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Department of Entomology, University of Maryland, 4112 Plant Sciences Building, College Park, MD 20742, USA
Correspondence
Raymond St. Leger
rl106{at}umail.umd.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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10-5) had their best BLAST hits among fungal sequences. These included chitinases and multiple subtilisins, trypsin, metalloprotease and aspartyl protease activities with the potential to degrade host tissues and disable anti-microbial peptides. Otherwise, compared to the ascomycete entomopathogen Metarhizium anisopliae, Con. coronatus produced many fewer types of hydrolases (e.g. no phospholipases), antimicrobial agents, toxic secondary metabolites and no ESTs with putative roles in the generation of antibiotics. Instead, Con. coronatus produced a much higher proportion of ESTs encoding ribosomal proteins and enzymes of intermediate metabolism that facilitate its rapid growth. These results are consistent with Con. coronatus having adapted a modification of the saprophytic ruderal-selected strategy, using rapid growth to overwhelm the host and exploit the cadaver before competitors overrun it. This strategy does not preclude specialization to pathogenicity, as Con. coronatus produces the greatest complexity of proteases on insect cuticle, indicating an ability to respond to conditions in the cuticle.
Present address: Institute of Plant Sciences, Biochemistry and Physiology of Plants, ETH Zurich/LFW E 51, Universitätsstr. 2, CH-8092 Zurich, Switzerland.
Present address: Bioinformatics Lead Identification, The Monsanto Company, 800 N. Lindbergh Blvd, St Louis, MO 63167, USA.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). Reflective of their ancient origins and probable polyphyly, the Zygomycota comprise 10 diverse orders (Voigt & Woestermeyer, 2001). However, the ubiquitous saprobes, the Mucorales, are a sister group to the almost equally ubiquitous insect pathogens, the Entomophthorales (Jensen et al., 1998). Members of the Entomophthorales are amongst the most common and potent pathogens of insects, frequently decimating insect populations in spectacular epizootics that have led to their being employed in biocontrol (Hajek, 1997).
Conidiobolus coronatus is the most basal and least specialized branch of the extant entomophthoralean fungi (Jensen et al., 1998) and probably resembles the forerunners of the more evolved genera and species (Evans, 1989). These are often highly host-adapted and show little or no growth on standard mycological media (Humber, 1984). In contrast, there is no evidence of host specificity in Con. coronatus; it has been isolated from various homopteran species, but also from Coleoptera, Lepidoptera or Diptera (see http://www.ppru.cornell.edu/Mycology/ARSEF_Culture_Collection.htm). Con. coronatus can probably attack any stressed insect (Papierok, 1986) and it is also an opportunistic pathogen of vertebrates (Benny et al., 2001). However, even stressed insects may deploy a range of physical, chemical and cellular defences that will need to be neutralized or evaded and currently there is no information for Con. coronatus as to the plasticity of its physiological responses to host behaviour or whether it has any requirement for host-related stimuli, such as insect cuticle, to produce virulence determinants. Indeed it is still not clear how Con. coronatus normally infects and colonizes host tissues.
A recent study on the ascomycetes Metarhizium anisopliae sf. anisopliae 2575 (wide host range) and Met. anisopliae sf. acridum 324 (specific to grasshoppers) demonstrated that utilizing expressed sequence tags (ESTs) multiple virulence factors and pathways can be viewed simultaneously, allowing the different lifestyles that exist in insect–fungus interactions to be understood from a broader perspective (Freimoser et al., 2003). Another advantage of using cDNA sequence data is that it represents a rapid and relatively efficient method for quickly discovering large numbers of genes in organisms which have little or no genetic research history, which include almost all orders within the Chytridiomycota and Zygomycota.
We have therefore adopted an EST strategy for gene discovery in C. conidiobolus. However, in the absence of biochemical and molecular data on the factors influencing the pathogenic habit of Con. coronatus, we used levels of protease production by this fungus as a marker to test culture conditions for maximum production of pathogenicity-related molecules, our rationale being that as most animal pathogens produce multiple proteases to degrade the proteinaceous outer integuments of their hosts, proteases may represent a ‘niche-specific trait’, that is, a trait shared by pathogens that occupy the same niche, irrespective of their phylogenetic position. We report here that Con. coronatus produces the greatest complexity of proteases on insect cuticle. Consequently, we targeted the EST project at genes expressed during growth on cuticle to identify potential candidate genes conditioning pathogenicity through a comparative analysis with sequences derived from other organisms, particularly Met. anisopliae.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Preparation and analysis of culture filtrate.
Cultures were inoculated with spores taken from 5–12-d-old agar plates or with standardized mycelial inocula (0·5 g wet wt per 10 ml) from 20 h Sabouraud-glucose cultures. The conical flasks were incubated with shaking (100 r.p.m) at 25 °C for up to 2 d in basal medium (BM) containing (l-1) 1 g KH2PO4, 0·5 g MgSO4, 0·7 mg Na2B4O7, 0·5 mg (NH4)Mo7O24, 10 mg Fe2(SO4) and 0·3 mg Zn2SO4, adjusted to pH 6·0 with NaOH, supplemented with 0·2 % yeast extract (YE) and additional nitrogenous nutrients. To compare protease production by diverse fungi, conidia (1x106) were inoculated into 50 ml BM plus yeast extract (0·2 %) supplemented with (1) insoluble protein (collagen at 1 %), (2) peptone (2 %) or (3) collagen (1 %) and peptone (2 %) (Table 1). The influence of nutrients on protease production by Con. coronatus was determined in BM plus yeast extract (0·2 %) supplemented with 1 % bovine serum albumin, 1 % peptone+2 % glucose, 1 % cellulose, 1 % collagen, 1 % chitin, 1 % chitosan, 1 % Manduca sexta cuticle or 1 % cockroach (Blaberus giganteus) cuticle. Manduca sexta and Blaberus giganteus are sources for unsclerotized and sclerotized cuticles, respectively. Cuticles were isolated and prepared as described previously (St. Leger et al., 1994).
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). Subtilisin-like protease activity (versus Suc-Ala-Ala-Pro-Phe-NA) was determined as previously described for Met. anisopliae and other entomopathogens; activities are expressed as nmol nitroanilide (NA) ml-1 min-1 (St. Leger et al., 1987). To determine dry weight of fungal biomass, mycelia harvested on filter paper were dried to constant weight under vacuum at 80 °C.
Analytical isoelectric focusing (IEF).
Samples were desalted and concentrated 50-fold using Centricon-10 ultrafiltration units. Two-microlitre aliquots of the concentrate were run on ultrathin polyacrylamide gels by using 1 % ampholytes (Bio-Lyte 3/10; Bio-Rad) (St. Leger et al., 1994). Immediately following IEF, gels were overlaid with X-ray film (gelatin overlay) or enzyme overlay membranes (EOM) to detect protease activity (St. Leger et al., 1994). The EOM was impregnated with Suc-Ala-Ala-Pro-Phe-AFC (AFC, 7-amino-4-trifluoromethyl coumarin).
Construction of cDNA library.
For cDNA library construction, Con. coronatus was first grown for 20 h in liquid SDB broth. The fungal biomass was then harvested on filter paper, washed with sterile distilled water and transferred for 18 h to basal medium supplemented with 1 % (w/v) Manduca sexta cuticle/0·2 % (w/v) peptone (insect cuticle was prepared as described previously; St. Leger et al., 1994). Total RNA was extracted from frozen fungus using TRI Reagent as described by Joshi et al. (1999) and a cDNA library was constructed in the unidirectional 
ZAP II vector (Stratagene), exploiting the EcoRI and XhoI restriction sites, according to the manufacturer's instructions.
Plasmid isolation and DNA sequencing.
Plasmid constructs were transformed into Escherichia coli TOP10 (Invitrogen). Individual transformants were picked, grown overnight in LB medium and plasmid DNA was isolated and purified using QIAprep Spin Miniprep Kits (Qiagen) following the manufacturer's protocols. cDNA inserts were sequenced from the 5' end by employing the M13 primer and ABI chemicals on ABI 377 DNA sequencers (DNA Sequencing Facility, Dupont, DE, USA).
Sequence analysis.
Vector sequences were removed by hand. Overlapping sequences were assembled into consensus sequences (contigs) by using the program CAP3 (Huang & Madan, 1999). The program BLASTX (Altschul et al., 1997) was used to search all ESTs against the non-redundant amino acid reference library (NCBI ‘nr’ database). Con. coronatus ESTs with significant BLAST matches (E10-5) were sorted into functional groups as outlined in Fig. 3 (see also Table of supplementary data available at http://mic.sgmjournals.org which contains a complete list of all best BLASTX matches for Con. coronatus sequences).
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). All fungi grew in each medium, raising the pH from an initial pH of 6 to above 7·5, consistent with breakdown of nitrogenous nutrients to ammonia (St. Leger et al., 1999). Also consistent with previous studies (St. Leger et al., 1988), production of proteases by Met. anisopliae was repressed when low-molecular-mass nitrogenous nutrients (1 % peptone) were added to media containing insoluble protein (collagen) showing that repression overrides the enhancing effect of polymeric substrates. In contrast, protease production by the entomophthoralean fungi (Basidiobolus ranarum, Basidiobolus haptosporus and Con. coronatus) was enhanced by peptone. Mucoralean fungi are reported to produce endocellular proteases and aspartyl (acid) proteases (rennin) (Gray et al., 1986). However, the mucoralean fungi (Mortierella hyalina, Sporodiniella umbellata, Mucor mucedo and Mucor rouxii) and the chytrid Allomyces macrogynus apparently do not secrete alkaline-active serine proteases, e.g. subtilisins, chymotrypsins and trypsins.
To investigate the link between fungal biomass and protease production, conidia of Con. coronatus and Met. anisopliae were inoculated into media containing from 0 to 4 % peptone (Fig. 1). At levels up to 1 % peptone, nutrients were a rate-limiting growth factor for Con. coronatus (Fig. 1a). Coincident with this, protease production peaked at 1 % peptone (Fig. 1a), implying that their primary role is in nutrient acquisition. Above 1 % peptone, growth rates levelled off and protease production declined, demonstrating catabolite repression at nutrient levels above that required for maximum growth rates (Fig. 1a). Production of proteases by Met. anisopliae was repressed at lower concentrations of peptone as compared to Con. coronatus (Fig. 1b), consistent with its lower growth rate.
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). Basic bands (pI
10) had the highest gelatinase activity, being visible in most cultures within 5 min of applying a gelatin overlay to the IEF gel (Fig. 2a). EOMs containing Suc-Ala-Ala-Pro-Phe-AFC confirmed that these bands hydrolysed a subtilisin substrate. The putative subtilisins were produced both during growth on multiple proteins and in the presence of solubilized readily accessible low-molecular-mass nutrients (peptone and glucose) (Fig. 2). The greatest complexity of proteinase bands was produced during growth on both unsclerotized (Manduca sexta) and sclerotized (cockroach) cuticles, with fewer isoforms being produced during growth on other proteins (bovine serum albumin or collagen) or chitin (a component of insect cuticle) (Fig. 2). With the caveat that different proteases may have the same isoelectric points, no new proteases appear to be produced during growth on non-cuticular substrates as compared to cuticle. The large number of proteinases produced in cuticle-containing media suggests an ability of Con. coronatus to respond to the environmental conditions in an insect host.
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10-5) for 58 % of the clones of which only 60 % had a fungal sequence as the best match (90 % from ascomycetes, 2 % from chytridiomycetes and 4 % each from zygomycetes and basidiomycetes). The large proportion (40 %) of the ESTs that were most similar to sequences from bacteria, protists, plants or animals probably reflects the very limited amounts of sequence data available from related zygomycete fungi.
Function of expressed Con. coronatus genes
The sorting of Met. anisopliae (Freimoser et al., 2003) and Con. coronatus genes expressed in medium that contains cuticle into the eight general-function categories in Fig. 3 (see also Table of supplementary data available at http://mic.sgmjournals.org which contains a complete list of all best BLASTX matches for Con. coronatus sequences) facilitated comparisons between the ascomycete and zygomycete pathogens. Thus Fig. 3 shows that unlike Met. anisopliae, metabolism-related genes were collectively the most prevalent among the identified cDNA clones from Con. coronatus. The high proportion of ESTs that encode enzymes of intermediary (energy) metabolism indicates that the rapid growth that is characteristic of Con. coronatus puts a high demand on central metabolic processes to furnish simple precursors and ATP. Complementary DNA clones encoding various ribosomal proteins were also abundant and contributed to the protein-synthesis-related gene category being the second most prevalent (Fig. 3). The oxidative deamination of amino acids also seems to be an important route in the degradation and assimilation of amino acid skeletons since at least three different dehydrogenase transcripts were represented by four ESTs. These enzymes play a key role in nitrogen metabolism in many organisms and free ammonia is released as a product of catalysis (Garnier et al., 1997). Ammonia alters ambient pH in fungal cultures and regulates the expression of virulence genes in Met. anisopliae (St. Leger et al., 1999). Con. coronatus also expresses a gene (BQ622518) that is similar (E=2x10-35) to a carbonic anhydrase from Schizosaccharomyces pombe that plays a key role in bicarbonate transport and regulation of pH (Sterling et al., 2001).
The Con. coronatus EST collection revealed that it produced considerable amounts of proteins generally referred to as stress-related proteins. The most conspicuous species were homologues to molecular chaperones that facilitate protein folding, e.g. the 70 kDa heat-shock protein. Although hsp70 genes fluctuate in response to cell stress they also function in normal cellular physiology and are developmentally regulated in Blastocladiella emersonii (Stefani & Gomes, 1995). Met. anisopliae also expresses high levels of protein chaperones during growth on insect cuticle, but the principal component is calnexin (Freimoser et al., 2003). The Met. anisopliae EST collection contained a variety of expressed genes encoding antioxidant proteins, including superoxide dismutase, catalase and peroxidase. These proteins are involved in the pathogenicity of animals and plants by fungi, in particular providing defence against active oxygen species produced by the host (Iwanaga & Kawabata, 1998; Wu et al., 1997). Con. coronatus has a different antioxidant profile, having homologues to glutathione-dependent antioxidant enzymes (
-glutamylcysteine synthetase, glutathione reductase) and lacking ESTs for catalase, superoxide dismutase and peroxidase. Recently, it was shown that synthesis of -glutamylcysteine is crucial for the survival of the malaria parasite in Plasmodium-infected blood cells (Meierjohann et al., 2002).
There is no evidence that Con. coronatus responds to light and, unlike in Met. anisopliae, we did not find ESTs for clock genes. However, BQ622207 shows similarity (E=8x10-22) with VIVID, a Neurospora crassa protein that is light-induced, clock-regulated and represses light-regulated processes (Heintzen et al., 2001). This implies that components of the Neurospora circadian system, to date only found in ascomycetes, have antecedents in the lower fungi.
The products of some pathogenicity genes will be involved in the exchange of signals between the pathogen and its environment, and activation of pathogenic mechanisms. Some of the Con. coronatus genes encoding sensors have homologues across a range of phyla. For example, BQ621736 shows similarity (E=3x10-30) to an osmosensor in Neurospora crassa that is also similar to osmosensors in bacteria (Schumacher et al., 1997). Likewise, BQ621855 has similarity (E=7x10-32) to notchless (Drosophila) and other proteins with WD40 repeats that are involved in protein interactions (Royet et al., 1998). BQ622041 is similar (E=7x10-22) to protein kinase C, involved in the developmental process in the human pathogenic ascomycete Sporothrix schenckii (Aquino-Pinero & Rodriguez del Valle, 1997). Production by Con. coronatus of a kinase (BQ622118) that is similar (E=1x10-25) to SNF1 is of special interest as SNF1 plays a key role in regulating expression of secreted cell-wall-degrading enzymes in the plant-pathogenic ascomycete Cochliobolus carbonum (Tonukari et al., 2000). This implies similarities between the regulatory circuitry of these unrelated pathogens with very different hosts.
Genes with putative roles in host invasion
The ESTs offered insight into components that may aid the fungus in the processes of physical ingress and nutrient solubilization, and thus may constitute quantitative factors that contribute to the overall virulence of the pathogen. Matches to secreted hydrolytic enzymes with substrates in insect hosts included proteases and chitinases. A subtilisin (BQ622771) that is highly similar (E=7x10-65) to the aqualysin 1 precursor from Thermus aquaticus comprised 5 % of all the sequences and is thus the most highly expressed gene in Con. coronatus grown on cuticle. The EST library revealed two additional subtilisins, a trypsin (BQ622656) similar (E=4x10-8) to Alp1 from Cochliobolus carbonum, a metalloprotease (BQ622216) similar (E=3x10-9) to MepB from Aspergillus fumigatus and an aspartyl protease (BQ622675) similar (E=5x10-30) to pepsinogen from Aspergillus niger.
Subtilisins have been intensively studied in insect–fungal interactions (St. Leger & Screen, 2001). Aspartyl and metalloproteases have been implicated in the infection processes of the plant pathogen Glomerella cingulata (Clark et al., 1997) and the human pathogen Aspergillus fumigatus (Sirakova et al., 1994), respectively. Trypsins are the most abundant proteases secreted by many plant-pathogenic ascomycetes (St. Leger et al., 1997). Likewise, a trypsin is the most abundant transcript among Met. anisopliae sf. anisopliae ESTs (Freimoser et al., 2003). The Con. coronatus trypsin EST was used as a probe to obtain a full-length cDNA (AF426410) that retained similarities with Alp1 (E=8x10-32) and demonstrated complete conservation of the active site components (unpublished data). It is the first trypsin isolated from a fungus that is not a pathogenic pyrenomycetous ascomycete. Trypsins are lacking in completed genomes from several ascomycetes, including yeast, Neurospora crassa and Magnaporthe grisea (http://www.genome.wi.mit.edu/annotation/fungi). This suggests that loss of trypsin genes has occurred independently in multiple lineages, indicating that gene loss may be an important factor in fungal evolution. Insertion elements could be a factor promoting genetic instability by increasing the frequency with which DNA sequence is gained or lost. However, in contrast to the Met. anisopliae EST project, no active transposable elements were detected in the Con. coronatus library.
The patchy distribution of trypsins is consistent with the theme of niche-specific traits, irrespective of their phylogenetic position. From an insect–microbe perspective such traits might include the ability to colonize insect surfaces and tissues, to render host tissues suitable for consumption and overcome cell- and peptide-mediated components of the insect immune system. Broadly dispersed, anti-insect virulence mechanisms might, therefore, include toxins and hydrolytic enzymes capable of degrading host tissues and disabling anti-microbial peptides. The proteases and chitinases produced by Con. coronatus could play this role in the same way as similar enzymes do for Met. anisopliae (St. Leger & Screen, 2000). Hits to endo- and exo-acting chitinases (BQ622770, BQ622772, BQ622421, BQ622341) indicate that like Met. anisopliae, Con. coronatus produces a chitinolytic complex to degrade cuticular components.
Conidiobolus spp. are reported to secrete toxins (Roberts & Humber, 1981). However, these were not characterized and the described symptoms (haemorrhaging, blackening of the blood) are also consistent with the action of fungal subtilisins on the prophenoloxidase system of the insect (St. Leger et al., 1996). In contrast to the Met. anisopliae strain 2575 EST project that identified many toxin-encoding genes (Freimoser et al., 2003), Con. coronatus appears to produce few if any toxic enzymes (e.g. no phopholipases) or secondary metabolites (although it is possible that novel toxin-encoding genes may be included in the hypothetical category). However, BQ622484 is similar (E=8x10-11) to L-ornithine N5-oxygenase involved in pyoverdin biosynthesis by Pseudomonas aeruginosa. Pyoverdin plays a predominant role in mobilizing iron from mammalian hosts (Leoni et al., 1996). Con. coronatus also expressed an EST (BQ622285) with similarity (E=4x10-32) to a ferritin subunit (iron storage protein) from the insect Galleria mellonella (Aisen et al., 2001). It is probable that iron is often a rate-limiting growth factor for Con. coronatus as is the case for many other pathogens (Weinberg, 1999).
Many pathogens produce a broad array of antimicrobials to defend against opportunistic colonizers of insect cadavers (Pirozynski & Hawksworth, 1988), e.g. Met. anisopliae strain 2575 has many ESTs encoding products with similarity to known antimicrobial agents, including lyzosyme and thaumatin. Con. coronatus expressed no such ESTs. Instead it produced sequences such as BQ622839, similar (E=3x10-24) to cephalosporin C acetylhydrolase from Acremonium chrysogenum which detoxifies bacterial products, and BQ622726 similar (E=2x10-18) to a xenobiotic metabolizing cytochrome p450 (monooxygenase) enzyme in tobacco. Zygomycetes apparently lack antibiotics and in consequence early death of the host can lead to the fungus being displaced from the cadaver by competing opportunistic micro-organisms (Evans, 1989).
Like Con. coronatus, the specific acridid pathogen Met. anisopliae strain 324 also expressed very few toxins and antimicrobials (Freimoser et al., 2003). This relates to lifestyles. Strain 2575 kills hosts quickly via toxins and grows saprophytically in the cadaver. In contrast, like many specialists, 324 causes a systemic infection of host tissues before the host dies. Unlike Met. anisopliae strain 324, Con. coronatus is an opportunistic pathogen of stressed insects. Assuming that the paucity of antimicrobials in the EST collection of Con. coronatus is a reflection of its survival strategies, this points to adaptation to environments with a low degree of combatative competition from other microbes. This is consistent with an R-selected strategy and exploitation of ephemeral habitats (Cooke & Rayner, 1984). Con. coronatus resembles the Mucorales (classic R-strategists) in being coenocytic with rapid spore germination and high mycelial extension rates, characteristics that permit Mucorales to utilize available soluble carbohydrates in advance of more slowly developing fungi. As would be expected from this lifestyle, Con. coronatus had a large number of ESTs for glucosidases and carriers, such as BQ622767 similar (E=5x10-16) to the monosaccharide transporter (Mst-1) of Amantia muscaria and BQ622015 similar (E=4x10-23) to the sucrose carrier (Sca1) of Pneumocystis carinii. These would permit uptake of the soluble carbohydrates that would be available to initial colonizers of recently living plant tissues. However, Con. coronatus also possesses amino acid transporters and secretes proteases that would permit it to solubilize recently living animal tissues and thus quickly access a longer lasting resource than simple sugars. This may guarantee a more prolonged existence and is a strategy presumably not available to the mucoralean fungi assayed in this study. This extra enzymic competence would allow niche differentiation between the Mucorales and the Entomophthorales that may have pre-adapted the latter to entomopathogenicity or may itself have derived from adaptation to entomopathogenicity. The ephemeral nature of Mucorales in a habitat is determined by their lack of combative ability as well as their inability to utilize carbon sources other than mono- or disaccharides (Cooke & Rayner, 1984). In the absence of an ability to combat other microbes, Con. coronatus is likewise not adapted to exploit an insect cadaver over a long period in the presence of competitors. However, production of high levels of proteases against a background of soluble nutrients, which would repress protease production by Met. anisopliae, will facilitate Con. coronatus to colonize wounds containing soluble nutrients, and hydrolyse and utilize other nutrient-rich insect resources such as the haemolymph in such a way that they are quickly exhausted.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 17, 3389–3402.
Aquino-Pinero, E. E. & Rodriguez del Valle, N. (1997). Different protein kinase C isoforms are present in the yeast and mycelium forms of Sporothrix schenckii. Mycopathologia 138, 109–115.[CrossRef][Medline]
Benny, G. L., Humber, R. A. & Morton, J. B. (2001). Zygomycota: Zygomycetes. In The Mycota 7A, pp. 113–146. Edited by D. J. McLaughlin, E. G. McLaughlin & P. A. Lemke. Berlin, Heidelberg: Springer.
Berbee, M. L. & Taylor, J. W. (2001). Fungal molecular evolution: gene trees and geologic time. In The Mycota 7A, pp. 229–245. Edited by D. J. McLaughlin, E. G. McLaughlin & P. A. Lemke. Berlin, Heidelberg: Springer.
Clark, S. J., Templeton, M. D. & Sullivan, P. A. (1997). A secreted aspartic proteinase from Glomerella cingulata: purification of the enzyme and molecular cloning of the cDNA. Microbiology 143, 1395–1403.[Abstract]
Cooke, R. C. & Rayner, A. D. M. (1984). Ecology of Saprotrophic Fungi. London: Longman.
Evans, H. C. (1989). Mycopathogens of insects of epigeal and aerial habitats. In Insect–Fungus Interactions, pp. 205–238. Edited by N. Wilding, N. M. Collins, P. M. Hammond & J. F. Webber. London: Academic Press.
Freimoser, F. M., Screen, S., Baga, S., Hu, G. & St. Leger R. J. (2003). Expressed sequence tag (EST) analysis of two subspecies of Metarhizium anisopliae reveals a plethora of secreted proteins with potential activity in insect hosts. Microbiology 149, 239–247.
Garnier, A., Berredjem, A. & Botton, B. (1997). Purification and characterization of the NAD-dependent glutamate dehydrogenase in the ectomycorrhizal fungus Laccaria bicolor (Maire) orton. Fungal Genet Biol 22, 168–176.[CrossRef][Medline]
Gray, G. L., Hayenga, K., Cullen, D., Wilson, L. J. & Norton, S. (1986). Primary structure of Mucor miehei aspartyl protease: evidence for a zymogen intermediate. Gene 48, 41–53.[CrossRef][Medline]
Hajek, A. E. (1997). Ecology of terrestrial fungal entomopathogens. Adv Micro Ecol 15, 193–249.
Heintzen, C., Loros, J. J. & Dunlap, J. C. (2001). The PAS protein vivid defines a clock-associated feedback loop that represses light input, modulates gating, and regulates clock resetting. Cell 104, 453–464.[CrossRef][Medline]
Huang, X. & Madan, A. (1999). CAP3: a DNA sequence assembly program. Genome Res 9, 868–877.
Humber, R. A. (1984). Foundations for an evolutionary classification of the Entomophthorales (Zygomycetes). In Fungus–Insect Relationships: Perspectives in Ecology and Evolution, pp. 166–183. Edited by Q. Wheeler & M. Blackwell. New York: Columbia University Press.
Iwanaga, S. & Kawabata, S. (1998). Evolution and phylogeny of defense molecules associated with innate immunity in horseshoe crab. Front Biosci 3, 973–984.
Jensen, A. B., Gargas, A., Eilenberg, J. & Rosendahl, S. (1998). Relationships of the insect-pathogenic order Entomophthorales (Zygomycota, Fungi) based on the phylogenetic analysis of small ribosomal sequences (SSU rDNA). Fungal Genet Biol 24, 325–334.[CrossRef][Medline]
Joshi, L. & St. Leger, R. J. (1999). Cloning, expression, and substrate specificity of MeCPA, a zinc carboxypeptidase that is secreted into infected tissues by the fungal entomopathogen Metarhizium anisopliae. J Biol Chem 274, 9803–9811.
Leoni, L., Ciervo, A., Orsi, N. & Visca, P. (1996). Iron-regulated transcription of the pvdA gene in Pseudomonas aeruginosa: effect of Fur and PvdS on promoter activity. J Bacteriol 178, 2299–2313.
Meierjohann, S., Walter, R. D. & Muller, S. (2002). Glutathione synthetase from Plasmodium falciparum. Biochem J 363, 833–838.[CrossRef][Medline]
Papierok, B. (1986). Some noteworthy biological problems within the genus Conidiobolus. In Fundamental and Applied Aspects of Invertebrate Pathology, pp. 197–200. Edited by R. A. Samson, J. M. Vlak & D. Peter. Wageningen: Foundation of the Fourth International Colloquium of Invertebrate Pathology.
Pirozynski, K. A. & Hawksworth, D. L. (1988). Coevolution of fungi with plants and animals: introduction and overview. In Coevolution of Fungi with Plants and Animals, pp. 1–29. Edited by K. A. Pirozynski & D. L. Hawksworth. London: Academic Press.
Roberts, D. W. & Humber, R. A. (1981). Entomogenous fungi. In Biology of Conidial Fungi, pp. 201–236. London: Academic Press.
Royet, J., Bouwmeester, T. & Cohen, S. M. (1998). Notchless encodes a novel WD40-repeat-containing protein that modulates Notch signaling activity. EMBO J 17, 7351–7360.[CrossRef][Medline]
Schumacher, M. M., Enderlin, C. S. & Selitrennikoff, C. P. (1997). The osmotic-1 locus of Neurospora crassa encodes a putative histidine kinase similar to osmosensors of bacteria and yeast. Curr Microbiol 34, 340–347.[CrossRef][Medline]
Sirakova, T. D., Markaryan, A. & Kolattukudy, P. E. (1994). Molecular cloning and sequencing of the cDNA and gene for a novel elastinolytic metalloproteinase from Aspergillus fumigatus and its expression in Escherichia coli. Infect Immun 62, 4208–4218.
St. Leger, R. J. & Screen, S. E. (2000). In vitro utilization of mucin, lung polymers, plant cell walls and insect cuticle by Aspergillus fumigatus, Metarhizium anisopliae and Haematonectria haematococca. Mycol Res 104, 463–471.[CrossRef]
St. Leger, R. J. & Screen, S. E. (2001). Prospects for strain improvement of fungal pathogens of insects and weeds. In Fungal Biocontrol Agents: Progress, Problems and Potential, pp. 219–238. Edited by T. M. Butt, C. Jackson & N. Morgan. Wallingford: CABI.
St. Leger, R. J., Charnley, A. K. & Cooper, R. M. (1986). Cuticle-degrading enzymes of entomopathogenic fungi: synthesis in culture on cuticle. J Invertebr Pathol 48, 85–95.[CrossRef]
St. Leger, R. J., Charnley, A. K. & Cooper, R. M. (1987). Characterization of cuticle-degrading proteases produced by the entomopathogen Metarhizium anisopliae. Arch Biochem Biophys 253, 221–232.[CrossRef][Medline]
St. Leger, R. J., Durrands, P. K., Cooper, R. M. & Charnley, A. K. (1988). Regulation of production of proteolytic enzymes by the entomopathogenic fungus Metarhizium anisopliae. Arch Microbiol 150, 413–416.[CrossRef]
St. Leger, R. J., Bidochka, M. J. & Roberts, D. W. (1994). Isoforms of the cuticle-degrading Pr1 proteinase and production of a metalloproteinase by Metarhizium anisopliae. Arch Biochem Biophys 313, 1–7.[CrossRef][Medline]
St. Leger, R. J., Joshi, L., Bidochka, M. J. & Roberts, D. W. (1996). Construction of an improved mycoinsecticide overexpressing a toxic protease. Proc Natl Acad Sci U S A 93, 6349–6354.
St. Leger, R. J., Joshi, L. & Roberts, D. W. (1997). Adaptation of proteases and carbohydrases of saprophytic, phytopathogenic and entomopathogenic fungi to the requirements of their ecological niches. Microbiology 143, 1983–1992.[Abstract]
St. Leger, R. J., Nelson, J. O. & Screen, S. E. (1999). The entomopathogenic fungus Metarhizium anisopliae alters ambient pH, allowing extracellular protease production and activity. Microbiology 145, 2691–2699.
Stefani, R. M. & Gomes, S. L. (1995). A unique intron-containing hsp70 gene induced by heat shock and during sporulation in the aquatic fungus Blastocladiella emersonii. Gene 152, 19–26.[CrossRef][Medline]
Sterling, D., Reithmeier, R. A. F. & Casey, J. R. (2001). A transport metabolon: functional interaction of carbogenic anhydrase II and chloride/bicarbonate exchangers. J Biol Chem 276, 47886–47894.
Tonukari, N. J., Scott-Craig, J. S. & Walton, J. D. (2000). The Cochliobolus carbonum SNF1 gene is required for cell wall-degrading enzyme expression and virulence on maize. Plant Cell 12, 237–248.
Voigt, K. & Woestermeyer, J. (2001). Phylogeny and origin of 82 zygomycetes from all 54 genera of the Mucorales and Mortierellales based on combined analysis of actin and translation elongation factor EF-alpha genes. Gene 270, 113–120.[CrossRef][Medline]
Weinberg, E. D. (1999). The role of iron in protozoan and fungal infectious disease. J Euk Microbiol 46, 231–238.[Medline]
Wu, G. S., Shortt, B. J., Lawrence, E. B., Leon, J., Fitzsimmons, K. C., Levine, E. B., Raskin, I. & Shah, D. M. (1997). Activation of host defense mechanisms by elevated production of H2O2 in transgenic plants. Plant Physiol 115, 427–435.[Abstract]
Received 22 January 2003;
revised 5 March 2003;
accepted 14 March 2003.
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