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Vol. 18, Issue 6, 2123-2136, June 2007
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*Department of Plant Pathology and Microbiology and Program in Biochemistry and Molecular Biology, University of California, Riverside, Riverside, CA 92521
Submitted March 23, 2006;
Revised March 6, 2007;
Accepted March 16, 2007
Monitoring Editor: Ralph Isberg
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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rrg-1 mutants is reminiscent of osmotic-sensitive (os) mutants, including nik-1/os-1 (a histidine kinase) and strains defective in components of a mitogen-activated protein kinase (MAPK) pathway: os-4 (MAPK kinase kinase), os-5 (MAPK kinase), and os-2 (MAPK). Similar to os mutants, rrg-1 strains are sensitive to hyperosmotic conditions, and they are resistant to the fungicides fludioxonil and iprodione. Like os-5, os-4, and os-2 mutants, but in contrast to nik-1/os-1 strains, rrg-1 mutants do not produce female reproductive structures (protoperithecia) when nitrogen starved. OS-2-phosphate levels are elevated in wild-type cells exposed to NaCl or fludioxonil, but they are nearly undetectable in rrg-1 strains. OS-2-phosphate levels are also low in rrg-1, os-2, and os-4 mutants under nitrogen starvation. Analysis of the rrg-1D921N allele, mutated in the predicted phosphorylation site, provides support for phosphorylation-dependent and -independent functions for RRG-1. The data indicate that RRG-1 controls vegetative cell integrity, hyperosmotic sensitivity, fungicide resistance, and protoperithecial development through regulation of the OS-4/OS-5/OS-2 MAPK pathway. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Two-component systems have been implicated in responses to light, osmolarity, cellular redox status, nutrient and oxygen levels, virulence, and other factors (for review, see Wolanin et al., 2002; Borkovich et al., 2004). A basic two-component system consists of a histidine kinase and a response regulator (for review, see West and Stock, 2001; Wolanin et al., 2002). Histidine kinases autophosphorylate on a conserved histidine residue in the histidine kinase domain. The phosphoryl group is then transferred to an aspartate residue in the receiver domain on the response regulator. Complex two-component signaling pathways (also termed phosphorelays) contain hybrid histidine kinases that have both a histidine kinase domain and a response regulator receiver domain in the same protein, thus facilitating an intramolecular phosphate transfer reaction. The phosphate group on the aspartate residue is transferred to a histidine phosphotransfer (HPT) protein. Subsequently, the histidine phosphotransfer protein phosphorylates the receiver domain on a response regulator. Phosphorelays are the major two-component pathways in eukaryotic systems; however, two-component systems are not present in animals, making them ideal candidate targets for antimicrobial drugs (for review, see Alex et al., 1998; Wolanin et al., 2002; Borkovich et al., 2004).
Many response regulators contain a transcriptional regulatory domain that is controlled by the phosphorylation status of the aspartate residue on the receiver domain, leading to changes in gene expression. The phosphorylation status of the response regulator can also determine its ability to interact with other regulatory proteins, such as mitogen-activated protein kinase (MAPK) cascades in eukaryotes (Maeda et al., 1994; Buck et al., 2001), and with proteins that control flagellar motor rotation in bacteria (Baker et al., 2006). Two-component systems have been well studied in the yeast Saccharomyces cerevisiae. The S. cerevisiae genome contains one histidine kinase (Sln1p), one HPT protein (Ypd1p), and two response regulators (Ssk1p and Skn7p). The Sln1p/Ypd1p/Ssk1p system regulates growth under hyperosmotic conditions through modulation of the Ssk2p (Ssk22p)/Pbs2p/Hog1p MAPK pathway (for review, see Saito and Tatebayashi, 2004). The Hog1p MAPK can also be regulated by the transmembrane protein Sho1p in response to hyperosmotic stress in a pathway that includes the Ste11p MAPK kinase kinase (MAPKKK) and the MAPK kinase (MAPKK) Pbs2p (Posas and Saito, 1997). The Ssk1p response regulator has a structure that includes the phospho-accepting receiver domain at the carboxy terminus. In addition to a carboxy-terminal receiver domain, the response regulator Skn7p also contains an amino-terminal heat-shock factor-like helix-turn-helix DNA binding domain. Various functions of Skn7p differ in their requirement for the conserved phospho-accepting aspartate residue Asp-427. For example, cell wall assembly and regulation of the cell cycle requires Asp-427 (Brown et al., 1994; Morgan et al., 1995), whereas the requirement for Skn7p during resistance to oxidative stress does not depend on Asp-427 (Morgan et al., 1997).
NIK-1 and NIK-2 from Neurospora crassa were the first histidine kinases to be identified in filamentous fungi (Borkovich et al., 2004). nik-1 strains are phenotypically similar to osmotic-sensitive (os) mutants, with swollen hyphae and asexual spores that lyse easily and are sensitive to hyperosmotic conditions. Subsequent work demonstrated that nik-1 is allelic with os-1 (Schumacher et al., 1997) and that three additional os genes (os-4 MAPKKK, os-5 MAPKK, and os-2 MAPK) comprise an MAPK pathway in Neurospora whose members are similar to those of the S. cerevisiae Hog1p system (Zhang et al., 2002; Fujimura et al., 2003). Mutation of os genes also leads to increased resistance to phenylpyrrole and dicarboximide fungicides (Fujimura et al., 2000a,b; Ochiai et al., 2001; Zhang et al., 2002). Of interest, wild-type S. cerevisiae strains are resistant to these fungicides (Zhang et al., 2002; Motoyama et al., 2005), but they are rendered sensitive by introduction of an OS-1/NIK-1–related histidine kinase gene from the filamentous fungus Magnaporthe grisea (Motoyama et al., 2005). In the yeast Candida albicans, deletion of the os-1/nik-1 homologue (cos-1) leads to defects in hyphal growth and reduced virulence (Alex et al., 1998; Selitrennikoff et al., 2001).
In contrast to results with nik-1, nik-2 mutants have no obvious phenotypes (Alex et al., 1996; Borkovich et al., 2004). However, analysis of the recently completed Neurospora genome sequence suggests that the lack of an identified function for NIK-2 may result from gene redundancy, because Neurospora possesses 11 genes encoding hybrid histidine kinases (Galagan et al., 2003; Borkovich et al., 2004). The Neurospora genome also predicts one HPT protein (HPT-1) and two response regulators, RRG-1 and RRG-2. RRG-1 and RRG-2 are most similar to the class of response regulators represented by S. cerevisiae Ssk1p and Skn7p, respectively. The expansion of histidine kinases in Neurospora relative to S. cerevisiae, coupled with the same number of HPTs and response regulators, suggests either a signaling bottleneck downstream of the histidine kinases or differential regulation of the HPT and response regulators due to time or tissue-dependent expression of the histidine kinases or downstream effectors (Galagan et al., 2003; Borkovich et al., 2004). It is also possible that histidine kinases regulate cellular functions independently of the HPT and response regulators in Neurospora (Galagan et al., 2003; Borkovich et al., 2004).
In this study, we characterize the cellular role of the response regulator RRG-1 in Neurospora. We determine the gene structure and pattern of expression of rrg-1 during the life cycle. We create gene replacement mutants as well as strains carrying a mutation at the presumed site of phosphorylation. We observe all strains for phenotypes during growth and development as well as for sensitivity to hyperosmotic conditions and fungicides. We also monitor the phosphorylation status of the OS-2 MAPK protein, and we identify downstream effects on gene expression. Our results demonstrate functions for RRG-1 in cell integrity, osmotic stress responses, fungicide sensitivity, and female fertility. We also present evidence that RRG-1 regulates the OS-2 MAPK pathway in Neurospora.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), whereas synthetic crossing medium (SCM) was used to induce the sexual cycle (Westergaard and Mitchell, 1947). For hyperosmotic conditions, VM solid medium was supplemented with 0.75 M NaCl, 0.75 M KCl, or 1.5 M sorbitol, whereas VM liquid medium was supplemented with 0.1 or 0.8 M NaCl. Sorbose-containing medium (FIGS or FGS) was used to facilitate colony formation on plates (Davis and deSerres, 1970). When required, hygromycin B (Calbiochem, EMD Biosciences, San Diego, CA) was added to media at a concentration of 200 µg/ml. Plasmids were maintained in Escherichia coli strain DH5
(Hanahan, 1983). Fludioxonil and iprodione (gifts from Drs. Allison Tally [Syngenta Crop Protection, Greensboro, NC] and Frank Wong [University of California, Riverside, CA]) were used at final concentrations of 10 or 100 µg/ml (see figure legends), from stock solutions prepared at 100 mg/ml in 100% dimethyl sulfoxide.
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) or with the TRIzol reagent according to the manufacturer's recommendations (Invitrogen, Carlsbad, CA) or by using the PureScript kit according to the manufacturer's directions for plant tissue (Gentra Systems, Minneapolis, MN).
rrg-1 Gene Structure, Intron Verification, and Expression Analysis
The rrg-1 open reading frame (ORF; NCU01895.1) was originally identified during homology searches of the N. crassa genome sequence (http://www.broad.mit.edu/annotation/fungi/neurospora/; Galagan et al., 2003) by using BLAST (Altschul et al., 1997) with response regulators from S. cerevisiae and Schizosaccharomyces pombe as queries. The predicted gene structure includes one 85-base pair intron near the 3' end of the ORF. The primer pair intron left and intron right (Table 2) was used with RNA isolated from SCM plate cultures in reverse transcriptase-polymerase chain reaction (RT-PCR) reactions (as described in Krystofova and Borkovich, 2005), to verify the existence and size of the intron. Amplification reactions containing cosmid pMOcosX G18F7 (Orbach et al., 1990; includes the entire rrg-1 region) were used to generate a genomic control fragment. Products were detected by subjecting samples to agarose gel electrophoresis, blotting and then Southern analysis. The same primers used in RT-PCR reactions were used to produce probe templates during PCR reactions with the pMOcosX G18F7 cosmid. All probe templates were labeled using the Prime-a-Gene labeling kit method according to the manufacturer's protocol (Promega, Madison, WI). The RT-PCR products were subsequently cloned into pGEMT (Promega) and sequenced.
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). This method was also used to check the rrg-1 and rrg-1D921N mutants and rrg-1, rrg-1+ complemented strains (Table 1) for the presence of the rrg-1 transcript by using 6-d-old SCM plates or 3-d-old liquid SCM cultures. Reaction samples were processed using the procedure described for intron verification above.
For Northern analysis of poi-2 (NCU05768.2), samples containing 25 µg of total RNA were analyzed as described previously (Krystofova and Borkovich, 2005). The poi-2 probe DNA was generated by PCR by using a cosmid template (Table 2) and then labeled as described above.
Construction of rrg-1, rrg-1D921N, and Rescued Strains
The rrg-1 gene replacement construct was generated using a modification of a yeast recombinational cloning procedure (Colot et al., 2006; Raymond et al., 1999). Fragments corresponding to the 2-kb regions just 5' and 3' to the rrg-1 ORF (5' and 3' flanks) were amplified by PCR by using genomic DNA from wild-type strain 74A as a template. Primers contained 45 bases of overlap to the SrfI gapped pCRG26 vector at the 5' end and 45 bases of overlap to the cassette at the 3' end, in addition to 20 bases of gene specific sequence (Table 2). RG1vL was paired with RG1cL, and RG1cR was paired with RG1vR to generate the 5' flank and 3' flank, respectively. Cycling conditions were as follows: 94°C for 5 min; 98°C for 20 s; 60°C for 30 s and 68°C for 2 min (35 cycles total), with a final incubation at 72°C for 10 min.
The antibiotic resistance cassette was generated by PCR of vector pAG32 by using the cassette 1 and cassette 2 primers (Table 2). The cassette contains the E. coli hph gene (conferring hygromycin B resistance) with the Ashbya gossypii TEF (translation elongation factor) promoter and terminator that are expressed in S. cerevisiae and N. crassa (Pitt, Colot, and Dunlap, personal communication). The cassette, 5' flank, 3' flank, and SrfI gapped pCRG26 were transformed into S. cerevisiae strain CRY1-2 for recombination via the overlapping 45mers into a circular plasmid. Colonies harboring correctly recombined constructs were selected by plating cells on YPAD containing 200 µg/ml hygromycin B. DNA was isolated from the population of yeast transformant colonies, and plasmids were recovered by transformation into E. coli and plating on ampicillin-containing medium. Plasmids were analyzed for the correct recombination event by restriction digestion and PCR. The resulting rrg-1 gene replacement construct was named pSP1.
The rrg-1 gene is located on linkage group 1 near the mating-type locus. To obtain the rrg-1 mutation in both mating types, the gene replacement construct was transformed into wild-type strains 74A and 74a and histidine auxotrophic strain his3a (Krystofova and Borkovich, 2005). Plasmid pSP1 was linearized by XhoI digestion, and 1 µg was used to electroporate 10-d-old macroconidia as described previously (Vann, 1995; Ivey et al., 1996). Transformants were suspended in regeneration agar (Case et al., 1979) and then plated on FIGS medium containing 150 or 200 µg/ml hygromycin B.
Genomic DNA was extracted from transformants using the PureGene kit according to the manufacturer's protocol (Gentra Systems). Homologous recombination events were identified by Southern analysis (Ivey et al., 1996) of genomic DNA from the transformants after digestion with HindIII (Supplemental Figure 1B; data not shown). A 4-kb fragment containing the hph cassette and 3' flank from the rrg-1 cassette (Supplemental Figure 1A) was used as the probe. Heterokaryotic strains containing the gene replacement event without ectopic integrations were crossed to the opposite mating type and homokaryotic progeny were selected on FIGS medium containing 150 µg/ml hygromycin B. The purity of strains was verified by Southern analysis as described above (Supplemental Figure 1, A and B). rrg-1 strains 11.4 (mat A) and 6.2 (mat a) were chosen for further analysis.
Two different approaches were used to complement the rrg-1 mutation in trans. In the first approach, a construct containing the rrg-1 gene and the bar dominant-selectable marker (confers resistance to Ignite; Pall, 1993) was integrated at a random ectopic site in the genome of a rrg-1 strain. The construct (pCJ2) was generated as follows. First, pTJK1 was made by inserting the XbaI–SpeI fragment containing the bar cassette from plasmid pBARGEM7-2 (Pall and Brunelli, 1993) into pBluescript II KS+ (Stratagene, La Jolla, CA). The pMOcosX G18F7 cosmid was digested with XmnI and AclI, which cuts 2.05 kb upstream and 0.87 kb downstream of the rrg-1 ORF, respectively, resulting in a 5.96-kb fragment. This fragment was subcloned into pTJK1 digested with ClaI and EcoRV (ClaI generates the same overhang as AclI, whereas EcoRV and XmnI create blunt ends) to yield pCJ2, the rrg-1+ bar+ rescue construct. Aliquots containing 300–600 ng of XmnI-linearized pCJ2 plasmid were transformed into 8-d old conidia from strain rrg-1 (11.4) via electroporation as described previously, by using water instead of 1 M sorbitol to suspend the conidia. Transformants were selected on FIGS medium supplemented with 400 µg/ml Ignite, and then they were screened for integration of the rescue construct by growth on VM plates containing 1 M sorbitol and by Southern analysis (using an XhoI–NotI digest of genomic DNA and with the 2-kb 5' flanking region of rrg-1 as the probe). Rescue strains produce a hybridizing fragment of 7.5 kb, whereas the rrg-1 gene replacement mutant produces a 4.8-kb fragment. Homokaryotic rrg-1 rrg-1+-complemented strains were obtained after cultivation of transformants with the desired integration event on microconidiation medium (Ebbole and Sachs, 1990) supplemented with 400 µg/ml Ignite.
The second approach for complementation involved targeting rrg-1+ to the his-3 locus of a rrg-1 his-3 strain. Because this rescue strain served as a control for the his-3-targeted rrg-1D921N allele, construction of both strains will be discussed together. First, the pMOcosX G18F7 cosmid was digested with NotI (made blunt with Klenow) and then SacI, releasing an 8.5-kb rrg-1 genomic fragment. This fragment was ligated into pTJK1 at StuI/SacI forming pCJ1. Separately, C-terminal rrg-1 gene fragments containing a FLAG tag and the RRG-1D921N point mutation were generated via PCR by using primers containing one copy of the FLAG sequence in place of the rrg-1 stop codon (for both constructs) and AAC in place of GAT at codon 921 (for the D921N mutation). All fragments were amplified using pCJ2 template and assembled using yeast recombinational cloning with yeast plasmid pRS426 as described previously (Raymond et al., 1999; Colot et al., 2006). Fragments A and B and D were used for the RRG-1D921N point mutation, whereas fragments C and D were used for the wild-type allele, Fragment A was generated using the RG1-1 and RG1-6 primers (Table 2), creating a 1.6-kb fragment extending from the EcoRV site in the rrg-1 ORF to amino acid 921. Fragment A also contained sequence homologous to pRS426 at the 5' end. Fragment B was amplified using RG1-5 and RG1-4 primers, generating a 0.6-kb fragment spanning from amino acid 921 to the position of the stop codon (a FLAG tag has been inserted upstream of a new stop codon). For the wild-type allele, fragment C was amplified using the RG1-1 and RG1-4 primers; this generates a 2.2-kb fragment extending from the EcoRV site in rrg-1 (with a pRS426 adaptor) to the end of the rrg-1 ORF (stop codon replaced with the FLAG sequence). Fragment D was generated using primers RG1-3 and RG1-2; the resulting 0.9-kb fragment extends from the FLAG tag to the XhoI site in pCJ2 (also includes sequence homologous to pRS426 vector). pCJ3 (D921N mutation with FLAG tag) and pCJ4 (wild-type allele, with FLAG tag) were then generated by yeast recombinational cloning of the appropriate fragments. pCJ2 was digested with XhoI/EcoRV, and the 7.2-kb fragment (5' end of rrg-1 ORF and promoter region) was ligated with the 3.1-kb fragment from the digestion of pCJ4 or pCJ3 with EcoRV/XhoI to generate pCJ5 and pCJ6, respectively. Finally, SmaI/SpeI-digested pBM61 (Margolin et al., 1997), the 2.7-kb XmnI/BamHI fragment from pCJ1, and the 3.3-kb BamHI/SpeI fragment from either pCJ5 or pCJ6 were ligated to generate the his-3-targeted rrg-1+ (pCJ7) or rrg-1D921N (pCJ8) vectors.
Eight-day-old conidia from the histidine auxotrophic strain rrg-1 his-3 mat a (1.12) were transformed with 300- to 600-ng aliquots of BssHII-linearized pCJ7 or pCJ8. Transformation was performed as described above for the bar+ rescue strains. Histidine prototrophs were selected on FGS medium and screened for successful his-3 targeting by Southern analysis by using a HindIII digest of genomic DNA and a 1.5-kb probe corresponding to of the 5' end of the rrg-1 ORF (amplified using primers RG1-7 and RG1-8; Table 2; see "probe his-3 targeting" in Supplemental Figure 1A). Strains with rrg-1 targeted to the his-3 locus produce a hybridizing fragment of 8.7 kb, whereas the rrg-1 his-3 recipient strain does not. Further Southern analysis by using alternative probes and enzymes confirmed the original results. Homokaryotic rrg-1 rrg-1+::his-3+ and rrg-1 rrg-1D921N::his-3+ strains were obtained after cultivation of transformants with the desired integration event on microconidiation medium (Ebbole and Sachs, 1990) and plating on FGS medium.
Phenotypic Testing and Western Analysis
Assessment of colony growth on VM plates, macroconidiation in slants, and protoperithecial and perithecial development during the sexual cycle were as described previously (Krystofova and Borkovich, 2005). Growth under hyperosmotic conditions was assayed as described previously (Ivey et al., 1996), with details in the figure legend. Photomicroscopy was performed using a SZX9 stereomicroscope or a BX41 compound microscope with a C-4040 digital camera (Olympus, Lake Success, NY).
For Western analysis, whole cell extracts were isolated from the tissues indicated in the figure legends by using a modified version of a published protocol (Borkovich et al., 1989). Neurospora tissues, ground in liquid nitrogen, were added to 1 ml of chilled 3 mM phenylmethylsulfonyl fluoride in 95% ethanol with 0.2 g of 0.5-mm glass beads in a 2-ml screw-top centrifuge tube. Samples were vortexed 3 times for 60 s with 60-s rests on ice in between. Extracts were chilled at –20°C for at least 16 h. Samples were then centrifuged at 14,000 rpm for 10 min at 4°C. The supernatant was removed, and precipitated proteins were dried on the beads for 30 min in a vacuum concentrator (Eppendorf, Westbury, NY). Samples were reconstituted in 250 µl of 1% SDS, heated at 85°C for 5 min, and centrifuged at 14,000 rpm for 5 min at room temperature. The protein supernatant was removed and saved. This step was repeated once, and the supernatants were combined. The combined supernatants were subjected to a final heating, centrifugation, and supernatant recovery as described above to remove residual cellular debris. Protein concentration was determined using the BCA protein assay (Pierce Chemical, Rockford, IL), and a volume containing 50 µg of protein was subjected to SDS-polyacrylamide gel electrophoresis as described previously (Krystofova and Borkovich, 2005).
The POI-2 antibody (Kim and Nelson, 2005) was used at a dilution of 1:1000. Commercially available antibodies against mammalian or S. cerevisiae MAPKs were used to detect phospho-OS-2 (anti-p38 9211; 1:1000 dilution; Cell Signaling Technology, Beverly, MA; Kawasaki et al., 2002), and OS-2 (anti-Hog1p sc-9079; 1:600 dilution; Santa Cruz Biotechnology, Santa Cruz, CA). Incubation with secondary antibody and chemiluminescent detection were performed as described previously (Krystofova and Borkovich, 2005).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Borkovich et al., 2004). The autocalled gene has one predicted intron in the carboxy terminus. Because there are no cDNA clones available for rrg-1, the existence of this intron was verified with RT-PCR. Primers were designed that would produce 220- and 305-base pair products from mRNA and genomic DNA, respectively, if an intron was present. A product of 220 base pairs was obtained using mRNA as a template in RT-PCR reactions (Figure 1A, left), and sequencing of this product verified the predicted position and size of the intron (data not shown).
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) predicts a carboxy-terminal response regulator receiver domain for RRG-1. RRG-1 belongs to the Ssk1p class of response regulators, implicated in resistance to hyperosmotic stress in other fungi. Under normal conditions, S. cerevisiae Ssk1p is phosphorylated at Asp-554, blocking activation of the Hog1 MAPK cascade (Maeda et al., 1994; Posas et al., 1996). Asp-921 in RRG-1 aligns with Asp-554 in Ssk1p, suggesting that Asp-921 is phosphorylated in RRG-1. The predicted 1114-amino acid sequence of RRG-1 is also similar to several predicted proteins in the genomes of other ascomycete filamentous fungi (Figure 1B and Supplemental Figure 2). Sequence conservation between these putative fungal response regulators is highest in and around the receiver domain (amino acids 869-1027 in RRG-1), and all contain the conserved aspartyl residue (indicated by an asterisk in Supplemental Figure 2). Areas of high similarity also exist 5' of the receiver domain; the first 17 amino acids of Neurospora RRG-1 are 82% similar to predicted response regulator proteins from other filamentous fungi. A region of 95% similarity spans from amino acids 485-1077, with highest conservation occurring between residues 527–642 and 688–743 (both regions are outside the response regulator domain). The rrg-1 mRNA is of relatively low abundance and cannot be detected using Northern analysis (data not shown). Therefore, quantitative RT-PCR was used to determine relative expression levels of rrg-1 during the Neurospora life cycle (Figure 1A, left). We tested tissues representing vegetative growth, asexual and sexual development (see below). The rrg-1 message was present in all tissues analyzed, but was most abundant in sexually differentiated (SCM) plate cultures.
The rrg-1 gene was mutated in Neurospora by using a gene replacement procedure. The knockout construct was made using a yeast recombinational cloning method (see Materials and Methods), with the selectable marker consisting of the E. coli hph gene under the control of a fungal promoter (Staben et al., 1989; see diagram in Supplemental Figure 1A). Southern analysis was used to verify the final homokaryotic rrg-1 gene replacement mutants (Supplemental Figure 1B). To ensure that the observed phenotypes resulted from deletion of the rrg-1 gene, the rrg-1 mutant was complemented via two different methods in trans with wild-type rrg-1+, by using the bar gene (Pall, 1993) or his-3+ as selectable markers (Margolin et al., 1997). Results from RT-PCR analysis of RNA showed that the rrg-1 transcript was absent from rrg-1 gene replacement mutants (Figure 1A, middle and right). Rescued rrg-1::rrg-1+ strains were similar to wild type with respect to all traits tested, with the exception of fungicide sensitivity in the rrg-1+::bar+ strain (data not shown; see below). Complemented rrg-1+::bar+ and rrg-1+::his-3+ targeted strains possessed reduced and comparable levels of rrg-1 transcript compared with wild type, respectively (Figure 1A, middle and right; data not shown). The lower level of expression in the rrg-1+::bar+ strain may explain the complementation of most, but not all phenotypes in these strains.
rrg-1 Strains Resemble os Mutants during Vegetative Growth
rrg-1 mutants were first analyzed for defects during vegetative growth. Neurospora grows vegetatively by apical extension of basal hyphae (for review, see Springer, 1993; Borkovich et al., 2004). Hyphae fuse and branch to form a multicellular web-like structure, the mycelium. A variety of environmental stimuli, including desiccation, heat, and carbon or nitrogen starvation, can induce Neurospora to commit to an asexual sporulation pathway, macroconidiation. The timing of macroconidiation is also controlled by a circadian rhythm. The macroconidiation pathway begins with differentiation of aerial hyphae from the mycelium. Vegetative spore-forming structures, conidiophores, develop from the tips of aerial hyphae. Constrictions in the aerial hyphae then form, yielding a chain of multinucleated spores, the macroconidia. Macroconidia are hydrophobic, and they are easily released by mechanical perturbation. Because of the requirement for desiccation, submerged liquid cultures of wild-type Neurospora do not develop aerial hyphae or macroconidia.
rrg-1 mutants have a hyphal apical extension rate similar to wild-type (data not shown). However, rrg-1 strains exhibit premature aerial hyphae development and macroconidiation on solid medium (data not shown). Under light exposure, Neurospora develops a characteristic orange color, which results from the production of carotenoid pigments. Macroconidia from rrg-1 strains eventually form a deep orange or red pigment due to lysis and leakage of cytoplasmic contents, including carotenoids (Figure 2A). This phenotype is enhanced when spores are collected in 1 M sorbitol (data not shown), greatly reducing the viability of rrg-1 macroconidia during preparation for electroporation. This trait made rrg-1 macroconidia poor candidates for complementation in trans with wild-type rrg-1 (see Materials and Methods; data not shown).
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rrg-1 strains on vegetative minimal medium are reminiscent of previously characterized Neurospora os mutants. As a group, the os mutants develop macroconidia that acquire a deep orange pigment and tend to lyse (Figure 2A; Perkins et al., 2001; Zhang et al., 2002). The os-1/nik-1, os-4, os-5, and os-2 genes encode a hybrid histidine kinase, MAPKKK, MAPKK, and MAPK, respectively. OS-4, OS-5, and OS-2 are similar to S. cerevisiae Ssk2p, Pbs2p, and Hog1p, which are required for the response to hyperosmotic stress in budding yeast (Maeda et al., 1994). OS-4, OS-5, and OS-2 also have homologues in S. pombe, making up a related MAPK pathway that controls resistance to oxidative stress (Degols et al., 1996). OS-1 does not have a close homologue in S. cerevisiae or S. pombe. In S. cerevisiae, the Sln1p hybrid histidine kinase operates upstream of the Hog1p MAPK cascade. The Mak2 and Mak3 hybrid histidine kinases phosphorylate response regulator Msc4 (similar to RRG-1) to control the oxidative stress response in S. pombe (Buck et al., 2001).
rrg-1 Mutants Are Sensitive to Hyperosmotic Stress and Resistant to Phenylpyrrole and Dicarboximide Fungicides
It was previously shown that os mutants are sensitive to hyperosmotic stress (Selitrennikoff et al., 1981; Perkins et al., 1982). The similar growth phenotypes of rrg-1 and os strains prompted comparison of the rrg-1 mutant to wild-type, os-5, os-4, and os-2 strains during growth on medium containing 0.75 M NaCl, 0.75 M KCl, or 1.5 M sorbitol (Figure 2B). Consistent with the previous studies, os-5, os-4, and os-2 mutants are more sensitive to all three hyperosmotic media than wild type. Similarly, growth of the rrg-1 mutant was severely restricted under osmotic stress, with the largest reduction on sorbitol-containing medium (Figure 2B).
There are conflicting reports regarding the sensitivity of os mutants to phenylpyrrole and dicarboximide fungicides (Grindle and Temple; 1982; Fujimura et al., 2000a,b). For example, one analysis reported that os-1/nik-1, os-2, os-4, and os-5 mutants are resistant to the phenylpyrrole, fludioxonil, and the dicarboximide iprodione (Grindle and Temple, 1982; Fujimura et al., 2000a,b), whereas a subsequent study showed that os-1/nik-1 and os-4 mutants were sensitive to fludioxonil (Zhang et al., 2002). These inconsistencies may be due to differences in the mutant alleles used for each analysis. Because rrg-1 strains and os pathway mutants exhibit osmotic sensitivity, and in light of the discrepancies in the literature, we analyzed the growth of wild-type, rrg-1, os-5, os-4, and os-2 mutants on solid medium containing iprodione or fludioxonil. The results show that rrg-1, os-5, os-4, and os-2 strains are indeed resistant to fludioxonil and iprodione (Figure 2C), suggesting that RRG-1 and proteins of the OS signaling cascade control fungicide sensitivity in Neurospora.
rrg-1 and Components of the os-2 MAPK Pathway Are Essential for Protoperithecial Development and Female Fertility
We next analyzed rrg-1 strains for defects in sexual fertility. Nitrogen starvation induces differentiation of female reproductive structures, protoperithecia, in Neurospora (for review, see Raju, 1992). Protoperithecia extend specialized hyphae called trichogynes that are positively chemotropic, growing toward male cells (any vegetative cell type; typically, macroconidia) of opposite mating type (Bistis, 1981). Successful attraction requires that the male cell produces pheromone and that the female contains the appropriate pheromone receptor (Bistis, 1983; Bobrowicz et al., 2002; Kim et al., 2002; Kim and Borkovich, 2004). Once physical contact is established, the trichogyne coils around and fuses with the male cell (Bistis, 1981; Kim and Borkovich, 2004). It is thought that the nucleus from the male then traverses the trichogyne tube and that fertilization takes place at the base of the protoperithecium where the female gametes are located. The diploid phase of the life cycle is transient, and nuclear fusion is followed immediately by meiosis and the onset of sexual spore (ascospore) development. Early during the process of fertilization and meiosis, the protoperithecium enlarges and becomes the perithecium. Once ascospore maturation is complete, the spores are ejected through a hole in the tip of the perithecium (ostiole). Under appropriate conditions (high temperature and available nutrients), ascospores can germinate to form a new mycelium.
rrg-1 mutants of either mating type are fertile as males when crossed with wild-type females of opposite mating type (data not shown). rrg-1 strains exhibit reduced mass accumulation on nitrogen-limited SCM compared with wild-type (data not shown). More importantly, protoperithecia cannot be observed in rrg-1 strains cultivated on SCM, and application of wild-type macroconidia does not result in production of perithecia or ascospores (Figure 3, A–C; data not shown). This defect is rescued by introduction of the wild-type rrg-1 gene in trans (Figure 3B). Thus, loss of rrg-1 leads to an early and complete block in female fertility in Neurospora. It has been reported that os-4 mutants are female sterile, but the exact defect was not specified (Fujimura et al., 2003). Therefore, we analyzed the os mutant strains for protoperithecial and perithecial development. Protoperithecia could not be detected in the os-4, os-5, or os-2 mutants, and perithecia were not observed after fertilization with wild-type macroconidia during our study (Figure 3A). This experiment was repeated several times, and an occasional rare perithecium could be observed in cultures of os-2 and os-5 mutants, but never in os-4 strains (data not shown). These results suggest that the OS-2 MAPK pathway is required for protoperithecial development and female fertility in Neurospora. This is in contrast with previous observations with nik-1/os-1 mutants, demonstrating that this histidine kinase is not required for protoperithecial or perithecial development (Alex et al., 1996). We analyzed a nik-1/os-1 mutant (allele Y256M209) during our study and observed that it was able to produce protoperithecia and perithecia, but at reduced numbers compared with wild type (Figure 3C). Further analysis showed that the os-1 mutant did not produce viable ascospores 10 d postfertilization (data not shown). The Y256M209 os-1 mutant allele was generated using UV light and contains a frameshift mutation at amino acid 294 (Ochiai et al., 2001). The difference in phenotypes observed for rrg-1 and os-2 versus os-1 mutants supports the hypothesis that the OS-2 MAPK pathway is regulated by multiple histidine kinases under different environmental conditions (e.g., osmotic stress vs. nitrogen starvation) in Neurospora.
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rrg-1 and os-2 strains to hyperosmotic conditions and their resistance to fludioxonil and iprodione prompted us to investigate whether 1) OS-2 phosphorylation varied after exposure to high salt or fungicide and 2) the possibility that rrg-1 mutants would have lower levels of phosphorylated OS-2 than wild-type cells. For these studies, we took advantage of commercially available peptide antibodies raised against the mammalian MAPK p38, and S. cerevisiae Hog1p, which are homologous to Neurospora OS-2. Anti-Hog1p recognizes both nonphosphorylated and phosphorylated forms of the protein, whereas anti-phospho p38 is specific for the phosphorylated MAPK. The phospho-p38 antibody was previously shown to cross-react with the homologous MAPK protein, SakA, from Aspergillus nidulans (Kawasaki et al., 2002), whereas both antibodies were shown to recognize the homologous MAPK protein OSC-1 from Colletotrichum lagenarium (Kojima et al., 2004). We tested the antibodies for binding to OS-2 via Western analysis of extracts from wild-type, rrg-1, and os-2 strains (Figure 4, A–C). The two antisera cross-react with a protein of the expected size of Neurospora OS-2 in wild-type extracts (41.3 kDa; Figure 4, A–C). The cross-reactive species is absent from the os-2 mutant, supporting the specificity of the antibodies for OS-2.
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rrg-1 strains during short-term exposure to different concentrations of NaCl (Figure 4A). We found levels of OS-2 protein are reduced two-fold in rrg-1 mutants compared with wild-type (Figure 4A, bottom). Furthermore, the amount of OS-2-phosphate is much lower in rrg-1 strains than in wild-type after a 10-min treatment with 0.8 M NaCl, and no reactive species were evident in rrg-1 mutants in the presence of 0 or 0.1 M NaCl (Figure 4A, top).
Similar results were obtained when we tested the effects of growth to different concentrations of the fungicide fludioxonil (Figure 4B) in wild-type and rrg-1 strains. Again, levels of OS-2 protein are reduced by 
50% in rrg-1 mutants (Figure 4B, bottom). In wild-type cells, the amount of phosphorylated OS-2 is maximal at 10 µg/ml fludioxonil, whereas OS-2-phosphate cannot be detected in rrg-1 mutants in the presence or absence of fludioxonil, even at 100 µg/ml (Figure 4B, top). During the preparation of this manuscript, another group reported that treatment with fludioxonil or iprodione leads to increased levels of phosphorylated OS-2 in wild-type Neurospora cells (Yoshimi et al., 2005). Taken together, these results suggest that RRG-1 is required for optimal phosphorylation of OS-2, particularly after fungicide exposure. The small amount of residual phosphorylated OS-2 in rrg-1 mutants treated with 0.8 M NaCl indicates that the RRG-2 response regulator or other proteins can activate phosphorylation of OS-2 to a limited extent in the absence of RRG-1 Neurospora.
The results mentioned above provide evidence that the OS-2 MAPK cascade is required for differentiation of female reproductive structures. Previous studies have demonstrated that mutation of nrc-1 (MAPKKK) or mak-2 (MAPK) abolishes formation of protoperithecia in Neurospora (Kothe and Free, 1998; Pandey et al., 2004; Li et al., 2005). MAK-2 is homologous to S. cerevisiae Fus3p/Kss1p (Pandey et al., 2004; Li et al., 2005). Other work has shown that phosphorylation of MAK-2 is dependent upon NRC-1 in vegetative cultures of Neurospora (Pandey et al., 2004). To explore the possible involvement of RRG-1 in regulation of OS-2 phosphorylation during sexual development and to investigate potential connections between OS-2 and MAK-2, we next analyzed levels of OS-2 protein and the extent of phosphorylation in extracts from wild-type, rrg-1, os-4, mak-2, and os-2 strains cultured under conditions that favor formation of protoperithecia (SCM plates; Figure 4C). We found that OS-2 protein levels were similar in all strains tested, except for os-2, where it is absent (Figure 4C, bottom). OS-2-phosphate was detected in wild-type and mak-2 strains but not in rrg-1, os-4, or os-2 mutants (Figure 4C, top), further supporting the hypothesis that RRG-1 regulates the OS-2 MAPK pathway.
rrg-1 Mutants Exhibit Reduced Expression of poi-2, a Gene Required for Protoperithecial Development
The poi-2 gene encodes a predicted secreted protein containing 16 tandem repeats consisting of 13–14 amino acids each. These tandemly repeated sequences are separated by apparent Kex2 protease sites (Kim and Nelson, 2005). POI-2 is required for normal growth and asexual and sexual development in Neurospora (Kim and Nelson, 2005). poi-2 mutants have reduced vegetative growth, form white conidia and few aerial hyphae, and they are prone to reversion (Kim and Nelson 2005; data not shown). poi-2 repeat-induced point mutants with truncated ORFs and complete gene replacement mutants do not produce protoperithecia and are thus female sterile (Kim and Nelson, 2005; Kim and Borkovich, unpublished data). Levels of poi-2 mRNA are also decreased in mak-2 strains (Li et al., 2005).
Based on the shared inability of mak-2 and poi-2 mutants to differentiate protoperithecia and the observed dependence of poi-2 expression on mak-2, we investigated expression of poi-2 in rrg-1 strains. We began our analysis by measuring poi-2 mRNA levels in VM and SCM plate cultures of rrg-1 and wild-type strains. poi-2 transcript levels were reduced in rrg-1 compared with wild type in SCM cultures and poi-2 transcript could not be detected in VM cultures of the rrg-1 mutant (Figure 5A). These results suggest that RRG-1 is required for normal expression of poi-2 in Neurospora.
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rrg-1, os-2, and os-1 strains by using a specific antiserum (Kim and Nelson, 2005). Consistent with previous results, POI-2 can be detected in wild type under both growth conditions, with highest levels in sexually differentiated cultures (Figures 5, B and C; data not shown; Kim and Nelson, 2005). POI-2 expression was greatly reduced in the rrg-1 mutant cultured on VM or SCM (Figure 5, B and C), similar to results with the nrc-1 and mak-2 mutants (data not shown). POI-2 levels were similar in os-2 mutants compared with wild type when cultured on VM (Figure 5B), but they were greatly reduced in SCM cultures (Figure 5C). In contrast, POI-2 could not be detected in VM cultures of os-1 mutants, but it was present at slightly reduced levels relative to wild-type strains in SCM plates (Figure 5, B and C). The residual level of POI-2 observed in os-1 mutants on SCM could be explained by functional redundancy to give the following model: NIK-1/OS-1 and other histidine kinases regulate expression of POI-2 (and protoperithecial development) through RRG-1 and the OS-2 MAPK pathway under nitrogen starvation conditions (SCM medium).
The wild-type amount of POI-2 observed in os-2 mutants in VM cultures suggests that the OS-2 MAPK pathway does not regulate POI-2 levels under conditions of ample nitrogen. However, the drastic reduction in POI-2 amount in VM cultures of os-1 and rrg-1 mutants suggests that the NIK-1/OS-1 hybrid histidine kinase and RRG-1 response regulator may regulate an alternative downstream pathway under conditions of nitrogen excess.
Analysis of the rrg-1D921N Allele, Containing a Mutation in the Predicted Phosphorylation Site
To determine the importance of the phosphorylation status of RRG-1D921, an rrg-1D921N strain was generated. Asp-921 is highly conserved among fungal response regulators, corresponding to Asp-554 in S. cerevisiae Ssk1p and Asp-427 in S. pombe Mcs4. S. pombe Mcs4 is required for activation of the Sty1 MAPK cascade, because deletion of mcs4 prevents phosphorylation of Sty1 under all conditions (Shieh et al., 1997). Although the Mcs4D421N protein does not support phosphorylation of Sty1 in response to hydrogen peroxide stress, phosphorylation is observed during salt or heat stress (Buck et al., 2001), suggesting that some functions of Mcs4 are independent of phosphorylation.
The Neurospora rrg-1D921N mutant and corresponding rrg-1+::his-3+ control strain possessed comparable levels of rrg-1 message (Figure 1A, right). Phenotypic analysis revealed that the rrg-1D921N strain differs from wild-type and the rrg-1 gene replacement mutant. In VM flask cultures, the rrg-1D921N strain is slower to conidiate (faint orange color) and has shorter aerial hyphae than wild type or rrg-1 (Figure 6A). The rrg-1D921N mutant exhibits greater growth than rrg-1 strains under hyperosmotic conditions (Figure 6B), and it is sensitive to fungicide (Figure 6C). However, the rrg-1D921N mutant grows considerably slower than wild-type or rrg-1 strains on normal medium (see fungicide – column in Figure 6C).
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We also observed that the rrg-1D921N mutant exhibits delays during sexual development. Protoperithecia are fewer but larger than those observed in wild type, and they take longer to develop (Figure 6D). After fertilization, perithecia from the rrg-1D921N strains often were deformed with a "hairy" appearance and were elongated either side to side or top to bottom. Crushing of perithecia showed that ascospores were produced 7 d after fertilization in wild-type strains, whereas the rrg-1D921N mutant did not form ascospores until 15 d postfertilization. Similarly, the wild-type strain shot ascospores 14 d postfertilization, whereas the rrg-1D921N mutants did not shoot ascospores until just after 30 d (data not shown). The observed delays in sexual development suggest that rrg-1D921N may be involved in nitrogen sensing. Other pathways, absent in the rrg-1 gene replacement mutant, must activate downstream effectors allowing protoperithecial development in the rrg-1D921N mutant. Analysis of OS-2 phosphorylation showed that the rrg-1D921N mutant supports production of phospho-OS-2 in 3-d liquid and 6-d solid SCM cultures (data not shown), suggesting that phosphorylation of RRG-1 is not absolutely essential for activation of the downstream MAPK cascade during sexual development.
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DISCUSSION |
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) in Neurospora versus yeast is also reflected in the greater number of phenotypes observed upon mutation of a single response regulator gene.
RRG-1 and Osmotolerance
The ability of fungi to respond to osmotic stress in their environment is pivotal to their survival. In Neurospora, the OS pathway is essential for osmotic stress responses. We found that rrg-1 mutants share many phenotypes with os mutants. For example, macroconidia from rrg-1 mutants develop a deep orange pigment and lyse easily and rrg-1 strains grow poorly in high concentrations of NaCl, KCl, and sorbitol. We determined that rrg-1 mutants were resistant to certain fungicides, a characteristic also associated with os mutants (Fujimura et al., 2000a,b; Ochiai et al., 2001; Zhang et al., 2002). We uncovered evidence suggesting that RRG-1 is upstream of the OS-4/OS-5/OS-2 MAPK pathway, in that OS-2-phosphate levels were greatly reduced in rrg-1 mutants relative to wild type. Previous results support OS-1/NIK-1 acting upstream of the OS-2 MAPK cascade with regard to hyperosmotic stress and fungicide resistance (Fujimura et al., 2003). Because there is only one HPT protein, we expect that NIK-1/OS-1 and perhaps all of the 11 histidine kinases operate upstream of HPT-1 to regulate the response regulators RRG-1 and RRG-2 in Neurospora (Figure 7).
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). This leads to cytosolic glycerol accumulation, restoring the osmotic gradient between the cell and the environment. A striking difference between Neurospora and the two yeast is that both the sln1 (hybrid histidine kinase) and ssk1D554N (response regulator) alleles are lethal in S. cerevisiae, due to constitutive phosphorylation of the Hog1p MAPK and subsequent build up of glycerol, causing cell lysis (Maeda et al., 1994). Conversely, the S. pombe Msc4 response regulator is not required for the response to osmotic stress (Shieh et al., 1997). Deletion of mcs4 prevents the phosphorylation of the Sty1 MAPK under all conditions (Shieh et al., 1997). However, the mcs4D412N allele supports phosphorylation of Sty1 in response to salt or heat stress but not in response to hydrogen peroxide (Buck et al., 2001). This suggests that the conserved aspartate residue is required for certain functions (i.e., the oxidative stress response) but not others (i.e., hyperosmotic stress, heat, etc.) in S. pombe. A similar situation may exist for Neurospora RRG-1 with respect to hyperosmotic stress and fungicide resistance versus female fertility. Furthermore, the observation that the rrg-1D921N and rrg-1 alleles are not equivalent supports the hypothesis that this mutant allele possesses at least partial activity during signaling.
OS-2-phosphate levels are greatly reduced, but still detectable, in rrg-1 mutants under hyperosmotic conditions (Figure 4A). This suggests that some other pathway can activate OS-2 phosphorylation in the absence of the NIK-1/HPT-1/RRG-1 system. Neurospora contains a Sho1p homologue (NCU08067.3) that is predicted to have four transmembrane helices and a Src homology 3 domain (http://www.broad.mit.edu/annotation/fungi/neurospora/). It is possible that the Neurospora Sho1p homologue and/or another pathway using the response regulator RRG-2 mediates the residual osmotic response via the OS-2 MAPK cascade.
Homologues of RRG-1 and the OS-2 MAPK also exist in A. nidulans and the human pathogens C. albicans and Cryptococcus neoformans. A. nidulans contains a Hog1p/OS-2 family MAPK, SakA. sakA mutants are not sensitive to hyperosmotic conditions, but they undergo premature sexual development and produce asexual spores that are sensitive to oxidative and heat stress (Kawasaki et al., 2002). The lack of an osmotic phenotype is of interest, because phosphorylation of SakA is induced by exposure to both 0.8 M KCl and 10 mM hydrogen peroxide. In C. albicans, deletion of ssk1 (a RRG-1 homologue) leads to avirulence in a mouse model of hematogenously disseminated candidiasis (Du et al., 2005) and sensitivity to oxidative stress but only partial sensitivity to hyperosmotic stress (Chauhan et al., 2003). Loss of skn7, another C. albicans response regulator, leads to sensitivity to hydrogen peroxide but not to hyperosmotic conditions (Singh et al., 2004). C. neoformans ssk1 deletion mutants also exhibit sensitivity to UV irradiation and oxidative and osmotic stress (Bahn et al., 2006). In contrast to C. albicans, mutation of the C. neoformans skn7 gene leads to reduced virulence in a mouse inhalation model as well as sensitivity to oxidative stress (Wormley et al., 2005; Bahn et al., 2006). Thus, although a response regulator gene is necessary for pathogenesis in C. albicans and C. neoformans, there is currently no clear pattern for predicting whether the ssk1 or skn7 homologue will fulfill this requirement.
The os-1, os-2, os-4, and os-5 mutants exhibit reduced glycerol accumulation under hyperosmotic stress conditions (Ellis et al., 1991; Fujimura et al., 2000b), which may explain why these mutants are unable to grow on hyperosmotic media. New evidence also suggests that, in addition to osmolyte regulation/accumulation, modulation of turgor pressure plays a role in this hyperosmotic adaptation. It has been shown that os-1 and os-2 mutants have lower turgor than wild-type Neurospora strains (Lew et al., 2006), suggesting that ion regulation/accumulation also contributes to the phenotype seen in os mutants.
We have demonstrated that hyperosmotic sensitivity and fungicide-resistant phenotypes place RRG-1 upstream of the OS-2 MAPK cascade. However, when treated with high concentrations of salts, rrg-1 mutants exhibit measurable, but severely reduced, levels of phosphorylated OS-2. In contrast, the rrg-1D921N mutant produced slightly lower but comparable levels of phosphorylated OS-2 in response to treatment with fungicide or NaCl compared with wild type. This suggests that an alternate pathway, possibly involving an alternative site of RRG-1 phosphorylation and/or the other response regulator RRG-2, may be capable of activating OS-2 under hyperosmotic stress conditions.
RRG-1 and Sexual Development
We found that rrg-1 mutants were unable to produce female reproductive structures during growth under nitrogen starvation and therefore were female sterile. In contrast, the rrg-1D921N mutant is not female sterile, but instead exhibits delayed female fertility and ascospore development. This is the first such report in two-component signaling pathways of filamentous fungi. The conidial lysis phenotype of rrg-1 strains coupled with the inability to produce protoperithecia may indicate that protoperithecial development is very closely tied to cell integrity. To further investigate this phenomenon, we measured levels of a candidate regulated gene, poi-2, in wild type versus rrg-1 strains. We found that poi-2 mRNA and protein levels were greatly reduced or absent in rrg-1 mutants. It is known that poi-2 expression is also controlled by the MAPK MAK-2 (Li et al., 2005), which is dependent on the MAPKKK NRC-1 for phosphorylation (Pandey et al., 2004). We found that mak-2 and rrg-1 mutants had greatly reduced levels of POI-2 under all conditions and that os-2 mutants had lower amounts of POI-2 on low-nitrogen medium. This shows that RRG-1 regulates poi-2. Other genes affecting Neurospora sexual development, such as the peptide pheromones mfa-1 and ccg-4 (Bobrowicz et al., 2002; Kim and Borkovich, 2006), were expressed at normal levels in rrg-1 mutants (data not shown). Because nik-1/os-1 mutants differentiate protoperithecia, whereas strains lacking rrg-1 and the downstream os mutants do not, additional histidine kinase(s) likely regulates RRG-1 to direct protoperithecial development in Neurospora.
Interestingly, when rrg-1 is deleted from the genome, the ability to induce protoperithecia in a nitrogen-limited environment is abolished; however, the rrg-1D921N mutant produces protoperithecia, which can undergo fertilization and eventually shoot viable ascospores. The slowing of maturation of female reproductive structures in the rrg-1D921N mutant suggests that phosphorylation of the conserved aspartate accelerates sexual development but is not essential for this process. The functions of RRG-1 during sexual development in Neurospora contrast with the known roles of two-component systems in the ascomycete yeast S. pombe and the basidiomycete yeast C. neoformans. S. pombe possesses three hybrid histidine kinases (Mak1-3p), one HPT protein and two response regulators (Mcs4 and Prr1). A triple mak1 mak2 mak3 mutant mates inappropriately in high nitrogen and enters the cell cycle precociously, producing cells that are smaller in size than wild type (Nakamichi et al., 2002). Of the two S. pombe response regulators, Mcs4 shares homology with Neurospora RRG-1, whereas Prr1 is most similar to RRG-2. Loss of either mcs4 or prr1 results in sexual sterility in S. pombe. Interestingly, deletion of the ssk1 or skn7 response regulator in C. neoformans results in enhanced mating ability during sexual reproduction (Bahn et al., 2006).
The relatively slow sexual development and conidiation observed in rrg-1D921N mutants relative to wild type suggests that the conserved aspartate residue, although not essential, is important for timely completion of these processes in Neurospora. RRG-1 may contain other unknown site(s) of phosphorylation that, if mutated, would eliminate the ability of RRG-1 to activate the OS-2 MAPK. Alternatively, RRG-1 may participate in a protein–protein complex with the presumed upstream protein HPT-1 and/or downstream MAPKKK OS-4; this interaction may be stronger when RRG-1 can be phosphorylated, leading to a higher rate of downstream MAPK activation.
We have shown that RRG-1 acts upstream of the OS-2 MAPK pathway with regards to hyperosmotic stress resistance, fungicide sensitivity, conidial integrity, and female fertility. Future investigations will reveal networking interactions that link upstream histidine kinases with the downstream response regulators and MAPK cascades or other effector pathways in Neurospora. Such studies will provide insight into the roles that two-component regulatory systems play during growth, development, and pathogenesis in filamentous fungi.
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ACKNOWLEDGMENTS |
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Footnotes |
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The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org). 
These authors contributed equally to this work.
Present address: Biology Department, Southwestern Adventist University, Keene, TX 76059.
Address correspondence to: Katherine A. Borkovich (katherine.borkovich{at}ucr.edu).
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REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Alex, L. A., Korch, C., Selitrennikoff, C. P., Simon, M. I. (1998). COS1, a two-component histidine kinase that is involved in hyphal development in the opportunistic pathogen Candida albicans. Proc. Natl. Acad. Sci. USA 95, 7069–7073.
Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W., Lipman, D. J. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25, 3389–3402.
Bahn, Y., Kojima, K., Cox, G., Heirman, J. (2006). A unique fungal two-component system regulates stress responses, drug sensitivity, sexual development, and virulence of Cryptococcus neoformans. Mol. Biol. Cell 17, 3122–3135.
Baker, M. D., Wolanin, P. M., Stock, J. B. (2006). Signal transduction in bacterial chemotaxis. Bioessays 28, 9–22.[CrossRef][Medline]
Bistis, G. N. (1981). Chemotropic interactions between trichogynes and conidia of opposite mating-type in Neurospora crassa. Mycologia 73, 959–975.[CrossRef]
Bistis, G. N. (1983). Evidence for diffusible, mating-type-specific trichogyne attractants in Neurospora crassa. Exp. Mycol 7, 292–295.[CrossRef]
Bistis, G. N., Perkins, D. D., Read, N. D. (2003). Cell types of Neurospora crassa. Fungal Gen. Newsl 50, 17–19.
Bobrowicz, P., Pawlak, R., Correa, A., Bell-Pedersen, D., Ebbole, D. J. (2002). The Neurospora crassa pheromone precursor genes are regulated by the mating type locus and the circadian clock. Mol. Microbiol 45, 795–804.[CrossRef][Medline]
Borkovich, K. A., et al. (2004). Lessons from the genome sequence of Neurospora crassa: tracing the path from genomic blueprint to multicellular organism. Microbiol. Mol. Biol. Rev 68, 1–108.
Borkovich, K. A., Farrelly, F. W., Finkelstein, D. B., Taulien, J., Lindquist, S. (1989). Hsp82 is an essential protein that is required in higher concentrations for growth of cells at higher temperatures. Mol. Cell Biol 9, 3919–3930.
Brown, J. L., Bussey, H., Stewart, R. C. (1994). Yeast Skn7p functions in a eukaryotic two-component regulatory pathway. EMBO J 13, 5186–5194.[Medline]
Buck, V., Quinn, J., Soto Pino, T., Martin, H., Saldanha, J., Makino, K., Morgan, B. A., Millar, J. B. (2001). Peroxide sensors for the fission yeast stress-activated mitogen-activated protein kinase pathway. Mol. Biol. Cell 12, 407–419.
Case, M. E., Schweizer, M., Kushner, S. R., Giles, N. H. (1979). Efficient transformation of Neurospora crassa by utilizing hybrid plasmid DNA. Proc. Natl. Acad. Sci. USA 76, 5259–5263.
Chauhan, N., Inglis, D., Roman, E., Pla, J., Li, D., Calera, J. A., Calderone, R. (2003). Candida albicans response regulator gene SSK1 regulates a subset of genes whose functions are associated with cell wall biosynthesis and adaptation to oxidative stress. Eukaryot. Cell 2, 1018–1024.
Colot, H. V., Park, G., Turner, G. E., Ringelberg, C., Crew, C. M., Litvinkova, L., Weiss, R. L., Borkovich, K. A., Dunlap, J. C. (2006). A high-throughput gene knockout procedure for Neurospora reveals functions for multiple transcription factors. Proc. Natl. Acad. Sci. USA 103, 10352–10357.
Davis, R. H. and deSerres, F. J. (1970). Genetic and microbiological research techniques for Neurospora crassa. Methods Enzymol 71A, 79–143.
Degols, G., Shiozaki, K., Russell, P. (1996). Activation and regulation of the Spc1 stress-activated protein kinase in Schizosaccharomyces pombe. Mol. Cell Biol 16, 2870–2877.
Du, C., Calderone, R., Richert, J., Li, D. (2005). Deletion of the SSK1 response regulator gene in Candida albicans contributes to enhanced killing by human polymorphonuclear neutrophils. Infect. Immun 73, 865–871.
Ebbole, D. and Sachs, M. S. (1990). A rapid and simple method for isolation of Neurospora crassa homokaryons using microconidia. Fungal Genet. Newsl 37, 17–18.
Ellis, S. W., Grindle, M., Lewis, D. H. (1991). Effect of osmotic stress on yield and polyol content of dicarboximide-sensitive and –resistant strains of Neurospora crassa. Mycol. Res 95, 457–464.[CrossRef]
Fujimura, M., Ochiai, N., Ichiishi, A., Usami, R., Horikoshi, K., Yamaguchi, I. (2000a). Fungicide resistance and osmotic stress sensitivity in os mutants of Neurospora crassa. Pestic. Biochem. Physiol 67, 125–133.[CrossRef]
Fujimura, M., Ochiai, N., Ichiishi, A., Usami, R., Horikoshi, K., Yamaguchi, I. (2000b). Sensitivity to phenylpyrrole fungicides and abnormal glycerol accumulation in os and cut mutant strains of Neurospora crassa. J. Pestic. Sci 25, 31–36.
Fujimura, M., Ochiai, N., Oshima, M., Motoyama, T. (2003). Putative Homologs of SSK22 MAPKK Kinase and PBS2 MAPK Kinase of Saccharomyces cerevisiae Encoded by os-4 and os-5 Genes for Osmotic Sensitivity and Fungicide Resistance in Neurospora crassa. Biosci. Biotechnol. Biochem 67, 186–191.[CrossRef][Medline]
Galagan, J. E., et al. (2003). The genome sequence of the filamentous fungus Neurospora crassa. Nature 422, 859–868.[CrossRef][Medline]
Geer, L. Y., Domrachev, M., Lipman, D. J., Bryant, S. H. (2002). CDART: protein homology by domain architecture. Genome Res 12, 1619–1923.
Grindle, M. and Temple, W. (1982). Fungicide-resistance of os mutants of Neurospora crassa. Neurospora Newsl 29, 16–17.
Gustin, M. C., Albertyn, J., Alexander, M., Davenport, K. (1998). MAP kinase pathways in the yeast Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev 62, 1264–1300.
Hanahan, D. (1983). Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol 166, 557–580.[Medline]
Ivey, F. D., Hodge, P. N., Turner, G. E., Borkovich, K. A. (1996). The Gi homologue gna-1 controls multiple differentiation pathways in Neurospora crassa. Mol. Biol. Cell 7, 1283–1297.[Abstract]
Kawasaki, L., Sanchez, O., Shiozaki, K., Aguirre, J. (2002). SakA MAP kinase is involved in stress signal transduction, sexual development and spore viability in Aspergillus nidulans. Mol. Microbiol 45, 1153–1163.[CrossRef][Medline]
Kim, H. and Borkovich, K. A. (2004). A pheromone receptor gene, pre-1, is essential for mating type-specific directional growth and fusion of trichogynes and female fertility in Neurospora crassa. Mol. Microbiol 52, 1781–1798.[CrossRef][Medline]
Kim, H. and Borkovich, K. A. (2006). Pheromones are essential for male fertility and sufficient to direct chemotropic polarized growth of trichogynes during mating in Neurospora crassa. Eukaryot. Cell 5, 544–554.
Kim, H., Metzenberg, R. L., Nelson, M. A. (2002). Multiple functions of mfa-1, a putative pheromone precursor gene of Neurospora crassa. Eukaryot. Cell 1, 987–999.
Kim, H. and Nelson, M. A. (2005). Molecular and functional analyses of poi-2, a novel gene highly expressed in sexual and perithecial tissues of Neurospora crassa. Eukaryot. Cell 4, 900–910.
Kojima, K., Takano, Y., Yoshimi, A., Tanaka, C., Kikuchi, T., Okuno, T. (2004). Fungicide activity through activation of a fungal signaling pathway. Mol. Microbiol 53, 1785–1796.[CrossRef][Medline]
Kothe, G. O. and Free, S. J. (1998). The isolation and characterization of nrc-1 and nrc-2, two genes encoding protein kinases that control growth and development in Neurospora crassa. Genetics 149, 117–130.
Krystofova, S. and Borkovich, K. A. (2005). The heterotrimeric G-protein subunits GNG-1 and GNB-1 form a G
dimer required for normal female fertility, asexual development, and galpha protein levels in Neurospora crassa. Eukaryot. Cell 4, 365–378.
Lew, R. R., Levina, N. N., Shabala, L., Anderca, M. I., Shabala, S. N. (2006). Role of a mitogen-activated protein kinase cascade in ion flux-mediated turgor regulation in fungi. Eukaryot. Cell 5, 480–487.
Li, D., Bobrowicz, P., Wilkinson, H. H., Ebbole, D. J. (2005). A mitogen-activated protein kinase pathway essential for mating and contributing to vegetative growth in Neurospora crassa. Genetics 170, 1091–1104.
Maeda, T., Wurgler-Murphy, S. M., Saito, H. (1994). A two-component system that regulates an osmosensing MAP kinase cascade in yeast. Nature 369, 242–245.[CrossRef][Medline]
Margolin, B. S., Freitag, M., Selker, E. (1997). Improved plasmid for gene targeting at the his-3 locus of Neurospora crassa by electroporation. Fungal Gen. Newsl 44, 34–36.
Morgan, B. A., Banks, G. R., Toone, W. M., Raitt, D., Kuge, S., Johnston, L. H. (1997). The Skn7 response regulator controls gene expression in the oxidative stress response of the budding yeast Saccharomyces cerevisiae. EMBO J 16, 1035–1044.[CrossRef][Medline]
Morgan, B. A., Bouquin, N., Merrill, G. F., Johnston, L. H. (1995). A yeast transcription factor bypassing the requirement for SBF and DSC1/MBF in budding yeast has homology to bacterial signal transduction proteins. EMBO J 14, 5679–5689.[Medline]
Motoyama, T., Ohira, T., Kadokura, K., Ichiishi, A., Fujimura, M., Yamaguchi, I., Kudo, T. (2005). An Os-1 family histidine kinase from a filamentous fungus confers fungicide-sensitivity to yeast. Curr. Genet 47, 298–306.[CrossRef][Medline]
Nakamichi, N., Yamada, H., Aoyama, K., Ohmiya, R., Aiba, H., Mizuno, T. (2002). His-to-Asp phosphorelay circuitry for regulation of sexual development in Schizosaccharomyces pombe. Biosci. Biotechnol. Biochem 66, 2663–2672.[CrossRef][Medline]
Ochiai, N., Fujimura, M., Motoyama, T., Ichiishi, A., Usami, R., Horikoshi, K., Yamaguchi, I. (2001). Characterization of mutations in the two-component histidine kinase gene that confer fludioxonil resistance and osmotic sensitivity in the os-1 mutants of Neurospora crassa. Pest. Manag. Sci 57, 437–442.[CrossRef][Medline]
Orbach, M. J., Sachs, M. S., Yanofsky, C. (1990). The Neurospora crassa arg-2 locus. Structure and expression of the gene encoding the small subunit of arginine-specific carbamoyl phosphate synthetase. J. Biol. Chem 265, 10981–10987.
Pall, M. L. (1993). The use of Ignite (Basta;glufosinate;phosphinothricin) to select transformants of bar-containing plasmids in Neurospora crassa. Fungal Gen. Newsl 40, 58.
Pall, M. L. and Brunelli, J. P. (1993). A series of six compact fungal transformation vectors containing polylinkers with multiple unique restriction sites. Fungal Gen. Newsl 40, 59–62.
Pandey, A., Roca, M. G., Read, N. D., Glass, N. L. (2004). Role of a mitogen-activated protein kinase pathway during conidial germination and hyphal fusion in Neurospora crassa. Eukaryot. Cell 3, 348–358.
Perkins, D. D., Radford, A., Newmeyer, D., Bjorkman, M. (1982). Chromosomal loci of Neurospora crassa. Microbiol. Rev 46, 426–570.
Perkins, D. D., Radford, A., Sachs, M. S. (2001). In: The Neurospora Compendium: Chromosonal Loci, San Diego, CA: Academic Press.
Posas, F. and Saito, H. (1997). Osmotic activation of the HOG MAPK pathway via Ste11p MAPKKK: scaffold role of Pbs2p MAPKK. Science 276, 1702–1705.
Posas, F., Wurgler-Murphy, S.M., Maeda, T., Witten, E. A., Thai, T. C., Saito, H. (1996). Yeast HOG1 MAP kinase cascade is regulated by a multistep phosphorelay mechanism in the SLN1-YPD1-SSK1 "two-component" osmosensor. Cell 86, 865–875.[CrossRef][Medline]
Raju, N. B. (1992). Genetic control of the sexual cycle in Neurospora. Mycol. Res 96, 241–262.[CrossRef]
Raymond, C. K., Pownder, T. A., Sexson, S. L. (1999). General method for plasmid construction using homologous recombination. Biotechniques 26, 140–141.
Sachs, M. S. and Yanofsky, C. (1991). Developmental expression of genes involved in conidiation and amino acid biosynthesis in Neurospora crassa. Dev. Biol 148, 117–128.[CrossRef][Medline]
Saito, H. and Tatebayashi, K. (2004). Regulation of the osmoregulatory HOG MAPK cascade in yeast. J. Biochem 136, 267–272.
Schumacher, M. M., Enderlin, C. S., Selitrennikoff, C. P. (1997). The osmotic-1 locus of Neurospora crassa encodes a putative histidine kinase similar to osmosensors of bacteria and yeast. Curr. Microbiol 34, 340–347.[CrossRef][Medline]
Selitrennikoff, C., Lilley, B., Zucker, R. (1981). Formation and regeneration of protoplasts derived from a temperature-sensitive osmotic strain of Neurospora crassa. Exp. Mycol 5, 155–161.[CrossRef]
Selitrennikoff, C. P., Alex, L., Miller, T. K., Clemons, K. V., Simon, M. I., Stevens, D. A. (2001). COS-l, a putative two-component histidine kinase of Candida albicans, is an in vivo virulence factor. Med. Mycol 39, 69–74.[Medline]
Shieh, J. C., Wilkinson, M. G., Buck, V., Morgan, B. A., Makino, K., Millar, J. B. (1997). The Mcs4 response regulator coordinately controls the stress-activated Wak1-Wis1-Sty1 MAP kinase pathway and fission yeast cell cycle. Genes Dev 11, 1008–1022.
Singh, P., Chauhan, N., Ghosh, A., Dixon, F., Calderone, R. (2004). SKN7 of Candida albicans: mutant construction and phenotype analysis. Infect. Immun 72, 2390–2394.
Springer, M. L. (1993). Genetic control of fungal differentiation: the three sporulation pathways of Neurospora crassa. Bioessays 15, 365–374.[CrossRef][Medline]
Staben, C., Jensen, B., Singer, M., Pollock, J., Schechtman, M., Kinsey, J., Selker, E. (1989). Use of a bacterial hygromycin B resistance gene as a dominant selectable marker in Neurospora crassa transformation. Fungal Genet. Newsl 36, 79–81.
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, 4673–4680.
Vann, D. C. (1995). Electroporation-based transformation of freshly harvested conidia of Neurospora crassa. Fungal Gen. Newsl 42A, 53.
Vogel, H. J. (1964). Distribution of lysine pathways among fungi: evolutionary implications. Am. Nat 98, 435–446.[CrossRef]
West, A. H. and Stock, A. M. (2001). Histidine kinases and response regulator proteins in two-component signaling systems. Trends Biochem. Sci 26, 369–376.[CrossRef][Medline]
Westergaard, M. and Mitchell, H. K. (1947). Neurospora V. A synthetic medium favoring sexual reproduction. Am. J. Bot 34, 573–577.[CrossRef]
Wolanin, P. M., Thomason, P. A., Stock, J. B. (2002). Histidine protein kinases: key signal transducers outside the animal kingdom. Genome Biol 3, REVIEWS3013.
Wormley, F. L. Jr, Heinrich, G., Miller, J. L., Perfect, J. R., Cox, G. M. (2005). Identification and characterization of an SKN7 homologue in Cryptococcus neoformans. Infect. Immun 73, 5022–5030.
Yoshimi, A., Kojima, K., Takano, Y., Tanaka, C. (2005). Group III histidine kinase is a positive regulator of Hog1-type mitogen-activated protein kinase in filamentous fungi. Eukaryot. Cell 4, 1820–1828.
Zhang, Y., Lamm, R., Pillonel, C., Lam, S., Xu, J.-R. (2002). Osmoregulation and fungicide resistance: the Neurospora crassa os-2 gene encodes a HOG1 mitogen-activated protein kinase homologue. Appl. Environ. Microbiol 68, 532–538.
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