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Department of Plant Pathology, University of California, 1415 Boyce Hall, 900 University Avenue, Riverside, California 92521
Received 28 April 2006/ Accepted 30 June 2006
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ABSTRACT |
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gpr-1 strains are weakly pigmented, small, and
submerged in the agar. The perithecia produced after fertilization have
deformed beaks that lack ostioles, the openings through which
ascospores are discharged. Localization studies using a GPR-1-green
fluorescent protein fusion protein showed that GPR-1 is targeted to
female reproductive structures. Genetic epistasis experiments with the
three G
genes were inconclusive due to the early block in
mating exhibited by gna-1 strains. Phenotypic
analysis of mutants from a high-throughput N. crassa knockout
project allowed identification of BEK-1, a homeodomain transcription
factor that is a potential target of GPR-1. The perithecial defects of
bek-1 strains are similar to those of the
gpr-1 strain, and epistasis analysis indicates that
bek-1 could function downstream of gpr-1 during
postfertilization events. The effect must be posttranscriptional, as
bek-1 transcript levels are not affected in
gpr-1 strains. The lack of ostioles in
gpr-1 and bek-1 mutants has an
undesirable effect on the ability to spread progeny (ascospores) by the
normal ejection mechanism and would severely compromise the fitness of
these strains in
nature. |
INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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G-protein-coupled receptors (GPCRs) are seven-transmembrane helix proteins that are the sensory components of heterotrimeric G protein signaling pathways. GPCRs are crucial for the swift response to extracellular stimuli, including light, odors, hormones, neurotransmitters, and other signals (48, 53). In fungi, heterotrimeric G proteins mediate responses to environmental agents (including nutrients and pheromones) to regulate growth, colony formation, cell-cell recognition and fusion, chemotropism, mating, and cell differentiation. The early stage of mating (pheromone attraction and fusion) in N. crassa is regulated by pheromone receptors localized in the plasma membrane of trichogynes that sense pheromones secreted from male cells of opposite mating type (44, 45; Kim and Borkovich, unpublished). N. crassa has two pheromone receptors, PRE-1, expressed in mat A cells and PRE-2, produced by mat a strains (44, 45). PRE-1 and PRE-2 share homology with the Saccharomyces cerevisiae pheromone receptors Ste2p and Ste3p (10, 31). Pheromone receptor GPCRs are well conserved and have been reported in other fungi, including Schizosaccharomyces pombe (66), Aspergillus nidulans (65), Ustilago maydis (11), Cryptococcus neoformans (18, 21), Schizophyllum commune (72), and Coprinus cinereus (54, 56). Besides the pheromone receptors, few other groups of GPCRs have been studied in detail in fungi. Carbon source and amino acid sensing receptors have been identified and characterized in S. cerevisiae (75, 78, 79), Candida albicans (49, 50, 52), S. pombe (71), C. neoformans (74), and N. crassa (48a). In the homothalic fungus A. nidulans, a GPCR (GprD) was found to function as a negative regulator of fruiting body development (32).
Ten putative
GPCRs, three G protein subunits (GNA-1, GNA-2, and GNA-3),
one Gß subunit (GNB-1), and one G
subunit (GNG-1) have
been identified in N. crassa
(12,
28,
43,
47,
68,
77). Loss of
gna-1 leads to trichogynes that grow randomly and are blind to
males, causing female (not male) sterility in both mating types
(39,
44). These observations
indicate that GNA-1 is involved in the pheromone response pathway and
is presumably coupled to the pheromone receptors. In contrast to
pre-1 mutants which form normal protoperithecia,
gna-1 strains produce fewer protoperithecia, which
have abundant fringe hyphae
(39). This shows that
GNA-1 is also required for normal protoperithecial development. The
observation that protoperithecia are normal in pre-1
mutants suggests that regulation of protoperithecial formation by GNA-1
occurs through coupling to a nonpheromone receptor GPCR. Deletion of
gna-2 or gna-3 does not result in female sterility
(4,
43,
44), and the female
infertility of gnb-1 and gng-1
strains very likely results from reduced protein levels of all three
G subunits (47,
77).
In this study,
we report isolation and characterization of a putative GPCR, GPR-1, in
N. crassa. GPR-1 is a member of a GPCR family consisting of
three closely related receptors, GPR-1, GPR-2, and GPR-3
(12,
28). Deletion of
gpr-1 leads to pleiotropic phenotypic defects during sexual
development. gpr-1 mutants form small, pale
protoperithecia, and perithecia are frequently ruptured, lack ostioles
(pores), and have defective beaks. Epistatic analysis between
gpr-1 and the three G genes indicates that GNA-1 may
function downstream of GPR-1. Using a GPR-1-green fluorescent protein
(GFP) fusion construct, we demonstrate that GPR-1 is localized in
protoperithecia. In addition, we show that a homeodomain transcription
factor, BEK-1, may function downstream of GPR-1 during perithecial
development.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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gpr-1 isolation and gene structure confirmation.
The N. crassa gpr-1 gene was
identified during homology searches (BLAST
[1]) of the Munich
Information Center for Protein Sequences (MIPS) Neurospora
database
(http://mips.gsf.de/projects/fungi/neurospora),
using the Dictyostelium discoideum cAR1 protein sequence, and
corresponds to hypothetical gene 90c4_060. The predicted GPR-1
protein sequence is designated NCU00786.2 at the Broad Institute
Neurospora genome database
(http://www.broad.mit.edu/annotation/fungi/neurospora_crassa_7/index.html).
A gpr-1 genomic clone was isolated from the pMOcosX cosmid
library (55) using PCR
amplification with specific primers (GPR-1-FW and GPR-1-RV) as the
screening procedure. One positive clone (G11B20) was obtained and shown
to contain the gpr-1 gene by sequence analysis (Core
Sequencing Facility, Department of Microbiology and Molecular Genetics,
University of Texas-Houston Medical School). Plasmids were
maintained in Escherichia coli strain DH5
(33).
Construction of N. crassa strains. The gpr-1 gene was mutated by gene replacement using the selectable marker bar. The gpr-1 gene replacement vector, pSVK21, was constructed as follows. Cosmid G11B20 was digested with XbaI, and a 9-kb fragment containing the gpr-1 open reading frame (ORF) was cloned into pBSSKII+ (Stratagene, La Jolla, CA), generating pSVK19. pSVK19 was subsequently digested with NcoI and SacI, leaving the 3-kb 3' flank and removing the gpr-1 ORF and 5' region. pSVK20 contains the 1.1-kb bar gene (58) under the control of the A. nidulans trpC promoter. pSVK20 was constructed by digestion of pBARGEM-7 (58) using XbaI and SpeI to release the bar gene cassette. The ends of the bar cassette fragment were filled in using DNA polymerase I (Klenow; Promega, Madison, WI) and the blunt-end fragment was subsequently cloned into pGEM5zf digested with EcoRV to yield pSVK20. The bar gene cassette was excised from pSVK20 using NcoI and SacI and a 1.5-kb 5' region of gpr-1 from pSVK19 using SacI. The bar gene cassette and 1.5-kb 5' flank of gpr-1 were ligated into pSVK19 digested with NcoI and SacI, yielding pSVK21. The correct orientation of the gpr-1 5' region was verified by sequencing.
Conidia from 10-day-old cultures of N. crassa wild-type strain 74a (Table 1) were electroporated with 1 µg of pSVK21 linearized with EcoRI, as described previously (39, 69). Transformants were selected on FIGS medium supplemented with phosphinothricin. To identify homologous and ectopic integrations, genomic DNA was isolated from transformants, digested with KpnI, and subjected to Southern analysis (39). Genomic DNA was extracted using a Puregene kit as described by the manufacturer (Gentra Systems, Minneapolis, MN) (47). The 1.5-kb 5' SacI-XbaI fragment from pSVK19 was labeled using the Prime-A-Gene method (Promega, Madison, WI) and used as a probe. The wild-type gpr-1 allele produced a 6-kb hybridizing fragment during Southern analysis, while a 2.7-kb fragment was detected only in strains with homologous insertion of the barR cassette at the gpr-1 locus (data not shown). Heterokaryotic strains with homologous recombination events were crossed to the wild-type strain 74A (Table 1). The progeny were selected on FIGS medium with phosphinothricin, and homokaryotic status of strains was confirmed by Southern analysis as described above.
To complement the gpr-1 mutation in
trans, the gpr-1 genomic clone was inserted into
vector pRAUW122 and targeted to the his-3 locus
(3). The rescue plasmid
pSVK22 was generated by cloning of the 9-kb XbaI fragment from pSVK19
into pRAUW122 digested with XbaI. In order to isolate a
gpr-1 his-3 recipient strain, a
gpr-1 mat A strain (28-6) was
crossed to a his-3 mat a strain (his-3a),
and progeny were plated on FIGS medium supplemented with histidine and
phosphinothricin. The progeny were tested for
his-3 prototrophy using VM medium without histidine. The
gpr-1 his-3 strain (28-6-1) was transformed
by electroporation with 1 µg of pSVK22, and transformants were
plated on histidine-free FIGS medium. Genomic DNA from heterokaryons
was digested with HindIII, and strains containing the wild-type
gpr-1 allele integrated at the his-3 locus were
identified by Southern analysis using the HindIII 8.8-kb his-3
fragment excised from pRAUW122 as a probe (data not shown).
Homokaryotic
gpr-1::bar+
gpr-1+::his-3+
rescued strains were isolated using the microconidiation procedure
(25).
Vector pMF280
contains the N. crassa histone H1 protein as a
carboxy-terminal GFP fusion, under the control of the ccg-1
promoter (27). Strain
N2276 contains this vector targeted to the his-3 locus in an
otherwise wild-type background
(27). pMF280 was also
electroporated into the gpr-1 his-3 strain 28-6-1
(Table 1). Transformants
were subjected to Southern analysis using the 8.8-kb HindIII fragment
from pRAUW122 as a probe (see above) (data not shown), and strains with
the vector integrated at the his-3 locus were purified using
the microconidiation procedure
(25).
The
gpr-1 his-3 strain 28-6-1 and his-3
strains were used as recipients to produce strains expressing a
GPR-1-GFP fusion protein. The pSVK52 gpr-1-GFP vector was
constructed as follows. A 2,388-bp PCR product was amplified from
pSVK19 using Turbo Pfu DNA polymerase (Stratagene) and
Gpr1gfp-Xba-FW and Gpr1gfp-Bgl-RV oligomers designed with XbaI
(5' end) and BglII (3' end) restriction sites. The
primary blunt-end PCR product was cloned into pGEM5zf digested with
EcoRV (Promega) yielding pSVK51. The 2,338-bp gpr-1 fragment
was subsequently released from pSVK51 with XbaI and BglII and cloned
into his-3 targeting vector pMF272
(27) (described above)
digested with XbaI and BamHI. Ten-day-old conidia from
gpr-1 his-3 strain 28-6-1 were transformed
by electroporation with 1 µg of pSVK52, and transformants
were plated on FIGS medium. Strains with homologous insertion
of the his-3::gpr-1-GFP fragment
were identified by Southern analysis of genomic DNA using the 8.8-kb
HindIII fragment from pRAUW122 as a probe (data not shown). Homokaryons
were purified using the microconidiation technique
(25).
Western analysis. For Western analysis, plasma membrane fractions from various tissues (see above) were isolated as described previously (68). The protein concentration was determined using the Bradford protein assay (Bio-Rad). Samples containing 25 µg of total protein were denatured and solubilized in sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis sample buffer (62.5 mM Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, 1% ß-mercaptoethanol, 0.005% bromophenol blue) by boiling for 5 min. To detect GNA-1, GNA-2, GNA-3, and GNB-1, protein samples were resolved using 10% SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (39, 68). The primary polyclonal rabbit antibodies against GNA-1, GNA-2, GNA-3, and GNB-1 were used at dilutions of 1:1,000, 1:3,000, 1:1,000, and 1:3,000, respectively (4, 39, 43, 77). A horseradish peroxidase conjugate (Bio-Rad) was used as the secondary antibody at a dilution of 1:10,000. The blocking solution contained 5% nonfat dry milk, 25 mM Tris-Cl (pH 7.6), 140 mM NaCl, 3 mM KCl, and 0.2% Tween-20. The membranes were incubated with the primary antibody in the blocking solution for 3 h at room temperature and for 1 h with the secondary antibody. The membranes were washed four times for 5 min each time between incubations with the primary and secondary antibodies and before the application of chemiluminescence detection reagents. Detection was performed using a Biochemi system (UVP, Upland, CA) with chemiluminescence detection reagents used according to the manufacturer's protocol (Pierce, Rockford, IL).
RT-PCR analysis. In order to investigate the expression profile of gpr-1, total RNA was extracted from various tissues (5), and 1 µg was used in quantitative reverse transcriptase PCR (RT-PCR) (43) using the Access RT-PCR System (Promega). RT-PCR products were electrophoresed on 1.5% agarose gels, blotted onto nylon membranes, and subjected to Southern analysis as described above. The 2.4-kb gpr-1 fragment excised from pSVK51 (XbaI-BglII) was used as the probe. The tissues used were conidia (harvested from 7-day-old flask cultures), 8- and 16-h submerged cultures, and cultures from 3-day-old VM medium plates and 6-day-old SCM plates prior to (protoperithecia) and 3 days after fertilization with opposite mating type conidia (perithecia).
Four sets of primers (Table 2) were designed to confirm the gpr-1 gene structure predicted by the Broad Institute automated gene caller using RT-PCR (Access RT-PCR; Promega). Total RNA was extracted from 6-day-old SCM plate cultures as previously described (5), and 1 µg was used in RT-PCRs (43). Genomic intron-containing DNA controls were amplified by PCR using LA Taq (Takara) with cosmid G11B20 as a template. The 2.4-kb gpr-1 fragment excised from pSVK51 (XbaI-BglII) was used as a probe.
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For trichogyne pheromone attraction assays (7, 8, 44), cultures were grown for 6 days on 2% water agar. Chemoattraction between trichogynes and microconidia from wild-type strains was observed using a BX41 fluorescent microscope with UM Plan Fluorite objective lenses as described (44).
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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GPCRs homologous
to GPR-1 are absent from the genomes of the ascomycete yeasts S.
cerevisiae and S. pombe
(28). Among ascomycete
filamentous fungi whose genome sequences are available, only
Fusarium graminearum has a GPCR gene family similar to GPR-1,
-2, and -3 and contains four members (24 to 36% identical to GPR-1)
(Fig. 1). In contrast,
Magnaporthe grisea and A. nidulans have only
a single GPCR similar to GPR-1 (28% and 25% identical, respectively).
The ClustalW alignment
(20) (Fig.
1) shows that these
proteins are similar in all seven transmembrane helices as well as in
the first and second extracellular loops that are important for ligand
binding. The proteins are less homologous in the third intracellular
loop and the carboxy-terminal tail that are required for the physical
interaction with G subunits. A GPCR with 27% identity to GPR-1
has been described in the basidiomycete yeast C.
neoformans (Gpr4)
(74) and is implicated in
sensing the amino acids proline and methionine.
The predicted gene structure of gpr-1 contains five exons and four introns (Fig. 2B). The presence of the introns was confirmed by RT-PCRs using four sets of oligonucleotides (Table 2) designed within the exons that flank each intron. Total RNA was used as the template in RT-PCR, whereas the genomic cosmid clone G11B20 was used for amplification of genomic fragment controls using the same sets of primers. The RT-PCR and genomic DNA PCR products were subjected to Southern analysis. The analysis confirmed the presence and sizes of the four introns as predicted by the automated gene caller (Fig. 2B) (data not shown). The A. nidulans gprH gene (Fig. 1) also contains four predicted introns (http://www.broad.mit.edu/annotation/fungi/aspergillus). In contrast, other GPR-1-related GPCRs have two predicted introns in their ORFs at very similar or identical positions as GPR-1. In particular, the position of the first intron is highly conserved in all GPR-1-related GPCRs and is located at the junction of the first extracellular loop and the third transmembrane helix (Fig. 1).
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gpr-1 strains exhibit phenotypic defects during sexual development.
A gpr-1 mutant was
isolated after electroporation of a wild-type strain with a construct
in which the gpr-1 coding region was replaced with a
phosphinothricin resistance gene cassette (Fig.
2B)
(58). Heterokaryotic
primary transformants were obtained by selection on medium containing
phosphinothricin (see Materials and Methods for details). Strains with
the proper homologous recombination event were crossed to a wild-type
strain of opposite mating type in order to produce homokaryotic
gpr-1 mutant ascospore progeny (data not shown). The
purity of homokaryons was verified by Southern analysis (data not
shown), and RT-PCR analysis confirmed that homokaryotic
gpr-1 strains lack gpr-1 mRNA (Fig.
2C). The
gpr-1 mutation was complemented in trans
with the original 9-kb gpr-1 genomic fragment targeted to the
his-3 locus (see Materials and Methods). The restoration of
gpr-1 mRNA expression in gpr-1 +
gpr-1+ complemented strains was confirmed
by RT-PCR (Fig.
2C).
gpr-1
strains were analyzed for phenotypes during the life cycle.
gpr-1 mutants do not possess any obvious defects
during asexual growth or development (data not shown). Traits tested
included hyphal extension rate under normal and hyperosmotic conditions
and various aspects of macroconidiation. Consistent with the results
from expression profile analysis showing that gpr-1 mRNA
levels are highest in unfertilized and fertilized female tissues,
deletion of gpr-1 leads to several defects during sexual
development. Protoperithecia are weakly pigmented, and a significant
number are small and submerged in the agar during growth on solid
medium (Fig. 3A).Perithecia from gpr-1 mutants possess a significantly
greater number of fringe hyphae on the surface in comparison to the
wild-type strain, giving them a "hairy" appearance. The
abundant formation of fringe hyphae has been reported previously in
gna-1 strains
(39) (see below). The
underlying cause of this phenomenon, which has been observed in other
mutants with defects in sexual reproduction, is not known. The
gpr-1 perithecia have deformed beaks and lack
ostioles (pores) at the tips that are essential for appropriate
ejection of ascospores. Moreover, they are frequently ruptured, leading
to the release of the perithecial contents (Fig.
3A and B; see also Fig.
6B). In contrast to wild
type, significantly fewer perithecia reach maturity in
gpr-1 mutants (Fig.
3A). Those
gpr-1 perithecia that are fully developed produce a
similar number of ascospores as wild-type strains. The viability of
ascospores was identical to that of wild type during both homozygous
and heterozygous crosses (data not shown). gpr-1
+ gpr-1+ rescued strains are
phenotypically identical to wild type (Fig.
3A and B), confirming that
the defects of gpr-1 mutants result from loss of the
gpr-1 gene product.
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gpr-1
mutant (H1GFP) strains with regard to missing ostioles and ruptures in
the perithecial wall (Fig.
3B). Fluorescence from
ascospores within intact perithecia was observed through the ostiole in
wild type (Fig. 3B). In
contrast, perithecia from gpr-1 mutants are ruptured,
with ascospores oozing from the fissures in the structure (Fig.
3B).
GPR-1 is not
the only GPCR that is highly expressed during sexual development in
N. crassa. A previous study has shown that the pheromone
receptor gene pre-1 is present at high levels in both
protoperithecial and perithecial tissues
(44). Deletion of
pre-1 in a mat A background leads to
defective chemoattraction towards mat a males,
subsequently resulting in a complete block in cell fusion and
perithecial development
(44). The trichogyne
attraction assay was used to investigate the possibility that
gpr-1 strains might also be affected during early
stages of mating. Chemotropism of trichogynes toward male cells of
opposite mating type, as well as the ability of male cells (conidia) to
attract trichogynes of opposite mating type, were not disrupted in
gpr-1 strains (data not shown). This result indicates
that although PRE-1 and GPR-1 are highly expressed during sexual
development, they very likely regulate different events. Furthermore,
contrary to pre-1, gpr-1 expression is not
mating-type specific (data not shown). We further probed the
relationship between pre-1 and gpr-1 through analysis
of gpr-1 pre-1 double mutants (Fig.
3C).
gpr-1 pre-1 strains possessed the
defects of both single mutants, confirming independent roles for these
receptors during sexual development (Fig.
3C). The
gpr-1 pre-1 double mutants formed
fewer protoperithecia than wild-type or gpr-1
strains, and protoperithecia from the double mutants were
"hairy." The mat A
gpr-1 pre-1 mutants did not form
perithecia after fertilization with mat a males,
reflecting the absence of PRE-1. Conversely, the mat
a gpr-1 pre-1 strains
formed perithecia without ostioles after fertilization with
mat A males, indicating the loss of GPR-1. Thus,
PRE-1 is essential for cell-cell recognition and fusion, while GPR-1 is
required for normal development of unfertilized (protoperithecial) and
fertilized (perithecial) female
structures.
Epistasis analysis between gpr-1 and the three N. crassa G subunits.
Previous work has
demonstrated that the G subunit GNA-1
(39,
44) and
Gß dimer GNB-1/GNG-1
(44,
47,
77) are essential for
female (but not male) fertility in N. crassa. The functions of
these proteins are mating type independent, since they are required
downstream of both pheromone receptors (PRE-1 and PRE-2) in
mat A and mat a strains,
respectively
(44; Kim and Borkovich,
unpublished observations). Trichogynes of
gna-1, gnb-1, and
gng-1 mutants exhibit severe defects in
chemoattraction and fusion with male cells, similar to those of
pre-1 mat A strains
(44,
47). It has been
hypothesized that GNA-1 is the G subunit that communicates the
pheromone signal from PRE-1 to downstream effectors
(44). During homozygous
crosses, gna-3 strains form a large number of
perithecia with short beaks that are embedded in the agar medium
(43; data not shown).
Such crosses also produce a large proportion of nonviable ascospores.
The gna-2 mutant has no obvious defects during either
asexual or sexual development, but gna-1
gna-2 and gna-2
gna-3 double mutants have more severe defects than
strains lacking only gna-1 or gna-3
(4,
42).
The results
demonstrate that gpr-1 is required for normal perithecial
development, but the phenotype of gpr-1 strains
differs from that observed in the single G mutants. Therefore,
we explored functional relationships between GPR-1 and the three
G proteins through examination of protoperithecial and
perithecial development in gpr-1 G single
and double mutants. gna-1 strains form
"hairy" protoperithecia that enlarge slightly after
fertilization but fail to develop mature perithecia. This phenotype is
similar to that observed for fringe hyphae production in perithecia of
gpr-1 mutants (Fig.
4A). The gpr-1 gna-1 double mutant
exhibits phenotypic defects identical to those of the
gna-1 mutant during protoperithecial and perithecial
development, leaving open the possibility that GNA-1 is coupled to
GPR-1 (Fig. 4A). However,
since the other two G subunits are also implicated in sexual
development, we could not exclude the possibility that either GNA-2 or
GNA-3 might be coupled to GPR-1 at a certain time during sexual
development. The gpr-1 gna-2 strain
has defects identical to those observed in gpr-1
single mutants (Fig. 4A),
but the epistatic relationship is unclear due to the absence of
phenotypes in gna-2 single mutants. Similar to
gpr-1 strains, gpr-1
gna-3 mutants form small perithecia with no ostioles.
However, perithecia from gpr-1
gna-3 mutants also have short beaks, like
gna-3 strains (Fig.
4A). The phenotype of
gpr-1 gna-3 mutants is a
combination of both gene mutations, and therefore the epistatic
analysis does not support coupling between GNA-3 and GPR-1 during
perithecial development.
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genes ruled out GNA-2 and GNA-3 as
downstream effectors of GPR-1 but left open the possibility that GNA-1
is coupled to GPR-1, we examined whether constitutive activation of
gna-1 could bypass the defects of the gpr-1
mutation. The predicted GTPase-deficient, dominant-activated
gna-1(Q204L) allele construct
(76) was targeted to the
his-3 locus of a gpr-1 his-3 strain, and
transformants were selected and purified as described in Materials and
Methods. gpr-1 strains expressing the
dominant-activated gna-1 allele showed an even greater
reduction in perithecial formation than observed in
gpr-1 strains, but ostiole production was not
recovered (data not shown). This outcome indicates that activation of
GNA-1 alone cannot rescue the perithecial defects of strains lacking
GPR-1.
To elucidate whether deletion of gpr-1 affects G
protein levels, we performed Western analysis using specific antibodies
raised against individual G protein subunits
(4,
39,
43,
77). The results show
that the three G proteins, GNA-1, GNA-2, and GNA-3, and the
Gß protein, GNB-1, are present at the same level in
gpr-1 and wild-type strains (Fig.
4B). Thus, the presence of
gpr-1 has no effect on the concentration of G protein subunits
associated with the plasma membrane, supporting the proposal that the
aberrations observed during sexual development in
gpr-1 mutants result from loss of
GPR-1.
GPR-1 is localized in female reproductive structures.
The
phenotypic defects identified during sexual development in
gpr-1 strains are consistent with the high expression
of gpr-1 in female reproductive structures. To determine
whether GPR-1 localization correlates with the presence of
gpr-1 mRNA, we constructed strains that express a
carboxy-terminal fusion of GFP to GPR-1. The fusion vector was
transformed into his-3 and gpr-1 his-3
strains, and transformants with homologous recombination events at the
his-3 locus were isolated (see Materials and Methods). The
GPR-1-GFP fusion protein did not have any obvious effect on sexual
development in the wild-type background and was able to partially
complement the defects of the gpr-1 mutant (data not
shown). Since perithecia are heavily melanized, we focused on
microscopic observation of unfertilized female reproductive tissues.
Fluorescence was observed in protoperithecia of strains containing the
GPR-1-GFP fusion, consistent with localization of GPR-1 in this
structure (Fig.
5).
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60-amino-acid long homeodomain sequence
(30). BEK-1 is similar to
members of the TALE (three-amino-acid loop extension) superfamily of
atypical homeodomain transcription factors characterized by an
extension of three amino acids between two helices within the
homeodomain (16). This
extension almost always consists of proline-tyrosine-proline (PYP) and
is followed by a serine or threonine and several acidic residues
(16). These amino acids
are crucial for the interaction with other homeobox proteins, such as
PBX and HOX (29). Members
of the TALE family include MEIS (myeloid ecotropic viral insertion
sites), TGIF, PBX, and IRO (Iroquois) in animals, KNOX and BEL in
plants, and M-ATYP and CUP in fungi
(16). Alignment analysis
of the homeodomain regions of BEK-1 with members of
the TALE family revealed that BEK-1 shares high identity (53
to 77%) with the homeodomains of these proteins and confirmed that
BEK-1 contains the PYP motif in the same position as other proteins in
the TALE superfamily (Fig.
6A).
A bek-1 N. crassa mutant was created during a
high-throughput reverse genetics project
(22). In contrast to
results with gpr-1 mutants, there were no visible
protoperithecial defects in bek-1 strains. However,
the bek-1 mutant shares several defects in common
with gpr-1 strains during perithecial development.
The bek-1 mutant forms small, "hairy"
perithecia that are frequently ruptured, and these perithecia also lack
ostioles (Fig. 6B).
However loss of bek-1 results in more severe deformation of
perithecial beaks than observed in gpr-1 strains.
Perithecia from bek-1 strains have very short
beaks (Fig.
6B), while those from
gpr-1 mutants form volcano-shaped beaks
during later stages of perithecial development. The more severe defects
observed in the bek-1 mutant could result from a
convergence of multiple signal transduction pathways, including GPR-1,
that regulate BEK-1 function. To corroborate a possible functional
relationship between GPR-1 and BEK-1, gpr-1
bek-1 double mutants were isolated.
gpr-1 bek-1 strains exhibit defects
very similar to those observed in the bek-1 mutant,
consistent with BEK-1 functioning downstream of GPR-1 during
perithecial development. The mechanism by which GPR-1 regulates BEK-1
is not currently known. However, deletion of gpr-1 does not
greatly affect expression of bek-1, and loss of bek-1
does not lead to a significant reduction in gpr-1 mRNA levels,
indicating that any regulation of BEK-1 function by GPR-1 is
posttranscriptional (Fig.
6C).
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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gpr-1 strains are not female
sterile, their ability to disperse progeny is severely compromised.
This is caused by the absence of ostioles at the tip of the perithecium
through which mature ascospores are discharged. Fungal GPCRs have been divided into four major groups: (i) the pheromone-sensing receptors Ste2 and Ste3 of S. cerevisiae, (ii) the glucose sensing Gpr1 receptor of S. cerevisiae, (iii) the putative nutrient sensing receptor Stm1 of S. pombe, and (iv) proteins related to the CRL receptors of D. discoideum (12, 28, 32). A fifth group of seven-helix proteins, the microbial opsins, have not been implicated in G protein signaling in fungi (6; data not shown). The CRL receptors constitute a functionally divergent group of GPCRs that are similar to cAMP receptor GPCRs from D. discoideum. In N. crassa, this receptor group consists of three proteins (GPR-1, GPR-2, and GPR-3). A. nidulans and M. grisea have only one CRL-related protein, while F. graminearum contains four CRL receptors (Fig. 1). The related protein from A. nidulans, GprH, has been described previously, but no function has yet been assigned (32).
CRL GPCRs
have been characterized phenotypically in slime mold D.
discoideum (61) and
the model plant A. thaliana
(19,
23,
41,
60). Although CRL GPCRs
are absent from both budding and fission yeasts, proteins with weak
similarity to this group of receptors have been identified in the
basidiomycete C. neoformans
(74). Despite their
sequence homology, CRL receptors appear to have very diverse functions
in different species. For example, although N. crassa GPR-1,
GPR-2, and GPR-3 possess significant amino acid identity, our analysis
indicates that GPR-1 does not share any obvious functions with GPR-2 or
GPR-3 (S. Krystofova and K. A. Borkovich, unpublished
observations). In D. discoideum, the CrlA receptor functions
as a negative regulator of cell growth and appears to be required for
prestalk cell differentiation
(61). GCR1 is the only
characterized GPCR from A. thaliana and physically interacts
with the lone A. thaliana G protein, GPA1
(59). Previous studies
investigating loss-of-function or overexpression gcr1 A.
thaliana lines, as well as in vitro experiments with BY2 tobacco
cell cultures, showed that GCR1 is required for seed germination and
that GCR1 regulates DNA synthesis through activation of
phosphatidylinositol-specific phospholipase C
(2,
19,
23). Deletion of the
CRL-related gene gpr4 in C. neoformans leads to
modest defects in cell fusion during mating and reduced capsule
formation (74).
The
shared sequence homology of CRL proteins in ascomycete fungi is
distributed in both extracellular and transmembrane regions, suggesting
that these GPCRs share structural or functional similarity, e.g., by
responding to similar stimuli (ligands) or coupling to comparable
downstream components. In addition, we examined the correlation between
intron positions and secondary protein structure in this group of
GPCRs. In our previous studies, we reported that the position of
introns in heterotrimeric G protein genes (G and G
subunits) was highly conserved in mammals and N. crassa
(47,
68). Since mammalian
GPCRs are for the most part intronless
(14) and do not contain
receptors related to GPR-1, we compared gene structure only within the
cAMP receptor-like family present in ascomycete fungi (Fig.
1). These receptors share
a very similar gene structure in which introns are found only in
nontransmembrane regions. It is worthwhile to point out that the
position of the first intron in all genes presented in our study is
highly conserved and located at the junction of the first extracellular
loop and the third transmembrane region (Fig.
1). The evolutionary
significance of the presence of an intron in a highly conserved region
of these proteins has not yet been established. The large number of
GPCRs identified in N. crassa and other fungal and nonfungal
organisms provides a unique opportunity to examine the evolution of a
specific intron on a larger scale.
It has been demonstrated that
gna-1 strains form fewer protoperithecia than wild
type and that these protoperithecia are "hairy," with
an abundance of fringe hyphae on their surfaces. Importantly, no
ascospore progeny are produced after application of opposite mating
type conidia (39). The
failure of gna-1 protoperithecia to develop into
mature perithecia with ascospores could be explained by involvement of
GNA-1 in a signaling pathway required for perithecial development. In
this aspect, gna-1 strains display a defect similar
to pre-1 mutants during development of perithecia
(44). PRE-1 is one of two
pheromone receptors required for chemotropism of female trichogynes
toward male cells in N. crassa. In comparison to
pre-1 mutants, the complexity of phenotypic defects
during protoperithecial development and the block in perithecia
formation observed in gna-1 strains indicate that
multiple receptors must be coupled to GNA-1 during sexual development.
The epistasis analysis between gpr-1 and G genes
rules out GNA-2 and GNA-3 acting downstream of GPR-1, leaving GNA-1 as
a potential candidate for coupling to GPR-1. The genetic analysis using
single and double mutants in the gna-1 background is
complicated by the fact that GNA-1 is coupled to the pheromone
receptors, and therefore deletion of gna-1 causes a
simultaneous block in the pheromone response pathway
(39,
44). Defects in
protoperithecial development in gpr-1
gna-1 strains were identical to those observed in
gna-1 mutants, consistent with GNA-1 acting
downstream of GPR-1 for this trait. It is also possible that GPR-1
exerts its action in N. crassa independently of heterotrimeric
G proteins, particularly during perithecial development. Accumulating
evidence points to G-protein-independent activities for GPCRs in
several systems (13,
15). Along these lines,
it is noteworthy that a heterotrimeric G-protein-independent function
has been proposed for A. thaliana GCR1 with respect to
hormonal regulation of seed germination
(19). Determination of
possible G-protein-independent functions for N. crassa GPR-1
will require additional study.
Six putative homeodomain
transcription factors have been annotated in N. crassa
(12). Homeodomain
proteins form a large superfamily of transcription factors fundamental
to cellular proliferation, differentiation, and cell death in various
species. Interestingly, deletion of the homeodomain transcription
factor gene bek-1 led to several defects in common with
gpr-1 mutants, including the formation of ruptured
perithecia lacking ostioles. Three TALE superclass homeobox genes have
been identified in the genome of S. cerevisiae and have been
grouped into the M-ATYPE and the CUP classes
(16). One of the yeast
homeobox proteins, Tos8p, targets promoter regions of genes that are
involved in bud growth during G1/S events
(36). A TALE member,
Pth12p, has been previously identified in the filamentous
fungus M. grisea and has been shown to be involved in
appressorium maturation (P. Hauck et al.
[http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db
= protein&val = 67005921]).
Some members of
the TALE homeodomain protein family have been implicated as downstream
targets of signal transduction pathways. It has been reported that
protein kinase A strongly activates expression through PBX-HOX binding
sites in HEK293 cells (human embryonic kidney epithelial cell line)
(64), and protein kinase
A transactivation domains were mapped to the MEIS1A and MEIS1B C
termini (38). The
MEIS protein forms stable heterodimers with the PBX
homeodomain protein and binds to DNA in cooperation
with a HOX partner (51).
In a recent study, the ERK1/2 mitogen-activated protein kinase was
shown to phosphorylate and negatively regulate the Arix/Phox2a
homedomain protein (37).
The epistasis analysis between gpr-1 and bek-1
indicates that BEK-1 possibly acts downstream of GPR-1 in N.
crassa. However, the relationship between these two proteins
remains unclear. We propose that the effect must be
posttranscriptional, as the bek-1 transcript level was not
greatly affected in gpr-1 strains or vice versa.
Further experimental work will be required to establish the molecular
mechanism by which BEK-1 is regulated by the GPR-1 signaling pathway,
including identification of potential partners, upstream regulators,
and genes targeted by BEK-1.
Sexual development in N. crassa is a complex process for which molecular mechanisms are not well understood. Unfertilized and fertilized female reproductive structures consist of numerous different cell types (9). The mature perithecium is filled with asci (unbranched nonseptate hyphae) that contain ascospores and paraphyses (branched, multinucleate nonascogenous hyphae). The internal contents of the perithecium are enveloped by the outer perithecial wall, consisting of thick-walled pseudoparenchymatous cells (63). The cell types of the perithecial neck differ from the cells of the perithecial body. The ostiolar canal is lined with short branched cells (periphyses) which differentiate into pseudoparenchymatous neck wall cells (63). Upon maturation, a single ascus extends to the ostiole to discharge the ascospores (35). The actual mechanism of ascospore ejection has not been determined either in N. crassa or other ascomycetes. A recent study in Giberella zeae showed that three major osmolytes, mannitol, K+, and Cl–, were present in the epiplasmic fluid that is discharged along with ascospores but that potassium and chloride ions are likely the main osmolytes driving ascospore ejection (67).
gpr-1
strains display unique morphological defects (deformed beaks with no
ostioles) that have not been previously reported in N. crassa.
However, the isolation of two mutants that lack ostioles, m
and n, has been reported for the ascomycete filamentous fungus
Sordaria macrospora
(26). The loss of
ostioles dramatically compromises the ability of these fungi to
disseminate progeny. In addition, gpr-1 mutants form
beaks that are defective in phototropism (unpublished data). We
speculate that the absence of ostioles stems from defects in
development of periphyses and wall cells of the perithecial neck
(63). Moreover, we cannot
exclude the possibility that the volcano-shaped beaks and ruptured
perithecia observed in gpr-1 mutants could result
from an excess of turgor pressure inside perithecia of this strain.
Therefore, future studies will require a multipronged approach to
identify the molecular mechanism(s) underlying GPR-1 action. This
includes identification and characterization of the cell types affected
by loss of gpr-1 during protoperithecial and perithecial
development by using microscopic approaches, determination of the genes
regulated by GPR-1 and BEK-1 during sexual development, and analysis of
possible changes in concentration of osmolytic compounds in developing
perithecia.
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ACKNOWLEDGMENTS |
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This work was supported by grant GM48626 from the National Institutes of Health to K.A.B.
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FOOTNOTES |
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REFERENCES |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MAT-specific pheromone receptor gene of
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