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Mycol. Res. 108 (6): 654–661 (June 2004).

DOI: 10.1017/S0953756204009906

f The British Mycological Society

Printed in the United Kingdom.

654

Development of a transformation system for the nematophagous fungus Pochonia chlamydosporia

Simon D. ATKINS*, Tim H. MAUCHLINE, Brian R. KERRY and Penny R. HIRSCH

Rothamsted Research, Harpenden, Hertfordshire, AL5 2JQ, UK. E-mail : simon.atkins@bbsrc.ac.uk

Received 7 August 2003; accepted 2 March 2004.

The nematophagous fungus Pochonia chlamydosporia is a potential biocontrol agent against root knot and cyst nematodes. Genetic transformation of the fungus to introduce visual marker genes, novel traits, or changes in expression levels of endogenous genes, would greatly enhance understanding of its behaviour on nematode-infested roots and of its interactions with other soil and rhizosphere microorganisms. A transformation system for the introduction of novel genes into P. chlamydosporia has been developed. Methods to generate protoplasts, introduce DNA and regenerate transformed viable fungal mycelium have been optimised, using plasmids carrying the green fluorescent protein marker gene gfp and the hygromycin resistance gene hph. Cultures of P. chlamydosporia were resistant to high levels of a range of fungal inhibitors, including hygromycin, that are commonly used with dominant selectable marker genes in the transformation of other fungi. However, regenerating protoplasts transformed with hph could be selected by their ability to grow through an agar overlay containing 1 mg ml x1 hygromycin. Green fluorescence was observed in protoplasts and regenerating mycelium after transformation with gfp, but the GFP phenotype was lost on subculture. Maintenance of introduced genes was not stable, and during subculture, PCR assays indicated that the transformants lost both hph and gfp. When these genes were introduced on the same plasmid, segregation of hph and gfp was observed prior to their loss. It was unclear whether the introduced plasmids were able to replicate autonomously in P. chlamydosporia, or if they integrated transiently into the fungal genome. Possible reasons for the instability of the transformants are discussed.

INTRODUCTION

Pochonia chlamydosporia (Zare, Gams & Evans 2001; syn. Verticillium chlamydosporium) is a widespread and naturally occurring facultative fungal parasite of cyst and root-knot nematode eggs and has shown potential as a biological control agent of these nematodes in pot and microplot (De Leij & Kerry 1991, De Leij, Dennehy & Kerry 1993) and field trials (Atkins et al. 2003). The fungus occurs naturally in soil and can col- onise the rhizosphere of a range of plants to a varying degree (Bourne, Kerry & De Leij 1994). This is an important criterion in the selection of isolates as bio- logical control agents. The potential of P. chlamydosporia as a nematode biological control agent will become increasingly important when methyl bromide, a major nematicide for agriculture and horticulture, is phased out in 2005 (Thomason 1987), and other methods of control are investigated and implemented. It is likely to form part

* Corresponding author.

of an integrated pest management strategy (Atkins et al. 2003). Methods for the isolation, enumeration and identi- fication of the fungus from root and soil samples have been developed (Hirsch et al. 2001). A recently designed competitive PCR assay (Mauchline, Kerry & Hirsch 2002) has allowed more accurate quantification from soil samples, but these methods cannot inform on the physiological or developmental status of the fungus, which are of primary importance in under- standing the ecology of different isolates. To fully understand the tri-trophic interactions between the fungus, plant and nematode hosts, methods for in situ visualisation of the fungus are needed. Monoclonal antibodies have been developed that allow visualisation of the fungus on root samples (Hirsch et al. 2001), but have showed some cross-reactivity with other soil fungi, and further work is necessary to improve their specificity. The development of a transformation system to generate targeted deletions of candidate genes, or trans- genic fungi stably expressing a visible marker gene

S. D. Atkins and others

such as green fluorescent protein (GFP), would enable a more detailed assessment of the interactions with plant and nematode hosts. The reason for choosing gfp as a visual marker gene over other reporter genes, such as GUS (b-glucuronidase), is that it does not require either exogenous substrates, co-factors or antibodies for detection. Problems in substrate uptake, the necess- ity to fix, and hence kill the treated tissue, and cell permealization, especially in multicellular organisms, sometimes limits the use of certain reporter genes (Maor et al. 1998). All of these techniques are un- necessary for the detection of GFP and makes it an ideal visual marker gene for in situ studies. With the range of fluorescent proteins now available (Chalfie & Kain 1998) a transformation system would enable a number of isolates to be transformed with marker genes for different fluorescent proteins, which could subsequently be co-inoculated into soil and used to monitor fungal interactions in the rhizosphere. The gene encoding GFP, cloned from the jellyfish Aequorea victoria (Prasser et al. 1992) has been suc- cessfully expressed in a wide range of organisms including filamentous fungi (Lorang et al. 2001). Expression of gfp in filamentous fungi requires an efficient transformation system, a fungal promoter that satisfies the requirements of a given experimental objective, and a gfp variant that is efficiently trans- lated in fungi. A range of gfp mutants are reported in the literature offering enhanced expression or site- directed expression and the types and uses are reviewed by Kendall & Badminton (1998) and Lorang et al.

(2001).

Here, the development of a transformation system for the nematophagous fungus P. chlamydosporia is described and the transformation of protoplasts to express gfp and resistance to hygromycin.

MATERIAL AND METHODS

General methods

Pochonia chlamydosporia isolate 10 was revived from the Rothamsted Research culture collection. The iso- late was grown from freeze dried stocks on potato dextrose agar PDA (Oxoid, Basingstoke) and incu- bated at 28 xC. Fungal plates were stored at 4 x until needed, then sub-cultures were taken and used to in- oculate fresh PDA plates and Czapek Dox liquid broth (Oxoid). Liquid cultures were shaken at 130 rpm unless stated. All data were subjected to analysis using one way analysis of variance (ANOVA) using the Genstat programme (Genstat 5 Committee 1993). Significance (P) is below the 5% level of significance and the stan- dard error of deviation (sed) is recorded in the text.

Plasmids

The plasmid pDH33, described by Smith et al. (1990), contained the hygromycin resistance gene (hph) from

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Table 1. List of fungal inhibitors, solvents and maximum concentrations tested for inhibition of Pochonia chlamydosporia.

Inhibitor

Solvent

Maximum concentration tested mg ml x1

Hygromycin Puromycin Paromomycin Geneticin (G418) Blasticidin Phosphinothricin (PPT) Bleomycin Phleomycin Cyclohexamide Oligomycin Benomyl Thiabendazole Carbendazim N-phenylcarbamate (MDPC)

Water

1000

Ethanol

100

Water

500

Water

500

Water

300

Water

1000

Water

100

Water

100

Water

500

Ethanol

200

Ethanol

200

Ethanol

50

Ethanol

50

Methanol

50

Escherichia coli fused to the Aspergillus niger glucamy- lase gene promoter, and the A. nidulans trpC terminator for selection in fungi. The construct was ligated into the pBR322 cloning vector that contains an ampicillin resistance gene for propagation in E. coli. The expression vector pTEFEGFP, described by van den Wymelenberg et al. (1997), contained a red shifted mutant GFP cDNA fused to the Aureobasidium pull- ulans translation elongation factor promoter, and a terminator region derived from the Aspergillus awamori glucoamylase gene for expression in fungi. The con- struct was ligated into the pBluescript cloning vector containing the ampicillin resistance gene and lacZ gene under the control of the lac promoter for propagation in E. coli. The expression plasmid pCT74, described by Lorang et al. (2001), contained the GFP expression vector SGFP-TYG under regulation by the toxA gene pro- moter, and the hygromycin resistance gene hph under the regulation of the trpC promoter, ligated into the cloning vector pBluescript for propagation in E. coli. Plasmids were isolated from 16 h cultures of E. coli grown under selection in LB media using a Qiagen plasmid midi prep (Qiagen, Crawley), quantified by spectrophometry, and quality was assessed by gel elec- trophoresis and digestion with restriction enzymes.

Sensitivity to fungal inhibitors

Fungal inhibitors were added to corn meal agar (Oxoid) after dissolving in an appropriate solvent (Table 1). Compounds were obtained from Sigma (Poole) with the exceptions of benomyl (Dupont, Stevenage) and hygromycin (Melford Laboratories, Ipswich). A range of inhibitor concentrations, up to the maximum concentration shown in Table 1, were tested. These levels were in excess of ED 50 levels that have affected other fungi (Urban, Bhargava & Hamer 1999). Spores were washed from a fresh culture of P. chlamydosporia and added to each plate, and

Transformation of Pochonia chlamydosporia

sensitivity to the solvent was tested using plates con- taining the solvent alone. To demonstrate that the selective agent was not affected by the agar, cultures of Verticillium dahliae, V. tricorpus and V. albo-atrum (supplied by Simon Foster, Rothamsted Research) were added to agar plates containing hygromycin at a con- centration range of 0–200 mg ml x1 . V. dahliae had been previously shown to be sensitive to hygromycin (Do- binson 1995), and all three fungal species are related to Pochonia, which was previously classified in the same genus, Verticillium.

Development of a system for protoplast generation

Each step in the process was tested to optimise the generation of protoplasts from fungal mycelium. Pro- toplasts were examined after each treatment using Evans Blue staining (Coury et al. 1993).

Effect of osmotic buffer on protoplast generation

Spores of Pochonia chlamydosporia were washed from an actively growing culture on an agar plate and used to inoculate 100 ml Czapek Dox broth (Oxoid) in a 250 ml conical flask. Previous work (Atkins 2000) had shown that germinating spores yielded the highest level of protoplasts. The culture was incubated at 28 x for 48 h and the mycelium collected by centrifugation at 8 K g for 15 min at 4 x in sterile centrifugation bottles. Aliquots (1 g wet weight mycelium) were added to 10 ml sterile Osmotic Buffer (OM–10 mM Tris-HCl, pH 7.5) containing one of the following reagents dissolved in water (0.8 M sorbitol; 1 M sorbitol; 1.2 M sorbitol; 0.6 M KCl; 1 M sucrose; 1.2 M MgSO 4 ; 0.6 M NaCl; 0.7 M NaCl). Filter sterilised Novozyme 234 (Sigma) was added to each buffer at a concentration of 5 mg ml x1 . Each buffer was incubated at 28 x with gentle shaking, 80 rpm.

Effect of pre-incubation with b-mercaptoethanol

Pre-incubation of mycelium with b-mercaptoethanol, before addition of the lysing enzyme, had been shown to increase protoplast generation in some fungi (Davies 1985). Fungal spores were collected and grown as above. Mycelium was preincubated in 50 ml Buffer 1 (10 mM sodium phosphate, pH 7.5, 10 mM EDTA) containing 1% v/v b-mercaptoethanol in a 250 ml conical flask and incubated at 28 x for 1 h, with shaking at 150 rpm. A control of Buffer 1 containing no b- mercaptoethanol was run in parallel. Mycelium was collected after 2 h incubation by centrifugation for 15 min at 8 K g at 4 x. Mycelium was re-suspended in 50 ml OM containing 5 mg ml x1 Novozyme 234.

Effect of Novozyme 234 concentration on protoplast generation

Mycelium was pre-incubated in Buffer 1 containing 1% v/v b-mercaptoethanol, collected as above and

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re-suspended in 50 ml OM. A range of Noyozyme 234 concentrations (1, 5, 10, and 15 mg ml x1 ) was added to aliquots of mycelium and incubated as above.

Effect of length of incubation in Novozyme 234 on protoplast regeneration

Protoplasts were prepared as above and incubated in 10 mg ml x1 Novozyme. At hourly intervals, for 3 h, aliquots were removed and observed.

Transformation

Pochonia chlamydosporia was co-transformed with pDH33 and pTEFEGFP, or transformed with pCT74

alone. The protoplast production and transformation systems were optimised from the results of the exper- iments outlined above. Fungal spores were washed from

a two week culture grown on corn meal agar plates,

into 200 ml Czapek Dox liquid broth (Oxoid) in 1 litre

flasks for 48 h. Mycelium was collected by filtration through a sterile 30 mm nylon mesh (Lockertex, Cheshire), washed twice with sdH 2 O, once with Buffer 1 before re-suspension in 50 ml Buffer 1 and incubated

at 28 x with gentle agitation (80 rpm) for 1 h. Mycelium

was collected by filtration as before, washed twice with sdH 2 O and once with OM, before re-suspension in 25 ml OM and incubation, with gentle agitation (60 rpm) at 28 x for 2 h. Filter-sterilised Novozyme 234 was added to the OM at a concentration of 10 mg ml x1 . Protoplasts were separated from mycelial debris by filtering through a 30 mm mesh, collecting the filtrate in a sterile centrifuge bottle, then pelleted by centrifugation in a swing-out rotor at 2.8 K g for 7 min. The pellet was resuspended and washed twice with STC (1 M sorbitol, 10 mM Tris-HCl, pH 7.5; 50 mM CaCl 2 ). The centrifugation step was repeated after each wash. Protoplasts were re-suspended in STC and counted using a haemocytometer and the concentration adjusted to 10 7 protoplasts ml x1 . Protoplasts were transformed by adding 50 ml PTC (25% PEGx8000 (Sigma) in STC) to 200 ml protoplast suspension. DNA was added at a concentration of 10 mg and gently mixed with the pipette tip and in- cubated on ice for 20 min. A further 2 ml of PTC was added and incubated for a further 20 min at room temperature. After incubation, 4 ml of Regeneration Media (RMx1 M sorbitol; 0.1% yeast extract; 0.1% di-potassium orthophosphate; 0.05% MgSO 4 ) was added and the protoplasts incubated at 28 x in an orbital shaker with agitation (100 rpm) for 2 h. The protoplasts were plated on osmotically buffered plates (PDA+1 M sorbitol) and overlaid with osmotically buffered PDA containing 1 mg ml xl hygromycin (Melford Laboratories), and incubated at 28 x. As controls, protoplasts were plated on unbuffered media, and untransformed protoplasts were plated onto selec- tive buffered agar. Stocks (200 ml) of transformed, or unused protoplasts were supplemented with 50 ml

S. D. Atkins and others

PTC and 2.5 ml dimethyl sulfoxide (DMSO, Sigma) and stored at x80 x, according to Bardi, Perotto & Bonfante (1999) for further analysis if necessary. All preparations were examined using a Zeiss Axioskop microscope (Welwyn Garden City) fitted with epi- fluorescence illumination with a 455 nm excitation filter and a 520 nm barrier filter. Samples were observed using r40 oil immersion lens and images collected with a Xillix Micro Image digital camera (Vancouver) attached to an OpenLab Image Analysis system (Improvision, Coventry).

Selection of transformants

Colonies that grew through the hygromycin-supple- mented overlay before untransformed colonies ap- peared on the control plates (a window of 12–24 h) were transfered onto fresh PDA agar supplemented with 1 mg ml xl hygromycin. DNA was extracted from individual colonies by the method described by Klimyuk et al. (1993). DNA was used in PCR reactions using primers for the detection of hph or gfp (see below). Colonies that showed positive for transform- ation with either of the two genes were sub-cultured twice more onto unselective PDA media at weekly intervals and colonies were tested once more for the presence of hph or gfp using PCR.

Detection of hph

Primers (HPHF: CGC ATA ACA GCG GTC ATT GAC TGG AGC; HPHR: GTC GGG GCG TCG GTT TCC ACT ATC) were taken from the GenBank/ EMBL database (accession numbers E16482 and E16481 respectively). Detection of the hph gene was performed by PCR optimized in a reaction volume of 20 ml containing 0.1 mM each primer, 2 mlr10 PCR reaction buffer (Roche, Lewes, 1.5 mM Mg 2+ ), 0.1 mM each dNTP, 1 U Taq polymerase (Roche), 1 ml DNA (20–60 ng). PCR conditions were optimised as follows: hot start hold of 95 x followed by 40 cycles of 94 x for 1 min, 60 x for 1 min, 72 x for 1 min, with a final incubation at 72 x for 5 min. PCR products were separated on 2% Nu-Seive agarose gels (FMC Bio- Products, Rockland, ME) in TBE buffer. The 123 bp DNA ladder (Invitrogen, Paisley) was used as a size marker. Positive control DNA was amplified from pure plasmid DNA. The primers generated a single fragment of 370 bp.

Detection of gfp

Primers (GFPF: GGG(C) GAA(G) TGG G(C)GA TGC A(C)AC A(C)TA CGG; GFPR: ACG CTG(T) CCT TCG(C) TCA(G) ATG TTG T(G) were designed by comparison of GFP sequences from the GenBank/ EMBL database. These were then tested for the detec- tion of a range of GFP variants (Atkins 2000). The primers generated a single fragment of 380 bp.

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100 90 80 70 60 50 40 30 20 10 0 Hygromycin Puromycin Paromomycin Geneticin
100
90
80
70
60
50
40
30
20
10
0
Hygromycin
Puromycin
Paromomycin
Geneticin
PPT
Bleomycin
Phleomycin
Cycloheximide
Oligomycin
Benomyl
% inhibition
Thiabendazole
Carbendazim
MDPC

Inhibitor

Fig. 1. Effect of fungicides (n=3) on colony growth of Pochonia chlamydosporia.

Conditions for PCR were the same as for the detection of the hph gene. Isolates were confirmed as Pochonia chlamydosporia by PCR diagnosis using species specific primers described by Hirsch et al. (2000).

RESULTS

Pochonia chlamydosporia showed an inherent resist- ance to a wide range of fungicides tested (Table 1, Fig. 1). This resistance was not shown by the other fungal isolates tested for their ability to grow on media supplemented with hygromycin with LD 50 levels of 12.5 mg ml x1 for V. albo-atrum and V. dahliae, and 50 mg ml x1 for V. tricorpus, demonstrating that the media did not affect sensitivity. Growth of P. chlamy- dosporia was inhibited by methyl-N(3,5-dichloro- phenyl)carbonate (MDPC), a compound that binds to the b-tubulin protein, but previous investigations of the b-tubulin gene (Hirsch et al. 2001) had demon- strated that the P. chlamydosporia genotype did not match the phenotypic response to this compound. Therefore, transformation with a b-tubulin gene vari- ant resistant to MDPC (as demonstrated by Blakemore 1990) was highly unlikely to change this phenotype and further investigation into the phenomenon is necessary.

Optimisation of protoplast generation

The optimum time for generation of mycelium was 48 h after inoculation of liquid broth with spores. After this time mycelium pellets formed that severely inhibited protoplast generation. The effect of the osmotic buffer on protoplast generation was drastic (Fig. 2). The use of 1 M sorbitol generated significantly greater numbers of protoplasts (P<0.001, SED¡3.8r10 4 , n=8) than the other buffers. Protoplasts generated were spherical and ranged in size, averaging 5 mm in diameter (hyphal diameter averaged 2.5 mm). Significantly greater num- bers of protoplasts (P<0.001, SED¡6.2r10 5 , n=3) were generated when mycelium was pre-washed in

Transformation of Pochonia chlamydosporia

7 6 5 4 3 2 1 0 0.8 M 1 M 1.2 M 0.6
7
6
5
4
3
2
1
0
0.8 M
1 M
1.2 M
0.6 M
1 M
1.2 M
0.6 M
0.7 M
sorbitol
sorbitol
sorbitol
KCl
sucrose
MgSO4
NaCl
NaCl
5
-1
10xProtoplasts
ml

Buffer

Fig. 2. Effect of osmotic buffer (n=8) on protoplast gener- ation.

Table 2. Optimised conditions for protoplast generation from Pochonia chlamydosporia.

Age of culture

Conidiospores germinated for 48 h in

2 h with gentle shaking, 80 rpm

Pre-incubation

Czapek Dox broth 1 h in Buffer 1 plus 1%

Osmotic buffer Lysing enzyme and concentration Incubation time

b-mercaptoethanol OM plus 1 M sorbitol Novozyme 234 (Sigma) at 10 mg ml x1

Buffer 1 containing 1% b-mercaptoethanol (mean 4.0r10 6 ¡7.2r10 5 ) than when b-mercaptoethanol was absent (mean 7.3r10 5 ¡8.3r10 4 ). Novozyme 234 concentration also affected protoplast generation. No protoplasts were generated at the lowest concentration of enzyme used (1 mg ml x1 ), whereas significantly greater numbers of protoplasts were generated (P<0.001, SED¡3.8r10 4 , n=16) at the higher con- centrations of 10 mg ml x1 (2.2r10 5 ¡3.9r10 4 ) and 15 mg ml x1 Novozyme (1.6r10 5 ¡3.2r10 4 ). No sig- nificant differences were recorded between treatments that had either 10 or 15 mg ml x1 Novozyme added, although fewer protoplasts were generated at the highest concentration. The incubation time in the lysing solution also affected protoplast generation. At 1 h the protoplast yield was low (3.8r10 5 ¡3.5r 10 4 ) but had increased at 2 h (7.2r10 5 ¡4r10 4 ) and 3 h (5.8r10 5 ¡3.1r10 4 ). This increase was significant (P<0.001, SED¡1.3r10 4 , n=16). The optimum con- ditions for protoplast generation from P. chlamydo- sporia mycelium are listed in Table 2, which gave a yield of protoplasts of 1.8r10 8 g x1 wet weight mycelium.

Fungal transformation

Expression of gfp was clearly seen in both protoplasts and regenerating mycelium transformed with pTE- FEGFP or pCT74, examples of which are shown in Fig. 3. Expression of gfp was observed in regenerating mycelium up to three weeks after transformation when kept in Czapek Dox liquid medium, but was not

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observed in colonies plated onto solid media, irrespec- tive of whether Czapek Dox agar or the richer corn meal agar was used. This was not due to an inability to observe fluorescence on agar media because mycelium expressing GFP in liquid medium continued to fluor- esce when transferred onto agar.

Co-transformation with pDH33 and pTEFEGFP

Of the 50 colonies selected after co-transformation with pDH33 and pTEFEGFP, 11 contained the hph gene from pDH33, but none contained the gfp gene from pTEFEGFP, although gfp expression was clearly visible in regenerating mycelium in liquid culture (Fig. 3) demonstrating that hph-transformed isolates were preferentially selected. All colonies were con- firmed to be Pochonia chlamydosporia when tested using specific primers for the identification of the species (Hirsch et al. 2000). The transformed colonies were sub-cultured onto fresh PDA plates containing 1 mg ml x1 hygromycin after 1 wk and screened again using the hygromycin primers. PCR analysis indicated that six of the remaining 11 colonies contained the hph gene. After a further week these colonies were subcultured onto fresh PDA plates with and without selection with hygromycin. PCR screening of these sub-cultures with the hph-specific primers demon- strated that only one isolate under hygromycin selec- tion retained hph, but had lost the gene on non-selective plates. Conversely, two other isolates had retained hph when cultured on non-selective plates, but had not retained it on the selective media. After one further sub-culture hph could not be detected in any of the colonies using PCR.

Transformation with pCT74

Of the 50 colonies taken from the selection plates inoculated with Pochonia chlamydosporia after trans- formation with pCT74, five colonies contained hph and gfp. These isolates were sub-cultured onto fresh PDA plates and DNA was extracted and tested again using the hph and gfp selective primers. Three isolates still retained hph, but only two contained gfp. The col- onies were sub-cultured again as described earlier. On the third sub-culture a spore suspension was made of all isolates and spread onto plates with and without hygromycin and DNA was extracted from the resulting colonies. One isolate still retained hph and gfp in all the resulting colonies sampled, whereas, the other iso- lates had retained only gfp. No gfp expression was present in the mycelia of any of the isolates.

DISCUSSION

The first report of a DNA-mediated transformation system of a fungus was in 1973 (Mishra, Szabo & Tatum 1973). Since then many fungal species have been

S. D. Atkins and others

(a)

S. D. Atkins and others ( a ) ( c ) ( b ) ( d

(c)

S. D. Atkins and others ( a ) ( c ) ( b ) ( d

(b)

S. D. Atkins and others ( a ) ( c ) ( b ) ( d

(d)

S. D. Atkins and others ( a ) ( c ) ( b ) ( d

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Fig. 3. Detection of GFP in protoplasts and regenerating mycelium in liquid media transformed with pTEFEGFP (a, c) and pCT74 (b, d). Bar=5 mm.

transformed and a range of protocols now exists (Fincham 1989, 1999, Hynes 1996) not only for CaCl 2 / PEG mediated transformation of protoplasts but also electroporation, biolistics and Agrobacterium mediated transformation. What is apparent from the literature is that a process that works for one fungus may not necessarily work for another and optimisation of all stages of protoplast generation and transformation is essential. Various transformation protocols based on biolistics, electroporation and Agrobacterium mediated transformation were described by Atkins (2000). None of these methods were successful in transforming Pochonia chlamydosporia. A reliable method for the generation and regeneration of viable protoplasts is fundamental to any transformation strategy. For the first time, a successful protocol for protoplast gener- ation, transformation, and transient gene expression in the nematophagous fungus P. chlamydosporia. This required optimisation of several factors that affect protoplast generation and are listed in Table 2. A pre- wash of the mycelium with a buffer containing b- mercaptoethanol was essential for a high yield of protoplasts. The b-mercaptoethanol destabilises the fungal membrane sufficiently to enable the lysing enzyme to function more effectively, resulting in a sig- nificantly greater number of protoplasts. The high level of resistance to a wide range of fungicides in P. chlamydosporia was not seen with the other fungi tested. Consequently, background growth of untransformed propagules on selective media would swamp any transformants with increased resistance. Nevertheless, selection of colonies that grew quickly through the hygromycin selective overlay yielded a high percentage of hph-transformed isolates as demon- strated by the PCR detection, and this method has been used previously to select for transformants of fungal isolates that have a natural resistance to hygro- mycin (Punt & Hondel 1992). The ability of trans- formed protoplasts to regenerate more quickly in the

presence of a high concentration of hygromycin in- dicates that protoplasts may be more susceptible to the compound than mycelium. Once the protoplasts had regenerated, this sensitivity was lost and main- taining positive selection of hph with the fungicide had no effect, resulting in loss of the hph gene over time. Pochonia chlamydosporia demonstrated inherent resistance to a wide range of fungal inhibitors, rep- resenting a variety of different modes of action. Pre- vious research (Hirsch et al. 2001) has demonstrated that the phenotypic resistance of the fungus to beno- myl, and sensitivity to MDPC, was not the result of a mutation in the b-tubulin gene, the site of action for the fungicide. Resistance to any one inhibitor may not be due to a specific change in the target, or a speci- fic detoxification mechanism, but rather may be the result of a more general mechanism such as a multidrug resistant pump (Pereira, Fachin & Martinezrossi 1998). However, the exact mechanism(s) of resistance in P. chlamydosporia needs further investigation. The protoplasts generated were able to take up and express exogenous DNA, a fundamental step in a transformation system. The results indicate that co- transformation of the fungus with two or more plas- mids may not be a feasible option as transformants selected on hygromycin contained only the hph, with no trace of gfp, although regenerating protoplasts were seen to express GFP in liquid culture. Trans- formation of protoplasts with a single vector carrying both the resistance gene and the marker gene yielded transformants containing both genes when screened using PCR. The gradual loss of the exogenous DNA over time and with sub-culturing points to the inability of the plasmid to stably integrate, or the ability of the fungus to excise unwanted DNA. PCR demon- strated that gfp was still present within the fungus, but the lack of expression in the mycelium from colonies grown on plates demonstrated that the gene was either not being expressed, or was being expressed at an

Transformation of Pochonia chlamydosporia

undetectable level. It is unclear whether any vector DNA had integrated into the fungal genomic DNA, or whether the autonomous plasmid was replicating. The high copy number of plasmids in the initial transfor- mants may be diluted during cell division following protoplast regeneration and gradually lost from the fungal mycelium during growth. P. chlamydosporia may have a mechanism for silencing or regulating gene expression from unwanted or exogenous DNA. This phenomenon, in which ex- pression of the transgene and of endogenous genes containing sequences homologous to the transgene can be blocked, is involved in virus resistance and genome maintenance and has been investigated in a number of fungal species (Cogoni & Macino 1999). Irelan & Selker (1996) investigated three methods of gene silencing in filamentous fungi, methylation in- duced premeiotically (MIP), repeat-induced mutation, and quelling of the gene. Each of the gene silencing processes involved the silencing of repeated sequences. Thus each has the potential for preventing proliferation of selfish DNA elements and silencing repeated se- quences that may arise in a transformation system where many exogenous gene copies may become in- corporated. Insertion of many copies of the exogenous vector into the genome may result in gene silencing mechanisms being triggered and further investigation of this phenomenon is necessary. PCR detection of only hph or gfp in the protoplasts transformed with the vector pCT74, indicated that the fungus was able to delete genes. Transformants contained either or both, of the genes, with no apparent preference for the gene removed. Although this paper demonstrates the production and transformation of protoplasts of the fungus P. chlamydosporia, it also indicates problems with ex- pression of GFP. Expression of GFP may be deleteri- ous to the fungus. For example, bacterial cells become osmotically unstable when the protein is over expressed (Brendan Cormack, pers. comm.). The GFP was seen to be clearly compartmentalised within regenerating mycelium and protoplasts (Fig. 3). This may indicate adverse effects on P. chlamydosporia, resulting in a strong counter selection and consequent loss of trans- formants containing the expression vector. The next stage in developing a transformation system for P. chlamydosporia is to make transformants more stable. A new range of fluorescent protein vectors are now available (Reef Coral Fluorescent Proteins, CloneTech, Bedford) and have been used in the successful transformation of fungi (Bourett et al. 2002, Mikkelsen et al. 2003). These vectors are re- ported to offer a higher degree of stability in trans- formants (CloneTech).Fluorescent activated cell sorting (FACS) of transformed protoplasts could yield a greater number of transformed protoplasts than the selection method outlined in this paper, and therefore, increase the likelihood of stable transformants being isolated. Vectors designed to facilitate site-directed

660

integration can result in a stable integration of the vector DNA (Goldschmidtclermont 1991) and this offers possibilities for future transformation of P. chlamydosporia. The system described in this paper can be used to investigate the expression of transgenes in Pochonia protoplasts, although the genes were not maintained in a stable manner after regeneration. A stable trans- formed isolate expressing a visible marker gene would greatly enhance not only the knowledge of how P. chlamydosporia functions and interacts in the rhizo- sphere, increasing our understanding of how this fungus can be developed as a biological control agent, it is also of fundamental importance in trying to understand how fungi interact in the rhizosphere in general.

ACKNOWLEDGEMENTS

Rothamsted Research receives grant-aided support from the UK Biotechnology and Biological Sciences Research Council. We also acknowledge support provided by EU grant Fair-Pl97-3444 as part of this work.

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Corresponding Editor: S. J. Assinder