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Genetics, Vol. 171, 81-89, September 2005, Copyright © 2005
doi:10.1534/genetics.105.042796
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-Subunit in Governing Growth and Development of Aspergillus nidulans
,2
* Department of Food Microbiology and Toxicology and Food Research Institute, Molecular and Environmental Toxicology Center, University of Wisconsin, Madison, Wisconsin 53706
2 Corresponding author: Department of Food Microbiology and Toxicology, 1925 Willow Dr., Madison, WI 53706-1187.
E-mail: jyu1{at}wisc.edu
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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), SfaD (Gß), and a presumed G
. Analysis of the A. nidulans genome identified a single gene named gpgA encoding a putative G-subunit. The predicted GpgA protein consists of 90 amino acids showing 72% similarity with yeast Ste18p. Deletion (
) of gpgA resulted in restricted vegetative growth and lowered asexual sporulation. Moreover, similar to the sfaD mutant, the gpgA mutant was unable to produce sexual fruiting bodies (cleistothecia) in self-fertilization and was severely impaired with cleistothecial development in outcross, indicating that both SfaD and GpgA are required for fruiting body formation. Developmental and morphological defects caused by deletion of flbA encoding an RGS protein negatively controlling FadA-mediated vegetative growth signaling were suppressed by gpgA, indicating that GpgA functions in FadA-SfaD-mediated vegetative growth signaling. However, deletion of gpgA could not bypass the need for the early developmental activator FluG in asexual sporulation, suggesting that GpgA functions in a separate signaling pathway. We propose that GpgA is the only A. nidulans G-subunit and is required for normal vegetative growth as well as proper asexual and sexual developmental progression.
, ß, and -subunits relay and propagate signals sensed by membrane-bound G-protein-coupled receptors (GPCRs) to a diverse group of regulatory proteins (effectors). Upon activation by agonists, GPCRs undergo conformational changes that promote the GDP-to-GTP exchange of the G-subunit. This exchange provokes the dissociation of GTP-G from the Gß heterodimer, and GTP-G, Gß, or both can mediate signals by modulating effectors. The signal is turned off when GTP is hydrolyzed to GDP, resulting in the formation of the inactive heterotrimer G-GDP:Gß. The rates of GTP hydrolysis by the intrinsic GTPase activity of the G-subunit determine the lifetime of the active G-proteins and thereby the intensity of the signal (reviewed in MORRIS and MALBON 1999).
A G-protein (FadA) in the filamentous fungus Aspergillus nidulans was first identified by studying a dominant activating mutation that caused undifferentiated hyphal growth followed by autolysis, i.e., a "fluffy autolytic" phenotype (YU et al. 1996). Genetic studies revealed that activated GTP-FadA (G) mediates signaling that promotes vegetative growth and inhibits both asexual and sexual development as well as production of the mycotoxin sterigmatocystin (ST; YU et al. 1996; HICKS et al. 1997). This FadA signaling is negatively controlled by a regulator of G-protein signaling (RGS) protein called FlbA, which is proposed to function by enhancing the intrinsic GTPase activity of FadA (YU et al. 1996). Loss of flbA function results in fluffy-autolytic phenotypes similar to those caused by constitutively active FadA mutant alleles (LEE and ADAMS 1994a; YU et al. 1996, 1999; WIESER et al. 1997). As if FadA is the primary target of FlbA function, the deletion () or dominant negative (G203R) FadA mutations suppressed the fluffy-autolytic phenotype caused by flbA and restored asexual development (conidiation) and ST production (YU et al. 1996; HICKS et al. 1997).
In addition to fadA, four other suppressor loci bypassing the requirement of flbA in conidiation have been previously isolated (YU et al. 1999). One of the suppressors, sfaD, was found to encode a Gß-subunit with the central conserved Trp-Asp sequence that is referred to as the "WD-40" motif (ROSÉN et al. 1999). SfaD is required for normal vegetative growth and proper downregulation of conidiation, as well as formation of sexual fruiting bodies (ROSÉN et al. 1999). The fact that sfaD suppressed the flbA-induced fluffy-autolytic phenotype led us to propose that SfaD (with the presumed G-subunit) functions in vegetative growth signaling that is negatively regulated by FlbA. However, elimination of FadA or SfaD could not bypass the need for fluG in conidiation, where FluG is proposed to trigger conidiation-specific events and to (indirectly) activate FlbA (LEE and ADAMS 1994a,b, 1996; YU et al. 1996). Taken together, we proposed that two antagonistic signaling pathways govern growth and asexual development of A. nidulans and that FlbA plays a pivotal role in fine-tuning the degree of FadA-SfaD-mediated vegetative growth signaling to allow both asexual and sexual development to occur.
As has been found for all eukaryotes (for review see MORRIS and MALBON 1999), it is presumed that the Gß-subunit (SfaD) functions as a heterodimer with the cognate G-subunit in A. nidulans. Recently, a putative G-subunit (GNG-1) was identified and shown to form the heterodimer with a Gß-subunit (GNB-1) in the filamentous fungus Neurospora crassa (KRYSTOFOVA and BORKOVICH 2005). This GNB-1::GNG-1 heterodimer is found to be necessary for normal female fertility, asexual development, and G-protein levels. In this study, we report the identification and characterization of a gene (gpgA) encoding a putative G-subunit in A. nidulans. Gene disruption, genetic, and expression studies indicate that GpgA is required for normal vegetative growth and developmental progression. We found that deletion of gpgA resulted in reduced vegetative growth and highly elevated formation of Hülle cells (sexual-development-specific cells) similar to that caused by sfaD. As if GpgA functions in the FadA-SfaD-mediated vegetative growth signaling pathway, deletion of gpgA suppressed the fluffy-autolytic phenotype resulting from flbA and restored conidiation at the wild-type level. No mutations were identified in the gpgA gene region of the three previously isolated, yet unidentified, flbA suppressors (sfaA, sfaC, and sfaE; YU et al. 1999), indicating that gpgA defines the sixth flbA-suppressor locus. As with sfaD, deletion of gpgA caused severely impaired sexual fruiting body formation. We present a model for the roles of GpgA in controlling vegetative growth and development.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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106 conidia of relevant strains were spread onto MM and the plates were sealed with plastic films and incubated at 37° for 7–10 days in the dark; (2) conidia of appropriate strains were point inoculated at the center of solid MM and incubated at 37° for 2–3 days and then the plates were sealed and further incubated at 37° for 7 days in the dark; and (3) 5 x 105 conidia/ml were inoculated into 100 ml liquid MM in 250-ml flasks and incubated at 37°, 250 rpm for 18 hr. Mycelia were then collected by filtering through Miracloth (Calbiochem) and transferred to solid MM. The plates were sealed and incubated at 37° for 7 days in the dark. For Northern blot analyses, samples from vegetative growth and postdevelopmental induction cultures were collected as described (HAN et al. 2004a). Briefly, 5 x 107 conidia of relevant strains were inoculated in 100 ml liquid MM with 0.1% yeast extract in 250-ml flasks and incubated at 37°, 250 rpm. For vegetative growth phases, samples were collected at designated time points of liquid submerged cultures, squeeze dried, and stored at –80° until subjected to total RNA isolation. For sexual and asexual developmental induction, 18-hr vegetatively grown mycelia were transferred to solid MM and the plates were either air exposed for asexual developmental induction or tightly sealed and blocked from light for sexual developmental induction.
Construction of the gpgA deletion mutant:
The gpgA deletion cassette was constructed via multiple cloning processes because of the incomplete development of a PCR-assisted technique at that time (YU et al. 2004). The 5' (590 bp) and 3' (597 bp) flanking regions of the gpgA open reading frame (ORF) were amplified with primer pairs of OJA22-OJA27 and OJA23-OJA28, respectively, and the amplicons were digested with XhoI. These restricted 5' and 3' flanking DNA fragments were ligated to give rise to a 1.2-kb joined fragment (gpgA), which was cloned into the pGEM-T Easy vector (Promega, Madison, WI). The argB+ marker (1.8 kb) was generated by cutting the argB+ plasmid pJW88 (J. K. WIESER and T. H. ADAMS, unpublished data) with XhoI and ligated with the XhoI-cut gpgA/pGEM-T Easy plasmid. The final gpgA construct (pJAG2) was composed of a 5' (590-bp) flanking region, argB+ (1.8 kb), and a 3' flanking region (597 bp) in the pGEM-T Easy vector. This pJAG2 plasmid was directly introduced to A. nidulans RMS011 to generate TJAG2.7 (Table 1). The gpgA genotype was confirmed by PCR amplification of the gpgA coding region with a primer pair of OJA26 and OJA29 followed by restriction enzyme digestion of the amplicons and Southern blot analysis (see YU et al. 2004). Phenotypic alterations caused by gpgA were 100% linked with the gpgA PCR amplicon size and digestion patterns.
Nucleic acid manipulation:
Genomic DNA or total RNA isolation and Northern blot analyses were carried out as previously described (SEO et al. 2003; HAN et al. 2004a). The DNA probes used to examine mRNA levels of gpgA were prepared by PCR amplification of the coding region of gpgA, using genomic DNA of FGSC4 as a template with OJA24 and OJA25 (Table 2). Genotypes of double-deletion mutants of gpgA, fadA, sfaD, flbA, and fluG were confirmed by PCR amplification of the coding region of individual genes. For instance, the primer pair OJA24 and OJA25 were used to differentiate the deletion or wild-type gpgA alleles present in the progeny of crosses and the amplicons of gpgA and gpgA+ were 2.0 kb and 0.6 kb, respectively. The PCR products from progeny analyses were confirmed by restriction enzyme digestion of the amplicons.
Microscopy:
Colonies were photographed with a SONY digital camera (DSC-F707). Photomicrographs were taken using an Olympus BH2 compound microscope installed with a Kodak MDS290 digital camera.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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-subunit:-subunit Ste18p (WHITEWAY et al. 1989) identified a single gene showing 72% similarity with Ste18p. We designated this gene as gpgA for a G-protein gamma subunit (mapped on chromosome VI; the Broad Institute). Direct sequencing of a reverse transcription PCR (RT-PCR) amplicon of gpgA revealed that it encodes an ORF of 391 bp with two short introns (68 and 48 bp) and the predicted GpgA protein consists of 90 amino acids (Figure 1A). It should be noted that GpgA is slightly different from the hypothetical protein AN2742.2 (XP_406879.1) annotated by the Broad Institute in that AN2742.2 is 95 aa long and its stop codon is located at 51 bp downstream of the GpgA stop and the ORF is intervened by three introns (68, 48, and 36 bp).
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20–40 aa (http://www.ch.embnet.org/software/COILS_form.html). Furthermore, GpgA has the conserved Ste18p-like Cys-A-A-X box, where A stands for any aliphatic amino acids. As with other fungal G-subunits, the CTIM aa sequence at the C-terminal end of GpgA indicates that it is likely to be farnesylated (SINENSKY 2000). Northern blot analysis demonstrated that 1.7-kb gpgA mRNA levels are constantly high throughout the life cycle of A. nidulans (Figure 1B). Besides Ste18p, GpgA shows high levels of similarity with a putative G-protein of N. crassa (GNG-1; 93 aa; 75% similarity) and with Gibberella zeae (XP_387411.1; 93 aa; 75% similarity; Figure 1C). Perhaps not surprisingly, GpgA shows 99% identity and 100% similarity with the corresponding G-subunit of A. fumigatus [The Institute for Genomic Research (TIGR): http://www.tigr.org/tdb/e2k1/afu1/].
GpgA is required for normal conidiation:
To characterize functions of this putative G-subunit, we generated the gpgA mutant by replacing its ORF with the argB+ marker. The gpgA mutant (multiple strains tested: Table 1, RJAG19.6, -19.8, and -19.9) exhibited not only reduced vegetative growth (Figures 2 and 3, see below) but also delayed-conidiation phenotypes. The gpgA mutant displayed a nonconidial fluffy phenotype for 2–3 days of growth (see Figure 2) before the production of conidiophores from the center of the colony, resulting in a slightly reduced number of conidia (per colony) in the gpgA mutant (Figure 3). On the contrary, levels of Hülle cell production in the gpgA mutant were much higher (2.26 x 106/ml) than those in wild type (WT) or other strains (Figures 2 and 3).
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sfaD and gpgAsfaD mutants produced conidiophores within 22 hr of liquid submerged culture, neither the gpgA mutant nor WT produced conidiophores (Figure 4). Taken together, these results suggest that SfaD (Gß) plays a crucial role in negative regulation of conidiation under submerged culture conditions (ROSÉN et al. 1999; HAN et al. 2004b) and GpgA may not be involved in this controlling process (see DISCUSSION).
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gpgA mutant. Despite elevated Hülle cell formation, the gpgA mutant was unable to produce cleistothecia under self-fertilizing (homothallic) conditions. These phenotypes are almost identical to those caused by sfaD (ROSÉN et al. 1999). Furthermore, deletion of sfaD or gpgA, but not fadA, resulted in severely impaired cleistothecia formation in outcrosses with WT or other mutants. This trait made genetic analyses of gpgA (and sfaD) extremely difficult. In most cases, while heterokaryotic mycelia could be formed between two strains, no cleistothecia were developed. Due to this (semi-) dominant nature of gpgA or sfaD, the gpgAsfaD, gpgAfadA, and gpgAflbA mutants were generated by repeated (approximately five to eight times) meiotic crosses of various (8–22 different) combinations of multiple gpgA, sfaD, and flbAfadA mutant strains (only representative strains are shown in Table 1). From these crosses only a few cleistothecia were produced and isolated. Collectively, as with SfaD (and possibly FadA), GpgA is required for normal sexual fruiting body formation.
GpgA is required for normal vegetative growth:
Our hypothesis was that a cognate G-subunit forms the heterodimer with SfaD and functions in vegetative growth signaling. The role of GpgA in vegetative growth signaling was examined by determining growth rates of the gpgA (RJAG19.9), sfaD (RSRB1.15), fadA (RJA71.4), and gpgAsfaD (RJA55.4) mutants (see Table 1). Although gpgA did not clearly affect radial growth rates on solid medium, gpgA and gpgAsfaD caused significantly reduced vegetative growth in liquid submerged culture, where hyphal branching and extension were reduced. Again, these phenotypes were almost identical to those caused by sfaD. Dry weights of the gpgA (RJAG19.9) and gpgAsfaD (RJA55.4) mutants grown in liquid MM were only 34 and 27% of that of WT, respectively (Figure 3). These are comparable with dry weights of the fadA and sfaD mutants grown in liquid MM, i.e., 10–35% of that of WT. Collectively, these results suggest that GpgA likely functions in FadA/SfaD-mediated vegetative growth signaling.
Deletion of gpgA bypasses the need for flbA in conidiation:
To further provide genetic evidence of the involvement of GpgA in FadA/SfaD-mediated vegetative growth signaling, we generated the gpgAflbA double mutant. It should be noted that deletion of fadA and/or sfaD could suppress the fluffy-autolytic phenotype caused by flbA (YU et al. 1996; ROSÉN et al. 1999). Theoretically, if GpgA is the G-subunit forming a heterodimer with SfaD and if SfaD-GpgA interaction is required for vegetative growth signaling, then the absence of GpgA function should suppress uncontrolled activation of vegetative growth caused by flbA. We found that, as with fadA or sfaD, deletion of gpgA suppressed fluffy-autolytic and developmental defect phenotypes of the flbA mutant in that the gpgAflbA mutant recovered conidiation at the WT level (Figure 5). These results indicate that GpgA functions in vegetative growth signaling controlled by FlbA.
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sfaA–sfaE) were isolated (YU et al. 1999). Among these, sfaB and sfaD defined FadA and a Gß-subunit, respectively (ROSÉN et al. 1999; YU et al. 1999). The fact that deletion of gpgA eliminated the need for flbA in conidiation led us to test whether gpgA could identify one of the sfaA1, sfaC67, or sfaE83 mutations. A region of the gpgA gene including its promoter (1 kb upstream of the ATG), ORF, and terminator (0.5 kb) was PCR amplified using the individual suppressor mutant genomic DNA as template and the resulting amplicons were directly sequenced. No mutations were identified in those amplicons, suggesting that the gpgA gene defines the sixth suppressor of flbA loss-of-function. This result confirms the prediction that the previous flbA suppressor screenings did not reach saturation (YU et al. 1999). The small size of the gpgA ORF (391 bp) probably reduced the probability of introducing mutation(s) in the gpgA region via random chemical mutagenesis carried out in the previous study.
FluG is required for conidiation in the absence of gpgA:
FluG is an early developmental regulator that is required for activation of downstream conidiation-specific events (reviewed in ADAMS et al. 1998). Previously, we showed that, while the deletion or dominant interfering mutation (G203R) of fadA mutation or sfaD restored conidiation in the flbA mutant, these mutations could not eliminate the need for FluG in conidiation (YU et al. 1996; ROSÉN et al. 1999). To test the requirement of FluG in conidiation in the absence of GpgA functions, we constructed the gpgAfluG mutant and found that deletion of gpgA could not suppress conidiation defects caused by fluG (Figure 6), supporting that the FadA/SfaD/GpgA vegetative growth signaling pathway and the asexual developmental cascade activated by FluG are separate and independent (see model in Figure 7). Moreover, the facts that fluG eliminated Hülle cell production in the sfaD and gpgA mutants indicate that enhanced vegetative growth by fluG might be sufficient to block inappropriate Hülle cell production caused by the absence of Gß functions.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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-subunit in A. nidulans. Although the G-subunits have been known to be more diverse than the Gß-subunits (MORRIS and MALBON 1999), GpgA, Ste18p, and other putative fungal G-subunits including recently reported GNG-1 in N. crassa (KRYSTOFOVA and BORKOVICH 2005) share high levels of similarity (Figure 1C). Furthermore, GpgA contains a coiled-coil domain at the N-terminal region, which is shown to be necessary for the interaction of a G with the cognate Gß to form a heterodimer (for review see CABRERA-VERA et al. 2003). After the thorough analyses of the A. nidulans genome, we have tentatively concluded that only one each of Gß- and G-subunit exists in A. nidulans. No genes encoding the products similar to the yeast Gpb1/Gpb2 proteins (HARASHIMA and HEITMAN 2002) have been identified.
One of the important findings of this study is that elimination of gpgA function could bypass the requirement of flbA, but not fluG, in conidiation. These results support the idea that GpgA functions in the vegetative growth signaling pathway that is independent and parallel to the FluG-activated asexual sporulation branch (see Figure 7; YU et al. 1996; ROSÉN et al. 1999). The facts that mutational inactivation of fadA, sfaD, or gpgA all resulted in reduced vegetative growth as well as suppression of fluffy-autolytic phenotypes caused by flbA strongly support the hypothesis that FadA, SfaD, and GpgA constitute a functional heterotrimeric unit, of which the primary role is to mediate vegetative growth signaling (Figure 7). However, as shown in the previous study, constitutive activation of FadA alone in the absence of SfaD function was sufficient to cause proliferation of undifferentiated hyphae (ROSÉN et al. 1999). This indicates that FadA might be the primary component responsible for vegetative proliferation.
If constitutively active FadA alone is sufficient to confer uncontrolled activation of vegetative signaling, how can deletion of sfaD or gpgA suppress flbA? This question might be answered by understanding the proposed roles of G- or Gß-subunits in G-protein signaling. In general, G-protein -subunits are found to play the following roles: (1) transducing signals by forming a heterodimer with Gß (WHITEWAY et al. 1989; GARRITSEN et al. 1993), (2) promoting G-protein activation by binding and modulating efficiency of receptor-G-protein coupling (LAMBRIGHT et al. 1994; YASUDA et al. 1996; RONDARD et al. 2001; CHINAULT and BLUMER 2003), (3) granting selective and discrete coupling of GPCRs with Gß (KISSELEV et al. 1995, 1999), and (4) promoting activation of the Gß-effectors that cocluster with receptors (CHINAULT and BLUMER 2003). Similarly, Gß-subunits were found to play an important role in providing a GPCR binding site for the G protein, which is critical for G-protein activation (TAYLOR et al. 1996; HAMM 1998; KISSELEV et al. 1999; CHINAULT and BLUMER 2003). It can be speculated that GpgA and SfaD may be necessary for the proper coupling of GPCR and G-protein, thereby activating the G protein. If this were the case, sfaD would suppress flbA but not the constitutively active fadA alleles, e.g., G42R, Q204L, or R178C (ROSÉN et al. 1999; YU et al. 1999), because dominant-activating FadA mutant alleles are locked in the GTP-bound (active) form and the interaction of the FadA-SfaD-GpgA heterotrimer with GPCR may not be needed to reactivate FadA.
Previous studies showed that, in addition to their role as a heterodimer, individual Gß- or G-subunits might play distinct roles in activation of G-proteins and/or downstream effectors (LANDRY and HOFFMAN 2001). In this study, we found that deletion of sfaD or gpgA resulted in certain dissimilar phenotypes. For instance, unlike sfaD, deletion of gpgA resulted in fluffy-reduced conidiation phenotypes during the early phase of growth and did not cause conidiophore formation in liquid submerged culture. However, the sfaDgpgA double mutant exhibited phenotypes that are identical to those of the sfaD mutant; i.e., sfaD is epistatic to gpgA. These results suggest that, even in the absence of GpgA, SfaD may be able to (partially) activate downstream effectors that directly/indirectly control inappropriate conidiation in liquid submerged culture. Taken together, hyperactive conidiation by fadAG203R (dominant negative allele), sfaD, and the absence of GanB (another G) functions suggest that GanB and SfaD play a role in negative regulation of conidiation under submerged culture conditions (ROSÉN et al. 1999; CHANG et al. 2004; HAN et al. 2004b).
Sexual development in A. nidulans involves the development of two distinct structures: Hülle cells and cleistothecia (reviewed in CHAMPE et al. 1994; BRAUS et al. 2002). It is important to note that, although Hülle cells are associated with the sexual reproductive cycle, production of Hülle cells and development of cleistothecia are distinct processes. One striking common phenotype of the fadA, sfaD, or gpgA deletion mutant is the lack (or defect) of cleistothecia formation in self-fertilization (homothallic conditions) but highly elevated production of Hülle cells (ROSÉN et al. 1999). However, the requirement of these genes in cleistothecia development in outcrosses (heterothallic conditions) is different in that deletion of sfaD or gpgA (but not fadA) resulted in severe impairment in cleistothecia development (but not heterokaryon formation) in outcrosses in a somewhat dominant manner. While sexual development is a highly delicate process, which requires a number of genes and fulfillment of various factors, thus far, no A. nidulans genes have been found to specifically affect cleistothecia development in outcrosses without altering the ability to form heterokaryons. The requirement of SfaD and GpgA in normal fruiting body formation in both self-fertilization and outcrosses suggests that, similar to yeast mating, the SfaD-GpgA heterodimer may play a vital role in relaying the signals for fertilization. This is somewhat consistent with the findings that the Gß-subunit is necessary for sexual development and the pheromone-response mating systems in N. crassa (YANG et al. 2002) and Ustilago maydis (MÜLLER et al. 2004). In addition, a recent study showed that GNG-1 (G) is necessary for normal female fertility in N. crassa (KRYSTOFOVA and BORKOVICH 2005). Deletion of ganA or ganB encoding additional G-subunits in A. nidulans is found to cause no effects in sexual development (CHANG et al. 2004; K.-Y. JAHNG, personal communication).
In our previous studies, we identified nine putative GPCRs in A. nidulans and showed that GprA and GprB are required for cleistothecia formation in self-fertilization, but not in outcrosses (HAN et al. 2004a; SEO et al. 2004). In these studies, we also demonstrated that GprA and GprB are not the GPCRs for FadA-mediated vegetative growth signaling. If SfaD-GpgA and FadA function in both vegetative growth and cleistothecia development signaling, how can the fungal cells determine their fate? This can be explained by differential expression of multiple GPCRs and variation of coclustering effectors. We found that, while mRNA levels of all G-protein subunits are relatively constant throughout the life cycle, those of GPCRs vary (Figure 1, our unpublished data; HAN et al. 2004a; SEO et al. 2004). Particularly, gprA and gprB were specifically expressed at 48 hr postsexual developmental induction (SEO et al. 2004). If the expression and activities of GPCRs are tightly controlled by growth and developmental phases, and if distinct effectors cocluster with a specific GPCR, then discriminated activation of separate signaling cascades by the same G-protein can be achieved. For instance, during the vegetative growth phase certain (yet unidentified) GPCR(s) may be abundantly expressed and sensitized that can activate FadA-SfaD::GpgA-mediated hyphal growth signaling that is, in part, amplified by PKA (SHIMIZU and KELLER 2001). When environmental and internal conditions are met, GprA and GprB are expressed, and sensitization of these GPCRs would exert activation of the FadA-SfaD::GpgA heterotrimer and the subsequent signaling cascade for cleistothecia development. This branch may be composed of SteC (MAPKKK) and SteA (a Ste12 homolog) in A. nidulans (VALLIM et al. 2000; WEI et al. 2003). Further genetic and biochemical studies must be carried out to understand the molecular mechanisms underlying signal transduction from GPCRs to G-proteins to downstream effectors that selectively determine cellular responses.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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FOOTNOTES |
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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ADAMS, T. H., J. K. WIESER and J.-H. YU, 1998 Asexual sporulation in Aspergillus nidulans. Microbiol. Mol. Biol. Rev. 12 : 3827–3833.
BRAUS, G. H., S. KRAPPMANN and S. E. ECKERT, 2002 Sexual development in Ascomycetes fruit body formation of Aspergillus nidulans, pp. 215–244 in Molecular Biology of Fungal Development, edited by H. D. OSIEWACZ. Marcel Dekker, New York.
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