1 Institute for Molecular Biology and Genetics, Basic Science Research Institute, Chonbuk National University, Jeonju, Chonbuk 561-756, Korea
2 Department of Agricultural Biology, Chungbuk National University, Cheongju, Chungbuk 361-763, Korea
Correspondence
Dae-Hyuk Kim
dhkim{at}chonbuk.ac.kr
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the cpmk2 gene sequence from Cryphonectria parasitica strain EP155/2 is AY262368.
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INTRODUCTION |
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Since the phenotypic changes in fungal hosts induced by hypoviruses are pleiotropic but occur in a coordinated and specific manner, it was suggested that hypoviruses disturb one or more regulatory pathways (Nuss, 1996). Several studies have demonstrated the implementation of a signal transduction pathway during viral symptom development. Genes modulated during this process encode components of various signal transduction pathways, including heterotrimeric G-proteins, a putative regulator of these G-proteins (Choi et al., 1995
; Kasahara & Nuss, 1997
; Kasahara et al., 2000
), a novel kinase (Kim et al., 2002
) and many others that are under investigation (Dawe & Nuss, 2001
). Recently, we have demonstrated the hypoviral regulation of components of a mitogen-activated protein kinase (MAPK) signalling pathway in C. parasitica (Park et al., 2004
).
The MAPK signal transduction pathway is utilized by eukaryotic cells to transduce a wide variety of cellular signals through a stepwise phosphorylation relay. This cascade, which occurs in a wide variety of organisms from yeast to humans (Herskowitz, 1995; Schaeffer & Weber, 1999
), consists of three functionally interlinked protein kinases: MEEK (MAP kinase kinase kinase), MEK (MAP kinase kinase) and MAPK. Fungal MAPKs are involved in pathways that are required for numerous processes related to growth and differentiation (Xu, 2000
). Moreover, MAPKs are involved in the pathogenicity of many plant-pathogenic fungi (Xu, 2000
). We previously demonstrated the presence of a HOG1-like MAPK, CpMK1, in C. parasitica, as well as hypovirus-mediated perturbation of its phosphorylation in response to hypertonic stress (Park et al., 2004
).
In this study, we isolated from C. parasitica a member of a different class of MAPK, CpMK2, which is an ERK homologue. In addition, we addressed the putative biological functions of CpMK2 that are related to hypovirulence-associated traits and examined whether CpMK2 is affected by hypoviral infection.
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METHODS |
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Isolation and characterization of the MAPK cpmk2 gene.
Degenerate primers specific for consensus nucleotide sequences corresponding to the most conserved amino acids within subdomains II/VIb of MAPKs (Kultz, 1998) were designed as described previously (Park et al., 2004
). The primers used were MK1-F1 (forward; 5'-GTNGCNATRAARAARAT-3') and MK1-R1 (reverse; 5'-GGYTTNANRTCNCKRTG-3'). PCR was conducted as described by Park et al. (2004)
. The 310 bp PCR amplicon was cloned into the pGEM-T vector (Promega). Inserts containing positive clones were sequenced using the dideoxynucleotide method before being used as hybridization probes for genomic
library screening according to standard procedures (Sambrook et al., 1989
).
To obtain a cDNA clone for cpmk2, RT-PCR was performed with the primers cMK2-F1 (forward; 5'-ATGTCACGGTCCCAAGCTCCC-3') and cMK2-R1 (reverse; 5'-CGCATGATCTCCTGATAGATC-3'). The cDNA was sequenced using the dideoxynucleotide method and synthetic oligonucleotide primers.
Southern and Northern blot analysis.
Genomic DNA from C. parasitica was extracted using the method of Churchill et al. (1990). DNA (10 µg) was digested with restriction enzymes, blotted onto nylon membrane and hybridized with a radioactively labelled cpmk2 fragment.
RNA was extracted from liquid cultures at 1, 3 and 5 days after inoculation and Northern blot analysis was conducted as described previously (Kim et al., 1995). RNA from cultures grown on solid medium was prepared using a previously described method (Park et al., 2004
). The expression of cpmk2 was compared with that of the C. parasitica glyceraldehyde-3-phosphate dehydrogenase (Gpd) gene as an internal control (Choi & Nuss, 1990
).
Heterologous expression of cpmk2 in Escherichia coli.
The full-length cpmk2 protein product CpMK1 was expressed in E. coli as a hexahistidine fusion protein and purified by nickel-affinity chromatography according to the manufacturer's instructions (Novagen). A full-length CpMK2 cDNA was amplified by PCR using the primers 5'-CACATATGTCACGGTCCCAAGCTCCC-3' (forward) and 5'-CGCGCGGCCGCCCGCATGATCTCCTGATAGATC-3' (reverse). The primers were modified to incorporate restriction sites (underlined) for NdeI and NotI, respectively. The full-length 1067 bp cpmk2 gene was fused between the NdeI and NotI sites in the expression vector pET-28a. The resulting recombinant plasmid was transformed into the E. coli strain BL21. Induction, purification and confirmation of recombinant CpMK2 were performed using an anti-hexahistidine antibody according to the manufacturer's instructions (Novagen). E. coli-derived inclusion bodies, in which the recombinant CpMK2 was expressed, were solubilized and then refolded by stepwise dilution of the denaturants by dialysis (Creighton, 1990).
Kinase activity of CpMK2.
MAPK activity of the E. coli-expressed CpMK2 was assayed by measuring the incorporation of 32P from [-32P]ATP into myelin basic protein (MBP) as described previously (Park et al., 2004
). To test for autophosphorylation, purified CpMK2 was used in a kinase assay without MBP. A kinase assay of cell-free extracts was conducted as described previously (Kim et al., 2002
).
Immunoblot analysis.
Anti-CpMK2 antibody was obtained as described previously (Kim et al., 2002). Purified recombinant CpMK2 (100 µg) was injected into an 8-week-old BALB/c mouse, which was boosted with the same amount of the CpMK2 emulsified in incomplete Freund's adjuvant 10 days after the initial injection. Polysera were obtained 5 days after the booster injection and Western blot analysis was conducted according to standard procedures (Sambrook et al., 1989
).
To determine whether mycoviruses specifically affect the signal cascade that involves CpMK2, levels of phosphorylated CpMK2 were examined by immunoblotting with an antibody specific for doubly phosphorylated p44/42 MAPK (phospho-p44/42 MAP kinase antibody) according to the manufacturer's instructions (Cell Signalling Technology). To examine the induction of phosphorylation owing to changes in the culture medium, mycelia grown in liquid culture were weighed, transferred onto PDAmb and harvested at the appropriate time to measure the phosphorylation level of CpMK2. The non-phosphorylation-specific p44/42 MAP kinase antibody (Cell Signalling Technology) was also applied to verify the equal loading of protein.
Construction of a replacement vector and fungal transformation.
The replacement vector pDmk2, which was designed to favour double-crossover integration events, was constructed as follows. A 2·0 kb KpnI fragment containing the full-length cpmk2 ORF was ligated into SalI-inactivated pBluescript II SK (+) and the resulting plasmid was used as a template for inverted PCR using the primers 5'-GTCGACGCAGACAACACAATAGGCACC-3' and 5'-GTCGACCCTTCCAACCTACTCCTCAAC-3', which incorporate the restriction site for SalI (underlined). The PCR amplicon was digested with SalI and religated. The resulting plasmid was further digested with SalI and fused with a 2·4 kb SalI fragment of pDH25 (Cullen et al., 1987) carrying the hygromycin phosphotransferase gene (hph) cassette. In the replacement vector pDmk2, the hph cassette was inserted between sites 113 and 553 of the cpmk2 gene relative to the start codon and was flanked by approximately 630 and 930 bp, respectively, of 5' and 3' sequences. KpnI-digested linear pDmk2 was then used to transform the virus-free EP155/2 strain.
Functional complementation of the cpmk2 mutant using a wild-type allele was performed. The complementing vector pCmk2 was constructed by the insertion of a 2·6 kb blunt-ended SalI fragment of pSV50 containing the benomyl resistance cassette (Orbach et al., 1986) into blunt-ended SphI-digested pCpmk2, which carried a 7·5 kb SalI fragment containing the full-length cpmk2 gene. The resulting vector was then used to transform the cpmk2 mutant.
Protoplast preparation and transformation were performed as described previously (Churchill et al., 1990; Kim et al., 1995
). Transformants were selected from agar plates that were supplemented with 150 µg hygromycin B ml1 (Calbiochem) or 1·5 µg benomyl ml1 (DuPont) as appropriate, passaged three or four times on selective media and single-spore-isolated, as described previously (Kim et al., 1995
, 2002
). PCR and Southern blot analysis were conducted with genomic DNA from the transformants to confirm the replacement and complementation of the cpmk2 gene in trans.
Characteristics of the cpmk2 mutant.
The phenotypic and molecular characteristics of the cpmk2 mutant were compared with those of the wild-type EP155/2 and the hypovirulent UEP1 strains. Phenotypic changes in pigmentation, conidiation and mating capability were measured as described previously (Kim et al., 1995; Park et al., 2004
). A virulence test using excised chestnut tree bark was conducted according to Lee et al. (1992)
. Laccase activity was gauged by growing the strains on Bavendamm's medium (0·05 % tannic acid, 1·5 % malt extract, 2·0 % agar) and assessing the resulting colouration of the medium (Rigling et al., 1989
). Laccase activity of culture filtrate was determined as described previously (Kim et al., 1995
). Expression of the virus-regulated cryparin (Crp), laccase (lac1) and mating pheromone (Mf2/1) genes was examined by Northern blot analysis (Kim et al., 1995
, 2002
).
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RESULTS |
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The predicted sequence of CpMK2 contains all 11 conserved protein kinase subdomains and the characteristic MAPK dual phosphorylation sites (TEY, residues 183185) upstream of the YRAPE domain (Nishida & Gotoh, 1993). A homology search indicated that CpMK2 is highly related to the fungal MAPKs CMK1 from Colletotrichum lagenarium (96 % identity), PMK1 from Magnaporthe grisea (94 %) and FUS3 from Saccharomyces cerevisiae (58 %) and belongs to the yeast extracellular signal-regulated kinase 1 (YERK1) subfamily. CpMK2 has only 45 % identity to C. parasitica CpMK1, the functional homologue of S. cerevisiae HOG1, which demonstrates the phylogenetic difference between the two C. parasitica MAPKs.
Northern blot analysis revealed that, similar to the cpmk1 gene, cpmk2 is expressed at very low levels in the C. parasitica strain EP155/2 and its isogenic hypovirulent strain UEP1. The low expression level complicates efforts to detect any transcriptional regulation by the hypovirus (data not shown).
Kinase activity of E. coli-expressed CpMK2
Full-length CpMK2 was expressed in E. coli. Following gentle purification and renaturation, a single 50·0 kDa CpMK2 band was observed in SDS-PAGE, slightly larger than the calculated mass owing to the addition of the hexahistidine tag for purification purposes. Autophosphorylation of the renatured CpMK2 and its phosphorylation of a common substrate of MAPK, MBP (Fig. 1), indicated that the protein has kinase activity and belongs to a subgroup of the MAPK proteins.
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To ensure that any phenotypic changes attributed to the cpmk2 mutation were owing to the gene replacement event, the altered growth rate of TdMK2-1 was complemented in trans on solid medium with a wild-type allele of cpmk2. The benomyl resistance cassette (benR) was introduced into the pBluescript II KS (+) vector containing the 7·5 kb SalI fragment with the entire cpmk2 gene. The resulting vector, pCmk2, was used to transform the cpmk2 mutant TdMK2-1. Benomyl-resistant transformants of the cpmk2 mutant that had received a wild-type cpmk2 gene showed a normal growth rate and abundant pycnidia on solid media (Fig. 4). PCR analyses revealed that all of the complemented transformants contained an additional wild-type allele of cpmk2 (data not shown). Thus, functional complementation using a wild-type cpmk2 gene confirmed unequivocally that the phenotypic changes in the mutant were due to the disruption of cpmk2.
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DISCUSSION |
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MAPKs constitute a family of Ser/Thr kinases that are highly conserved in organisms ranging from yeast to humans. Three MAPKs exist in the model phytopathogenic fungus M. grisea. Two of these, PMK1 and MPS1, were identified as orthologues of FUS3/KSS1 and SLT2 of S. cerevisiae, respectively, and have been shown to be important for the pathogenicity of this fungus. A sequence comparison suggests that CpMK2 is a homologue of PMK1. To date, all plant-pathogenic fungal PMK1/FUS3 homologues that have been functionally analysed have been implicated in pathogenicity but not in defects in vegetative growth in axenic culture, implying that the PMK1/FUS3 homologue is part of a general pathogenicity-related MAPK signal chain in pathogenic fungi. The cpmk2 mutant produces drastically reduced canker areas, a phenotype that is even more severe than that of the hypovirulent strain. If pathogenic determinants are defined as factors that affect only pathogenicity, CpMK2, which when disrupted results in defective vegetative growth on solid surfaces, may not be a specific pathogenic determinant in the strict sense. The C. parasitica CpMK2 pathway might differ from those of other pathogenic fungi and may be essential for appropriate fungal growth on solid surfaces.
In addition to phenotypic changes, pheromone gene expression was severely repressed in the cpmk2 mutant, and this effect was not due to growth inhibition, as a similar reduction in pheromone gene expression was also observed when the mutant was grown in liquid culture. Although pheromone-related gene expression and the pheromone-responsive pathway are well-characterized in yeast, few details exist on the regulation of pheromone gene expression in filamentous fungi. The structures of all of the pheromones in the ascomycetes are of the two types described in S. cerevisiae. Genes of two classes of pheromones were identified recently from three heterothallic filamentous fungi, C. parasitica, M. grisea and Neurospora crassa, and from the homothallic fungus Sordaria macrospora (Zhang et al., 1998; Shen et al., 1999
; Poggeler, 2000
; Bobrowicz et al., 2002
; Turina et al., 2003
). The first complete set of pheromone precursor genes to be identified in a filamentous ascomycete was from C. parasitica (Zhang et al., 1998
), and three pheromone precursor genes, Mf1/1, Mf2/1 and Mf2/2, have been reported to exist in both C. parasitica mating types (Zhang et al., 1998
; Turina et al., 2003
). The expression of the pheromone precursor genes is mating-type-specific; for example, Mf1/1 is expressed only by Mat-1 strains, and the genes display transcriptional suppression, characteristic of a virus-containing hypovirulent strain. The downregulation of pheromone gene expression owing to alterations in the expression of CpMK2, which belongs to a family of pheromone-responsive MAPKs, could imply that pheromone gene expression is dependent on the MAPK pathway, as in S. cerevisiae, and that a MAPK cascade similar to the S. cerevisiae FUS3/KSS1 MAPK pathway exists in C. parasitica. Based on the significant similarities in the structures and expression patterns of the pheromone precursor genes of C. parasitica and the ascomycetous yeasts, it is expected that the features of the mating system of yeasts are conserved in C. parasitica and that pheromone expression in C. parasitica is regulated by the mating-type locus, which encodes transcriptional regulators (Zhang et al., 1998
; Shen et al., 1999
). Therefore, the finding that disruption of CpMK2 results in inhibited pheromone gene expression could provide clues to intriguing connections between possible mating signals and a MAPK pathway in a phytopathogenic fungus.
In fungal signalling pathways, interactions frequently exist between the cAMP signalling and MAPK pathways involved in mating, morphogenesis, virulence and stress responses (Kronstad et al., 1998). Although the cAMP signalling and MAPK pathways act in parallel during filamentous growth in S. cerevisiae and appressorium formation in M. grisea, many examples of antiparallel cooperation have also been observed, such as negative regulation by the cAMP pathway but positive regulation by the MAPK pathway in the induction of ste11 in S. pombe and prf1 in Ustilago maydis (Kronstad et al., 1998
). Moreover, there has been considerable progress in the understanding of the regulation of intracellular cAMP levels in filamentous fungi, including M. grisea and C. parasitica. Briefly, a mutation in the M. grisea pmk1 gene, a homologue of cpmk2, results in defects in appressorium formation and invasive growth in rice plants, causing avirulence. Since the pmk1 mutants remain responsive to exogenous cAMP but do not form appressoria, it was suggested that cooperative signalling between the cAMP- and MAPK-dependent pathways is required for surface recognition and infection structure formation (Xu & Hamer, 1996
). In addition, of the three G
subunit genes in M. grisea, mutants with disruptions in magB but not magA or magC show reduced appressorium formation, conidiation and virulence (Liu & Dean, 1997
). These effects can be suppressed by exogenous cAMP, implicating MAGB in the cAMP signalling pathway (Xu, 2000
). Therefore, one can speculate that the regulatory cascade from the G-protein to the two independent but tightly coordinated and cooperative cAMP- and MAPK-dependent pathways is required for appressorium formation and that this cooperative signalling might be a common feature in fungal pathogens (Xu & Hamer, 1996
; Kronstad et al., 1998
). Likewise, targeted disruption of the genes for two C. parasitica G
subunits, cpg-1 and cpg-2, showed that the G
subunit encoded by cpg-1, but not cpg-2, has roles in fungal reproduction, virulence and vegetative growth. Moreover, CPG-1 cosuppression resulted in constitutively elevated cAMP levels, consistent with the prediction that CPG-1 negatively regulates adenylyl cyclase (Chen et al., 1996
). Since the phenotype of the cpmk2 mutant does resemble the cpg-1 mutant in two interesting ways, complete loss of conidiation and differential growth on solid and liquid media, it is conceivable that CpMK2 may be subject to regulation through the G-protein-coupled signalling pathway. Accordingly, it can again be speculated that the cAMP signalling and MAPK pathways interact cooperatively during the processes of conidiation and surface sensing in C. parasitica. It will be of interest to disrupt the adenylyl cyclase gene to examine the resulting phenotypic changes.
Fungal growth is the consequence of many metabolic processes, including various sensing and response processes. Therefore, the absence of signalling can result in severe growth defects through disruption of a required signal relay. Based on the time of induction of elevated phosphorylation of CpMK2, this phosphorylation event may be implicated in the fungal response to environmental stimuli, such as the sensing of hard surfaces. However, the lack of differences in the basal and induction levels of phosphorylated CpMK2 between the virus-free EP155/2 and virus-containing UEP1 suggests that the normal regulation of the phosphorylation level of CpMK2 in C. parasitica is independent of hypoviral infection.
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ACKNOWLEDGEMENTS |
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Received 29 November 2004;
revised 25 January 2005;
accepted 31 January 2005.
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