) lost its ability to grow on selective media, probably due to loss of the plasmid.

FIG. 2. Southern blot analyses of eight glufosinate ammoniumresistant isolates of Paecilomyces fumosoroseus transformed with linearized pBARGEM7-2 and grown for five consecutive generations on selective (A) and nonselective media (B). DNA was digested with EcoRI and separated by electrophoresis on 0.8% agarose gels. After transfer to Nytran membranes, DNA was hybridized with linearized pBARGEM7-2 labeled with 32P. DNA molecular size markers (in kb) are indicated on the left.

TRANSFORMATION AND MUTAGENESIS OF P. fumosoroseus

The addition of a restriction enzyme during the transformation procedure (restriction enzyme-mediated integration or REMI) results in the cleavage of chromosomal DNA. This is followed by ligation repair at the recognition sites that are occasionally interrupted by insertion of the plasmid (Kupsa and Loomis, 1992; Schiestl and Petes, 1991). The REMI procedure has significantly increased the number of transformants recovered (Kupsa and Loomis, 1992; Lu et al., 1994; Schiestl and Petes, 1991; Shi et al., 1995) in other fungal systems studied. Additionally, it has been used to target the transforming plasmid to corresponding restriction sites to achieve tagged mutagenesis (Bo¨lker et al., 1995; Kupsa and Loomis, 1992; Lu et al., 1994; Schiestl and Petes, 1991; Shi et al., 1995). We tested this modified transformation procedure with EcoRIlinearized pBARGEM7-2 along with the restriction enzyme EcoRI. Because ATA prevents the activity of restriction enzymes in vitro (data not shown), it was excluded from the REMI transformation mixtures. In the first experiment, addition of high concentrations of EcoRI restriction enzyme (200 units/reaction) decreased transformation frequencies compared to lower concentrations of enzyme (50 units) and non-REMI treatments (Table 1, Experiment 1). When we decreased concentrations of EcoRI we obtained up to 50-fold higher transformation rates than in non-REMI transformation without ATA (Table 1, Experiment 3). REMI with EcoRI included at 10 or 25 units/reaction generally yielded numbers of transformants comparable to those seen with ATA included in the transformation mix (Table 1, Experiments 2 and 3). Similarly, Bo¨lker et al. (1995) observed no change in transformation frequency in Ustilago maydis when the enzyme BamHI was included in their transformation reactions.

TABLE 1 The Number of Transformants of Paecilomyces fumosoroseus Obtained Following Transformation with Linearized pBARGEM7-2 Transformation method REMI a

Plasmid alone Experiment a

With ATA b

Without ATA

10 c

25

50

200

1 2 3 4 5

69 d 73 110 82 89

ND e ND 2 3 6

ND 96 27 10 30

ND 70 102 5 23

44 31 ND ND ND

16 ND ND ND ND

a The enzyme used to linearize the plasmid was the same enzyme used for REMI. EcoR1 was used in Experiments 1–3; BamH1 was used in Experiment 4; Xho1 was used in Experiment 5. b Aurintricarboxylic acid, 1 mM (nuclease inhibitor). c Units of enzyme per reaction. d Number of transformants per µg plasmid DNA. e Not determined.

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We also tested the addition of the restriction enzymes BamHI and XhoI at 10 or 25 units/reaction and observed that REMI transformation rates were increased several fold over those treatments without ATA. However, unlike REMI with EcoRI, we were unable to achieve efficiencies similar to ATA-supplemented reactions (Table 1, Experiments 4 and 5). Perhaps the concentrations of BamHI and XhoI enzymes used were still too high, resulting in higher numbers of putative transformants that contained gross chromosomal rearrangements (e.g., translocations, deletions) leading ultimately to cell death (King et al., 1993; Schiestl and Petes, 1991). Southern hybridization analysis revealed that, for transformants generated with REMI in the presence of XhoI, all but one appeared to integrate at single sites within the genome (Fig. 3A). Five of the transformants yielded hybridizing fragments the size of pBARGEM7-2, indicating that insertions at XhoI sites were restored upon integration (Fig. 3A, lanes 3 and 5–8). The remaining transformants displayed hybridizing bands larger than pBARGEM7-2, suggesting that one or both restriction sites were destroyed upon integration. The hybridization pattern displayed in Fig. 3A, lane 3 illustrated by the presence of a pBARGEM7-2sized band along with a single larger fragment probably arose from a tandem integration in which the XhoI site of one flank was not maintained. Alternatively, independent integration events may have occurred at two sites, one of which lost the XhoI restriction site. In lane 10 (Fig. 3A) the transformant either lost the plasmid during growth in nonselective medium or arose spontaneously. Similar types of integrations with restoration of restriction sites (Fig. 3B, lanes 5 and 9; Fig. 3C, lane 6) or without (Fig. 3B, lanes 2–4, 6–8, and 10; Fig. 3C, lanes 2–5 and 9) were obtained with transformants generated in the presence of BamHI and EcoRI. REMI transformation in the presence of EcoRI resulted in several transformants (Fig. 3C, lanes 7, 8, and 10) with multiple sites of plasmid integration in the genome. The appearance of bands larger than pBARGEM7-2 in REMI transformations may have resulted from degradation of plasmid ends before ligation repair (Bo¨lker et al., 1995) or may have occurred because of recombination (Schiestl and Petes, 1991). Despite the failure to regenerate restriction sites, integrations may have occurred at genomic sites corresponding to the REMI enzymes as observed in Magnaporthe grisea (Sweigard, 1996). The analysis of transformants generated with linear plasmid DNA under non-REMI conditions revealed illegitimate integration in nearly all of the transformants tested (Figs. 3D–3F). Two transformants displayed plasmid-sized fragments upon hybridization (Fig. 3E, lane 9; Fig. 3F, lane 8), whereas several transformants exhibited multiple integrations (Fig.

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FIG. 3. Southern blot analyses of glufosinate ammonium-resistant colonies of Paecilomyces fumosoroseus transformed with linearized pBARGEM7-2 under REMI (A, B, and C) or non-REMI (D, E, and F) conditions. With REMI the same enzyme was used to linearize the plasmid, transform protoplasts, and digest the genomic DNA. With non-REMI the same enzyme was used to linearize the plasmid and digest the genomic DNA. DNA (10 µg) from individual transformants was digested with XhoI (A and D), BamHI (B and E), or EcoRI (C and F) and separated by electrophoresis on 0.8% agarose gels. After transfer to Nytran membranes DNA was hybridized with linearized pBARGEM7-2 labeled with 32P. In each panel, Lane 1 contains pBARGEM7-2, lanes 2–11 contain DNA from 10 unique transformants, and Lane 12 contains DNA from untransformed wild-type strain 5540, all digested with the respective enzymes indicated above. DNA molecular size markers (in kb) are indicated on the left.

3D, lanes 3, 5, and 10; Fig. 3F, lanes 7 and 9) Figure 3F, lane 8 shows a complex hybridization pattern, perhaps resulting from rearrangement of the plasmid at the insertion sites. Although we obtained higher REMI transformation efficiencies in the presence of EcoRI than with the other enzymes tested, in a very small proportion of transformants the restriction enzyme sites flanking the insertion sites were preserved. In contrast, REMI transformations in the presence of XhoI gave us proportionally higher numbers of integrations in which restriction sites were maintained, compared with transformation efficiency. These results agree with Shi et al. (1995), in which transformation efficiency in M. grisea was correlated with the frequency of integration events yielding plasmid-sized fragments and with the particular enzyme used. However, the most valuable feature of the REMI procedure may be the targeting of single copies of transforming plasmid to recognition sites, as observed with U. maydis (Bo¨lker et al., 1995). The inactivated, tagged gene can be recovered by digesting the genomic DNA with an enzyme that cuts outside the plasmid. This is followed by ligation of the fragment ends and transformation of the plasmid into Escherichia coli. Recovery of genomic sequences flanking the single inserted plasmid is greatly enhanced because of known

enzyme sites, restriction sites used to target and retrieve the plasmid, surrounding the tagged gene (Shi et al., 1995; Sweigard, 1996). This results in the generation of a pre-made disruption vector that can be used to transform the wild-type strain to disrupt specific gene function. In this way one can verify that the original insertion caused the specific aberration in the phenotype via mutation—an important consideration for fungi lacking a sexual stage (Sweigard, 1996). Additionally, restriction enzymes with different specificities can be incorporated in the transformation reaction to extend the number of genes that can be tagged. We successfully used this transformation procedure to introduce random mutations into P. fumosoroseus. Two mutants resistant to glufosinate ammonium, 20-A5 and 55-A3, exhibited changes in colony morphology, growth rate, sporulation, and virulence for the Russian wheat aphid. The two transformants produced very little aerial hyphae and displayed significant infolding on the surface of colonies growing on SDAY, in contrast to the floccose appearance of wild type 5540 (Fig. 4). The pink/wine shaded pigments of the wild type were altered to a tan or brownish shade in the transformants. Both transformants growing on SDAY sporulated significantly less than wild type 5540 (Table 2). Percentage mortality for Russian wheat aphids did

TRANSFORMATION AND MUTAGENESIS OF P. fumosoroseus

FIG. 4. Colonies of Paecilomyces fumosoroseus wild type 5540 (right) and transformants 20-A5 (top left) and 55-A3 (bottom left) grown for 21 days on Saboraud’s dextrose agar medium supplemented with 1% yeast extract.

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killed by 20-A5 and 55-A3, hyphal growth and subsequent sporulation was slower than with wild type (Fig. 5), and the transformants supported fewer conidia per cadaver (Table 2). Although we did not observe an alteration in pathogenicity, the results indicate reduced virulence of the two transformants and imply potential epizootiological changes in the disease. The increased time to sporulate and the lower pathogen density on cadavers decrease the probability of contact between a pathogen and a host, thus reducing the rate of horizontal transmission and diminishing the efficacy of the biocontrol agent (Fuxa, 1987; Glare, 1991; Tanada and Kaya, 1993). Southern hybridization analysis of the two transformants revealed that pBARGEM7-2 integrated at single sites in each genome (data not shown). The appearance of multiple phenotypic alterations in our transformants does not appear to be uncommon as Upchurch et al. (1994) also observed pleiomorphic changes with Cercospora kikuchii resulting from mutations of single genes. In conclusion, we have shown that protoplasts of P. fumosoroseus can be transformed to produce cultures resistant to glufosinate ammonium. The transforming plasmid integrates within the chromosomes, in single and multiple copies, and transformants appear to be stable under selective and nonselective growth conditions. Although the REMI procedure did not enhance transformation rates as with other fungi, it did appear

not vary significantly among the three strains (range 49 to 52% for two assays) but aphid survival times were significantly shorter for those inoculated with the wild type versus 20-A5 and slightly shorter for wild type versus 55-A3 (Table 2). Additionally, on aphid adults TABLE 2 Selected Phenotypic Traits of Wild Type and Transformed Strains of Paecilomyces fumosoroseus Strain

Sporulation in vitro a

Sporulation in vivo b

Aphid survival time c

Wild type 5540 Transformant 55A3 Transformant 20A5

10.1 (1.5) a 1.1 (0.1) b 1.4 (0.8) b

31.9 (2.6) a 7.1 (0.9) b 4.5 (0.6) b

4.4 (0.2) a 5.0 (0.2) b 4.8 (0.2) ab

a Average (SE) conidia ⫻ 103 /mm2 of culture surface for three replicate dishes. Analysis done using log10-transformed counts. Means followed by the same letter are not significantly different by analysis of variance, P ⬍ 0.05. b Average (SE) conidia ⫻ 105 per Russian wheat aphid cadaver for eight replicate cadavers. Analysis done using log10-transformed counts. Means followed by the same letter are not significantly different by analysis of variance, P ⬍ 0.05. c Average (SE) days to death for Russian wheat aphid adults inoculated with a dosage of approximately 100 spores/mm2. Combined results of two experiments and 120 aphids per isolate. Means followed by the same letter are not significantly different by analysis of variance, P ⬍ 0.05. Percentage mortality did not vary significantly among strains (average 51%).

FIG. 5. Cadavers of the Russian wheat aphid infected with Paecilomyces fumosoroseus wild type 5540 (1 and 2) and transformants 20-A5 (3 and 4) and 55-A3 (5 and 6) at 2 (odd-numbered panels) and 4 (even-numbered panels) days post-inoculation. Size marker in 1 indicates 1 mm. Insects in each panel were photographed at identical magnifications.

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to increase the number of single-site integrations of plasmid DNA, depending on the enzyme used. Two strains with altered phenotypes were recovered using this approach. We are currently using this transformation-based manipulation of P. fumosoroseus for insertional mutagenesis of additional mutants. ACKNOWLEDGMENTS We acknowledge Jennifer Williams, Jennifer McManus, and Mark Ramos for their excellent technical assistance; Dr. Alice Churchill and Jim Sweigard for their suggestions and valuable discussions; and Drs. Larry Dunkle, Robert Proctor, and Richard Staples for providing comments on the manuscript. We also thank Dr. John Lydon for discussion concerning glufosinate ammonium resistance and the AgrEvo USA Co. for donating a sample of Ignite.

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