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1 Department of Biological Sciences, Brock University, 500 Glenridge Avenue, St Catharines, ON L2S 3A1, Canada
2 Biotechnology Research Center, Southwest University, Beibei Chongqing 400716, P. R. China
Correspondence
Michael J Bidochka
bidochka{at}brocku.ca
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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flbA, which cannot conidiate, with M. anisopliae cag8 restored conidiation. Gene disruption of cag8 in M. anisopliae resulted in the lack of conidia on agar plates and on infected insects, reduced mycelial growth, decreased virulence, lysis during growth in liquid medium as well as lack of pigmentation and irregularly shaped blastospores. Transcript levels of ssgA (hydrophobin-encoding gene) were markedly reduced in a cag8 strain, while pr1A (subtilisin-like protease) transcription was unaffected. These results suggest that cag8 is involved in the modulation of conidiation, virulence and hydrophobin synthesis in M. anisopliae.
The GenBank/EMBL/DDBJ accession number for the sequence reported in this paper is DQ826044.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). To enhance their virulence, detailed knowledge of the mechanisms of fungal pathogenesis is needed. The conidium is the infective propagule, which comes into contact with, and adheres to, the insect cuticle via hydrophobic mechanisms (Boucias et al., 1988) mediated by conidial surface proteins called hydrophobins (Bidochka et al., 2001). Once attached, the conidium germinates and penetrates the insect cuticle via mechanical pressure and cuticle-degrading enzymes (St Leger et al., 1996). Hyphae proliferate within, and emerge from, the dead insect and subsequently conidiate on the cadaver. Under favourable conditions, the newly produced conidia will disperse and infect other insects. Thus, the conidium is the propagule that initiates pathogenesis and is involved in disease transmission. An understanding of the regulatory processes involved in conidiation is essential to the commercial development and improvement of this biocontrol fungus.
In several filamentous fungi, the heterotrimeric G protein signalling pathway is involved in conidiation, as well as morphogenesis and pathogenesis (Bölker, 1998; Borkovich et al., 2004; Lengeler et al., 2000). The genetic regulatory circuit for conidiation is initiated when the heterotrimeric G protein pathway is activated. This allows the G
subunit of the heterotrimeric G protein complex to substitute GDP for GTP, resulting in the dissociation of the G and G
subunits and the activation of downstream effectors by both G and G (Hamm, 1998). G and G subunits have been identified in several filamentous fungi and their implication in fungal conidiation is well established. The signal amplitude of G protein signalling is determined by the balance of the rates of GDP/GTP exchange (activation) and the rates of GTP hydrolysis (deactivation), which is achieved by the intrinsic GTPase of the G subunit. However, the GTPase of the G subunit requires a regulator of G protein signalling (RGS) to enhance GTPase activity (Ross & Wilkie, 2000).
Altogether, six RGS protein families (orthologues of RgsA, RgsB, RgsC, RgsD, FlbA and Gprk) have been identified from Aspergillus spp. and other fungal RGS proteins fall into one of these families (Lafon et al., 2006). The first RGS protein-encoding gene identified in a filamentous fungus was flbA (fluffy low brlA) in Aspergillus nidulans (Lee & Adams, 1994). FlbA negatively regulates signalling for vegetative growth and activated conidiation, mediated through the G subunit, FadA (Yu et al., 1996), and the G subunit, SfaD (Rosen et al., 1999). Han et al. (2004) described a second RGS protein in A. nidulans, RgsA, which down-regulated stress responses and stimulated conidiation through attenuation of GanB (G) signalling. Several RGS proteins with domain organizations similar to FlbA have been identified and implicated in conidiation in other filamentous fungi. The RGS protein encoded by the thn-1 gene of Schizophyllum commune is necessary for fruit body formation in dikaryons and aerial hyphae formation in monokaryons (Fowler & Mitton, 2000). Most recently, an RGS protein, CPRGS-1, from the phytopathogenic fungus Cryphonectria parasitica has been implicated in fungal conidiation as well as virulence and hydrophobin synthesis (Segers et al., 2004).
Metarhizium anisopliae is arguably one of the best studied insect-pathogenic fungi. Several genes implicated in cuticle penetration and pathogenesis have been characterized (Bogo et al., 1998; Joshi & St Leger, 1999; Screen et al., 1998, 2001; St Leger et al., 1992); however, to our knowledge, genes regulating conidiation have not been identified. Here, we isolated an RGS protein gene, cag8, from M. anisopliae that showed significant homology to flbA (Lee & Adams, 1994). The characterization of cag8 using gene disruption mutants established a role for its product in modulating conidiation, virulence and hydrophobin synthesis in M. anisopliae.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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flbA (yeA flbA : argB methG pyro veA) was kindly provided by Dr Nancy Keller, Department of Plant Pathology, University of Wisconsin-Madison, Madison, WI, USA) and Dr J. H. Yu (Department of Food Microbiology and Toxicology & Molecular and Environmental Toxicology Center, University of Wisconsin-Madison, Madison, WI, USA). On complete medium supplemented with methionine and pyridoxin, A. nidulans flbA cannot conidiate and the colony undergoes autolysis as it matures. Yellow conidia of A. nidulans flbA were obtained by culturing on complete medium agar containing 0.8 M NaCl (Yu et al., 1996). Escherichia coli DH5 was employed for DNA manipulations and transformations. Agrobacterium tumefaciens AGL-1 was used for fungal transformations.
Gene cloning.
Fungal genomic DNA was isolated by using the DNeasy Isolation Kit (Qiagen). RNA was prepared by using the RNeasy Mini Kit (Qiagen); genomic DNA was eliminated by loading RNase-free DNaseI onto the filter column (Qiagen). Nucleic acid extractions were performed according to manufacturer's instructions. Nucleic acid concentrations were measured with GeneQuantII (Pharmacia Biotech).
To clone a fragment of the RGS protein gene from M. anisopliae by PCR, consensus degenerate primers RGSF1 and RGSR1 (Table 1) were designed based on regions of homology to A. nidulans FlbA (Lee & Adams, 1994), Schizophyllum commune THN-1 (Fowler & Mitton, 2000) and Saccharomyces cerevisiae SST2 (Dohlman et al., 1996). PCR was performed using hot start Taq DNA polymerase (Qiagen). The PCR cycles were 95 °C for 15 min, followed by 35 cycles of 1 min denaturation at 94 °C, 45 s annealing at 48 °C and 1 min polymerization at 72 °C, followed by 72 °C for 10 min. The resultant PCR product was shown to be a fragment of an RGS protein gene. The full length of this gene as well as the upstream and downstream regulatory sequence was subsequently cloned by PCR walking using the Y-shaped adaptor-dependent extension (YADE) method as described previously (Fang et al., 2005). Restriction enzymes DraI, EcoRV, ScaI, SmaI, XbaI, SpeI, NheI, BamHI and BglII were used to digest the M. anisopliae genomic DNA. Three rounds of PCR walking were conducted for the isolation of the upstream sequences and one round for downstream sequences. Primers used for YADE are listed in Table 1. Hot start Taq DNA polymerase (Qiagen) was used and the reaction mixtures were prepared according to manufacturer's instructions. For YADE, all linear amplifications were performed at 95 °C for 15 min, followed by 40 cycles of 30 s denaturation at 94 °C, 30 s annealing at 60 °C and 3 min polymerization at 72 °C, followed by 72 °C for 3 min. All exponential amplifications were conducted at 95 °C for 15 min, followed by 35 cycles of 30 s denaturation at 94 °C, 30 s annealing at 60 °C and 2 min polymerization at 72 °C, followed by 72 °C for 10 min. All PCR products were cloned into the A-T cloning vector pGEM-T (Promega), according to manufacturer's instructions, and subsequently sequenced. The RGS protein-encoding gene from M. anisopliae was named cag8.
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) were designed to clone the cDNA sequence of this gene using Second Generation 5' and 3' RACE (rapid amplification of cDNA ends) kits (Roche).
Reverse-transcribed PCR (RT-PCR).
RT-PCR was conducted with the One-Step RT-PCR kit (Qiagen). Total RNA (250 ng) was subjected to RT-PCR and the reaction components were prepared according to manufacturer's instructions. RT-PCR cycles were 50 °C for 30 min, 95 °C for 15 min, followed by 30 cycles of 45 s denaturation at 94 °C, 30 s annealing at 60 °C and 45 s polymerization at 72 °C, followed by 72 °C for 10 min. RT-PCR primers for cag8 and gpd are listed in Table 1. Expression of cag8 was investigated during growth in broth culture (three developmental stages), agar medium (four developmental stages), as well as during insect pathogenesis (three developmental stages). In YPD broth (0.2 % yeast extract, 1 % peptone, 2 % glucose), the developmental stages were (i) conidium, (ii) swollen conidia (7 h) after inoculation and (iii) conidial germination (16 h) with germ tube lengths of 40–100 µm. In YPD agar (0.2 % yeast extract, 1 % peptone, 2 % glucose, 1.5 % agar), the developmental stages were (i) mycelial growth (2 days), (ii) early conidiophore development with no conidia (3 days), (iii) late conidiophore development with few conidia (4 days) and (iv) fully mature conidiophore with conidia (6 days). During insect pathogenesis, the developmental stages were (i) emergent mycelia from insect (Galleria mellonella) cadavers (3 days after insect death), (ii) emergent mycelia with few conidia (5 days after insect death) and (iii) insect cadavers mummified with mycelia and conidia (10 days after insect death). Insects (G. mellonella) were infected by immersion in a conidial suspension (2x107 conidia ml–1). At death, the insect was surface-sterilized in 1 % sodium hypochlorite (1 min) and rinsed twice in sterile distilled water. The sterilized cadavers were then placed in a Petri dish with moist paper to promote fungal emergence from the cadaver. RNA was extracted from emergent mycelia and conidia from infected insects.
Real-time RT-PCR.
First-strand cDNA was synthesized using hexamers and oligo-dT primers with iScript reverse transcriptase, according to manufacturer's instructions (Bio-Rad). One microgram of total RNA was used for cDNA synthesis and the cDNA was subsequently diluted with nuclease-free water (Qiagen) to 10 ng µl–1. Real-time RT-PCR amplification mixtures (25 µl) contained 10 ng template cDNA, 2xSYBR GreenI master mix buffer with fluorescein for dynamic well factor collection (12.5 µl) (Bio-Rad) and 200 nM each of the forward and reverse primers (Sigma). The reaction was performed on the iCycler system (Bio-Rad). PCR was accomplished after a 1.5 min activation/denaturation step at 95 °C, followed by 40 cycles of 15 s at 95 °C, 30 s at 60 °C and 30 s at 72 °C. Fluorescence was detected and recorded at each polymerization step. The expression of cag8, ssgA (Bidochka et al., 2001) and pr1A (St Leger et al., 1992) were analysed using gpd, tef and try as reference genes, and expression levels were calculated using geNORM (Fang & Bidochka, 2006). Primers are listed in Table 1.
Disruption of cag8.
To disrupt cag8 in M. anisopliae based on homologous recombination, the herbicide resistance gene bar was inserted into cag8. The 5' end of cag8 was cloned by PCR with primers Hcag8-1 and Hcag8-2 (Table 1) using Jumpstart AccTaq LA DNA polymerase (Sigma). The resultant PCR product was digested with SpeI and PmlI, and inserted into XbaI and PmlI sites of pBARGPE1 (Ponting & Bork, 1996) to form pGPE-cag8-5 : bar. The 3' end of cag8 was amplified with primers Hcag8-3 and Hcag8-4 with Jumpstart AccTaq LA DNA polymerase (Sigma). The PCR product was then digested with EcoRI and NotI and cloned into the corresponding sites of pGPE-cag8-5 : bar to form pGPE-cag8-5 : bar : cag8-3. From this, the cag8-5 : bar : cag8-3 portion was excised using EcoRI and PmlI and inserted into EcoRI/EcoRV-digested binary vector pPK2 (McCluskey, 2003) in which an hph cassette was deleted using KpnI and XbaI to form pPK-cag8-5 : bar : cag8-3. The gfp cassette was excised from TEFGFP (Spear et al., 1999) using EcoRI and subsequently inserted into the same site of pPK-cag8-5 : bar : cag8-3 to form pPKGFP-cag8-5 : bar : cag8-3, which was then mobilized into Agr. tumefaciens AGL-1 for fungal transformation (Fang et al., 2006).
cag8-deficient mutants were selected as herbicide-resistant and observed for GFP expression. PCR with primer combinations Dcag8-1/Dcag8-2 and Dbar/Dcag8-2 as well as Southern blotting was used to confirm disruption of cag8 in M. anisopliae transformants.
Complementation experiments.
To complement flbA of A. nidulans with cag8, a plasmid vector containing cag8 was constructed. The ORF of cag8, as well as the upstream and downstream sequences, were amplified with primers cag8F and cag8R (Table 1) using High-Fidelity Taq DNA polymerase (Qiagen) and sequenced for verification. The PCR product was digested with EcoRV and inserted into the SmaI site of vector pUG11-41 which contains the methionine gene (kindly provided by Dr Nancy Keller) to form the vector pUG11-41-cag8.
Conidia of A. nidulans flbA were collected from complete medium agar supplemented with 0.8 M NaCl and cultured overnight in complete medium broth at 37 °C. Protoplast preparation and transformations were performed as described previously (Ballance et al., 1983; Ballance & Turner, 1985). Plasmids pUG11-41 and pUG11-41-cag8 were used for transformations. Transformants were selected based on methionine utilization and further confirmed by PCR with primers Hmacag8F and Hmacag8R (Table 1) as well as Southern hybridization. Expression of cag8 in transformants was verified by RT-PCR with primers cag8RTF and cag8RTR (Table 1).
Bioassays.
Virulence of fungal blastospores was tested against G. mellonella by injecting 3 µl blastospore suspension (104 ml–1) or by immersion of insects into a blastospore suspension (3x106 ml–1). Blastospores were obtained from wild-type transformants with cag8-5 : bar : cag8-3 integrated ectopically, and cag8-deficient mutants which were cultured in 100 ml YPD broth. After a 4 day incubation at 27 °C with 200 r.p.m. shaking, cultures were filtered through glass wool to obtain blastospores. Mortality was recorded daily and LT50 values were estimated by probit analysis.
Each treatment was replicated three times with 10 insects per replicate. All bioassays were repeated three times.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The putative coding sequence was identified using BLASTX and based on this, primers (described in Methods) were designed to clone the concordant cDNA using 5' and 3' RACE. A 1933 bp cDNA fragment was resolved based on RACE products, which contained a 1326 bp ORF and a 123 bp 5' UTS and 484 bp 3' UTS. A protein of 441 aa with a predicted molecular mass of 49.9 kDa and a pI of 8.7 was deduced. Two introns of 451 and 122 bp, respectively, were identified. Southern blotting showed that cag8 existed as a single copy in the M. anisopliae genome (data not shown). Three stress-responsive element (STRE) domains were identified in the 2838 bp upstream regulatory sequence. STREs have previously been shown to mediate transcriptional activation in response to various stresses, particularly heat stress, osmotic stress, low pH and nutrient starvation in Saccharomyces cerevisiae (Siderius & Mager, 1997).
RT-PCR analysis demonstrated that cag8 was constitutively expressed throughout various growth stages in broth culture and agar medium as well during emergence of fungal mycelia and conidiation on the infected insect cadaver (data not shown).
Homology and phylogenetic analysis of CAG8
Six RGS protein families have been identified from Aspergillus spp. (Lafon et al., 2006). They are orthologues of RgsA, RgsB, RgsC, RgsD, FlbA and GprK. Other fungal RGS proteins show homology to one of these six Aspergillus RGS proteins (Lafon et al., 2006). Fig. 1(a) shows that CAG8 is most closely related to FlbA with a well supported bootstrap value of 100 %. Also, FlbA and CAG8 feature two DEP domains and one RGS domain. Fig. 1(b) shows the relationship of FlbA-like proteins from various ascomycetous and basidiomycetous fungi. The ascomycetous clade contains A. nidulans and M. anisopliae, Gibberella zeae, Neurospora crassa and Cryphonectria parasitica and is well supported (bootstrap 100 %). The RGS protein, CAG8, from M. anisopliae is most closely related to the RGS protein from the plant pathogen Gibberella zeae. The other major clade consists of the basidiomycetes Schizophyllum commune, Ustilago maydis, Cryptococcus neoformans and Cryptococcus bacillisporus.
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cag8), D1–D5 and D7, were utilized for further study. These six cag8 strains, which were randomly selected from the transformants, were confirmed by PCR and Southern blotting and showed identical patterns of genomic integration (Fig. 2).
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cag8 strain did not produce conidia (Fig. 3a) and mycelial growth was slower than the wild-type strain or transformants with the cag8-5 : bar : cag8-3 integrated ectopically (Bar). In YPD broth, cag8 produced fewer blastospores than the wild-type strain (Table 2). Blastospores of cag8 (mean length 19.1±1.3 µm, n=100) were about twice as long as the wild-type (mean length 8.9±0.9 µm, n=100) (Fig. 3b). After 8 days of growth in YPD broth, the cultures of the wild-type strain or transformants with cag8-5 : bar : cag8-3 integrated ectopically had many blastospores, while blastospores were absent in cag8 cultures. Furthermore, cag8 cultures were relatively unpigmented in YPD broth while wild-type cultures were typically dark green (Fig. 3c).
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cag8 strains. However, when PDA was supplemented with 0.8 M NaCl, cag8 strains did produce some conidia, but the yield was significantly less than the wild-type strain or transformants with cag8-5 : bar : cag8-3 integrated ectopically (Table 2). Therefore, high osmolarity in PDA agar can partially restore conidiation in a cag8 strain, similar to flbA in A. nidulans (Lee & Adams, 1994).
Since cag8 strains did not produce conidia on agar media, blastospores were used for bioassays. The cag8 strains showed a significant decrease in virulence compared to the wild-type strain when insects were infected by immersion in a blastospore suspension (Table 2). However, cag8 strains had similar virulence to the wild-type strain when blastospores were injected into insects (Table 2). Cadavers infected by the wild-type strain were firm and showed emergent mycelia with conidia, while insect cadavers infected by cag8 strains showed no conidiation, sparse mycelial growth and the cadavers were soft (Fig. 3D).
Relationship between cag8, ssgA and pr1
Transcript levels of the hydrophobin-encoding gene ssgA and the subtilisin-like protease-encoding gene pr1A were analysed by real-time RT-PCR. In the wild-type strain, ssgA transcript levels were relatively greater during the late stages of conidiation and pr1A transcripts were relatively greater during early and late stages of pathogenesis. Real-time PCR showed that the transcript level of ssgA was markedly reduced (>200-fold) in the cag8 strains (Fig. 4). Using RNA derived from mycelia from insect cadavers as the template, pr1A transcript levels were similar in the wild-type and cag8 strains (data not shown).
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flbA with cag8 from M. anisopliaeflbA. Sixty meth+ colonies transformed with pUG11-41-cag8 and 75 meth+ colonies transformed with pUG11-41 were selected on minimal medium lacking methionine. All pUG11-41 transformant colonies were similar to A. nidulans flbA in colony development and conidiation. Transformants, with pUG11-41-cag8, were found to conidiate similar to A. nidulans wild-type. Insertion of cag8 into the genome of the transformants was confirmed by PCR and Southern hybridization in five transformants with identical results (data not shown). RT-PCR showed that cag8 was expressed in the transformant (data not shown). On complete medium, conidiation of the transformant was similar to the wild-type, but, starting in the centre, the colony underwent autolysis after approximately 14 days incubation at 37 °C, but autolysis ceased thereafter (Fig. 5). The A. nidulans flbA strain never produced conidia and the colony underwent autolysis in the centre after 3.5 days growth; by day 7, the entire colony underwent autolysis. We concluded that cag8 was able to restore conidiation of A. nidulans flbA, but was unable to completely halt colony autolysis.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Similar to FlbA, CAG8 also featured two DEP domains. The DEP domain was initially identified in dishevelled (Drosophila melanogaster), Egl-10 (Caenorhabditis elegans) and human pleckstrin (Rosen et al., 1999), which are all involved in G-protein signalling. DEP domains have been shown to be necessary for membrane localization (Martemyanov et al., 2003) and are also involved in regulating the expression of stress-responsive genes through interaction with other proteins (Burchett et al., 2002). Several putative STREs were identified in the upstream regulatory sequence of cag8, the DEP-domain-containing RGS protein gene itself.
Despite the similarities of RGS proteins from various fungi, the knockout phenotypes can sometimes be quite different. In M. anisopliae, cag8 resulted in reduced growth and the presence of aerial mycelia without conidia, which is similar to the phenotype of cprgs-1 in the plant-pathogenic fungus Cryphonectria parasitica (Segers et al., 2004). flbA in A. nidulans results in a fluffy colony phenotype with abundant mycelial growth, reduced conidiation and autolysis as the colony ages (Lee & Adams, 1994).
From the gene complementation experiments we found that cag8 can restore conidiation, but cannot completely halt autolysis in A. nidulans flbA. This could possibly be due to the different flanking sequences of the RGS and DEP domains in these two RGS proteins. FlbA is also 278 aa longer than CAG8, mainly in the flanking sequences of the RGS and DEP domains. Functional domains and motifs in the flanking sequence of the RGS domain are considered to contribute to specific activity, subcellular localization or regulation, resulting in different RGS proteins contributing to different signalling pathways (Ross & Wilkie, 2000). The unique flanking sequence in FlbA affords control over mycelial proliferation as well as conidiation and, thus, RGS protein genes from different fungi may not be fully complementary.
The association between RGS proteins and hydrophobin synthesis was shown in cag8 where real-time RT-PCR showed significantly reduced ssgA gene expression. Similarly, thn-1 Schizophyllum commune and cprgs-1 Cryphonectria parasitica were deficient in expression of their hydrophobin-encoding genes Sc3 and cryparin, respectively (Fowler & Mitton, 2000; Segers et al., 2004).
Mutations in RGS genes have diverse effects on fungal pathogens of insects, plants or humans. In this study, cag8 mutants had virulence similar to that of the wild-type strain when blastospores were injected into insects. However, when insects were infected by immersion into a blastospore suspension, the virulence of cag8 mutants was significantly lower than that of the wild-type strain. The disruption of the RGS gene cprgs-1 resulted in complete loss in virulence in the phytopathogenic fungus Cryphonectria parasitica (Segers et al., 2004). On the other hand, mutations in crg1 increased virulence in Cryptococcus neoformans, a human-pathogenic fungus (Wang et al., 2004).
The identification of an RGS gene involved in the regulation of conidiation is a potentially important step in the further development of this fungus as a biocontrol agent. Several genetic manipulations have previously been performed to improve the efficacy of M. anisopliae (St Leger et al., 1996). However, there are potential negative ecological implications in the dissemination of genetically modified fungal conidia from insect cadavers (Hu & St Leger, 2002). The information generated in this study allows for the control of conidiation and the restriction in the dissemination potential of a genetically modified biocontrol fungus.
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ACKNOWLEDGEMENTS |
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Edited by: B. A. Horwitz
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Received 1 September 2006;
revised 21 December 2006;
accepted 26 December 2006.
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