| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 32611, USA
Correspondence
Nemat O. Keyhani
keyhani{at}ufl.edu
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
10 kDa protein found in boiling SDS-insoluble, trifluoroacetic acid-soluble extracts from aerial conidia identified the major component of the B. bassiana rodlet layer to be the hyd2 gene product. These results reveal the differential regulation of the isolated hydrophobins and indicate that phage display represents a novel approach to cDNA cloning of hydrophobins.
The GenBank/EMBL/DDBJ accession numbers for the hyd1 and hyd2 sequences of B. bassiana are EF452344 and EF520285, respectively.
Figures showing the sequences of hydrophobin genes used in phylogenetic analysis, atomic force micrographs and SDS-PAGE results obtained during rodlet extraction, MS results for a peptide fragment resulting from in-gel tryptic digestion, and BLAST matches of peptide fragment data are available with the online version of this paper.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Wessels, 1997; Wosten & de Vocht, 2000). Members of this protein family contain eight cysteine residues with conserved spacing and display characteristic hydropathy patterns. Hydrophobins are often major protein components of the fungal conidial cell envelope, polymerizing to form bundle or rodlet-like structures, or they can be secreted into the medium, where they self-assemble at the water–air interface, lowering the water surface tension, thus enabling the fungi to breach the interphase to form aerial structures (Kwan et al., 2006; Talbot, 1999; Wosten et al., 1993). Although the role of hydrophobins in some cell processes remains unclear, they have been implicated in a variety of developmental processes, including pathogenesis, fruit body formation, and sporulation, possibly by contributing to key structural features required for differentiation (Beckerman & Ebbole, 1996; Bell-Pedersen et al., 1992; Ebbole, 1997; Girardin et al., 1999; Kazmierczak et al., 2005; Kershaw & Talbot, 1998; Lugones et al., 1996; Nishizawa et al., 2002). Due to their unique biophysical properties, hydrophobins have also been suggested for use in a wide variety of medical and technical applications, such as tissue engineering (by increasing the biocompatibility of medical implants and devices), drug delivery (using hydrophobin-stabilized vesicles), and nanotechnology-based surface patterning and alteration (Corvis et al., 2005; Scholtmeijer et al., 2002, 2004).
Beauveria bassiana is an important entomopathogenic fungus that displays a broad host range, and is able to target a diverse range of arthropod species (Clarkson & Charnley, 1996; Leathers et al., 1993; Maurer et al., 1997). Strains of B. bassiana have been selected for the control of insects and other arthropods that act as disease vectors, including mosquitoes and ticks, crop pests such as whiteflies and borers, and even ecologically hazardous, invading pests such as fire ants and termites (Blanford et al., 2005; Brownbridge et al., 2001; Cruz et al., 2006; Kirkland et al., 2004; Klinger et al., 2006; Marti et al., 2005; McCoy, 1990).
Phage display technology is based upon the expression of protein or peptide sequences on the surface of phage particles by genetic fusion with phage capsid proteins. Applications include biopanning for antibody-, protein-, peptide-, DNA-, RNA- and oligonucleotide-binding phage-fusion particles (particularly using peptide libraries), as well as cDNA cloning of proteins that act as allergens (Danner & Belasco, 2001; Mullen et al., 2006; Paschke, 2006; Rhyner et al., 2004; Sergeeva et al., 2006). Phage display has also been used to clone a protein with carbohydrate affinity, namely galectin-3 from a HeLa cell display library by mucin affinity selection (Yamamoto et al., 1999).
In this report, we describe the successful use of phage display for the isolation of hydrophobins. A phage display cDNA library was constructed using RNA from B. bassiana grown in the presence of insect cuticle. Biopanning experiments were performed using a number of carbohydrate affinity resins, including N,N'-diacetylchitobiose–, fucose–, lactose–, maltose– and melibiose–agarose beads. Hydrophobins appeared to be selectively enriched by melibiose–agarose beads, which yielded two different hydrophobin proteins highly represented in the final library enrichment, and to a lesser extent by lactose–agarose beads, which yielded one of the hydrophobins. Expression studies were performed using RNA isolated from a variety of fungal developmental stages, including: (i) single-cell types such as aerial conidia, in vitro blastospores and submerged conidia; (ii) during fungal mycelial growth; and (iii) during sporulation on chitin and insect cuticle. Additionally, extraction and mass spectrometric analysis of the major protein constituent of the hydrophobic aerial conidia rodlet layer revealed it to be composed of the hyd2 gene product.
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Biopanning protocol.
N,N'-Diacetylchitobiose-, fucose-, lactose-, maltose- and melibiose-coupled agarose beads were obtained from Seigakagu (Tokyo). Beads were washed three times with PBS (50 mM sodium phosphate, pH 7.4, 0.15 M NaCl) before being resuspended in PBS+1 % BSA before use. The phage stock (10 ml of 1010 p.f.u. ml–1) was precipitated using PEG-8000 (4 % final concentration) and NaCl (3 % final concentration) and resuspended in 1 ml PBS+1 % BSA. Beads (200 µl) were mixed with 0.5 ml precipitated phage and incubated at 4 °C for 2 h with gentle shaking, before being washed five times with 1 ml PBS containing 0.5 M NaCl. Bound phage was eluted (twice) with a 0.2 M solution (300 µl total volume, incubated at room temperature for 30 min) of the carbohydrate moiety bound to each respective agarose-coupled bead. The titre of the eluted phage was amplified by passage through an Escherichia coli host, and the resultant phage stock was used for the next round of biopanning. Five rounds of the biopanning procedure were performed successively with each affinity resin.
Molecular manipulations.
cDNA inserts of phage plaques were analysed by PCR amplification and sequencing (ICBR Sequencing Facility, University of Florida) using vector primers (T7Up and T7Down). Genomic DNA was extracted from fungal mycelium using hexamethyltrimethyl ammonium bromide (CTAB), as described by Talbot (2001). DNA hybridization probes were labelled using the BioPrime chemiluminescent DNA labelling system (Invitrogen). Plasmid preparation, restriction enzyme digestion, agarose gel electrophoresis and Southern blotting were performed using standard procedures (Ausubel et al., 1996).
RNA extraction.
Total RNA was extracted from B. bassiana cells using either RNAwiz or TRI Reagent (Ambion) according to the manufacturer's recommendations, including the high-salt precipitation step for removal of proteoglycans and polysaccharides. Culture conditions for RNA extraction were as follows: B. bassiana was grown on PD agar (PDA) or on Sabouraud dextrose+1 % yeast extract either on agar plates (SDAY) or in liquid broth (SDY). Plates were incubated at 26 °C for 10–12 days and aerial conidia were harvested by flooding the plate with sterile dH2O. Conidial suspensions were filtered through Miracloth and final spore concentrations were determined by direct counting using a haemocytometer. Liquid broth cultures were inoculated (1 : 50, v/v) with conidia harvested from plates to a final concentration of 0.5–5x105 conidia ml–1. Cultures were grown for 3–4 days at 26 °C with aeration. Cultures were filtered through glass wool or Mira cloth to remove mycelia, and the concentration of blastospores was determined by direct counting. Submerged conidia were isolated from TKI broth (per litre: 50.0 g fructose, 10.0 g KNO3, 5.0 g KH2PO4, 2.0 g MgSO4 . 7H20, 50.0 mg CaCl2, 50.0 mg yeast extract), as previously described (Cho et al., 2006b). A time course of growing mycelia was prepared by growing B. bassiana on PDA. At the desired time points (3, 5, 10, 18 and 28 days), conidia were removed by washing the plates by flooding two to three times with dH2O, and the mycelium was obtained by lightly scraping off the fungal biomass from the resultant agar plates. Mycelia were examined by light microscopy for the presence of conidia, and samples containing less than 1 % conidia were used for further experimentation. Chitin and insect cuticle liquid broth cultures (50–100 ml), consisting of 1 : 4 diluted Sabouraud dextrose broth supplemented with 1 % (w/v) chitin, 1 % powdered, sterilized Manduca sexta cuticle (kind gift of D. Boucias, Department of Entomology and Nematology, University of Florida), or 1 % chitin+1 % M. sexta culticle, were inoculated with conidia harvested from plates to a final concentration of 0.5–5x105 conidia ml–1 and were grown for 3 days at 26 °C with aeration, after which the total fungal cell culture was harvested by centrifugation (10 000 g, 10 min), washed twice with dH2O, flash-frozen in liquid nitrogen, and stored at –70 °C.
Semi-quantitative RT-PCR analysis.
Total RNA isolated as described above was precipitated once with LiCl before being DNase-treated using the DNA-free reagent (Ambion). RNA samples were then treated with SUPERase-In (RNase inhibitor, Ambion) and stored at –70 °C until use. Total RNA concentration was quantified for each sample preparation using the Ribogreen RNA quantification kit (Molecular Probes). cDNA for each sample was synthesized using 1.0 µg of total RNA plus Superscript III reverse transcriptase with oligo dT18 priming, following the manufacturer's protocol (Invitrogen). PCR reactions were as follows: 1.0 µl of a twofold dilution of the cDNA sample was PCR-amplified using 6.0 µl DNA polymerase MasterMix (Eppendorf), 0.3 µl of 0.01 mM of each primer, and dH2O to a final volume of 17.0 µl. PCR amplification was performed at 94 °C for 30 s, 64 °C for 30 s, and 72 °C for 35 s. Samples (10 µl) were taken and analysed by agarose gel electrophoresis after 15, 20, 25 and 30 PCR cycles. Amplifications of portions of the B. bassiana actin and β-tubulin genes (see Table 2) were performed as controls during all reactions, and were used as internal standards to normalize the expression levels of the hyd1 and hyd2 genes in the various RNA samples. The relative intensity of the bands was determined after densitometric scanning using Adobe Photoshop. Primer sets used for the amplification of hyd1, hyd2, actin and β-tubulin are presented in Table 2. In all experiments, controls containing no template or no enzyme were performed. Each PCR reaction was performed in duplicate with duplicate biological samples and duplicate cDNA preparations for each sample.
|
). Briefly, aerial conidia were resuspended in water, and sonicated at 140 W (3 mm diameter microtip, 50 % duty cycle) twice for 10 min using a Sonifier cell disrupter B-30 (Branson Ultrasons). Unlysed cells and cell debris were removed by low-speed centrifugation (10 000 g, 10 min), and the supernatant was centrifuged for 30 min at 50 000 g. The resultant pellet was boiled in SDS-PAGE sample buffer (2 % SDS, 5 % β-mercaptoethanol, 10 % glycerol, v/v, in 62 mM Tris/HCl, pH 6.8), and washed twice with sample buffer and three times with distilled water. The final pellet was lyophilized, then treated with 100 % trifluoroacetic acid (TFA) for 10 min at room temperature. The acid was removed under a stream of nitrogen, and dried extracts were stored at room temperature under dry air and resuspended in water prior to analysis. Aliquots of protein sample were mixed with 4x lithium dodecyl sulfate (LDS) sample buffer plus DTT and run on a 10 % Bistris NuPAGE gel with MES-SDS gel running buffer together with standards (Invitrogen). Protein bands were visualized using either Sypro Ruby Red (Biorad) or Coomassie Blue (Biorad). For MS fingerprinting, proteins were separated by SDS-PAGE, and a protein with an apparent molecular mass of 10 kDa was subjected to in-gel tryptic digestion. Capillary reversed-phase HPLC separation of protein digests was performed on a 10 cmx75 µm internal diameter PepMap C18 column (LC Packings) in combination with a home-built capillary HPLC system operated at a flow rate of 200 nl min–1. Inline mass spectrometric analysis of the column eluate was accomplished by a quadrupole ion trap instrument (LCQ, ThermoFinnigan) equipped with a nanoelectrospray source. Fragment ion data generated by data-dependent acquisition via the LCQ were searched against a local sequence database using the SEQUEST (ThermoFinnigan) database search engine. The score for SEQUEST protein identification was considered significant when dCn was equal to 0.08 or greater and the cross-correlation score (Xcorr) was greater than 2.2.
Rodlet layer extracts were electrophoresed on SDS-PAGE and electroblotted onto Immobilon-P PVDF membranes. After transfer, the blot was stained with Coomassie Blue, an 10 kDa protein band was cut out, and the N-terminal sequence determined using an Applied Biosystems Procise Sequencer (Protein CORE Facility, University of Florida).
Phylogenetic analysis.
A phylogenetic analysis was performed using nearest-neighbour joining analysis of 56 hydrophobin sequences found in GenBank, along with the B. bassiana sequences using the CLUSTALW (EMBL-EBI, 1000 bootstrap trails, 500 random number generator) multiple sequence alignment program. The resultant tree was visualized using the online program Phylodendron phylogenetic tree printer (http://iubio.bio.indiana.edu/treeapp/treeprint-form.html).
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
After five rounds of enrichment, inserts from 20 plaques of each library selected by biopanning were amplified by PCR and examined by agarose gel electrophoresis, revealing that >95 % of the plaques tested had inserts ranging in size from 0.4 to 1.5 kb. Using heat-denatured phage lysate from each cycle of the biopanning procedures, cDNA inserts were amplified by PCR, and enrichment of certain phages was observed, as judged by the increased band intensity through the rounds of selections (data not shown). Sixteen plaques from each library were randomly selected and their insert nucleotide sequences determined. Sequencing reads averaged 450 bp per read, and their translated amino acid sequences were analysed by BLAST searches of the National Center for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov/blast/). Sequences derived from enrichment using two of the five affinity resins, lactose–agarose and melibiose–agarose, corresponded to cDNAs encoding two different B. bassiana hydrophobins. Four cDNA clones contained almost identical sequence inserts, which were used to construct a full-length unambiguous cDNA sequence termed hyd1. Three additional phage-display-enriched cDNA sequences that shared no homology to hyd1 clustered to yield hyd2.
In order to obtain the positions of possible introns within the coding sequences of the B. bassiana genes, primer oligonucleotides covering the start and stop codons of each hydrophobin gene were used in PCR reactions performed on genomic DNA. A summary of the gene and deduced protein characteristics is listed in Table 1. Based upon the genomic sequence, hyd1 contained two small (53 and 56 nt) introns, whereas hyd2 contained a single intron (64 nt). Putative promoters and polyadenylation signals were also detected in the isolated gene sequences. The ORFs corresponding to hyd1 and hyd2 coded for proteins of 136 and 116 aa, respectively, with both proteins containing putative export signal peptides. Southern blot analysis using the cloned genes as probes indicated that only one allele of each gene was present in the genome (data not shown).
|
), whereas Hyd2 was most closely related to the Magnaporthe grisea mpg1 and Metarhizium anisopliae ssgA gene products (also class I hydrophobins) (St Leger et al., 1992; Talbot et al., 1993) (Fig. 1). The phylogenetic analysis also revealed the ancient ancestry of the different hydrophobin genes, suggesting that the two genes are not derived from a recent duplication event.
|
). These data are consistent with that reported for other hydrophobins, and confirm the amphipathic nature of the proteins.
|
The relative expression level of the two genes was assessed in RNA pools derived from aerial conidia, blastospores, submerged conidia, growing mycelia, and from cells grown on insect cuticle, chitin (the main carbohydrate constituent of insect cuticles), and insect cuticle+chitin (Fig. 3). The data presented were determined to be within the linear portion of the PCR analysis (25 cycles), with the actin concentration closely correlating to the amount of RNA quantified for each sample under test. Hydrophobin expression was normalized to the quantification of the actin band. B. bassiana hyd1 expression was detected in all developmental stages and media conditions tested. Hyd1 levels appeared to increase during mycelial growth, with the highest level of expression observed after 28 days of growth on agar plates, although caution should be taken in any interpretation of these results, as they derived from semi-quantitative measurements. On agar plates, cells began sporulating (i.e. producing aerial conidia) after 14 days; however, conidia were washed from the plates of all the mycelial samples before RNA isolation. This was confirmed by microscopic visualization of the mycelial samples, in which <1 % of contaminating aerial conidia were visible in the mycelial preparations. Thus, the observed hyd1 expression appears to be derived from the mycelia. Expression in aerial conidia was similar to that observed in either blastospores or submerged conidia, and was equivalent to that observed in 18 day mycelia. Cells growing in the presence of 1 % chitin, 1 % cuticle, and 1 % chitin+1 % cuticle also showed robust expression of hyd1, similar to levels seen in blastospores and submerged conidia, and 5–10 day mycelia. In contrast to hyd1, hyd2 appeared to be constitutively expressed and at about the same level as actin throughout the growing mycelia stages (3–28 days). Little or no hyd2 was observed in either blastospores or aerial conidia. Some hyd2 transcript was detected in submerged conidia, which corresponded to about 5–10 % of the levels seen in the mycelial samples. Intriguingly, hyd2 was expressed (at approximately the same levels as actin) in fungal cells growing on 1 % chitin or 1 % cuticle, but almost no transcript could be detected when the cells were grown on 1 % chitin+1 % cuticle.
|
). A protein with an apparent molecular mass of 10 kDa was identified by SDS-PAGE and used for in situ tryptic digestion followed by MS peptide fingerprinting (SDS-PAGE and MS data are available as supplementary data with the online journal). Two peptide fragments, TGDICGNGNTMHCCNDESVTNK and LTGPSVLSDLDLR, were obtained. In addition, N-terminal amino acid sequencing of the 10 kda band resulted in the sequence (P/H)-GPSHGPXVKTGDI, where (P/H) represents poorly identified residues and X an unresolved residue. The amino acid fingerprints were consistent with the translated ORF of the hyd2 sequence cloned by phage display, and the N-terminal amino acid sequence matched the sequence of the putative mature Hyd2 sequence: (APH)GPSHGP(S)VKTGDI. These data indicate that the hyd2 gene product constitutes the rodlet layer hydrophobin that surrounds B. bassiana conidia. |
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
, 1999; Wosten & de Vocht, 2000). These approaches are limited by expression levels and the success of protein purification protocols. Lectin-like activity has been reported for several hydrophobins. Both monomeric and assembled forms of the SC3 hydrophobin from the basidiomycete Schizophyllum commune agglutinate rat blood cells, whereas the SC4 hydrophobin from the same organism agglutinates horse red blood cells (van Wetter et al., 2000). The class II hydrophobin of the plant pathogen Cryphonectria parasitica, named cryparin, agglutinates rabbit, and to a lesser extent rat and chicken red blood cells, but not those of ox, horse, human blood group A or guinea pig (Kazmierczak et al., 1996; McCabe & Van Alfen, 1999). This behaviour is considered typical of lectins; however, although crude cell extracts were able to inhibit agglutination, no sugar tested appeared to inhibit agglutination. A lectin has been reported from B. bassiana that recognizes the Thomsen–Friedenrich antigen and related structures, although neither the primary amino acid nor the nucleotide sequence of the gene encoding the protein has been reported (Kossowska et al., 1999). Based upon a comparison of the amino acid content of the described B. bassiana lectin, it does not appear to be one of the hydrophobins characterized in this report.
Phage display technologies represent a method for isolating proteins and/or peptides that bind molecules of interest. (Danner & Belasco, 2001; Mullen et al., 2006; Paschke, 2006; Rhyner et al., 2004; Sergeeva et al., 2006). Phage display has been used to isolate human galectin-3, a galactose/lactose-specific lectin and member of the S-type lectin family, using blood group glycoproteins during a biopanning selection (Yamamoto et al., 1999). In this report, we demonstrate the feasibility of phage display to isolate cDNAs coding for hydrophobins. Initial attempts using hydrophobic substrata for biopanning were unsuccessful, due to non-specific adsorption of the phage. However, it may be possible to alter the conditions to minimize this background. Using various carbohydrate-derivatized beads, cDNAs encoding two hydrophobins were enriched from a B. bassiana phage display library constructed using mRNA isolated from fungal cells grown in vitro in the presence of insect cuticle. The interaction between the hydrophobins and the carbohydrate-derivatized beads appeared to be specific, since (1) not all carbohydrate-derivatized beads tested enriched for the B. bassiana hydrophobins, and (2) the elution step during each round of enrichment was competitive, using 0.2 M of the cognate carbohydrate. Further experiments, using the purified hydrophobin preparations, are however needed to conclusively demonstrate lectin-like activity for the isolated proteins.
The translated amino acid sequences of hyd1 and hyd2 did not match the 16 aa N-terminal sequence of a putative hydrophobin isolated from B. bassiana reported elsewhere, although the N terminus of mature hyd2 did show some homology (50 %) to the reported protein (Bidochka et al., 1995b). Similarly, although not an exact match, the N terminus of mature Hyd1 showed homology (40 %) to the N terminus (as determined by amino acid sequencing) of what was termed an inner cell wall protein (cwp1) of B. bassiana (Bidochka et al., 1995a). Although our results indicate that Hyd2 is a major constituent of the spore rodlet layer, additional experiments are needed to determine the cellular localization of Hyd1. Expression analysis indicated that hyd1 was highly expressed under almost all conditions. These results are consistent with our previous EST analyses of B. bassiana grown under different developmental conditions (Cho et al., 2006a, b). Notably, hyd1 transcript was abundant in all the single-cell spore types (aerial conidia, in vitro blastospores and submerged conidia), whereas hyd2 transcripts were essentially absent from these cells. Thus, even though the spore coat of aerial conidia is composed of the Hyd2 protein, no hyd2 transcript was detected in these cells. This is perhaps not too surprising, since it is the conidiogenic cells that need to make hyd2 and its protein product (in order to make the aerial conidia), and therefore one would not expect to see hyd2 transcript in the final product of this process, namely the aerial conidia themselves. Transcripts corresponding to hyd2 were also detected in cells grown in 1 % chitin or 1 % insect cuticle, but were not detected in cultures containing chitin+cuticle, each at 1 % concentration. This result was observed in two separate biological samples in which actin was used as a control. Altough it is unclear why hyd2 transcript was not detected under the latter growth conditions, it could be due to the relative nutrient levels in the cultures. Cells growing under 1 % chitin or 1 % cuticle conditions may be nutrient-limited, which would result in sporulation (i.e. conidiogenesis and hence hyd2 transcript production), whereas cultures containing both nutrient sources may grow in a vegetative state for a longer period of time. Further experiments delineating the expression of hyd1 and hyd2, especially during growth on insect hosts, i.e. during pathogenesis, as well as gene knockouts, are likely to give important insights into the role of these hydrophobins in fungal development and virulence.
Edited by: S. D. Harris
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Beckerman, J. L. & Ebbole, D. J. (1996). MPG1, a gene encoding a fungal hydrophobin of Magnaporthe grisea, is involved in surface recognition. Mol Plant Microbe Interact 9, 450–456.[Medline]
Bell-Pedersen, D., Dunlap, J. C. & Loros, J. J. (1992). The Neurospora circadian clock-controlled gene, ccg-2, is allelic to eas and encodes a fungal hydrophobin required for formation of the conidial rodlet layer. Genes Dev 6, 2382–2394.
Bidochka, M. J., St Leger, R. J., Joshi, L. & Roberts, D. W. (1995a). An inner cell wall protein (cwp1) from conidia of the entomopathogenic fungus Beauveria bassiana. Microbiology 141, 1075–1080.
Bidochka, M. J., St Leger, R. J., Joshi, L. & Roberts, D. W. (1995b). The rodlet layer from aerial and submerged conidia of the entomopathogenic fungus Beauveria bassiana contains hydrophobin. Mycol Res 99, 403–406.[CrossRef]
Blanford, S., Chan, B. H., Jenkins, N., Sim, D., Turner, R. J., Read, A. F. & Thomas, M. B. (2005). Fungal pathogen reduces potential for malaria transmission. Science 308, 1638–1641.
Brownbridge, M., Costa, S. & Jaronski, S. T. (2001). Effects of in vitro passage of Beauveria bassiana on virulence to Bemisia argentifolii. J Invertebr Pathol 77, 280–283.[CrossRef][Medline]
Cho, E. M., Boucias, D. & Keyhani, N. O. (2006a). EST analysis of cDNA libraries from the entomopathogenic fungus Beauveria (Cordyceps) bassiana. II. Fungal cells sporulating on chitin and producing oosporein. Microbiology 152, 2855–2864.
Cho, E. M., Liu, L., Farmerie, W. & Keyhani, N. O. (2006b). EST analysis of cDNA libraries from the entomopathogenic fungus Beauveria (Cordyceps) bassiana. I. Evidence for stage-specific gene expression in aerial conidia, in vitro blastospores and submerged conidia. Microbiology 152, 2843–2854.
Clarkson, J. M. & Charnley, A. K. (1996). New insights into the mechanisms of fungal pathogenesis in insects. Trends Microbiol 4, 197–203.[CrossRef][Medline]
Corvis, Y., Walcarius, A., Rink, R., Mrabet, N. T. & Rogalska, E. (2005). Preparing catalytic surfaces for sensing applications by immobilizing enzymes via hydrophobin layers. Anal Chem 77, 1622–1630.[Medline]
Cruz, L. P., Gaitan, A. L. & Gongora, C. E. (2006). Exploiting the genetic diversity of Beauveria bassiana for improving the biological control of the coffee berry borer through the use of strain mixtures. Appl Microbiol Biotechnol 71, 918–926.[CrossRef][Medline]
Danner, S. & Belasco, J. G. (2001). T7 phage display: a novel genetic selection system for cloning RNA-binding proteins from cDNA libraries. Proc Natl Acad Sci U S A 98, 12954–12959.
Ebbole, D. J. (1997). Hydrophobins and fungal infection of plants and animals. Trends Microbiol 5, 405–408.[CrossRef][Medline]
Fuchs, U., Czymmek, K. J. & Sweigard, J. A. (2004). Five hydrophobin genes in Fusarium verticillioides include two required for microconidial chain formation. Fungal Genet Biol 41, 852–864.[CrossRef][Medline]
Girardin, H., Paris, S., Rault, J., Bellon-Fontaine, M. N. & Latgé, J. P. (1999). The role of the rodlet structure on the physicochemical properties of Aspergillus conidia. Lett Appl Microbiol 29, 364–369.[CrossRef][Medline]
Holder, D. J. & Keyhani, N. O. (2005). Adhesion of the entomopathogenic fungus Beauveria (Cordyceps) bassiana to substrata. Appl Environ Microbiol 71, 5260–5266.
Kazmierczak, P., Pfeiffer, P., Zhang, L. & Van Alfen, N. K. (1996). Transcriptional repression of specific host genes by the mycovirus Cryphonectria hypovirus 1. J Virol 70, 1137–1142.[Abstract]
Kazmierczak, P., Kim, D. H., Turina, M. & Van Alfen, N. K. (2005). A hydrophobin of the chestnut blight fungus, Cryphonectria parasitica, is required for stromal pustule eruption. Eukaryot Cell 4, 931–936.
Kershaw, M. J. & Talbot, N. J. (1998). Hydrophobins and repellents: proteins with fundamental roles in fungal morphogenesis. Fungal Genet Biol 23, 18–33.[CrossRef][Medline]
Kirkland, B. H., Westwood, G. S. & Keyhani, N. O. (2004). Pathogenicity of entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae to Ixodidae tick species Dermacentor variabilis, Rhipicephalus sanguineus, and Ixodes scapularis. J Med Entomol 41, 705–711.[Medline]
Klinger, E., Groden, E. & Drummond, F. (2006). Beauveria bassiana horizontal infection between cadavers and adults of the Colorado potato beetle, Leptinotarsa decemlineata (Say). Environ Entomol 35, 992–1000.
Kossowska, B., Lamer-Zarawska, E., Olczak, M. & Katnik-Prastowska, I. (1999). Lectin from Beauveria bassiana mycelium recognizes Thomsen–Friedenreich antigen and related structures. Comp Biochem Physiol B Biochem Mol Biol 123, 23–31.[CrossRef][Medline]
Kwan, A. H., Winefield, R. D., Sunde, M., Matthews, J. M., Haverkamp, R. G., Templeton, M. D. & Mackay, J. P. (2006). Structural basis for rodlet assembly in fungal hydrophobins. Proc Natl Acad Sci U S A 103, 3621–3626.
Kyte, J. & Doolittle, R. F. (1982). A simple method for displaying the hydrophobic character of a protein. J Mol Biol 157, 105–132.[CrossRef][Medline]
Leathers, T. D., Gupta, S. C. & Alexander, N. J. (1993). Mycopesticides: status, challenges, and potential. J Ind Microbiol 12, 69–75.[CrossRef]
Linder, M. B., Szilvay, G. R., Nakari-Setala, T. & Penttila, M. E. (2005). Hydrophobins: the protein-amphiphiles of filamentous fungi. FEMS Microbiol Rev 29, 877–896.[CrossRef][Medline]
Lugones, L. G., Bosscher, J. S., Scholtmeyer, K., de Vries, O. M. & Wessels, J. G. (1996). An abundant hydrophobin (ABH1) forms hydrophobic rodlet layers in Agaricus bisporus fruiting bodies. Microbiology 142, 1321–1329.
Marti, G. A., Scorsetti, A. C., Siri, A. & Lastra, C. C. (2005). Isolation of Beauveria bassiana (Bals.) Vuill. (Deuteromycotina: Hyphomycetes) from the Chagas disease vector, Triatoma infestans (Hemiptera: Reduviidae) in Argentina. Mycopathologia 159, 389–391.[CrossRef][Medline]
Maurer, P., Couteaudier, Y., Girard, P. A., Bridge, P. D. & Riba, G. (1997). Genetic diversity of Beauveria bassiana and relatedness to host insect range. Mycol Res 101, 159–164.[CrossRef]
McCabe, P. M. & Van Alfen, N. K. (1999). Secretion of cryparin, a fungal hydrophobin. Appl Environ Microbiol 65, 5431–5435.
McCoy, C. W. (1990). Entomogenous fungi as microbial pestidides. In New Directions in Biological Control, pp. 139–159. Edited by R. R. Baker & P. E. Dunn. New York: A.R. Liss.
Mullen, L. M., Nair, S. P., Ward, J. M., Rycroft, A. N. & Henderson, B. (2006). Phage display in the study of infectious diseases. Trends Microbiol 14, 141–147.[CrossRef][Medline]
Nishizawa, H., Miyazaki, Y., Kaneko, S. & Shishido, K. (2002). Distribution of hydrophobin 1 gene transcript in developing fruiting bodies of Lentinula edodes. Biosci Biotechnol Biochem 66, 1951–1954.[CrossRef][Medline]
Paschke, M. (2006). Phage display systems and their applications. Appl Microbiol Biotechnol 70, 2–11.[CrossRef][Medline]
Rhyner, C., Weichel, M., Fluckiger, S., Hemmann, S., Kleber-Janke, T. & Crameri, R. (2004). Cloning allergens via phage display. Methods 32, 212–218.[CrossRef][Medline]
Scholtmeijer, K., Janssen, M. I., Gerssen, B., de Vocht, M. L., van Leeuwen, B. M., van Kooten, T. G., Wosten, H. A. & Wessels, J. G. (2002). Surface modifications created by using engineered hydrophobins. Appl Environ Microbiol 68, 1367–1373.
Scholtmeijer, K., Janssen, M. I., van Leeuwen, M. B., van Kooten, T. G., Hektor, H. & Wosten, H. A. (2004). The use of hydrophobins to functionalize surfaces. Biomed Mater Eng 14, 447–454.[Medline]
Sergeeva, A., Kolonin, M. G., Molldrem, J. J., Pasqualini, R. & Arap, W. (2006). Display technologies: application for the discovery of drug and gene delivery agents. Adv Drug Deliv Rev 58, 1622–1654.[CrossRef][Medline]
St Leger, R. J., Staples, R. C. & Roberts, D. W. (1992). Cloning and regulatory analysis of starvation-stress gene, ssgA, encoding a hydrophobin-like protein from the entomopathogenic fungus, Metarhizium anisopliae. Gene 120, 119–124.[CrossRef][Medline]
Talbot, N. J. (1999). Fungal biology. Coming up for air and sporulation. Nature 398, 295–296.[CrossRef][Medline]
Talbot, N. (2001). Nucleic acid isolation and analysis. In Molecular and Cellular Biology of Filamentous Fungi, pp. 23–26. Edited by N. Talbot. Oxford: Oxford University Press.
Talbot, N. J., Ebbole, D. J. & Hamer, J. E. (1993). Identification and characterization of MPG1, a gene involved in pathogenicity from the rice blast fungus Magnaporthe grisea. Plant Cell 5, 1575–1590.[Abstract]
van Wetter, M. A., Wosten, H. A. & Wessels, J. G. (2000). SC3 and SC4 hydrophobins have distinct roles in formation of aerial structures in dikaryons of Schizophyllum commune. Mol Microbiol 36, 201–210.[CrossRef][Medline]
Wessels, J. G. (1997). Hydrophobins: proteins that change the nature of the fungal surface. Adv Microb Physiol 38, 1–45.[Medline]
Wessels, J. G. (1999). Fungi in their own right. Fungal Genet Biol 27, 134–145.[CrossRef][Medline]
Wosten, H. A. & de Vocht, M. L. (2000). Hydrophobins, the fungal coat unravelled. Biochim Biophys Acta 1469, 79–86.[Medline]
Wosten, H., De Vries, O. & Wessels, J. (1993). Interfacial self-assembly of a fungal hydrophobin into a hydrophobic rodlet layer. Plant Cell 5, 1567–1574.[Abstract]
Yamamoto, M., Kominato, Y. & Yamamoto, F. (1999). Phage display cDNA cloning of protein with carbohydrate affinity. Biochem Biophys Res Commun 255, 194–199.[CrossRef][Medline]
Received 28 March 2007;
revised 14 May 2007;
accepted 30 May 2007.
This article has been cited by other articles:
|
|
 |
|
  Y. Zhang, J. Zhao, W. Fang, J. Zhang, Z. Luo, M. Zhang, Y. Fan, and Y. Pei Mitogen-Activated Protein Kinase hog1 in the Entomopathogenic Fungus Beauveria bassiana Regulates Environmental Stress Responses and Virulence to Insects Appl. Envir. Microbiol., June 1, 2009; 75(11): 3787 - 3795. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
