Department of Microbial Ecology, Lund University, Ecology Building, SE-223 62 Lund, Sweden
Correspondence
Anders Tunlid
Anders.Tunlid{at}mbioekol.lu.se
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Microarray raw data are available at the EBI-EMBL ArrayExpress database (http://www.ebi.ac.uk/arrayexpress) (accession no. E-MEXP-250).
A comparison of the primary structure of the M. haptotylum gph1 protein with the phosphorylases of yeast (Saccharomyces cerevisiae) and rabbit (Oryctolagus cuniculus) is available in Supplementary Fig. S1a, and a phylogeny of glycogen phosphorylases in Supplementary Fig. S1b, with the online version of this paper at http://mic.sgmjournals.org/.
Present address: European Bioinformatic Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD, UK.
These authors contributed equally to this work.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The ultrastructure of the nematode-trapping structures has been examined extensively. These studies have shown that despite the large variation in morphology, the adhesive types of trap (branches, nets and knobs) have a unique ultrastructure that clearly distinguishes them from vegetative hyphae (Dijksterhuis et al., 1994). One feature, which is common to all of these traps, is the presence of numerous cytosolic organelles, the so-called dense bodies. Although the function of these organelles is not yet clear, the fact that they exhibit catalase and D-amino acid oxidase activity indicates that the dense bodies are peroxisomal in nature (Dijksterhuis et al., 1994
). Another feature common to the trap cells is the presence of extensive layers of extracellular polymers, which are thought to be important for attachment of the traps to the surface of the nematode (Tunlid et al., 1991
).
In this study we have analysed the global pattern of gene expression in knobs and mycelium of the fungus Monacrosporium haptotylum (syn. Dactylaria candida) (Fig. 1). The advantage of using M. haptotylum is that during growth in liquid cultures with heavy aeration, the connections between the traps (knobs) and mycelium can be broken easily and the knobs can be separated from the mycelium by filtration (Friman, 1993
). The isolated knobs retain their function as infection structures: they can capture and infect nematodes. In order to characterize overall gene expression, we have conducted expressed sequence tag (EST) analysis with 8466 clones from four different cDNA libraries. These include libraries made from mycelium and knobs, and two libraries from knobs infecting the nematode Caenorhabditis elegans. Since the genome of C. elegans has been sequenced, transcripts expressed by the fungus were easily separated from those of the host. From the EST information assembled, we obtained a unique set of 3036 clones, of either fungal or worm origin, which was used for the construction of cDNA microarrays. Here we describe the analysis of gene expression patterns in knobs and mycelium.
|
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
RNA isolation and construction of cDNA libraries.
Total RNA was extracted from the vegetative mycelium, the knobs and infected C. elegans using the RNeasy plant mini kit (Qiagen) with the guanidinium isothiocyanate buffer provided. The integrity of the total RNA was checked by denaturating formaldehyde agarose gel electrophoresis and the quantity was assessed by UV spectrophotometry. Four unidirectional cDNA libraries were constructed from total RNA using the PCR-based SMART cDNA Library Construction kit (BD Clontech). After ligation of size-fractionated cDNA (>100 bp) into the TriplEx2 arms (BD Clontech) and lambda phage packaging (GigaPack III Gold extract; Stratagene), the primary titres for the unamplified libraries were determined to be 5·9x106 p.f.u. for mycelium, 1·6x106 p.f.u. for knob, 2·5x106 p.f.u. for the early stage of C. elegans infection (4 h) and 2·9x106 p.f.u. for the late stage of C. elegans infection (24 h). In order to amplify the libraries, phage suspensions were used to transduce Escherichia coli XL-1 Blue, which resulted in titres of more than 5·0x109 p.f.u. ml1. The
TriplEx2 libraries were converted to plasmid libraries by in vivo excision in E. coli BM25.8 (Johansson et al., 2004
). PCR amplification followed by gel electrophoresis demonstrated that more than 90 % of the randomly collected clones contained insert. Based on the analysis of 96 individual clones from each of the four libraries, the average sizes of the inserts in the mycelium, knob, 4 h infection and 24 h infection cDNA libraries were found to be approximately 1100, 1000, 750 and 1200 bp, respectively. The size of the transcripts ranged from 120 to 2400 bp in all four libraries.
EST analysis.
Plasmid clones were randomly collected from each of the four libraries. The colonies were transferred to 96-well plates, and the bacterial lysates were used as starting material for PCR amplification (Johansson et al., 2004). PCR was performed using pTriplEx2-specific universal primers P104 (5'-GGGAAGCGCGCCATTGTGTT-3') and P105 (5'-AGTGAGCTCGAATTGCGGCC-3') in a total volume of 10 µl, followed by assessment of the size and quality of the products by gel electrophoresis. Partial nucleotide sequences of amplified cDNA inserts were determined using the P104 primer and the dideoxy chain-termination method employing the BigDye Terminator Cycle Sequencing kit (Applied Biosystems). The sequencing reaction products were analysed using either a 377 or 3100 ABI sequencer (Applied Biosystems). cDNA clones representing genes that are discussed in more detail were verified and completely sequenced by using universal and gene-specific primers (Table 1
).
|
To separate fungal sequences from worm sequences, a local BLASTN search was performed against 22 055 C. elegans cDNA sequences downloaded from the ENSEMBLE website (http://www.ensembl.org/Caenorhabditis_elegans/). Sequences with an E value <1·0x104 were annotated as C. elegans and sequences with an E value >1·0x104 were annotated as fungal. Sequences of <50 bp were annotated as unknown.
Construction of cDNA arrays.
A single EST cDNA clone was selected to represent each assembled sequence (i.e. putatively unique transcript). For an assembled contig represented by multiple ESTs, the rule followed was to select the clone with the most extensive DNA sequencing read length. These clones were collected from the master EST library stock contained as bacterial lysate plates and aliquots were transferred into new 96-well plates. Plasmid inserts were amplified by PCR, purified and concentrated as previously described (Johansson et al., 2004). In total, 3518 clones representing approximately 3036 contigs were processed successfully. cDNA PCR products were printed on CMT-GAPSII-coated slides (Corning Glass) using a 16-pin configured MicroGrid II array printer (BioRobotics) controlled by the MicroGrid TAS Application Suite (version 2.2.0.6). The general design of the microarrays was two identical tool arrays, each containing 16 blocks (sub-arrays) and with each clone replicated twice within each block, which provided on-chip quadruplicates for each clone. Additionally, various positive and negative control reporters were replicated in various positions within each block, such as positive homologous controls, that is, M. haptotylum and C. elegans genes expected to be highly expressed. In addition, eight different heterologous and commercial PCR reporters (ArrayControl; Ambion) for which complementary RNAs were spiked into the amplification and labelling process of targets were also included in the blocks. Each control reporter was present in up to 64 per-chip replicates. DNA was cross-linked to the slides by baking at 80 °C for 2 h in glass jars, followed by UV cross-linking at 90 mJ cm2. Spot and print quality were assessed by staining with POPO3 dye (Molecular Probes), and subsequent scanning showed that no spots were missing or joined on the slides.
Microarray analysis.
To compare the expression pattern of genes in mycelium and knobs of M. haptotylum, our microarray experiments were designed as two-samples comparisons (Kerr & Churchill, 2001; Wu, 2001
) (i.e. knobs versus mycelium) using three independent biological replicates, which also included technical and dye-swapped control hybridizations. Nine cDNA microarrays were hybridized; four of these were hybridizations of knobs versus mycelium and five were mycelium against mycelium. cDNA probes were prepared from total RNA isolated from vegetative mycelium and knobs, grown under identical conditions to those used in the construction of the cDNA libraries. Total RNA was extracted according to Stiekema et al. (1988)
and purified further on an RNeasy plant mini kit spin column (Qiagen). To obtain the required amounts of RNA from limited amounts of starting material, two rounds of antisense RNA (aRNA) amplification were performed using the MessageAmp kit (Ambion). The integrity of aRNA was analysed by denaturating formaldehyde agarose gel electrophoresis and quantified using the RiboGreen RNA Quantification kit (Molecular Probes). The aRNA samples were divided into aliquots and stored at 80 °C. A total of 2 µg aRNA for each target, including ArrayControl RNA exogenous spikes (Ambion), was labelled (Cy3 or Cy5) using the CyScribe post-labelling kit (Amersham Biosciences). The labelled cDNA was purified with the CyScribe GFX Purification kit (Amersham Biosciences). The slides were hybridized, rinsed and scanned as previously described (Johansson et al., 2004
). The microarray raw data are available at the EBI-EMBL ArrayExpress database (http://www.ebi.ac.uk/arrayexpress) (accession no. E-MEXP-250).
Statistical analysis.
After scanning, data images were inspected manually and low-quality spots were excluded before further analysis. Reporters of unknown origin and related to C. elegans were excluded, and only data for 2822 reporters representing fungal genes were extracted. For those spots remaining, the raw fluorescence intensities for each channel on each slide were collected. The mean background fluorescence was calculated for each channel. After local background correction for each spot, the reporters yielding intensities below twice the mean background were excluded and the fluorescence for the remaining reporters was multiplied to give a common channel mean of 5000 fluorescence units. As a result, data for 2534 fungal genes remained in the dataset. The statistical approach we used, the mixed-model analysis of variance (ANOVA), served two purposes: (1) normalization of the data to remove systemic biases that may have affected all genes simultaneously, such as differences in the amount of RNA that was labelled for a particular replicate of a treatment; (2) assessment of the contribution of biological and experimental sources of error to the variation in expression of each individual gene (Wolfinger et al., 2001). This procedure uses differences in normalized expression levels, rather than ratios, as the unit of analysis of expression differences. We subjected the corrected log2-transformed measures (ygij) for the fungal gene g (g=1,..., 2534) which included scores for 107 445 fungal spot measures to a normalization model of the form ygij=µ+Ai+Dj+(AxD)ij+
gij, where µ is the sample mean, Ai is the effect of the ith array (i=19), Dj is the effect of the jth dye (Cy3 or Cy5), (AxD)ij is the arraydye interaction (channel effect), and
gij is the stochastic error. We then subjected the residuals from this model, which can be regarded as crude indicators of relative expression level (and which are referred to in the text as normalized gene expression levels) to 2534 gene-specific models of the form rijlm=µ+Ai+Tl+Dj+
ijlm, where Tl is the lth tissue (knob or mycelium; 1 degree of freedom, d.f.). In the gene models, which were fitted using PROC MIXED in SAS (SAS/STAT software version 8, SAS Institute), the Ai variable controls for spot effects and is random (8 d.f., leaving 7 d.f. for the residual error).
Real-time quantitative PCR.
aRNA was reverse transcribed into cDNA using the SYBR Green RT-PCR Reagents kit (Applied Biosystems). The reverse transcription (RT) reactions were normalized to contain equivalent amounts (2 µg) of aRNA. RT reactions were primed by random hexamers and were carried out in a total volume of 10 µl according to the manufacturer's instructions. The cDNA product was treated with 2 units of RNase H (Invitrogen) for 20 min at 37 °C, followed by heat inactivation at 75 °C for 10 min.
Gene-specific oligonucleotide primers were designed by using the web-based primer picking service (Primer3) (http://fokker.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) (Rozen & Skaletsky, 2000). The following criteria were applied: melting temperatures 5860 °C, primer lengths 2022 nt, GC content 4080 %, PCR amplicon length 100200 bp (Table 1
). The PCR reactions were performed with an Mx3000P Real-Time PCR system (Stratagene) using SYBR Green PCR Master Mix (Applied Biosystems) to monitor dsDNA synthesis. The reactions contained 12·5 µl 2x SYBR Green PCR Master Mix (including AmpliTaq Gold DNA Polymerase, dNTPs with dUTP, SYBR Green I Dye, Passive Reference and optimized buffer components), 1 µl cDNA (diluted 1 : 10), 1 µl gene-specific forward and reverse primers (0·5 µmol l1 of each), in a final volume of 25 µl. The following thermal profile was used for all PCRs: an initial denaturation step at 95 °C for 10 min, followed by 100 cycles at 95 °C for 30 s, 58 °C for 1 min, and 72 °C for 1 min, and finally extended elongation at 72 °C for 30 s. Reactions were set up in triplicate, including a control with no template. Amplification of gene-specific products was analysed by melting-curve analysis followed by agarose gel electrophoresis (cf. Fig. 3
).
|
Phylogenetic analysis.
To construct the phylogenetic trees of GTPases, gEgh16 homologues and glycogen phosphorylases, homologous sequences were retrieved from the GenBank nr protein database (Benson et al., 2004) using BLAST searches (Altschul et al., 1990
). Protein alignments were made using CLUSTAL W (Thompson et al., 1994
). Phylogeny was done using the Neighbour-Joining method (Saitou & Nei, 1987
) with 500 bootstrap replications implemented in the MEGA 2.1 program (Kumar et al., 2001
).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In total, 8466 ESTs were obtained from the four cDNA libraries. The sequences were assembled into 3121 contigs that putatively represent unique genes/transcripts (Table 2). Of the assembled sequences, 723 were represented in the mycelium library, 574 in the knob library, 682 in the 4 h infection library and 1531 in the 24 h infection library. Between 56 and 71 % of the contigs were singletons: they were represented by only one EST clone. Between 5 and 37 % of the assembled sequences displayed a high degree of similarity (FASTA score >299) to sequences in the GenBank nr protein database (Table 2
). The fraction of ESTs showing high-score homology was largest in the 4 h infection library. This is due to the fact that this library contained a large proportion of C. elegans sequences: 67 % of the contigs in this library were identified as being of C. elegans origin, while 21 % were of fungal origin and 12 % of unknown origin. The fraction of C. elegans transcripts in the 24 h library was considerably lower (3·7 %), whereas 88 % were of fungal and 8·2 % of unknown origin. A large fraction (3860 %) of the assembled sequences showed no homology (orphans) to protein sequences in the GenBank nr protein database, and the proportion of orphans was highest in the knob (58 %) and 24 h infection (60 %) libraries.
|
|
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The rho and ras family of small GTPases, including rho, rac and cdc42, play an important role in regulating the actin part of the cytoskeleton (Pruyne & Bretscher, 2000a), and these molecules act as molecular switches through their ability to hydrolyse GTP. In the GTP-bound form, these signalling proteins are active and exert a positive signal on proteins associated with polarized growth. We identified and characterized three small GTPases, including a rho1, a rac and a ras homologue (Fig. 5
) that were differentially expressed in knobs and mycelium. Phylogenetic analysis showed that the EST clone CN800249 (fold change on microarray 2·03 and by real-time RT-PCR 0·25) was a rho1 homologue, and the EST clone CN795728 (fold change on microarray 1·32) was a rac1 homologue (Fig. 5
). The rho1 homologue of M. haptotylum (AY635989) encodes a protein of 193 aa. The deduced peptide rho1 contains all the signatures that have been found to be characteristic of the small G protein superfamily, including five GTP-binding and GTP hydrolysis domains (G1, G2, G3, G4 and G5) as well as the C-terminal prenylation site for rho GTPases (CXXL, C indicates cystein, X any amino acid and L leucin) involved in membrane associations (Bourne et al., 1991
; Finegold et al., 1991
). Rho1 of M. haptotylum shows high sequence similarity to the rho1 protein of other fungi, including that from Aspergillus nidulans (89 % aa identity), Schizosaccharomyces pombe (79 %), S. cerevisiae (76 %) and Cryptococcus neoformans (76 %).
|
The rac1 homologue of M. haptotylum (AY635990) was predicted to encode a peptide of 194 aa, and contained the conservative G1, G2, G3, G4 and G5 domains as well as the prenylation site (CXXL) characteristic of rac1 proteins (Bourne et al., 1991). The protein showed 80 % sequence identity (aa) to a rac GTPase (cflB) of the dimorphic ascomycete Penicillium marneffei (Boyce et al., 2003
). Interestingly, cflB co-localizes with actin at the tips of the vegetative hyphal cells. Deletion of cflB results in growth defects in vegetative hyphae, depolarization and inappropriate septation (Boyce et al., 2003
).
The ras1 homologue of M. haptotylum (AY635992, fold change on microarray +2·67 and by real-time RT-PCR +2·19) was predicted to encode a 198 aa protein, and showed a significant sequence similarity to ras-like proteins from Colletotrichum trifolii (46 % identity), A. nidulans (46 %), S. cerevisiae (42 %) and Ustilago maydis (44 %), especially in regions corresponding to the GTP-binding and GTP hydrolysis domains (G1, G2, G3, G4 and G5) as well as the C-terminal prenylation site (CAAX, C indicates cysteine, A an aliphatic amino acid and X any amino acid) (Bourne et al., 1991).
Comparison of gene expression in knobs and appressoria
There are several similarities in the structure and function of knobs of nematode-trapping fungi and those of appressoria formed by plant-pathogenic fungi. Like a knob, an appressorium is a specialized infection structure, which develops as a spherical cell at the tip of a hypha (germ tube). Both structures contain an adhesive layer on the outside, which binds to the surface of the host. Furthermore, both appressoria and knobs form a hypha that penetrates the host using a combination of physical force and extracellular enzymic activities (Tucker & Talbot, 2001). Comparison of data from the transcriptional profiling of knobs with that of appressoria in Magnaporthe grisea and Blumeria graminis shows that there are also many similarities in the patterns of gene regulation in the infection structures of nematode-trapping and plant-parasitic fungi (Table 6
).
|
|
A number of genes involved in protein synthesis (such as homologues for ribosomal proteins and translation elongation factor), protein destination and degradation (such as homologues for ubiquitin, ubiquitin-conjugating enzyme and proteasome components) were differentially expressed in both knobs and appressorium (Tables 4 and 5). This suggests that development of the infection structures in both nematode-trapping and plant-pathogenic fungi is associated with an extensive synthesis and turnover of proteins (McCafferty & Talbot, 1998
).
Glycogen and carbon metabolism
One of the most upregulated genes in the knobs was a gene (EST clone CN795977, fold change on microarray +4·36 and by real-time RT-PCR +2·49) showing similarity to glycogen phosphorylases (-D-glucosyltransferase, EC 2.4.1.1). Complete sequence analysis of this cDNA clone (AY635994) revealed an ORF corresponding to a putative protein of 874 aa. The entire aa sequence of M. haptotylum phosphorylase (gph1) showed 59 % identity to the S. cerevisiae glycogen phosphorylase (Hwang & Fletterick, 1986
) and 45 % identity to rabbit muscle glycogen phosphorylase (Nakano et al., 1986
). Multiple sequence alignment showed extensive sequence similarity in regions corresponding to the catalytic domain and C-terminal domain, while the regulatory or N-terminal domain appeared more variable (Supplementary Fig. S1a with the online version of this paper at http://mic.sgmjournals.org/).
Extensive biochemical and crystallographic studies of the rabbit glycogen phosphorylase have identified seven functional domains, including the active site, the glycogen storage site, the purine nucleoside inhibitor site, the cofactor (pyridoxal phosphate, PLP) binding site, the phosphorylation site, the AMP binding site, and the subunit dimerization point (Hwang & Fletterick, 1986). Four of these regions, the active site (labelled as g in Supplementary Fig. S1a) (1 substitution in 17 residues), the glycogen storage site (s) (2 substitutions in 8 residues), the purine nucleoside inhibitor site (c) (1 substitution in 4 residues) and the PLP binding site (v) (1 substitution in 8 residues) were highly conserved between the M. haptotylum and the rabbit enzymes. The N-terminal regulatory domain is important for allosteric regulation, and carries the regulatory phosphorylation site (p) of glycogen phosphorylases. In the rabbit enzyme, this domain is located within the first 80 aa of the N-terminal region (Palm et al., 1985
). Apart from the phosphorylation site, the N-terminal region of the phosphorylases contains the AMP binding site (a) and the subunit dimerization point (d). Considerable amino acid sequence identity (76 %) was observed in the N-terminal region of the yeast and the M. haptotylum phosphorylases, whereas the rabbit enzyme appeared more divergent. The yeast enzyme is phosphorylated at a specific threonine residue (Thr-19) by a phosphorylase kinase, and additionally by a cyclic AMP-dependent protein kinase (Hwang & Fletterick, 1986
). The Thr-19 residue, as well as adjacent residues, were well conserved between the yeast and the M. haptotylum phosphorylase.
A phylogenetic analysis of phosphorylases from different organisms showed that the gph1 protein of M. haptotylum was found in a clade with high bootstrap support (value 100), containing sequences from several ascomycetes, including a putative glycogen phosphorylase of the human pathogen Aspergillus fumigatus and hypothetical proteins from the rice blast fungus Ma. grisea (Supplementary Fig. S1b).
Glycogen is an important storage carbohydrate in eukaryotes, and glycogen phosphorylase catalyses and regulates the degradation of glycogen to glucose 1-phosphate (Fletterick & Madsen, 1980). Glycogen is accumulated in incipient appressoria of Ma. grisea, but is rapidly degraded before generating the turgor pressure needed for penetration of the plant cuticle (Thines et al., 2000
). The turgor pressure results from a rapid accumulation of glycerol, and there are different lines of evidence suggesting that the production of glycerol is achieved by the mobilization of energy reserves, such as glycogen and neutral lipids (Thines et al., 2000
). The glucose 1-phosphate generated from the degradation of glycogen is metabolized in the glycolytic pathway. Glycerol can be synthesized from several of the intermediates in this pathway. Unfortunately, we did not identify any putative homologues in the M. haptotylum EST database of enzymes known to be involved in these pathways in S. cerevisiae (see references in Thines et al., 2000
). Thus, whether or not the genes encoding these enzymes are regulated in the knobs is not known. Alternatively, the breakdown of glycogen can lead to the formation of pyruvate and the production of energy (ATP). However, this part of the pathway does not appear to be upregulated in the knobs. The homologues of enzymes in this part of the glycolytic pathway that were identified, including glyceraldehyde-3-phosphate dehydrogenase (CN796696) and enolase (CN797534), were down-regulated in the knobs compared to mycelium (fold change on microarray, 1·59 and 2·07, respectively) (Table 4
). Studies have also shown that the degradation of glycogen in Ma. grisea is under the control of the cAMP-dependent protein kinase A (PKA) and MAP kinase (MAPK) pathway (Thines et al., 2000
). In yeast, phosphorylase activity is regulated by reversible phosphorylation/dephosphorylation mediated by the cAMP regulatory cascade (Hwang & Fletterick, 1986
). The fact that the N-terminal domain, which contains regions important for allosteric regulation and carries the regulatory phosphorylation site (Thr-19), was well conserved between the yeast and M. haptotylum enzymes, suggests that the two phosphorylases may be regulated by similar mechanisms.
Peroxisomal associated proteins
The presence of numerous dense bodies, which are related to peroxisomes, is typical for traps of nematode predatory fungi (Dijksterhuis et al., 1994). Interestingly, one transcript (CN796915, fold change on microarray +0·72) with similarity to a peroxisomal membrane protein in Candida boidiini (PMP30B) was significantly upregulated. There is a homologue in S. cerevisiae (PMP27, PEX11, peroxin) which is involved in peroxisomal proliferation. Deletion of PMP27 generates a phenotype containing a few large peroxisomes, as if peroxisomal fission was inhibited (Erdmann & Blobel, 1995
). Other genes that were regulated in knobs and displayed sequence similarities to genes encoding proteins associated with peroxisomes included homologues to D-amino acid oxidase (CN796046, fold change on microarray 3·09), 2,4-dienoyl-CoA reductase (SPS19, S. cerevisiae) (CN797536, fold change on microarray +1·05), and fatty acid transporter and very long-chain fatty acyl-CoA synthetase (FAT1, S. cerevisiae) (CN796073, fold change on microarray 2·30).
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ahrén, D., Ursing, B. M. & Tunlid, A. (1998). Phylogeny of nematode-trapping fungi based on 18S rDNA sequences. FEMS Microbiol Lett 158, 179184.[CrossRef][Medline]
Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. (1990). Basic local alignment search tool. J Mol Biol 215, 403410.[CrossRef][Medline]
Ayscough, K. R. (1998). In vivo functions of actin-binding proteins. Curr Opin Cell Biol 10, 102111.[CrossRef][Medline]
Barron, G. L. (1977). The Nematode-Destroying Fungi. Guelph, Canada: Lancester Press.
Benson, D. A., Karsch-Mizrachi, I., Lipman, D. J., Ostell, J. & Wheeler, D. L. (2004). GenBank: update. Nucleic Acids Res 32, 2326.
Bourne, H. R., Sanders, D. A. & McCormick, F. (1991). The GTPase superfamily: conserved structure and molecular mechanism. Nature 349, 117127.[CrossRef][Medline]
Boyce, K. J., Hynes, M. J. & Andrianopoulos, A. (2003). Control of morphogenesis and actin localization by the Penicillium marneffei RAC homolog. J Cell Sci 116, 12491260.
Brenner, S. (1974). The genetics of Caenorhabditis elegans. Genetics 77, 7194.
Dijksterhuis, J., Veenhuis, M., Harder, W. & Nordbring-Hertz, B. (1994). Nematophagous fungi: physiological aspects and structure-function relationships. Adv Microb Physiol 36, 111143.[Medline]
Emmons, S. W., Klass, M. R. & Hirsh, D. (1979). Analysis of the constancy of DNA sequences during development and evolution of the nematode Caenorhabditis elegans. Proc Natl Acad Sci U S A 76, 13331337.[Abstract]
Erdmann, R. & Blobel, G. (1995). Giant peroxisomes in oleic acid-induced Saccharomyces cerevisiae lacking the peroxisomal membrane protein Pmp27p. J Cell Biol 128, 509523.[Abstract]
Ewing, B., Hillier, L., Wendl, M. C. & Green, P. (1998). Base-calling of automated sequencer traces using Phred. I. Accuracy assessment. Genome Res 8, 175185.
Finegold, A. A., Johnson, D. I., Farnsworth, C. C., Gelb, M. H., Judd, S. R., Glomset, J. A. & Tamanoi, F. (1991). Protein geranylgeranyltransferase of Saccharomyces cerevisiae is specific for Cys-Xaa-Xaa-Leu motif proteins and requires the CDC43 gene product but not the DPR1 gene product. Proc Natl Acad Sci U S A 88, 44484452.
Fletterick, R. J. & Madsen, N. B. (1980). The structures and related functions of phosphorylase a. Annu Rev Biochem 49, 3161.[CrossRef][Medline]
Friman, E. (1993). Isolation of trap cells from the nematode-trapping fungus Dactylaria candida. Exp Mycol 17, 368370.[CrossRef]
Grell, M. N., Mouritzen, P. & Giese, H. (2003). A Blumeria graminis gene family encoding proteins with a C-terminal variable region with homologues in pathogenic fungi. Gene 311, 181192.[CrossRef][Medline]
Huang, X. (1992). A contig assembly program based on sensitive detection of fragment overlaps. Genomics 14, 1825.[CrossRef][Medline]
Hwang, P. K. & Fletterick, R. J. (1986). Convergent and divergent evolution of regulatory sites in eukaryotic phosphorylases. Nature 324, 8084.[CrossRef][Medline]
Johansson, T., Le Quéré, A., Ahrén, D., Söderström, B., Erlandsson, R., Lundeberg, J., Uhlén, M. & Tunlid, A. (2004). Transcriptional responses of Paxillus involutus and Betula pendula during formation of ectomycorrhizal root tissue. Mol PlantMicrobe Interact 17, 202215.[Medline]
Justesen, A., Somerville, S., Christiansen, S. & Giese, H. (1996). Isolation and characterization of two novel genes expressed in germinating conidia of the obligate biotroph Erysiphe graminis f.sp. hordei. Gene 170, 131135.[CrossRef][Medline]
Kerr, M. K. & Churchill, G. A. (2001). Experimental design for gene expression microarrays. Biostatistics 2, 183201.
Koch, G., Tanaka, K., Masuda, T., Yamochi, W., Nonaka, H. & Takai, Y. (1997). Association of the Rho family small GTP-binding proteins with Rho GDP dissociation inhibitor (Rho GDI) in Saccharomyces cerevisiae. Oncogene 15, 417422.[CrossRef][Medline]
Kumar, S., Tamura, K., Jakobsen, I. B. & Nei, M. (2001). MEGA2: molecular evolutionary genetics analysis software. Bioinformatics 17, 12441245.
Lappalainen, P. & Drubin, D. G. (1997). Cofilin promotes rapid actin filament turnover in vivo. Nature 388, 7882.[CrossRef][Medline]
Larsen, M. (2000). Prospects for controlling animal parasitic nematodes by predacious microfungi. Parasitology 120, 121131.[Medline]
Livak, K. J. & Schmittgen, T. D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2Ct method. Methods 25, 402408.[CrossRef][Medline]
McCafferty, H. R. & Talbot, N. J. (1998). Identification of three ubiquitin genes of the rice blast fungus Magnaporthe grisea, one of which is highly expressed during initial stages of plant colonisation. Curr Genet 33, 352361.[CrossRef][Medline]
Mewes, H. W., Frishman, D., Gruber, C. & 7 other authors (2000). MIPS: a database for genomes and protein sequences. Nucleic Acids Res 28, 3740.
Nakano, K., Hwang, P. K. & Fletterick, R. J. (1986). Complete cDNA sequence for rabbit muscle glycogen phosphorylase. FEBS Lett 204, 283287.[CrossRef][Medline]
Nordbring-Hertz, B., Jansson, H.-B., Friman, E. & 5 other authors (1995). Nematophagous fungi. Film No C 1851. Göttingen, Germany: Institut für den Wissenschaftlichen Film.
Palm, D., Goerl, R. & Burger, K. J. (1985). Evolution of catalytic and regulatory sites in phosphorylases. Nature 313, 500502.[Medline]
Pearson, W. R. (1994). Using the FASTA program to search protein and DNA sequence databases. Methods Mol Biol 24, 307331.[Medline]
Pruyne, D. & Bretscher, A. (2000a). Polarization of cell growth in yeast. I. Establishment and maintenance of polarity states. J Cell Sci 113, 365375.
Pruyne, D. & Bretscher, A. (2000b). Polarization of cell growth in yeast. II. The role of the cortical actin cytoskeleton. J Cell Sci 113, 571585.
Rauyaree, P., Choi, W., Fang, E., Blackmon, B. & Dean, R. A. (2004). Genes expressed during early stages of rice infection with the rice blast fungus Magnaporthe grisea. Mol Plant Pathol 2, 347354.
Rozen, S. & Skaletsky, H. (2000). Primer3 on the WWW for general users and for biologist programmers. In Bioinformatics Methods and Protocols, pp. 365386. Edited by S. Krawetz & S. Misener. Totowa, NJ: Humana Press.
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406.[Abstract]
Stiekema, W. J., Heidekamp, F., Dirkse, W. G., van Beckum, J., de Haan, P., ten Bosch, C. & Louwerse, J. D. (1988). Molecular cloning and analysis of four potato tuber mRNA. Plant Mol Biol 11, 255269.
Takano, Y., Choi, W., Mitchell, T. K., Okuno, T. & Dean, R. A. (2004). Large scale parallel analysis of gene expression during infection-related morphogenesis of Magnaporthe grisea. Mol Plant Pathol 4, 337346.[CrossRef]
Thines, E., Weber, R. W. & Talbot, N. J. (2000). MAP kinase and protein kinase A-dependent mobilization of triacylglycerol and glycogen during appressorium turgor generation by Magnaporthe grisea. Plant Cell 12, 17031718.
Thomas, S. W., Glaring, M. A., Rasmussen, S. W., Kinane, J. T. & Oliver, R. P. (2002). Transcript profiling in the barley mildew pathogen Blumeria graminis by serial analysis of gene expression (SAGE). Mol PlantMicrobe Interact 15, 847856.[Medline]
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 46734680.[Abstract]
Tucker, S. L. & Talbot, N. J. (2001). Surface attachment and pre-penetration stage development by plant pathogenic fungi. Annu Rev Phytopathol 39, 385417.[CrossRef][Medline]
Tunlid, A., Johansson, T. & Nordbring-Hertz, B. (1991). Surface polymers of the nematode-trapping fungus Arthrobotrys oligospora. J Gen Microbiol 137, 12311240.[Medline]
Viaud, M. C., Balhadere, P. V. & Talbot, N. J. (2002). A Magnaporthe grisea cyclophilin acts as a virulence determinant during plant infection. Plant Cell 14, 917930.
Wolfinger, R. D., Gibson, G., Wolfinger, E. D., Bennett, L., Hamadeh, H., Bushel, P., Afshari, C. & Paules, R. S. (2001). Assessing gene significance from cDNA microarray expression data via mixed models. J Comput Biol 8, 625637.[CrossRef][Medline]
Wu, T. D. (2001). Analysing gene expression data from DNA microarrays to identify candidate genes. J Pathol 195, 5365.[CrossRef][Medline]
Xue, C., Park, G., Choi, W., Zheng, L., Dean, R. A. & Xu, J. R. (2002). Two novel fungal virulence genes specifically expressed in appressoria of the rice blast fungus. Plant Cell 14, 21072119.
Received 14 July 2004;
revised 8 November 2004;
accepted 10 November 2004.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |