| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Corresponding author: Evelyne Coppin, Institut de Génétique et Microbiologie, Bâtiment 400, Université Paris Sud, F-91405 Orsay Cedex, France., coppin{at}igmors.u-psud.fr (E-mail)
Communicating editor: P. J. PUKKILA
 | ABSTRACT |
|---|
 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
In the heterothallic filamentous fungus Podospora anserina, four mating-type genes encoding transcriptional factors have been characterized: FPR1 in the mat+ sequence and FMR1, SMR1, and SMR2 in the alternative mat- sequence. Fertilization is controlled by FPR1 and FMR1. After fertilization, male and female nuclei, which have divided in the same cell, form mat+/mat- pairs during migration into the ascogenous hyphae. Previous data indicate that the formation of mat+/mat- pairs is controlled by FPR1, FMR1, and SMR2. SMR1 was postulated to be necessary for initial development of ascogenous hyphae. In this study, we investigated the transcriptional control of the mat genes by seeking mat transcripts during the vegetative and sexual phase and fusing their promoter to a reporter gene. The data indicate that FMR1 and FPR1 are expressed in both mycelia and perithecia, whereas SMR1 and SMR2 are transcribed in perithecia. Increased or induced vegetative expression of the four mat genes has no effect when the recombined gene is solely in the wild-type strain. However, the combination of resident FPR1 with deregulated SMR2 and overexpressed FMR1 in the same nucleus is lethal. This lethality is suppressed by the expression of SMR1, confirming that SMR1 operates downstream of the other mat genes.
THE mating-type locus of the filamentous ascomycete Podospora anserina appears to be a master regulatory locus, mainly controlling self-nonself recognition between cells at fertilization and between nuclei after fertilization. Four genes assumed to encode transcriptional factors were characterized (see Fig 1): FPR1 in the mat+ haplotype and FMR1, SMR1, and SMR2 in the alternative mat- haplotype corresponding to completely different DNA sequences (
|
IR is a brief event, occurring in the early stage of fruiting-body development. That implies evident difficulties in observation and analysis. On the assumption that vegetative expression of mating-type genes may mimic IR in the mycelium and aid in its analysis, we investigated the control of expression of mating-type genes in wild-type strains and the effects of deregulated IR genes (FMR1, SMR2, FPR1) and SMR1 during the vegetative phase. Expression studies showed that FMR1 and FPR1 are active during both the vegetative and sexual reproduction phase of P. anserina, while SMR1 and SMR2 are not vegetatively transcribed. Deregulated SMR2 and SMR1 and vegetatively overexpressed FMR1 transgenes have been associated in various combinations by crossing. The association of FMR1 and SMR2 was found to lead to ascospore lethality in mat+ genetic context. Germination of ascospores was shown to be restored by introducing a vegetatively expressed SMR1 transgene. In the frame of the functional model of the mating types, we will discuss how these results can be interpreted and exploited for further investigations.
| MATERIALS AND METHODS |
|---|
| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
P. anserina: genetic analysis, strains, transformation:
The ascus of P. anserina normally contains four ascopores that develop around two nonsister nuclei after a single postmeiotic mitosis. However, 2 to 5% of asci contain five ascospores, two of which are smaller and uninucleate, yielding homokaryotic mycelium. Tetrad analysis is routinely performed on five-spored asci. The 
mat strain used in the study is derived from a mat+ strain deleted for the mat locus (
Bacterial strains, plasmids, and plasmid constructions:
Cloning and plasmid preparations were performed with Escherichia coli HB101 (
Plasmids pPUTUL, pFUTUL, pSUTUL1, and pSUTUL2 contain the ble gene (
Plasmids pGFMR1, pGSMR1, and pGSMR2 (Fig 1A) are based on the pUL vector and contain the A. nidulans gpd promoter on a 2.3-kb EcoRI-NcoI fragment of pUT703 (
Determination of the phleomycin resistance of the transformants carrying the 5'mat::ble gene fusion:
The 5'mat::ble fusions cloned in the pUL plasmid were introduced into the leu1-1 mat- strain. Transformants (leu+) were then tested on minimal medium containing 20 µg/ml phleomycin. They were considered resistant if growth was observed after 3 days of incubation, since growth of the wild-type strain was totally abolished during this period. A total of 30 to 100 primary (leu+) transformants obtained with each fusion were tested. The phleomycin phenotype was then more accurately determined, using two or three purified transformants issued from crosses of selected primary transformants with a leu1-1 mat+ strain. Since the transformants also contained leu1-1, the segregation of the integrated transforming vector was easily scored as (leu+) phenotype, independent of expression of the 5'mat::ble fusion. In these transformants, the integrity of the fusion transgene was ascertained by PCR analysis. The minimal inhibitory concentration (MIC) of phleomycin was determined on mat+ and mat- progeny carrying the construct. An identical MIC was obtained for strains of opposite mating type. At 10 µg/ml phleomycin, growth of the wild-type strain was inhibited for ~6 days; a residual growth was then observed. The time lag before residual growth was increased when phleomycin was used at 20 µg/ml, and some implants showed zero growth. Transformants with different levels of phleomycin resistance were recovered, depending on the construct introduced. MIC higher than 100 µg/ml phleomycin were not tested.
Genetic analysis of the deregulated mat gene associations:
First, each mat+ SMR2 transformant containing a constitutively transcribed ectopic SMR2 gene was crossed with the mat- GFMR1-5 transformant carrying the pGFMR1 plasmid (gpd::FMR1, leu1). At least 18 five-spored asci from each cross were submitted to genetic analysis and screened for sexual phenotype (mating type and self-fertilization) and hygromycin resistance. Second, each SMR2 transformant was crossed with transformants carrying the pPgFMR1 plasmid (gpd::FMR1, ble) or the pPaFMR1 plasmid (FMR1, ble). At least 20 five-spored asci from each cross were screened for sexual phenotype (mating type and self-fertilization) and hygromycin and phleomycin resistance. In such crosses involving three genetic loci—the mating-type locus, the integration locus of the (SMR2, hph) transgenes, and the integration locus of the (gpd::FMR1, leu1, or ble) transgenes (provided that these are not genetically linked)—the eight genotypes of homokaryotic progeny listed in Table 3 and Table 4 are expected to be equivalent. When a phenotypic class is lacking, tetrad analysis allows us to determine whether it corresponds to ascospores that have not germinated, genotypes of which can possibly be deduced from segregation of the genetic markers in the remaining viable ascospores of the same ascus.
|
|
|
|
Construction of mat+ SMR2 gpd::FMR1 gpd::SMR1 strain:
The mat+ GSMR1-4 and mat+ GSMR1-5 were crossed with the mat- SMR2-19 GFMR1-1 and mat- SMR2-19 GFMR1-2 strains. In the four crosses, 15 five-spored asci were submitted to genetic analysis. In three crosses an additional sample of 60 to 100 homokaryotic ascospores from five-spored asci were also analyzed. The segregation of the gpd::SMR1 in mat+ progeny was scored by ability to restore fertility in sexual cross with the mat- (SMR1::ura5) mutant. Since no simple functional test for mat- progeny was possible, when necessary the presence of the gpd::SMR1 transgene was established by PCR analysis.
DNA procedures:
Genomic DNA of transformants was prepared as described previously (
To confirm the genotype of transgenic progeny from sexual crosses, the presence of the mat transgenes was tested by PCR analysis. The position of pairs of primers is indicated in Fig 1B, and their sequence is as follows: FPR1: E3 [5'-GTCACTGGAACACACTCAAG-3']; F10 [5'-TTGACCGAAGATTTGGGC-3']. FMR1: 267352 [5'-GGCGGGAATCAACAGTATTTTGC-3']; 2544 [5'-CATCCAAGGGCTTCCATGTA-3']. SMR1: 247109 [5'-CGCGCATATAATGAATATCACGG-3']; 7317 [5'-CCCTCCAACTGAGATGCCAC-3']; SMR2: 246738 [5'-GGATGTCTCCAACTCCACTC-3']; 3293 [5'-CGTTGAGATCCGCGGTGGTC-3'] .
To analyze the structure of the integrated 5'mat::ble fusions, PCR amplifications were performed using the ble2 primer [5'-CACGAAGTGCACGCAGTT-3'], localized close to the stop codon in the ble gene, in association with a primer specific to the 5' UTR of the concerned mat gene: 573 [5'-CTAATAAGAATAATGTAATG-3'], which is 540 nucleotides upstream of FMR1 start codon, 246738, close to the SMR2 start codon, the reverse primer flanking the 5'-SMR1 sequence in pSUTUL1.
The structure of the integrated gpd::mat fusions was analyzed using the 39048 primer [5'-CCATCCTTCCCATCCCTTATTCC-3'] localized in the gpd promoter 100 nucleotides upstream of the initiation codon in association with a primer localized at the 3' end of the relevant mat gene.
Standard procedures for Southern blotting on Hybond N nylon filters (Amersham, France) were used. The probes were prepared using a random primer labeling kit (Roche Diagnostics).
RNA extraction:
To prepare RNA from mycelium, fungal cultures were made on a cellophane disk placed on agar minimal medium. After 2 days at 27°, the mycelium was recovered and transferred to a microcentrifuge tube. To prepare RNA from perithecia, cultures of mat+ and mat- strains were fertilized, respectively, with mat- and mat+ microconidia on separate petri dishes. Two hundred perithecia samples were collected 3 days after fertilization (production of mature ascospores begins on the fourth day). Perithecia were crushed with a conical grinder in 4 M guanidium thiocyanate, 50 mM TRIS HCl pH 8, 10 mM EDTA pH 8, 2% N-lauroylsarcosine (sodium salt), and 1% ß-mercaptoethanol. The suspension was treated three times with phenol-chloroform (1:1) and nucleic acids were precipitated with 1 volume of isopropanol. After centrifugation the pellet was resuspended in water. LiCl was added to a final concentation of 2 M, the solution was centrifuged, and the pellet was resuspended in water; sodium acetate pH 5.2 was added to a final concentration of 0.3 M and total RNA precipitated with 2 volumes of ethanol and recovered by centrifugation. The RNA pellet resuspended in water was purified on a RNeasy Plant minikit (QIAGEN, Hilden, Germany) according to the manufacturer's indications. Contaminating DNA was eliminated by Dnase digestion or centrifugation on a CsCl2 cushion. Complete degradation of DNA was ascertained by PCR reaction seeking the internal transcribed sequences of RNA ribosomal genes, with one primer [5'-CCGTTGGTGAACCAGCGGAGGGATC-3'] localized at the end of the 18S gene and the other [5'-TCCGCTTATTGATATGCTTAAG-3'] at the beginning of the 28S gene. For quantitative competitive RT-PCR, RNA were purified with High Pure RNA kit (Roche Diagnostics).
The reverse transcriptase polymerase chain reaction method (RT-PCR):
Two micrograms of total RNA were used for RT-PCR with the Titan one Tube RT-PCR Kit (Roche Diagnostics) according to the manufacturer's specifications. The following pairs of primers were used (DNA sequence of some primers is cited in DNA procedures): FPR1: E3/2551 [5'-GATCTCAGAAGATCGACGAGG-3']; FMR1: 267352/2544; SMR1 247109/7317; and SMR2 246738/3293. The gpd::SMR1 transcript was sought using the 2302 primer [5'-GATTGACCTGGGGGTTGAGG-3'] localized downstream of the first intron in association with the gpd specific primer 39048. The localization of the primers is shown in Fig 1B.
QC-RT-PCR:
Quantification of FMR1 mRNA was done by competitive RT-PCR (reviewed in
| RESULTS |
|---|
| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
Search for the mat transcripts in vegetative and sexual phases:
To determine if the mat genes are differentially transcribed throughout the life cycle of P. anserina, total RNAs were isolated from growing mat+ and mat- mycelial cultures and from perithecia of mat+ x mat- crosses. Mat genes encode regulatory proteins and only low levels of transcripts were expected. In fact, we were unable to detect mat mRNA on Northern blots. Therefore, we used the highly sensitive RT-PCR technique. Aliquots of each RNA preparation were used as templates for reverse transcription of RNA, followed by DNA amplification with primers specific for each mat gene. The primers indicated in Fig 1B were chosen such that they allowed amplification of a cDNA spanning one or more introns. A PCR product with the expected size for FPR1 and FMR1 cDNA was obtained in reactions performed with RNA extracted from either mat+ or mat- mycelial cultures, respectively. No mature or primary SMR1 and SMR2 transcripts were detected in RT-PCR reactions from mat- mycelial cultures, although several RNA preparations were tested with different pairs of primers. The cDNA of the four mat genes were detected in RT-PCR reactions performed on RNA from 3-day perithecia (data not shown). The fragments were cloned and DNA sequencing demonstrated that they actually corresponded to cDNA with proper intron splicing (
Transcriptional activity of the 5' UTR of the mat genes during vegetative growth:
FMR1 and FPR1 RNA detected in whole mycelium extracts can be attributed to contamination of the extracts by sexual organs (protoperithecia or microconidia) or to the expression of FMR1 and FPR1 in vegetative hyphae. To test the expression of FMR1 and FPR1 in vegetative hyphae, we constructed fusions between FPR1 and FMR1 and the ble reporter gene (MATERIALS AND METHODS). Fusions of SMR1 and SMR2 with ble were also tested to confirm the negative results obtained in RT-PCR assays. Expression in vegetative hyphae could be easily measured by determining the resistance level to phleomycin conferred by the fusions (MATERIALS AND METHODS). The constructs were made in the pUL transformation plasmid containing the leu1 gene as selective marker. The 5' UTR and the origin of the coding region of FPR1 (mat+), FMR1, SMR1, and SMR2 (mat-) were fused in-frame with the entire coding sequence of the ble gene, leading to pPUTUL, pFUTUL, pSUTUL1, and pSUTUL2 plasmids, respectively (Fig 1A and Table 1). The in-frame fusions were reexamined by DNA sequencing.
Two series of controls were performed. First, we determined if the 5' UTR sequences were or were not competent to promote transcription of their native gene. Therefore, each mat gene with the upstream sequence used for the fusion with ble was introduced by transformation in the suitable recipient and was shown to be functional in assays similar to those described for the gpd::mat fusions (see below). Second, as plasmids mainly integrate by heterologous recombination in P. anserina, we determined the frequency of downstream integration of a promoter-like sequence. For this control, we used the pFLUT plasmid containing the ble coding region fused to a plasmid sequence assumed to be devoid of promoter activity. Data on the transformation experiments are presented in Table 1. One phleomycin-resistant transformant among the 60 tested (leu+) transformants was obtained with the pFLUT control plasmid. Insertion of the ble gene downstream of a genomic promoter-like sequence expressed in the mycelium can therefore be considered as a rare event in comparison with the high frequency of phleomycin-resistant transformants recovered upon transformation with pPUTUL, pFUTUL, and pSUTUL2. Phleomycin-resistant transformants were recovered at a higher frequency with pPUTUL (75%) and pSUTUL2 (83%) than with pFUTUL (37%). Primary transformants were expected to manifest a heterogeneity of basic resistance to phleomycin. First, they contained variable proportions of transformed and untransformed nuclei because protoplasts are often plurinucleate. Second, transforming DNA integrates at random chromosomal locations that may have different cis-effects on the transcription activity of the ble gene. Third, inactivation of an active transgene during integration of the transforming DNA is a frequent event. The potential heterogeneity was examined by testing 30–100 primary (leu+) transformants obtained with each fusion for their resistance to phleomycin, and the MIC was determined for two or three purified transformants (Table 1). Purified transformants carrying the FPR1::ble fusion (pPUTUL) and SMR2::ble fusion (pSUTUL2) could still grow at 100 µg/ml phleomycin while transformants carrying the FMR1::ble fusion were inhibited at 30 µg/ml. These data suggest that the expression level of the FPR1::ble and SMR2::ble fusions is higher than the expression level of the FMR1::ble fusion. In contrast, all transformants carrying SMR1::ble fusion (pSUTUL1) were as sensitive to phleomycin as the wild-type strain. PCR assays (see MATERIALS AND METHODS) and Southern analysis of DNA from three purified strains transformed with pSUTUL1 were carried out and confirmed that the 5'SMR1::ble transgenes were not rearranged (data not shown).
Vegetative repression of SMR2 transcription requires an upstream cis element:
The two above-cited methods gave conflicting data with respect to SMR2, since SMR2 mRNA were not detected in mycelium by RT-PCR analysis whereas the 5'SMR2::ble fusion was found to be expressed vegetatively. These data suggest that the vegetative transcription of the 5'SMR2::ble fusion results from the loss of a regulatory cis element involved in transcriptional repression or in transcript instability during the vegetative phase. RT-PCR assays were performed on RNA prepared from mycelia of mat+ and mat- transformants bearing different transgenic constructs containing SMR2. SMR2 cDNA was not detected in mat+ transformants bearing the SMR2 coding sequence and 4.7 kb upstream of the SMR2 translation initiation covering the entire mat- region at an ectopic position (see Fig 1A). In contrast, SMR2 mRNA were detected in mycelium of mat+ and mat- strains that contain the SMR2 coding sequence and 1.4 kb upstream of the SMR2 first codon. These data suggest that an element required for the repression of SMR2 transcription is present in the region between 1.4 kb and 4.7 kb upstream of the SMR2 translation start.
Deregulated mat genes complement mat mutants and do not alter vegetative phenotype:
To promote expression or overexpression of mat genes during vegetative growth, we constructed gene fusions between the glycero-phosphate-3-dehydrogenase (gpd) promoter of A. nidulans and the coding region of the four mat genes, including the initiation codon. Constructs were cloned in the pUL plasmid carrying the leu1 selective marker, giving rise to pGFPR1, pGFMR1, pGSMR1, and pGSMR2 plasmids (Fig 1A). Each plasmid was introduced into a strain suitable for examining the functions of the gpd::mat fusion, generally the mat mutant deleted for mat information (
mat strain pGFMR1 and pSUT12 containing the two other mat- genes (SMR1 and SMR2) with the ble selective marker. The cotransformants displayed a wild-type mat- activity (fertilization of a mat+ partner giving rise to fertile perithecia producing asci).
The data demonstrate that the four mat genes are active when driven by the foreign gpd promoter. Moreover, during these tests, no gpd::mat fusion was found to produce an effect on viability, growth, or morphology of the recipient strain.
The functional assays allowed us to determine whether the gpd::mat fusions were active during sexual reproduction but did not allow us to determine whether they were expressed vegetatively. In particular, replacement of the native promoter of SMR1 by the gpd promoter was expected to induce its vegetative expression. To check the presence of gpd::SMR1 mRNA, RT-PCR assays were performed on RNA prepared from mycelia of one transformant bearing a functional gpd::SMR1 fusion. A product of the expected size was detected using a primer localized close to the stop codon in association with a gpd specific primer, indicating that the gpd::SMR1 fusion was vegetatively transcribed.
The combination of deregulated mat genes produces a lethal phenotype:
With the help of the transgenic strains obtained by transformation with the different mat constructs, we performed numerous crosses to obtain different combinations of the deregulated mat genes. In the course of the genetic analysis of some crosses, we observed a high frequency of ascospores unable to germinate. Tetrad analysis allowed us to determine their genotype: the lethal ascospore carried the mat+ resident mating type (FPR1) and both gpd::FMR1 and SMR2 transgenes, that is to say an artificial association of the three IR genes. To further investigate this phenomenon, new transformants more suitable for genetic analysis were constructed. For that purpose, the gpd::FMR1 fusion was cloned in a plasmid carrying the ble gene as selective marker (pPgFMR1). A plasmid containing the native FMR1 gene with its own 5' UTR (pPaFMR1) was also constructed to examine the role of the promoter. A SMR2 gene with a 1.4-kb 5' UTR was cloned in a plasmid carrying the hph gene, conferring resistance to hygromycin (pCBSMR2). The segregation of FMR1 and SMR2 transgenes could thus be easily followed through resistance to phleomycin (phleoR) and hygromycin (hygroR), respectively.
Transformants (hygroR) were recovered upon transformation of the mat+ recipient with the pCBSMR2 plasmid. Introduction of the SMR2 transgene into a mat+ strain was previously found to induce the enlargement of female organs that do not develop if they are not fertilized (S. ARNAISE, personal communication). This phenotype was used to determine whether the (hygroR) transformants carried a functional SMR2. Such transformants were genetically purified by crossing with a mat- wild-type strain, and homokaryotic mat+ and mat- progeny harboring the SMR2 and hph transgenes were recovered. Three homokaryotic transformants were generated from three independent primary transformants in which the plasmid had integrated at different chromosomal locations. They are named SMR2-4, SMR2-16, and SMR2-19: in the nomenclature SMR2-x, x specifies the integration locus of the SMR2 transgene. The same nomenclature is used to design each mat gene introduced by integrative transformation. Transformants carrying the pCBSMR2 construct must express SMR2 in hyphae because the gene does not contain the negative cis element that represses its transcription in mycelium. This prediction is confirmed by the detection of a fragment with the expected size for cDNA in RT-PCR assays performed on RNA extracts from the mat+ SMR2-19 strain (data not shown). SMR2c will subsequently designate this constitutive SMR2 transgene.
In the same way, (phleoR) mat+ primary transformants obtained with the pPgFMR1 or the pPaFMR1 plasmids were used to generate homokaryotic mat+ and mat- progeny carrying the gpd::FMR1 fusion (GFMR1-1, GFMR1-2) or the FMR1 transgene (FMR1-1, FMR1-3, FMR1-7). Introduction of these plasmids into a mat+ recipient induced self-fertilization and ability to fertilize a mat+ tester provided the FMR1 transgene was active. Only primary transformants exhibiting this phenotype were selected. QC-RT-PCR detection of FMR1 transcripts in total RNA extracted from GFMR1-1, FMR1-1, and wild-type mat- strains indicated that transcription of FMR1 is at least 10 times higher in GFMR1-1 strain than in FMR1-1 and wild-type strains (MATERIALS AND METHODS). The GFMR1-5 transformant was previously obtained with the pGFMR1 plasmid carrying the gpd::FMR1 fusion associated with the leu1 gene.
Crosses were performed between mat+ strains carrying a SMR2c transgene and mat- strains carrying a gpd::FMR1 fusion and submitted to genetic analysis. Data are presented in Table 3 and in Table 4 (first four columns). The most important finding is that no viable mat+ SMR2c GFMR1 homokaryotic progeny was recovered, while the seven other possible genotypes were obtained at equivalent frequency (except in crosses with SMR2-16 because the transgene is linked to mat+). Tetrad analysis (MATERIALS AND METHODS) indicated that the absent genotype was attributable to immature ascospores that had not germinated. This genetic association of mat genes was thus responsible for an autonomous ascospore lethal phenotype, that is, a phenotype controlled by the nuclei within the ascospore itself. Abundant asci with morphologically normal ascospores were produced in all crosses, indicating that proper sexual development occurred before ascospore delimitation. Heterokaryotic ascospores interpreted as containing both mat+ SMR2c GFMR1 and mat- wild-type nuclei were unable to germinate, which indicated that the lethality was dominant. Contrary to the mat+ SMR2c GFMR1 ascospores, the mat- SMR2c GFMR1 ascospores were viable. Nevertheless, on growth medium they gave rise to an unpigmented and flat mycelium that grew as well as the wild-type strain but failed to form aerial hyphae and rarely differentiated female organs (at least 200 times less than the wild type). Consequently, when used as female parent in a cross, only 10 to 100 fruiting bodies were produced on a petri dish in contrast to the thousands produced by a wild-type cross. The six other genotypes listed in Table 3 and Table 4 did not confer any particular phenotype. Finally, a mat- SMR2-19 GFMR1-2 strain issued from this analysis was crossed with a mat+ strain. As previously, mat+ SMR2-19 GFMR1-2 progeny were not obtained. The data indicated that the lethality phenomenon occurs whatever the initial association of the SMR2 and GFMR1 transgenes in the parental strains (one transgene in each parent as in Table 3 and Table 4 or both in the same parent).
Crosses were also performed between mat+ SMR2c strains and mat- FMR1 strains (Table 4, last six columns). By contrast to crosses with mat- GFMR1 strains, mature mat+ SMR2c FMR1 ascospores giving rise to viable mycelium were recovered. Although the SMR2-19 FMR1-3 and SMR2-19 FMR1-7 associations did not confer an ascospore lethal phenotype in a mat+ strain, they nonetheless impaired the growth rate and morphology of the mycelium, which was devoid of aerial hyphae. In a mat- strain, they conferred the phenotypic alteration (unpigmented female sterile mycelium) already observed in mat- SMR2c GFMR1 strains (Table 5). By contrast, SMR2c FMR1-1 associations did not impair the phenotype, whatever might be the mating-type resident haplotype (Table 5). One may assume that the vegetative effects are less drastic because the FMR1 transgenes are less expressed than are the gpd::FMR1 fusions as indicated by QC-RT-PCR experiments. Moreover, the integration site of the ectopic FMR1 copy may influence its expression level, which could be lower in FMR1-1 than in FMR1-3 and FMR1-7 transformants. The same rationale can be applied to SMR2 to explain the phenotypic differences between mat+ FMR1-3 SMR2-4 and mat+ FMR1-3 SMR2-19 strains (Table 5).
|
In conclusion, the association of a FPR1 (mat+) resident gene with a vegetatively expressed SMR2 transgene and a gpd::FMR1 transgene appears to be lethal. Replacement of the gpd::FMR1 by the FMR1 transgene results in viable strains that nevertheless exhibited impaired growth rate and mycelium morphology. Vegetative expression of SMR2 was confirmed by detection of a fragment with the expected size for cDNA in RT-PCR assays performed on RNA extracts from the mat- SMR2-19 GFMR1-2 strains (data not shown).
To examine the role of the resident FPR1 gene, the SMR2 and gpd::FMR1 transgenes were introduced into a mat strain by crossing the mat- SMR2-19 GFMR1-1 strain with a mat strain carrying the 6.3-kb EcoRI-SalI fragment from the mat+ locus (see Fig 1A). Progeny with the mat SMR2-19 GFMR1-1 genotype were recovered that displayed a normally pigmented mycelium that grew well. Viability of the mat SMR2-19 GFMR1-1 ascospores definitely confirms that the deregulated mat- genes must be associated with the resident FPR1 (mat+) gene to confer the ascospore lethal phenotype.
Observations made in the course of our study show that P. anserina transgenic strains exhibit some instability. In particular, sectors with increased growth rate appeared frequently from the poorly growing mat+ SMR2-19 FMR1-3 or mat+ SMR2-19 FMR1-7 mycelium. This phenomenon was investigated by crossing several sectors with the mat- wild-type strain. Although resistance to hygromycin (SMR2, hph) and phleomycin (FMR1, ble) segregated normally in the offspring, the sexual phenotype associated with either SMR2 or FMR1 transgene was lost. PCR reactions were performed on DNA extracted from sectors and from some of their progeny with a pair of primers specific to the inactive transgene: no band was detected, whereas the specific band was present in assays with DNA from the original mycelium (data not shown). The growth improvement in the sectors is thus caused by the loss of one of the transgenes without concomitant loss of the associated resistance marker. Consequently, to avoid any misinterpretation of a phenotype, the presence of a transgene was never deduced solely from the phleR or hygroR phenotype, but also using a functional test when possible and/or a PCR assay when necessary. To date, excision of an integrated plasmid or transgene in P. anserina transformants was considered a rare event, perhaps mistakenly. The situation we report is particularly well adapted to reveal such an event since the transgenes are "toxic." First, there is a selection pressure in favor of nuclei that have lost one transgene. Second, the loss of a transgene is associated with an increased growth rate and aerial hyphae production and thus with a directly observable phenotypic change.
Integration of a gpd::SMR1 fusion restores viability to the mat+ SMR2-19 GFMR1-2 strain:
FPR1, FMR1, and SMR2 control a sexual development-specific function, internuclear recognition. As explained in the Introduction, the fourth mat gene, SMR1 (mat-), is assumed to act downstream of FPR1, FMR1, and SMR2. We therefore tested the effect of the gpd::SMR1 fusion on the phenotype resulting from the expression of FPR1, FMR1, and SMR2 in the same nucleus. The gpd::SMR1 transcriptional fusion was used to force expression of SMR1 in vegetative mycelium since transcription of SMR1 was observed only in perithecia (see above). The mat- SMR2-19 GFMR1-1 and mat- SMR2-19 GFMR1-2 strains were crossed with mat+ strains harboring the gpd::SMR1 fusion (GSMR1-4 and GSMR1-5). These were obtained in two steps. First, the mat+ leu1-1 strain was transformed with the pGSMR1 plasmid and (leu+) primary transformants were recovered. Second, the transformants were crossed with the mat- (SMR1::ura5) strain. The sterility of this strain was complemented by the gpd::SMR1 transgene carried by the mating partner, allowing these crosses to produce progeny among which we identified the expected mat+ gpd::SMR1 genotype. Data of the crosses mat- SMR2-19 GFMR1-1/GFMR1-2 x mat+ GSMR1-4/GSMR1-5 are presented in Table 6. The segregation of the gpd::SMR1 in mat+ progeny was scored by ability to restore fertility in sexual cross with the mat- (SMR1::ura5) mutant. Since no simple functional test for gpd::SMR1 in mat- progeny was possible, the presence of the gpd::SMR1 transgene was established by PCR analysis when necessary. If the gpd::SMR1 fusion gene acts as a suppressor of ascospore lethality, (mat+ hygroR phleoR) mycelium corresponding to the mat+ SMR2-19 GFMR1 GSMR1 genotype should be recovered at a 6.25% frequency of the homokaryotic ascospores (16 genotypes at equivalent frequency were expected in a cross involving four unlinked genetic markers). Progeny with this phenotype were not obtained in crosses with mat+ SMR2-19 GFMR1-1 (two final columns of Table 6). In contrast, in crosses of mat+ SMR2-19 GFMR1-2 with mat+ GSMR1-4 and mat+ GSMR1-5 (first two columns of Table 6), 8.4% and 6.7%, respectively, of viable homokaryotic ascospores generating mycelium with the expected phenotype were found. The presence of the gpd::SMR1, SMR2, and GFMR1 transgenes deduced from functional tests was also confirmed by genomic DNA PCR analysis in some progeny. Although the mat+ SMR2-19 GFMR1-2 GSMR1 ascospores germinated well, the colonies displayed a slight morphological alteration on the germination medium as compared to wild type (smaller thallus with a less regular margin). Moreover, after transfer on minimal medium, mycelial growth is very slow and irregular. The (mat- hygroR phleoR) progeny were submitted to PCR analysis to test the presence of the gpd::SMR1 transgene. In fact these progeny corresponded to two genotypes, mat- SMR2-19 GFMR1-2 and mat- SMR2-19 GFMR1-2 GSMR1, which gave the same altered phenotype: unpigmented female sterile mycelia with no aerial hyphae. These genetic data indicated that SMR1 did not suppress the mutant phenotype conferred by the mat- SMR2-19 GFMR1-2 association.
|
Finally, to confirm the suppression of lethality by SMR1, a mat- SMR2-19 GFMR1-2 GSMR1-5 progeny was crossed with a mat+ GSMR1-5 strain. The gpd::SMR1 transgene was present in both parents, and only viable ascospores were recovered. Among the 80 homokaryotic ascospores analyzed, 11% were (mat+ hygroR phleoR) and 9% were (mat- hygroR phleoR), in agreement with the 12.5% expected for each class.
| DISCUSSION |
|---|
| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
We have studied the means by which mating-type genes are regulated during the life cycle of P. anserina to choose a strategy to deregulate them. We have further examined the physiological consequences of forcing their expression in vegetative hyphae.
RT-PCR analyses and fusions of mating-type genes with reporter genes revealed that during the vegetative phase FMR1 and FPR1 are expressed whereas SMR1 and SMR2 are transcriptionally silent. The vegetative expression of FMR1 and FPR1 genes is in agreement with their role in fertilization (
A comparison of the transcription pattern of Neurospora crassa (
Since transcriptional regulation is the primary regulatory control of expression of the P. anserina mat genes, we deregulated them by replacing their natural promoter with the A. nidulans gpd promoter. Each of the four gpd::mat fusions was found to be functional since it can complement a null or a mutated allele. No effect was noticed when one fusion was introduced in a mat+ or mat- wild-type strain. Crosses were performed between transgenic strains to associate the three IR genes in the same nucleus. We were unable to construct mat+ (FPR1) strains containing a constitutively transcribed SMR2c gene and a gpd::FMR1 fusion (Table 5). Ascospores with this genotype were nevertheless recovered but they were unable to germinate, which indicated that this genetic association was lethal. By contrast, homokaryotic mat+ (FPR1) strains containing a constitutively transcribed SMR2c transgene and a FMR1 transgene driven by its own promoter were obtained (Table 5). They exhibited a flat mycelium with reduced growth. The gpd::FMR1 fusion was found to be at least 10 times more transcribed than FMR1 driven by its own promoter, suggesting that the overexpression of FMR1 in a SMR2c mat+ strain is lethal, while a lower expression of FMR1 has a less drastic effect. Coexpression of the three IR genes thus results in a partial or complete inhibition of growth. We propose that this phenomenon mimics the events that occur during sexual reproduction. A cross between a mat+ strain and the mat- (SMR1::ura5) mutant, which contains functional FMR1 and SMR2 genes, displays a phenotype in agreement with this interpretation. This cross is sterile, although fertilization has occurred. Cytological observations indicate that development of perithecia is blocked before formation of ascogenous hyphae (
The hypothesis that recently proposed that IR is mediated by a pheromone response pathway (
protein results in a haploid-specific lethal phenotype due to cell-cycle arrest (
The association of the SMR2c and the gpd::FMR1 transgenes, lethal in the mat+ strain, is viable in the mat- strains but leads to phenotypic alterations. The mycelium of all mat- SMR2c GFMR1 and some mat- SMR2c FMR1 strains is unpigmented, devoid of aerial hyphae, and almost totally female sterile (Table 5). However, these strains grow as well as the wild-type strain in contrast to the mat+ SMR2c FMR1 strains. The finding that the mat SMR2-19 GFMR1-1 strain has a wild-type phenotype in contrast to the mat- SMR2-19 GFMR1-1 strain that displayed a mutant phenotype demonstrates that the resident mat- information is important in determining the morphological alterations. It has been established that differentiation of sexual reproductive structures is not controlled by the mating-type genes, since it occurs in the mat mutants (
All available lines of evidence suggest that vegetative expression of mating-type genes results in the activation of their target genes and mimics the transitory events occurring within the fruiting body. Ectopic expression of the mating-type genes is a valuable tool for the comprehension of fruiting-body development. The characterization of vegetative growth inhibition triggered by coexpression of the three IR genes opens a new field of investigation. This phenotype provides a powerful genetic screen for selecting suppressors and identifying possible target genes of the transcription factors encoded by the mating-type genes. Moreover, in nonlethal associations leading to a reduced mycelial growth rate, we have available biological material for the identification of specific mRNA by differential hybridization with RNA from a wild-type strain.
| ACKNOWLEDGMENTS |
|---|
We are grateful to H. D. Osiewacz for providing the pRP81-1 plasmid. We thank S. Arnaise, V. Berteaux-Lecellier, M. Chablat, and M. Picard for critical reading of the manuscript.
Manuscript received August 24, 1999; Accepted for publication March 1, 2000.
| LITERATURE CITED |
|---|
| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
ARNAISE, S., R. DEBUCHY, and M. PICARD, 1997 What is a bona fide mating-type gene? Internuclear complementation of mat mutants in Podospora anserina. Mol. Gen. Genet. 256:169-178[Medline].
BOYER, H. W. and D. ROULLAND-DUSSOIX, 1969 A complementation analysis of the restriction and modification of DNA in Escherichia coli.. J. Mol. Biol. 41:459-472[Medline].
CALMELS, T., M. PARRICHE, H. DURAND, and G. TIRABY, 1991 High efficiency transformation of Tolypocladium geodes conidiospores to phleomycine resistance. Curr. Genet. 20:309-314[Medline].
CARROLL, A. M., J. A. SWEIGARD, and B. VALENT, 1994 Improved vectors for selecting resistance to hygromycin. Fungal Genet. Newsl. 41:22.
COPPIN, E., S. ARNAISE, V. CONTAMINE, and M. PICARD, 1993 Deletion of the mating-type sequences in Podospora anserina abolishes mating without affecting vegetative functions and sexual differentiation. Mol. Gen. Genet. 241:409-414[Medline].
COPPIN-RAYNAL, E., M. PICARD, and S. ARNAISE, 1989 Transformation by integration in Podospora anserina III. Replacement of a chromosome segment by a two-step process. Mol. Gen. Genet. 219:270-276[Medline].
CROSS, F., L. H. HARTWELL, C. JACKSON, and J. B. KONOPKA, 1988 Conjugation in Saccharomyces cerevisiae.. Annu. Rev. Cell Biol. 4:429-457.
DEBUCHY, R., 1999 Internuclear recognition: a possible connection between euascomycetes and Homobasidiomycetes. Fungal Genet. Biol. 27:218-223[Medline].
DEBUCHY, R. and E. COPPIN, 1992 The mating types of Podospora anserina: functional analysis and sequence of the fertilization domains. Mol. Gen. Genet. 233:113-121[Medline].
DEBUCHY, R., S. ARNAISE, and G. LECELLIER, 1993 The mat- allele of Podospora anserina contains three regulatory genes required for the development of fertilized female organs. Mol. Gen. Genet. 241:667-673[Medline].
DROCOURT, D., T. CALMELS, J. P. REYNES, M. BARON, and G. TIRABY, 1990 Cassettes of the Streptoalloteichus hindustanus ble gene for transformation of lower and higher eukaryotes to phleomycin resistance. Nucleic Acids Res. 18:4009
FERREIRA, A. V. B., S. SAUPE, and N. L. GLASS, 1996 Transcriptional analysis of the mt A idiomorph of Neurospora crassa identifies two genes in addition to mt A-1.. Mol. Gen. Genet. 250:767-774[Medline].
FREEMAN, W. M., S. J. WALKER, and K. E. VRANA, 1999 Quantitative RT-PCR: pitfalls and potential. Biotechniques 26:112-125[Medline].
HERSKOWITZ, I., 1988 Life cycle of the budding yeast Saccharomyces cerevisiae.. Microbiol. Rev. 52:536-553
MCCULLOCH, R. K., C. S. CHOONG, and D. M. HURLEY, 1995 An evaluation of competitor type and size for use in the determination of mRNA by competitive PCR. PCR Methods Appl. 4:219-226[Medline].
MIYAJIMA, I., M. NAKAFUKU, N. NAKAYAMA, C. BRENNER, and A. MIYAJIMA et al., 1987 GPA1, a haploid-specific essential gene, encodes a yeast homolog of mammalian G protein which may be involved in mating factor signal transduction. Cell 50:1011-1019[Medline].
PICARD, M., R. DEBUCHY, and E. COPPIN, 1991 Cloning the mating types of the heterothallic fungus Podospora anserina: developmental features of haploid transformants carrying both mating types. Genetics 128:539-547[Abstract].
PUNT, P. J., M. A. DINGEMANSE, M. B. JACOBS, P. H. POUWELS, and C. VAN DEN HONDEL, 1988 Isolation and characterization of the glyceraldehyde-3-phosphate dehydrogenase gene of Aspergillus nidulans.. Gene 69:49-57[Medline].
RAJU, N. B. and D. D. PERKINS, 1994 Diverse programs of ascus development in pseudohomothallic species of Neurospora, Gelasinospora and Podospora.. Dev. Genet. 15:104-118[Medline].
RIDDER, R. and H. D. OSIEWACZ, 1992 Sequence analysis of the gene coding for glyceraldehyde-3-phosphate dehydrogenase (gpd) of Podospora anserina: use of homologous regulatory sequences to improve transformation efficiency. Curr. Genet. 21:207-213[Medline].
SIMONET, J.-M. and D. ZICKLER, 1972 Mutations affecting meiosis in Podospora anserina.. Chromosoma 37:327-351[Medline].
THOMPSON-COFFE, C. and D. ZICKLER, 1994 How the cytoskeleton recognizes and sorts nuclei of opposite mating type during the sexual cycle in filamentous ascomycetes. Dev. Biol. 165:257-271[Medline].
TURCQ, B., 1989 Clonage direct de genes par complémentation chez le champignon Podospora anserina : application à l'étude de gènes d'incompatibilité. Ph.D. Thesis, University of Bordeaux II, France.
YANISCH-PERRON, C., J. VIEIRA, and J. MESSING, 1985 Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103-109[Medline].
ZICKLER, D., S. ARNAISE, E. COPPIN, R. DEBUCHY, and M. PICARD, 1995 Altered mating-type identity in the fungus Podospora anserina leads to selfish nuclei, uniparental progeny, and haploid meiosis. Genetics 140:493-503[Abstract].
This article has been cited by other articles:
 |
|
 |
|
  M. Karlsson, K. Nygren, and H. Johannesson The Evolution of the Pheromonal Signal System and Its Potential Role for Reproductive Isolation in Heterothallic Neurospora Mol. Biol. Evol., January 1, 2008; 25(1): 168 - 178. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Paoletti and C. Clave The Fungus-Specific HET Domain Mediates Programmed Cell Death in Podospora anserina Eukaryot. Cell, November 1, 2007; 6(11): 2001 - 2008. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K.A. Aronstein, K.D. Murray, J.H. de Leon, X. Qin, and G.M. Weinstock High mobility group (HMG-box) genes in the honeybee fungal pathogen Ascosphaera apis Mycologia, July 1, 2007; 99(4): 553 - 561. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Pinan-Lucarre, A. Balguerie, and C. Clave Accelerated Cell Death in Podospora Autophagy Mutants Eukaryot. Cell, November 1, 2005; 4(11): 1765 - 1774. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. H. Sellem, C. Lemaire, S. Lorin, G. Dujardin, and A. Sainsard-Chanet Interaction Between the oxa1 and rmp1 Genes Modulates Respiratory Complex Assembly and Life Span in Podospora anserina Genetics, March 1, 2005; 169(3): 1379 - 1389. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Coppin, C. de Renty, and R. Debuchy The Function of the Coding Sequences for the Putative Pheromone Precursors in Podospora anserina Is Restricted to Fertilization Eukaryot. Cell, February 1, 2005; 4(2): 407 - 420. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
|
