National Institute of Chemistry, Department for Biotechnology and Industrial Mycology, Hajdrihova 19, SI-1000 Ljubljana, Slovenia1
Wageningen University, Section Molecular Genetics of Industrial Micro-organisms, Dreijenlaan 2, 6703HA Wageningen, The Netherlands2
Author for correspondence: George Ruijter. Tel: +31 71 5262967. Fax: +31 71 5266876. e-mail: g.j.g.ruijter{at}lumc.nl
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ABSTRACT |
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Keywords: cAMP-dependent protein kinase, regulatory subunit, signal transduction, morphology
Abbreviations: PKA, cAMP-dependent protein kinase; PKA-R, regulatory subunit of PKA; PKA-C, catalytic subunit of PKA
a The EMBL accession number for the sequence reported in this paper is AJ296317.
b Present address: Wageningen University, Laboratory of Phytopathology, PO Box 8025, 6700 EE Wageningen, The Netherlands.
c Present address: Dr Van Haeringen Laboratorium, Agro Business Park 100, 6708 PW Wageningen, The Netherlands.
d Present address: FGT Consultancy, PO Box 396, 6700AJ Wageningen, The Netherlands.
e Present address: Department of Pediatrics, Leiden University Medical Centre, Building 1 P3-P, PO Box 9600, 2300RC Leiden, The Netherlands.
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INTRODUCTION |
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cAMP signalling controls a number of developmental events such as growth polarity in the filamentous fungus Neurospora crassa (Bruno et al., 1996 ), cell development of Blastocladiella emersonii (de Oliveira et al., 1997
) and sexual development of Schizosaccharomyces pombe (Maeda et al., 1994
). The influence of cAMP via PKA on dimorphic transition was demonstrated for several dimorphic fungi, e.g. Candida albicans (Niimi, 1996
) and Mucor rouxii (Orlowski, 1991
). For the plant-pathogenic fungi Ustilago maydis (Gold et al., 1994
) and Magnaporthe grisea (Mitchell & Dean, 1995
) and for the human pathogen Cryptococcus neoformans (Kronstad et al., 1998
), cAMP signalling is directly connected to fungal virulence (reviewed by Borges-Walmsley & Walmsley, 2000
). PKA is also involved in metabolic regulation, activating glycolysis (Goncalves et al., 1997
) and diauxic transition in Saccharomyces cerevisiae (Boy-Marcotte et al., 1998
). The inactivation of Sac. cerevisiae fructose-1,6-bisphosphatase (Jiang et al., 1998
), the stability of Sac. cerevisiae neutral trehalase (Zahringer et al., 1998
) and the activation of 6-phosphofructo-1-kinase from Mytilus galloprovincialis (Fernandez et al., 1998
) are results of PKA phosphorylation. In Aspergillus nidulans conidia PKA is proposed to be involved in mobilization of trehalose by phosphorylation of trehalase (dEnfert et al., 1999
).
In most cases the inactive form of PKA is a tetrameric protein composed of two regulatory and two catalytic subunits. Upon binding of cAMP, inactive PKA dissociates into two active catalytic subunits and a dimer of regulatory subunits. Two types of PKA regulatory subunits (PKA-R), type I and type II, have been isolated and four different genes encoding PKA-R (I, Iß, II
, IIß) have been identified in higher eukaryotes (Taylor et al., 1992
). Fungi usually posses a single gene for PKA-R, which encodes a protein similar to either the mammalian type I or type II regulatory subunits. In N. crassa, Sch. pombe, Mag. grisea, Sac. cerevisiae, U. maydis and B. emersonii type II regulatory subunits have been described (Bruno et al., 1996
; de Voti et al., 1991
; Adachi & Hamer, 1998
; Kunisawa et al., 1987
; Gold et al., 1994
; Marques & Gomes, 1992
). PKA from Dictyostelium discoideum (Mutzel et al., 1987
) and Paramecium tetraurelia (Carlson & Nelson, 1996
) have a different structure. The regulatory subunit of these enzymes does not contain dimerization domains they are heterodimeric proteins composed of only one catalytic and one regulatory subunit.
In the filamentous fungus Aspergillus niger, a transient increase in cAMP levels during the early stage of growth in a medium with high initial sucrose concentration has been observed (Legia et al., 1981
; Gradi
nik-Grapulin & Legi
a, 1997
). Simultaneously with the cAMP peak, a change in morphology from bulbous cells to filamentous hyphae took place (Legi
a & Gradi
nik-Grapulin, 1995
; Gradi
nik-Grapulin & Legi
a, 1997
). Growth tests indicated that even moderate overproduction of PKA-C affected growth and sporulation characteristics of A. niger transformants (Ben
ina et al., 1997
). During germination of wild-type A. niger the level of expression of pkaC and the specific PKA activity steadily decreased (Ben
ina & Legi
a, 2000
). These data suggest that PKA is involved in morphogenesis. To analyse the A. niger PKA enzyme in more detail and study its role in the regulation of morphological and cellular development, we cloned A. niger pkaR and constructed A. niger strains with disrupted or overexpressed pkaC and/or pkaR.
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METHODS |
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Plasmid vectors pBluescript KS (Stratagene) and pEMBL19 (Roche) were used for subcloning. Plasmid pGW635, containing A. niger pyrA (Goosen et al., 1987 ), and plasmid pIM650, containing A. nidulans argB (Johnstone et al., 1985
), were used for construction of pkaR and pkaC disruption plasmids. Phage ExAssist (Stratagene) was used as a helper phage for phagemid excision.
The following gene fragments were used to generate probes for screening A. niger genomic and cDNA libraries: a 1·51 kbp BglII/BamHI fragment from Mag. grisea pkaR (Adachi & Hamer, 1998 ) and a 1·45 kbp HindIII fragment from A. nidulans pkaR [accession no. AF043231 (gi: 3170247)].
Culture media and growth conditions.
For the preparation of conidiospores, A. niger strains were grown at 30 °C for 34 days on complete medium (CM), originally described for A. nidulans (Pontecorvo et al., 1953 ), using 1% (w/v) glucose as a carbon source, with appropriate supplements and solidified with 1·5% (w/v) agar. Since disruption of the pkaR gene in A. niger resulted in an inability to form conidiospores, we could not maintain a
pkaR mutant in the form of conidiospores. Instead, mycelium of an A. niger
pkaR mutant was picked from a plate and stored in 30% (v/v) glycerol at -70 °C. For submerged growth, medium was inoculated with 106 conidiospores ml-1. One litre of minimal medium (MM) contained 6 g NaNO3, 1·5 g KH2PO4, 0·5 g KCl, 0·5 g MgSO4.7H2O, pH 6·0, and 0·2 ml trace metal solution (Visniac & Santer, 1957
). Glucose was used as a carbon source. For the growth of auxotrophic strains, appropriate supplements were added: 10 mg nicotinamide l-1, 200 mg leucine l-1, 1220 mg uridine l-1 and 200 mg arginine l-1. Mycelium was grown at 30 °C in a rotary shaker at 250 r.p.m.
Germination kinetics were analysed by microscopic examination of slides coated with minimal medium containing 2% glucose and 1·5% agar and spot-inoculated with 2x104 conidiospores. Spores were incubated at 30 °C. The percentage of germinated spores was followed in time by examining 50100 spores at 30 min intervals.
DNA manipulation.
A. niger chromosomal DNA was isolated as described by de Graaff et al. (1988) . Propagation and isolation of plasmid DNA, Southern blot analysis and other DNA manipulations were essentially done as described by Sambrook et al. (1989)
. [
-32P]dATP-labelled probes were synthesized using random hexamer primers (Sambrook et al., 1989
). Automated sequencing based on the dideoxy chain-termination procedure (Sanger et al., 1977
) was done with ALFexpress (Amersham Pharmacia Biotech) using the T7 DNA polymerase sequencing kit (Amersham Pharmacia Biotech). DNA sequence was analysed with PC/GENE and WINSTAR computer program packages. Further analysis of the nucleotide sequence was performed using EDITSEQ and MAPDRAW from the PC/GENE software package, MATINSPECTOR v2.2 (Quandt et al., 1995
) and Prosite and InterPro databases.
The A. niger pkaR gene and the corresponding cDNA clone were isolated from an A. niger N400 genomic library in the replacement vector EMBL4 (Promega) and a cDNA library from A. niger N400 in
ZAP II (Stratagene), respectively. Plasmids containing the cloned cDNAs were obtained from the
ZAP II phages by in vivo excision, according to the manufacturers instructions.
Mitotic recombination was performed as described by Bos et al. (1988) . Heterokaryon formation between NW276 and NW275 was induced by protoplast fusion (Van Diepingen et al., 1998
). Haploidization was induced with benomyl.
To construct a plasmid for simultaneous overexpression of pkaC and pkaR, pIM492, an 8 kbp EcoRI fragment, containing pkaR, was inserted into pEMBL19. After removal of one EcoRI site, a 5 kbp EcoRI pkaC fragment from pPKAC1 (Benina et al., 1997
) was inserted into the other EcoRI site giving pIM492 (Fig. 1
).
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To construct the pkaC disruption plasmid, pKOG10, a SmaI/NcoI fragment of pyrA was ligated into the NsiI (blunt end)/NcoI sites of pkaC in pPKAC1 (Benina et al., 1997
). A 6·4 kbp EcoRI fragment of the resulting plasmid (pKOG10, see Fig. 1
) was used for pkaC disruption by REMI transformation of A. niger strain NW219.
Northern analysis.
Mycelium grown in liquid complete medium (CM) for 24 h was transferred for 1 h into MM medium with 1 or 15% glucose, harvested by filtration, washed briefly with ice-cold 0·05 M potassium phosphate buffer pH 7·0, frozen in liquid nitrogen and ground using a micro dismembrator (Braun Biotech). RNA was extracted using the TRIzol reagent (Life Technologies). After electrophoresis, RNA was transferred to nylon membranes (Hybond-N+; Amersham Pharmacia Biotech) by capillary blotting in 10xSSC. Pre-hybridization and hybridization were performed at 42 °C in formamide hybridization buffer containing 50% (w/v) formamide, 0·75 M NaCl, 50 mM NaH2PO4 (pH 7·4), 10 mM EDTA, 2x Denhardts, 0·1% (w/v) SDS and 10% (w/v) dextran sulphate. Northern blots were washed at 65 °C in 4xSSC, 0·5% SDS and 2xSSC, 0·5% SDS. The probes used for Northern analysis were as follows: a 0·8 kbp BglII/XhoI fragment from pIM490 for pkaR mRNA (probe RI, Fig. 1), a 1·2 kbp EcoRI/KpnI fragment from pIM492 for pkaC mRNA (probe C, Fig. 1
) and a 0·7 kbp EcoRI fragment of A. niger 18S rRNA. The pkaR and pkaC probes are also depicted in Fig. 1
.
Transformation of A. niger.
Mycelium was obtained by growing A. niger for 1618 h in liquid culture on complete medium with appropriate supplements. Preparation of protoplasts and subsequent transformation of A. niger were performed as described by Kusters-van Someren et al. (1991) . The A. niger pyrA gene and A. nidulans argB gene were used as selection markers. For co-transformation of A. niger NW219, 1 µg pGW635 DNA and 27 µg of the co-transforming plasmid pIM491 (containing pkaR) or pIM492 (containing a copy of both the pkaR and the pkaC genes) were added to 2x107 protoplasts. To disrupt pkaR, 5 µg of the 7·1 kbp EcoRI pkaR fragment from pIM493 was used to transform A. niger NW245. To disrupt pkaC, 5 µg of the 5 kbp EcoRI pkaC fragment from pKOG10 was used to transform A. niger NW219. The transformants were selected and purified by replating at low spore densities on selective medium without uridine or arginine.
Enzyme assays.
Frozen mycelium (0·5 g) obtained as described for RNA isolation, was ground and suspended in 1 ml extraction buffer (350 mM KH2PO4, pH 7·5, 0·1 mM DTT, 10% glycerol). After 15 min extraction, the homogenate was centrifuged at 10000 g for 10 min. Supernatant was used for the enzyme assay. Cell extracts (with protein concentration of approximately 2 mg ml-1) were diluted two- to tenfold before measuring enzyme activity. The activity of PKA was detected by the non-radioactive PepTag test method with dye-labelled Kemptide as a substrate, according to the manufacturers protocol (Promega) or by SpinZyme (Pierce), according to Benina et al. (1997)
. The incubation time for the enzymic reaction was 30 min at 30 °C. One unit of enzyme activity was defined as the amount of enzyme required to transfer 1 pmol phosphate from ATP to the substrate (Kemptide) per min at 30 °C. The catalytic subunit of bovine heart PKA (Promega) was used as a standard. Protein concentrations in cell-free extracts were determined with the Bicinchoninic-acid protein kit (Sigma), according to the suppliers instructions, and using BSA fraction V (Roche) as a standard.
RT-PCR.
RT-PCR was performed according to Benina & Legi
a (1999)
. Total RNA (2 µg) and primers PKAC14 [5'-AGAAAGGCGTGAAACCACACAG-3' (Ben
ina et al., 1997
)] and PEPC2 [5'-TATCACGGTGAGAGATACGAGC-3' (Frederick et al., 1993
)] (Ransom Hill Bioscience) were used. cDNA product (2 µl) was transferred to 20 µl PCR reaction mixture containing primers PKAC13 [5'-TGGTCATGGACTTCGTAGAGGG-3' (Ben
ina et al., 1997
)], PKAC14, PEPC1 [5'-CTATCTGGGTCTCAAGAACACC-3' (Frederick et al., 1993
)], PEPC2 (Ransom Hill Bioscience). PCR was performed in a Perkin Elmer thermal cycler GeneAmp PCR System 2400.
Twenty microlitres of the PCR products were separated by 1% (w/v) agarose gel electrophoresis, transferred to Hybond-N+ nylon membrane (Amersham Pharmacia Biotech) and subsequently the biotin-labelled fragments were detected by the avidin alkaline phosphatase conjugate (Tropix) using CDP-Star (Tropix) as a substrate. Signals were determined by imaging film X-OMATAR (Eastman Kodak). The expected sizes of the PCR products were 230 and 420 bp. The intensity of the signals on the film was measured with an Imaging Densitometer model GS-670 (Bio-Rad) and analysed by Molecular Analyst Software (Bio-Rad).
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RESULTS |
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The nucleotide sequence of a 3330 bp fragment of pIM490 was determined on both strands. The sequence from position -319 to -326 could not be identified unambiguously due to high GC content. The nucleotide sequence was deposited in the EMBL database under the accession no. AJ296317. In addition, a 2002 bp cDNA fragment of pkaR was isolated from an A. niger cDNA library using stringent hybridization conditions and a 0·6 kbp BamHI/SalI fragment of pIM490 as a probe.
By comparing the cDNA sequence with the sequence of the genomic clone, a single intron of 700 bp was found from position -112 to -811 with borders 5'-GTAAGG and 3'-CAG. Three putative CCAAT boxes were found upstream from the start codon at positions -361, -426 and -473. Analysis of the promoter region by the MATINSPECTOR v2.2 software package (core and matrix similarity parameters: 1·0 and 0·9) (Quandt et al., 1995 ) revealed several putative transcription elements, including stress response elements (Stre), a binding site for heat-shock factors (Hsp), a target site for an activator of nitrogen-regulated genes (Nit1), the positive transcriptional regulator Gcr1, the transcriptional activator for control of development, AbaA, and the regulator of asexual reproduction and differentiation of hyphae, StuA. In addition, the promoter region contains several E-boxes (CANNTG) (Murre et al., 1989
) and nine C4T or reverse complementary (AGGGG) sequences (Treger et al., 1998
), five of which are inside the intron. No obvious TATA sequence was found in the 5' non-coding region.
The cDNA contained an ORF of 1233 nt encoding a protein of 411 aa with a calculated molecular mass of 44527 Da, which is fairly similar to the apparent molecular mass determined for the purified protein, 48 kDa (Legia & Ben
ina, 1994
). The coding sequence showed a strong bias for A in the third position of the codon, which was also found in the A. niger pkaC gene (Ben
ina et al., 1997
). Three putative structural features were present within PKA-R (Leon et al., 1997
; Su et al., 1995
). First, a dimerization domain located at the N-terminal third of the protein. Second, a site for interaction between the regulatory and catalytic subunits [(117)RRTSVSAE] containing an autophosphorylation site (Ser-120). Third, two cAMP-binding sites: (227)VgSvgpGGSFGELALmYnaPRAATV and (348)VksykrgDYFGElALLddkPRAAsI (conserved amino acid residues are underlined). The deduced amino acid sequence suggests that the PKA-R of A. niger is most closely related to type II PKA-R, showing an overall identity of 75% with A. nidulans pkaR [accession no. AF043231 (gi: 3170247)], 69% with N. crassa (Bruno et al., 1996
) and 66% with Mag. grisea (Adachi & Hamer, 1998
).
Downstream of the stop codon the mRNA still contains 459 nt. Putative poyladenylation signals, ATAAA, were found at 16 and 366 nt downstream of the stop codon.
Construction of strains modified in pkaR and pkaC
To understand the role of the PKA-R in cell physiology, transformants containing multiple copies of pkaR and/or pkaC, and strains in which these genes were disrupted were prepared.
Multicopy pkaR strains (mcR) were isolated after co-transforming A. niger with pIM491 using pyrA as a selection marker. Similarly, transformants with increased copy numbers of both pkaC and pkaR (mcRC strains) were produced using pIM492.
Several transformants were analysed for copy number of pkaR and pkaR/pkaC by Southern analysis. Four transformants were found with multiple copies of pkaR (strain 10mcR, one extra copy; strain 38mcR, approximately 10 copies; strains 42mcR and 39mcR contained more than 20 copies). Five transformants contained an increased number of copies for both genes (strain 35mcRC, one extra copy for each gene; strain 7mcRC, 45 copies; strain 44mcRC, approximately 10 copies; strains 1mcRC and 18mcRC had at least 20 copies). Increased copy numbers of pkaC and pkaR resulted in overexpression of the two genes (Fig. 2).
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Specific PKA activities were measured to correlate it to modifications in the copy number of pkaC and pkaR. PKA activity was significantly higher in transformants with multiple copies of both pkaR and pkaC (Fig. 2). The activity correlated qualitatively to copy number and mRNA level (Fig. 2
), e.g. transformant 44mcRC had about 10 copies of both genes and 0·6 mU mg-1 (wild-type activity was 0·1 mU mg-1). In transformants with an increased copy number of only pkaR, PKA activity remained at the wild-type level (Fig. 2
). PKA activity was measured in one pkaR disruption strain, 9
R, and was found to be similar to wild-type activity (0·175 mU mg-1). Finally, in the pkaC disruptant PKA activity was below the detection level (Fig. 2
).
Growth and morphology of A. niger strains modified in pkaR and pkaC
The role of PKA activity in morphology and hyphal development was monitored by observing the phenotypic features of individual transformants growing on solid medium and in liquid cultures. By comparing growth of wild-type, various multicopy transformants and the different disruptants on solid medium, three morphological classes were identified on the basis of colony size and the ability to form spores (Fig. 4). Strains in the first group were similar to wild-type and included a pkaR multicopy transformant (38mcR), a pkaC multicopy transformant (13mcC with a three- to fourfold increased PKA-C level) and a strain with an increased copy number of both genes (44mcCR). The second morphological class included strains with reduced or abolished PKA activity. Transformants in which pkaC (
C) or both pkaC and pkaR (
R
C) were disrupted formed colonies with a two- to threefold smaller diameter than the wild-type (Fig. 4
). A pkaR disruptant (9
R) formed a third morphological class and developed colonies of only about half the size of wild-type and no sporulation could be observed after 3 days growth on agar plates.
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Germination kinetics were similar for the wild-type strain and strains overexpressing pkaR or both pkaR and pkaC (Fig. 5). Germination of conidiospores from the pkaC disruption strain (
C) and the pkaC pkaR double disruptant (
R
C) was slightly delayed (30 min to 1 h), but conidia from strain 13mcC, overproducing PKA-C , germinated considerably slower than the wild-type strain (1 to 2 h). Like the results obtained for colony morphology, strains
C and
R
C behaved similarly in germination experiments, indicating that disruption of pkaC was epistatic to deletion of pkaR.
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DISCUSSION |
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The presence of an intron within the 5' non-coding region of A. niger pkaR has also been reported for the PKA-R genes of B. emersonii (Marques & Gomes, 1992 ), N. crassa (Bruno et al., 1996
) and Mag. grisea (Adachi & Hamer, 1998
). Similar to intron I of B. emersonii, the A. niger pkaR intron (700 bp) is unusually large compared to the normal size (50100 bp) of fungal introns (Unkles, 1992
). Its role in the promoter region of pkaR is not clear, but it might play a role in the expression of the gene, as reported for rat cytochrome c (Evans & Scarpulla, 1988
). Analysis of the A. niger pkaR promoter region revealed no typical GC sites or binding sites for transcription factor Sp1 as could be found in the promoter of A. niger pkaC (Ben
ina et al., 1997
) and the pkaR promoter of B. emersonii (Marques & Gomes, 1992
). However, the promoter regions of the pkaC and pkaR genes of A. niger bear some similarities which are indicative of common transcription control mechanisms. Both promoters contain stress response elements (C4T) and E-motifs. Such elements have been found in the multistress response genes of Sac. cerevisiae (Treger et al., 1998
).
To analyse the involvement of PKA in morphogenesis, growth and morphology of strains with increased pkaC and/or pkaR expression as well as strains lacking PKA-C and/or PKA-R were examined on solid and in liquid medium. Strains overexpressing pkaR or both pkaC and pkaR showed higher mRNA levels as expected, but the strains could otherwise not be distinguished from the wild-type, suggesting that regulation of PKA-C activity is normal in these strains. With simultaneous overexpression of pkaC and pkaR, one might expect a relatively high PKA-C activity upon binding of cAMP to PKA-R and release of PKA-C, but this does not appear to interfere with regulation of processes which determine morphology. These data suggest that an increased level of PKA-C is not harmful to the cells as long as its activity is properly regulated by PKA-R.
A three- to fourfold increase in the PKA-C level in pkaC transformant 13mcC did not affect surface growth, but in submerged cultures, young mycelium of this transformant had thicker hyphae. In a later stage, however, newly formed hyphae were like wild-type. Apparently, overproduction of PKA-C only affects morphology at an early growth stage. This observation agrees with the finding that pkaC transcription in A. niger significantly decreased between 19 and 28 h after inoculation (Benina & Legi
a, 2000
). Thus, if overproduction of PKA-C results in increased hyphal diameter, a decrease in pkaC transcription might get the PKA-C/PKA-R balance back to normal, resulting in proper regulation of PKA activity and consequently in a normal hyphal diameter. Alternative mechanisms to control PKA-C level or activity in pkaC multicopy transformants may be at the translational level or by inducing the synthesis of PKA-R, but we have no proof that such mechanisms work in A. niger.
A pkaC disruption strain lacked PKA activity and developed smaller colonies than the reference strain on plates. Germination of C conidiospores was slightly delayed, but hyphal morphology in liquid culture and sporulation was normal. As expected, silencing of pkaR, in addition to pkaC, did not change the phenotype. Clearly, PKA activity is required for optimal growth and metabolism.
Disruption of pkaR gave the most dramatic phenotype, i.e. very small colonies on plates, absence of sporulation and complete loss of growth polarity during submerged growth. Compact colony morphology and inhibition of conidiation was also observed for a wild-type strain grown in the presence of 5 mM cAMP (Benina et al., 1997
). The morphological and developmental effects of both disruption of pkaR and continuous presence of cAMP are presumably due to unrestrained PKA activity. We did not, however, provide actual evidence that PKA activity is not regulated at all in the
pkaR strain. We cannot, for example, exclude the possibility that A. niger possesses another PKA-R-type protein that may, at least partially, control PKA activity.
Loss of growth polarity and absence of sporulation has also been described for the N. crassa mcb mutant, which is deficient in PKA-R (Bruno et al., 1996 ). Likewise, a key role for cAMP and PKA is reported for morphogenesis in dimorphic fungi (Borges-Walmsley & Walmsley, 2000
). For example, in U. maydis disruption of ubc1, encoding PKA-R, caused a multiple budding phenotype (Gold et al., 1994
). Thus, elevated or unrestrained PKA activity gives a budding growth in dimorphic fungi and loss of growth polarity in filamentous fungi. Filamentous fungi have a short period of isotropic growth during spore germination and PKA appears to be important for the switch of isotropic to polarized growth (dEnfert, 1997
). Interestingly, the A. niger
pkaR strain is able to form hyphae during growth on plate, although to a very limited extent. Surface growth may trigger a signal transduction pathway, also involved in hyphal extension, which is to some extent independent of the PKA pathway.
In summary, disruption of pkaR interferes with proper growth and development of A. niger. Our data show that cAMP-dependent protein phosphorylation in A. niger is involved in developmental processes such as growth polarity in germinating conidia and asexual reproduction, i.e. formation of conidiospores. The availability of a pkaR mutant will hopefully enable a study aiming to identify the PKA targets directly involved in growth and morphology.
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ACKNOWLEDGEMENTS |
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Received 28 March 2002;
accepted 24 April 2002.