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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. Note added in proof
  9. References

Homeobox-containing genes are widely described among eukaryotic species other than filamentous ascomycetes. We describe here the isolation and characterization of the first homeobox gene (pah1) identified in a filamentous ascomycete. It encodes a putative protein of 610 amino acids containing a typical homeodomain with 60 amino acids. Deletion of the pah1 gene enhances the number of male gametes (microconidia), whereas overexpression of pah1 results in a decrease in microconidia. These results led us to suppose that pah1 may be a repressor of genes involved in the microconidiation process. Moreover, pah1 is involved in hyphal branching and possibly in the development of female organs.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. Note added in proof
  9. References

Homeobox genes encode regulatory proteins acting as transcription factors (activators and/or repressors), which bind to specific cis-regulatory regions of their target genes. These proteins contain a conserved amino acid sequence motif, the homeodomain, which represents the DNA-binding domain. This class of proteins was first recognized in Drosophila melanogaster in which they cause homeotic transformations such as a fly with four wings instead of two (Lewis, 1978). They were then identified in most animal groups, as well as in plants, in fungi (Duboule, 1994) and, recently, in the unicellular alga Chlamydomonas reinhardtii (Kurvari et al., 1998). Most of these genes appear to be associated with developmental control or cell type regulation. In Hemiascomycetes, homeobox genes occupy a key position in the determination of the mating type in budding and fission yeast cells (Astell et al., 1981; Kelly et al., 1988). In Homobasidiomycetes, homeobox genes govern the synchronized division of nuclei inside dikaryotic cells resulting from mating of compatible strains and the formation of a specialized structure at each septum, the clamp connection, through which one nucleus must pass (for a review, see Casselton and Olesnicky, 1998). In the hemibasidiomycete Ustilago maydis, homeobox genes are involved in the control of filamentous growth, pathogenicity and the sexual cycle (Schultz et al., 1990). A homeobox gene, PHO2, which activates transcription of the regulated phosphatase gene PHO5 and possibly a phosphatase permease, has also been reported in Saccharomyces cerevisiae (Sengstag and Hinnen, 1987; Burglin, 1988).

In this report, we describe the isolation and characterization of the first homeobox gene (pah1) identified in a filamentous ascomycete. To address the question of homeodomain protein function, we have generated a pah1-deficient mutant by deleting the gene and have overexpressed the gene by replacing its 5′ untranslated region (UTR) with a strong fungal promoter. Sexual reproduction after fertilization was not altered in the PAH1-deficient mutant. Nevertheless, the number of male gametes was greatly enhanced in the deleted strain, and male and female fertility was decreased in the strains in which pah1 was overexpressed. In these two mutant strains, hyphal morphology was severely affected. These results indicated that the pah1 gene was involved in mycelium morphogenesis and in the development of sexual organs.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. Note added in proof
  9. References

PAH1 is more closely related to animal and yeast homeodomains than to fungal mating-type homeodomains

We performed reverse transcriptase–polymerase chain reaction (RT–PCR) experiments using degenerate primers (HOM1 and HOM2) designed from conserved amino acid sequences in the first and third helix of homeodomains, based on an alignment of homeodomains (Burglin, 1994) with total RNA extracted from fruiting bodies 3 days after fertilization. This yielded an amplification product of the expected size, 120 bp. A similar low-stringency PCR with HOM1 and HOM2 allowed us to obtain the same product from genomic DNA. A similar PCR protocol was used with HOM2 and HOM3, designed for the amplification of a homeobox sequence related to basidiomycetes homeobox. We did not detect any sequence related to homeobox genes among 10 cloned PCR products. The 120 bp fragment obtained with HOM1 and HOM2 was used as a probe to screen a Podospora anserina cosmid library and allowed us to isolate a 3.9 kb PstI fragment, which was cloned in pUC18 resulting in pPAH1. The sequence of the 3.9 kb PstI fragment revealed the presence of a discontinuous open reading frame (ORF) encoding a protein containing a homeodomain motif. This gene was named pah1 (P.anserinahomeobox). Its ORF is split by two putative introns of 56 bp and 58 bp exhibiting 5′ and 3′ consensus sequences typical of Ascomycete introns (Gurr et al., 1987). The pah1 transcripts could be detected by RT–PCR during the vegetative phase. The sequence of the cDNA confirms the location of the two proposed introns. The first ATG codon of the ORF has the conserved purine residue at position −3, which is present in most filamentous fungi. The size of the putative protein is 610 amino acids, with a calculated molecular weight of 67 kDa. The region flanking the pah1 ORF shows a possible TATA box at position −73. A putative CAAT box was found at position −143. Further examination of the polypeptide sequence was performed with the psort program to determine the possible location of PAH1 in the cell. The results indicate the presence of two nuclear targeting signals (RKSTLTQQQKNQKRQRA and PGHKRQR).

The genomic DNA of a wild-type strain was digested with five different restriction enzymes and probed with the entire pah1 gene. Only the fragments of the predicted size if pah1 is present in one copy have been obtained (data not shown).

Database search analysis using the blast program indicated that PAH1 has a region that shares significant sequence similarity with a variety of proteins that contain homeodomains. The homeodomains are defined as 60–63 amino acids and consist of a flexible stretch of nine residues, referred to as the N-terminal arm, followed by three α-helices (Burglin, 1994) (Fig. 1A). The homeodomains with 63 amino acids, with additional amino acids between helix 1 and helix 2, are considered to be atypical homeodomains. Moreover, additional amino acids might be found in basidiomycete homeodomains between helix 2 and helix 3. The PAH1 homeodomain is a typical homeodomain with 60 amino acids, more closely related to animal and yeast homeodomains than to fungal mating-type homeodomains (Fig. 1B).

image

Figure 1. Alignment and dendrogram of the PAH1 homeodomain with fungal, animal and plant homeodomains.

A. Alignment of the PAH1 homeodomain with homeodomains from the following fungal, animal and plant proteins (accession numbers in parentheses): Hoy1 of Yarrowia lipolytica (Q99160); putative Q10328 gene of Schizosaccharomyces pombe (Q10328); Otp of Mus musculus (O09113); PHO2 of S. cerevisiae (P07269); Antp of D. melanogaster (P02833); Ydr451c putative gene of S. cerevisiae (U33007); Aα2-1 of Coprinus cinereus;Aβ1-1 of C. cinereus (P40333); MATα2 of S. cerevisiae (P01367); MATa1 of S. cerevisiae (P101366); Hat1 of Arabidopsis thaliana (P46600); Stm of A. thaliana (Q38874); Kn1 of Zea mays (Q41330); Gsp1 of C. reinhardtii (AAD23383); MAT1P of S. pombe (X07643). Identical amino acids are shaded. The three α-helices derived from a composite of the structures of the Antp, engrailed and MATα2 homeodomains are shown above the sequences (from Burglin, 1994). *, percentage identity of each homeodomain with the PAH1homeodomain.

B. Dendrogram of PAH1 and fungal, animal and plant homeodomain sequences (see above). The dendrogram was generated by the pileup program. Sequences are grouped by similarity, and thus the dendrogram does not necessarily reflect evolutionary relationships.

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The blast program also revealed a similarity between the putative PAH1 protein and a ORF in Magnaporthe grisea. This ORF corresponds to the translation of a BAC end sequence, which starts shortly before the end of the putative homeodomain. In order to clone the sequence adjacent to the BAC end, primers PAH-MGH2 and MGH3 (see Experimental procedures) were used with genomic DNA of M. grisea as template. MGH3 corresponded to the BAC sequence of M. grisea, and PAH-MGH2 was designed from PAH1 downstream of the homeodomain and was expected to hybridize to a sequence similar to pah1 in M. grisea. A major PCR product of 0.4 kb was amplified, and its sequencing revealed that it encodes a homeodomain highly similar to the PAH1 homeodomain. It contains a putative 68 bp intron at a conserved position in an arginine (R) codon. This homeobox gene of M. grisea was termed mgh1 (M.griseahomeobox 1). A second intron was deduced from comparison with the pah1 gene and exhibits 5′ and 3′ consensus sequences typical of Ascomycete introns. As shown in Fig. 2, the two proteins show 72% identity in a 243-amino-acid overlap. Remarkably, the DNA sequences of the two genes are also very similar (73% identity). The two putative introns of the mgh1 gene were omitted in these comparisons. Pah1 and mgh1 are likely to define a new class of homeo-box genes, highly conserved among the filamentous fungi.

image

Figure 2. Alignment of the deduced proteins from pah1 (bottom line) and from a sequence of M. grisea (this paper for residues 1–52, accession number AQ255231for residues 53–261). Identical amino acids are shaded. The numbers refer to amino acid positions in the PAH1protein. There is no numbering for the M. grisea peptide, as the complete protein is not known. Triangles indicate the position of the two introns in the two genes; overlining indicates homeodomain.

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Deletion of the pah1 gene affects hyphal morphology and enhances microconidiation

To determine whether PAH1 is important for fungal development, the resident gene was deleted by substitution with a recombinant gene, in which the entire coding region was replaced by the bacterial hygromycin resistance gene hph under the control of the Aspergillus nidulans trpC promoter. Among 560 hygromycin transformants, 21 were easily distinguished from wild type. They were small and compact (colonial morphology on protoplast regeneration medium) compared with the filamentous growth of the other transformants. Evidence that this colonial morphology resulted from gene disruption was obtained by PCR analysis of genomic DNA from 10 of the primary colonial transformants (for details, see Experimental procedures). Crosses of these transformants with a wild-type strain showed that the [hygR] phenotype segregated with the colonial phenotype. Four of these were purified by crossing, and the deletion of pah1 was confirmed by Southern hybridization (for details, see Experimental procedures). The corresponding mutant strain was termed Δpah1.

Δpah1 is a morphological mutant. It has a wave of growth, first extremely compact and pigmented, then the mycelium becomes flat with very few hyphae; it then again becomes very compact (Fig. 3A). Mycelial growth in a Δpah1 mutant strain is two times less than in a wild-type strain. This reduction in the rate of hyphal elongation is accompanied by the appearance of numerous branches (Fig. 3B). Cyclic AMP influences growth and shape in Neurospora crassa (for a review, see Gadd, 1995). Mutants affected in the intracellular cAMP level display abnormal morphology that can be corrected by growth of the mutants in the presence of exogenous cAMP (Rosenberg and Pall, 1979). The addition of 2.5 mM cAMP or 10 mM dibutiryl cAMP to the growth medium of a Δpah1 mutant does not rescue the morphological phenotype, indicating that the defect in the Δpah1 mutant is not the result of a lack of cAMP. Dibutiryl cAMP is taken up by the cells, as demonstrated by Loubradou et al. (1999). In A. nidulans, morphological mutants defective in cell wall integrity show wild-type growth when extra osmoticum is added to the medium (Borgia and Dodge, 1992; Momany et al., 1999). The addition of 0.6 M sucrose or 0.6 M NaCl to the growth medium of a Δpah1 mutant does not restore wild-type growth, suggesting that there is not a general loss of cell integrity in the Δpah1 mutant. Nuclear distribution and positioning in the mycelium are not affected in the Δpah1 mutant (based on DAPI staining; data not shown).

image

Figure 3. Comparison of the wild-type and pah1 mutants with respect to mycelium morphology.

A. Vegetative growth on synthetic medium after 4 days. (a) Wild type; (b) Δpah1 mutant; (c and d) two gpd::pah1 mutants corresponding to two integration events of the gpd::pah1 recombinant gene.

B. Micrographs of peripheral regions of mycelium grown on HO solid medium. (e) Wild type; (f) Δpah1 mutant. Bar = 200 µm. The number of branches in hyphae is increased dramatically in the Δpah1 mutant.

C. Micrographs of hyphae grown on M2 medium. (g) Wild type; (h) gpd::pah1 mutant. Bar = 10 µm.

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In addition to its vegetative phenotype, the Δpah1 mutant also displays a sexual phenotype: it produces more microconidia than a wild-type strain (Table 1). The differentiation of female organs is not affected; the density of protoperithecia is of the same order in a Δpah1 mutant strain as in a wild-type strain. To test whether the pah1 gene is required for sexual reproduction after a fertilization event, we performed matings between two Δpah1 mutants. In these crosses, the progeny is normal 4 days after fertilization. The development of perithecia, analysed as described previously (Zickler et al., 1995), is the same as in a wild-type cross (data not shown).

Table 1. Microconidial production of the Δpah1mutant and wild-type strains.
StrainsNumber of microconidiaaNumber of peritheciab
6 daysc14 days6 days14 days
  • a . Microconidia were recovered by washing the surface (≈ 60  cm 2) of one Petri dish from each culture. They were counted using a haemocytometer. The numbers correspond to 1 ml of suspension.

  • b

    . Perithecia were counted after fertilization of the wild-type strain used as the female partner with 1 ml of microconidia suspension from the relevant strain. The numbers are the mean values of four Petri dishes; in brackets, the dilution factor of the microconidia suspension.

  • c

    . Only one Petri dish was observed.

Wild-type mat+6.5 × 1052 × 106164 (103)294 ± 10 (104)
Wild-type mat5.2 × 1051.3 × 106122 (103)173 ± 9 (104)
Δpah1 mat+6.5 × 1067.2 × 107323 (104)290 ± 31 (105)
Δpah1 mat1.2 × 1077.3 × 107221 (104)279 ± 15 (105)

A wild-type copy of the gene complements all defects of the Δpah1 strain. This confirms that the mutant phenotype is caused by the deletion of the pah1 gene and indicates that the Δpah1 mutation is recessive.

Overexpression of pah1 affects mycelial morphology and female and male fertility

A 2.8 kb transcript corresponding to pah1 has been detected in total RNA extracted from the mycelium of a wild-type strain (Fig. 4), indicating that pah1 is transcribed during the vegetative phase. We have tested the effect of pah1 overexpression by fusing the glyceraldehyde-3-phosphate dehydrogenase (gpd) promoter of P. anserina (Ridder and Osiewacz, 1992) with the coding region of the pah1 gene. We constructed a gene fusion between the gpd promoter of P. anserina and the coding region of the pah1 gene. The construct was cloned in the pCB1004 plasmid carrying the hygromycin-selective marker, giving rise to plasmid pCBGPAH1 (for details, see Experimental procedures). pCBGPAH1 was introduced by transformation into a recipient wild-type strain, and 13 out of 18 hygromycin transformants tested displayed a vegetative phenotype. Growth was affected, and the mycelium was totally apigmented. Five transformants displaying an apigmented mycelium and three transformants displaying a wild-type mycelium were tested by PCR for a complete, untruncated copy of the gpd::pah1 gene fusion. All five transformants displaying a vegetative phenotype have an intact gpd::pah1 gene fusion, and the three wild type do not, suggesting that all the transformants displaying an apigmented mycelium contain an intact gpd::pah1 gene fusion. We confirmed that the gpd::pah1 recombinant genes in these five transformants were active by introducing them by crossing in a Δpah1 strain and verifying the complementation of the colonial phenotype. The Δpah1 gpd::pah1 strains displayed the same morphological phenotype as the gpd::pah1 strain. Northern blot analysis of pah1 transcripts in total RNA extracted from one gpd::pah1 transformant and a wild-type strain confirms that pah1 transcription is increased in this gpd::pah1 transformant (Fig. 4). Female and male fertility was tested for all transformants. Ten of the 13 gpd::pah1 strains, including the transformant used for the Northern blot analysis, displayed a decrease in male and female fertility, and the other three exhibited a decrease in female fertility. Three of the gpd::pah1 strains with a decrease in male and female fertility have been observed microscopically (Table 2 and Fig. 5). No microconidia, either protoperithecia or ascogonia (protoperithecia at an early stage of development), were observed after 3 days of culture, although these sexual organs are present in a wild-type strain. Nevertheless, when the gpd::pah1 strains are incubated for a longer time, very few microconidia and some ascogonia and protoperithecia develop. This delayed development of sexual organs may result from the instability of the gpd::pah1 transgene or is a phenotype of the overexpression (see below and Experimental procedures). These results suggest strongly that overexpression of the pah1 gene impairs the formation of male cells and female organs.

image

Figure 4. Transcription of pah1 in wild type and gpd::pah1 transformant. Northern blot containing total RNA (20 µg and 1 µg) isolated from a wild-type strain (wt) and 1 µg of total RNA obtained from a pah1-overexpressing strain (gpd::pah1). The blot was probed sequentially with pah1 and AS1. AS1 was used for comparison of total RNA loaded in each lane, in addition to spectrophotometer quantification. Sizes of ribosomal RNAs are given in kb to the left. RNA from the wild-type strain has been loaded in high quantities in order to detect the transcript of pah1 and in low quantities for comparison with the gpd::pah1 strain, which yielded little RNA because of poor growth. The size of the wild-type pah1 transcript is 2.8 kb. The size of the AS1 transcript is 0.85 kb, in agreement with the size previously obtained by Dequard-Chablat and Sellem (1994).

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Table 2. Sexual organ development in wild-type and gpd:: pah1 strains.
StrainaNumber of protoperithecia and ascogoniabMicroconidiac
  • a

    . The three gpd::pah1 strains correspond to three independent transformants obtained by transforming a wild-type mat– strain with the gpd::pah1 gene fusion.

  • b . The number corresponds to the number of protoperithecia and ascogonia counted on a 2  mm 2 surface at 7 cm from the front of growth at 3 days of growth.

  • c . The microconidia were observed at 3 days of growth. NC, present but not counted; microconidia are clustered, which prevents accurate counting (see Fig. 5).

Wild-type mat+18NC
Wild-type mat149NC
gpd::pah1-1300
gpd::pah1-6 00
gpd::pah1-7 00
image

Figure 5. Comparison of the wild-type and pah1 mutants with respect to microconidium formation. The microconidia were observed after 3 days of growth (see Table 2).

A. Wild type.

B. Δpah1 mutant.

C. gpd::pah1 mutant.

The arrows indicate microconidia in clusters, much greater in the Δpah1 mutant strain than in the wild type. No microconidia are observed on the gpd::pah1 mutant strain. Bar = 600 µm.

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The heterogeneity of the male and female fertility phenotype can be explained either by variations in expression of the transgene depending on its integration site or by instability of the transgenes. Five of the gpd::pah1 transformants were purified by crossing. The heterogeneity of the phenotypes was greater than that in the primary transformants and was observed in different crosses for the same gpd::pah1 mutant. We concluded that the gpd::pah1 transgenes were unstable. This instability was verified as described in Experimental procedures. This instability prevented us from determining the exact step at which the development of female organs was blocked. A similar instability has been observed previously; the mating-type genes of P. anserina under the control of the A. nidulans gpd promoter are frequently lost during vegetative growth (E. Coppin, personal communication).

In the gpd::pah1 mutant strains, the morphology of the mycelium is strongly affected. During growth, the hyphae first present swelling (Fig. 3C), which then decreases, and the hyphae begin as twisted as vine shoots. The gpd::pah1 mutant strains grow more slowly than wild type (Fig. 3A), but their instability did not permit us to determine the growth rate.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. Note added in proof
  9. References

A new class of homeobox genes in filamentous fungi

In this report, we describe a homeobox-containing gene in the euascomycete P. anserina, which is, to our knowledge, the first identified in a filamentous ascomycete. It was obtained by PCR experiments using degenerate primers corresponding to regions conserved in most homeodomains. The search for homeobox genes with degenerate primers deduced from the alignment of homeodomains specific to fungal mating-type proteins has failed. The PAH1 homeodomain is a typical homeodomain with 60 amino acids, which is more closely related to animal and yeast homeodomains than to those filamentous fungal homeodomains already described. Gene inactivation experiments have demonstrated that the pah1 gene is not involved in sexual development after fertilization. This is in contrast to the function of all known homeobox genes in Homobasidiomycetes, which encode regulators of sexual development. In U. maydis, homeobox genes control filamentous growth and pathogenicity. The completion of the sexual cycle is dependent upon plant infection; therefore, homeobox genes are, at least indirectly, involved in the sexual cycle. Southern hybridization of genomic DNA of P. anserina points to the existence of a single pah1 gene. Further PCR experiments or Southern hybridizations at low hybridization stringency with the pah1 gene as a probe did not permit us to identify other homeobox genes in P. anserina. These results and the strong similarity between the putative PAH1 and MGH1 proteins are consistent with the idea that they define a new class of homeoproteins in filamentous fungi.

Pah1 is involved in the regulation of hyphal branching

The null mutant Δpah1 exhibited severe alteration in cell morphogenesis, notably hyphal extension, morphology and branching. The Δpah1 strain grew slowly with numerous branches. It has been proposed that, once the production of materials for wall extension exceeds the quantity that can be used at the pre-existing apex, a signal is generated promoting hyphal branching and the concomitant formation of a new growing tip (Yarden et al., 1992). We do not know whether hyphal branching in the Δpah1 mutant results from an excess production of wall materials or from a reduction in hyphal elongation. When pah1 is overexpressed, hyphal branching is not altered, although hyphal morphology is affected.

The mechanisms controlling branch initiation in filamentous fungi are still unknown, but several factors that control the production and frequency of branches have been identified. They are both environmental and genetic. Calcium and choline affect hyphal branching (Markham et al., 1993; Pera and Callieri, 1997). Mutations conferring a colonial morphology associated with a hyperbranched mycelium have been described in genes involved in the signal transduction pathway (Yarden et al., 1992; Loubradou, 1997), in genes encoding components of the cytoskeleton (Robb and Wilson, 1995) or in microtubule-associated motor proteins (Seiler et al., 1997; Inoue et al., 1998), as well as in genes directly or indirectly involved in cell wall structure (Borgia et al., 1996). Normal patterns of hyphal development can also be disrupted by mutations in samB, which encodes a novel Zn finger-containing protein in A. nidulans, and deletion in MUB1, the homologue of samB in S. cerevisiae, led to increased bud formation (Krüger and Fischer, 1998).

Pah1 is a regulator of microconidiogenesis in P. anserina

Inactivation of pah1 leads to overproduction of microconidia; in contrast, overexpression of pah1 results in a strong decrease in microconidiation. This leads us to propose that the pah1 gene acts as a repressor of genes involved in microconidium formation in P. anserina. Genes that are homologous to pah1 may control the formation of microconidia or related organs in other Ascomycetes. In M. grisea, one-celled microconidia have not been described. The rice blast fungus produces three-celled conidia showing one nucleus per cell (Yaegashi and Hebert, 1976), which serve both as male cells and as a major source of inoculum for the spread of disease during an epidemic. Mgh1 may be a regulatory gene for conidium formation in M. grisea. Although the conidiation process of A. nidulans is not considered to be similar to microconidiation, two genes required for microconidiation in P. anserina have homologues involved in conidiation in A. nidulans. The P. anserina genes are ami1 (Graïa et al., 2000) and fle1 (E. Coppin, personal communication), which are similar to apsA (Fischer and Timberlake, 1995) and flbC respectively (accession number AF083468). Ami1 and apsA are essential for nuclear distribution during microconidiogenesis in P. anserina and conidiogenesis in A. nidulans. Fle1 has been shown to be essential for microconidium formation (E. Coppin and Y. Brygoo, personal communication). FlbC is a regulatory gene that controls the decision to initiate conidiophore development in A. nidulans and is involved in the activation of brlA (for a review, see Adams et al., 1998). These similarities point to a possible common regulatory pathway between microconidiation in P. anserina and conidiation in A. nidulans and may suggest that a homologue of pah1 could control conidiation in A. nidulans.

Mutations in the pah1 homeobox gene are highly pleiotropic, as reported for other homeobox genes

Pah1 acts as a repressor of microconidium development and is involved in mycelial morphogenesis and, possibly, in female organ development. These results suggest that pah1 plays an important upstream regulatory role in the cascade of molecular events that leads to normal development in P. anserina by different developmental pathways. There are comparable examples in other organisms, such as the Ubx genes of D. melanogaster (Duncan, 1996) or the Gsh-1 gene of mouse (Li et al., 1996), in which homeobox genes are required at different steps to co-ordinate developmental events. Moreover, it has been reported that 25–50% of genes in D. melanogaster are directly or indirectly regulated by homeodomain proteins such as Eve, Ftz and Ubx (Mannervik, 1999).

Experimental procedures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. Note added in proof
  9. References

RNA extraction from perithecia

Five thousand perithecia (fertilized female organ) were picked up individually from mycelium with pliers 3 days after fertilization (production of mature ascospores begins on the fourth day). Perithecia were crushed with a conical grinder in 4 M guanidine thiocyanate, 50 mM Tris, pH 8, 10 mM EDTA, pH 8, 2% N-lauroylsarcosine (sodium salt) and 1% β-mercaptoethanol. The suspension was treated three times with phenol–chloroform (1:1), and nucleic acids were precipitated with one volume of isopropanol. After centrifugation, the pellet was resuspended in water. LiCl was added to a final concentration 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 was precipitated with two volumes of ethanol and recovered by centrifugation. The RNA pellet resuspended in water was purified on an RNeasy Plant minikit (Qiagen) according to the manufacturer's instructions. Contaminating DNA was eliminated by DNase digestion (Bauer et al., 1997).

RNA extraction and RNA blotting for overexpression analysis

Wild-type and gpd::pah1 strains were grown on minimal medium agar plates covered with cellophane sheets for 2 and 4 days respectively. The growth of gpd::pah1 strains is strongly impaired, and they require more time than wild-type strains to produce enough mycelium for RNA extraction. The mycelium was collected from the surface of the cellophane sheet and crushed in a mortar as described for RNA extraction from perithecia (see above), except that the RNA was not purified on a column. Denaturing gel for RNA, RNA blotting and all other molecular techniques were as described by Sambrook et al. (1989). The pah1 template for probe preparation was obtained by PCR from a plasmid containing pah1 with primers SA8 (5′-CAGACATGGAGAGCGTGTG-3′) and SA13 (see below). Purified PCR product (10 ng) was labelled with the T7QuickPrime kit (Amersham Pharmacia Biotech). AS1 encodes the ribosomal protein S12 (Dequard-Chablat and Sellem, 1994) and was not expected to be regulated by pah1. The AS1 transcription has been used as a reference to assess the quantity of RNA loaded in each well. The AS1 template for probe preparation was obtained by PCR from a plasmid containing AS1 with primers OI1 (5′-GCTGACATGACATGCAAC-3′) and MS (5′-GCACCGATACCTGGTCTAC-3′). Purified PCR product (10 ng) was labelled with the T7QuickPrime kit (Amersham Pharmacia Biotech).

Amplification, cloning and sequencing of the pah1 gene and RT–PCR experiments

The pah1-specific 120 bp fragment was obtained by RT–PCR from RNA of wild-type P. anserina perithecia. The RT–PCR experiment was performed using the pairs of primers HOM1 (5′-GGGATCCGARYTNGARAARGARTTY-3′) and HOM2 (5′-AACTGCAGCKNCKRTTYTGRAACCA-3′), which correspond to the ELEKEF and WFQN(R/S)R amino acid sequences respectively. Reverse transcription and amplification were performed with the Titan One Tube kit (Roche Diagnostics) in 50 µl reaction volumes containing 1 µg of mRNA and 100 pmol of each primer, according to the manufacturer's instructions, under the following cycle profiles: 30 min at 50°C, an initial denaturation step of 2 min at 94°C, 1 cycle of 30 s at 94°C, 30 s at 37°C, followed by a slope rate of 20°C min−1, 1 min at 68°C and 40 cycles of 30 s at 95°C, 30 s at 50°C, 1 min at 68°C. The last cycle was followed by an additional 10 min at 68°C. PCR fragments were separated on an agarose gel, and a band of the expected size (120 bp) was extracted from the gel with GFX columns (Amersham Pharmacia Biotech) and cloned into pGEM-T (Promega). After transformation of the ligation mix into E. coli DH5α, four recombinant plasmids were recovered and sequenced. They encode an identical sequence with significant similarities to the homeodomain proteins. The PCR amplification obtained with the forward and reverse sequencing primers on one of the recombinant pGEM-T plasmids was used to probe a P. anserina cosmid library (a gift from M. Chablat). Three overlapping cosmids containing a 3.9 kb PstI fragment hybridizing with the 120 bp fragment were obtained. The 3.9 kb fragment was subcloned in the pUC18 plasmid (Yanisch-Perron et al., 1985). The insert of one recombinant plasmid, pPAH1, was entirely sequenced using the ABI PRISM Ready Reaction DyeDeoxy terminator cycle sequencing kit (Applied Biosystems), with an automatic sequencing machine (373A DNA sequencer; Applied Biosystems). Universal primer and synthetic oligonucleotides were used to start the reactions.

Primers HOM1 and HOM2 have been used to amplify pah1 from genomic DNA. The PCR reaction mixture contained about 50 ng of genomic DNA in 50 µl of reaction buffer [1 ×Taq DNA polymerase incubation mix (Appligene Oncor), 0.1 mM dNTPs, 2 µM each primer and 2 U of Taq DNA polymerase (Appligene Oncor)]. Denaturation at 94°C for 2 min was followed by one cycle of 94°C for 30 s; 37°C for 30 s; a slope rate of 20°C min−1 to 68°C; 68°C for 1 min and 40 cycles of 94°C for 30 s; 50°C for 30 s; 72°C for 1 min. Reactions were held at 72°C for 5 min for final extension. PCR products were then treated as indicated above. Four clones have been sequenced, and all were identical to those isolated by RT–PCR from total RNA. The degenerate primer HOM3 (5′-GGGATCCYWYAAYSCNTAYCCNWS-3′), deduced from the alignment of fungal homeoproteins [(Y/F/H)N(P/A)YP(T/S)] (Kues and Casselton, 1992), and HOM2 were used for the amplification of a DNA sequence encoding a homeobox gene related to the mating-type homeobox genes of basidiomycetes. The protocol was as described above for the amplification of pah1 with HOM1 and HOM2 from genomic DNA. The size of the cloned fragment after amplification was 120–170 bp. Ten clones were sequenced. None corresponded to a homeobox gene.

The cDNA used to amplify the cDNA of the pah1 gene was a kind gift from M. Chablat and was prepared from RNA extracted from mycelium (Dequard-Chablat and Sellem, 1994). The following pah1 primers were used: primer SA13, 5′-GCTACCGACGTGGTCAAGC-3′; primer SA12, 5′-TCGGGATGGTTGAATGAGAG-3′. The amplified fragments were purified and sequenced using specific primers deduced from the DNA sequence, according to the same procedure as pah1 sequencing.

The protocol used to amplify the homeobox of M. grisea was as follows. Primer PAH-MGH2 (5′-AARWSNACNYTNACNCARCARCAR-3′) was deduced from the peptide region (KSTLTQQQ) of PAH1 immediately downstream of the homeodomain. Primer MGH3 (5′-ACCTTTACCAGACTCCATAGCCTG-3′) corresponded to the sequence of the BAC end. The template was genomic DNA from isolate Guy11 of M. grisea (Leung et al., 1988). PCR reaction conditions and cycle profile were as described above. PCR products were separated on an agarose gel, extracted with GFX columns (Amersham Pharmacia Biotech) and sequenced on each strand with PAH-MGH2 and MGH3.

P. anserina strains, growth conditions and transformation

P. anserina is an ascomycete whose life cycle and general methods of genetic analysis have been described previously (Rizet and Engelmann, 1949). All strains are derived from the wild-type S strain. The HO medium contains only H2O supplemented with 50 g l−1 agar. NaCl and cAMP were added to the minimal medium when indicated. Protoplasts were prepared and transformed as described previously (Berteaux-Lecellier et al., 1995). When necessary, hygromycin (Roche Diagnostics) or phleomycin (Cayla) were added to the protoplast regeneration medium at a concentration of 100 µg ml−1 and 5 µg ml−1 respectively. Segregation of antibiotic resistance in the sexual crosses was scored on minimal medium containing either 75 µg ml−1 hygromycin or 20 µg ml−1 phleomycin. Crosses were performed by spermatization (spraying of microconidia onto strains of opposite mating type). The resulting perithecia were analysed as described previously (Zickler et al., 1995).

Counting of microconidia and perithecia

The relevant strains were grown on Petri dishes containing minimal synthetic medium (M2) and incubated at 27°C in the dark. The microconidia were recovered at different times (6 or 14 days) by washing the surface of the mycelia with 2 ml of sterile water. This permitted the recovery of 1 ml of microconidial suspension, which was counted by microscope with a haemocytometer. To test fertilization ability, 1 ml of microconidial suspension (after dilutions when required) was spread on wild-type mycelia used as female partners, which had been grown previously on M2 medium at 27°C for 6 days in the light to allow the formation of female organs. Perithecia were counted 5 days after fertilization.

Bacterial strains, plasmids and plasmid construction

Cloning and plasmid preparations were performed in either E. coli HB101 (Boyer and Roulland-Dussoix, 1969) or DH5α (Hanahan, 1983).

The 3.9 kb PstI fragment containing the pah1 gene was cloned into the pUC18 vector (pPAH1 plasmid). The gene disruption vector pDPAH1 was constructed by replacing the 2 kb StyI–BsrGI fragment of pPAH1 with the 1.4 kb XbaI–Acc65.1 fragment containing the trpC::hph construct from pUCHYGRO. pUCHYGRO was the source of the hph gene, encoding hygromycin phosphotransferase, which confers hygromycin resistance (Gritz and Davies, 1983). This gene was driven by the A. nidulans trpC promoter region (Mullaney et al., 1985) and was used as a dominant selectable marker in the isolation of P. anserina transformants. pUCHYGRO (a gift from G. Ruprich-Robert) was generated by cloning the 1.4 kb HpaI fragment carrying the trpC::hph construct from pCB1004 at the SmaI site of pUC18 (Carroll et al., 1994).

The pCBGPAH1 plasmid was generated by cloning in pCB1004 the 4.5 kb EcoRI–PstI fragment encompassing the gpd::pah1 fusion prepared from pGPAH1. pGPAH1 was constructed by ligation of the following three fragments: (i) the 350 bp EcoRI–NcoI fragment containing the gpd promoter of P. anserina obtained by digestion of the pRP81 plasmid (Ridder and Osiewacz, 1992); (ii) the 350 bp NcoI–AgeI fragment beginning at the ATG and containing the 5′ end of the pah1 gene obtained through PCR amplification with the SA26 (5′-CGACGCCATGGCTACCGACGTGGTCAAGCAA-3′) and SA3 (5′-CCAACACGGGTCCATCTTC-3′) primers on pPAH1 and digestion of this fragment with NcoI and AgeI; (iii) the 5.4 kb AgeI–EcoRI fragment containing the 3′ end of the pah1 gene and the pUC18 sequences from pPAH1. The PCR-amplified region of the pah1 gene in pGPAH1 has been sequenced and checked for the absence of mutation.

DNA procedures

Genomic DNA was prepared using the rapid Petri dish-grown mycelium method (Lecellier and Silar, 1994). Standard procedures for Southern blotting on neutral membrane (Appligene) were used. The probes were prepared using a T7 QuickPrime kit (Pharmacia). The pah1 probe was obtained by PCR amplification of the pPAH1 plasmid with two primers flanking the ORF (SA13, 5′-GCTACCGACGTGGTCAAGC-3′; and SA17, 5′-TCCTCTCAAAACGCCGCAG-3′).

To analyse the structure of the integrated gpd::pah1 fusions, PCR amplifications were performed using two pairs of primers: the forward sequencing primer flanking the 5′pah1 sequence in pCBGPAH1 in association with the SA8 primer (5′-CAGACATGGAGAGCGTGTG-3′) at 1141 nucleotides downstream of the pah1 start codon and the reverse primer flanking the 3′pah1 sequence in pCBGPAH1 in association with the SA7 primer (5′-GTTCATGCACCGTCA TACCG-3′) at 1065 nucleotides downstream of the pah1 start codon.

The two pairs of primers used for analysis of the disruptant are as follows: one primer is localized in the hph gene, SA27 (5′-AGCACTCGTCCGAGGGCAA-3′) or SA28 (5′-ACGTCGCGGTGAGTTCAGGC-3′), and the other primer hybridizes to genomic sequences flanking the PstI fragment, which contains either the wild-type or the deleted pah1 gene, SA21 (5′-ATGATGGGAGTGAGTGAGC-3′) or SA22 (5′-ACATCA GATCTCACACTTC-3′). In the 10 primary transformants tested, a PCR fragment of the expected size was obtained with the two pairs of primers, indicating that the disrupted gene of the pDPAH1 plasmid has integrated at its homologous locus by a double crossing over, resulting in the replacement of the wild-type pah1 gene by the disrupted copy.

The deletion of pah1 in the four purified colonial transformants was confirmed by Southern hybridization as follows: DNAs were digested with PstI and probed with the 3.9 kb PstI fragment containing the pah1 gene and its flanking regions. In wild type, there was one band of 3.9 kb, which was missing in the colonial transformants and replaced by a band of the expected size 3.4 kb.

Instability of the gpd::pah1 constructs

Five of the primary gpd::pah1 transformants were purified by crossing. They were crossed with a wild-type strain, either as female partner (1) or as male partner (2). The crosses were also performed by confronting the mycelia of the two strains (3). In this last case, the vegetative hyphae may act as male gametes. For each cross, 14–32 asci were analysed for segregation of the apigmented phenotype (phenotype of the strain containing the gpd::pah1 construct, see Results). In case (1), 107 asci were tested and contained ascospores giving an apigmented mycelium phenotype. In case (2), 39 asci among 79 and, in case (3), 18 asci among 79 presented the segregation of the apigmented mycelium phenotype. PCR assays were performed on DNA extracted from the mycelium issued from ascospores coming from wild-type asci with the two pairs of primers specific for the gpd::pah1 construct. No amplification was detected (10 asci analysed). As expected, fragments were amplified with DNA extracted from apigmented mycelium (five analysed). These data clearly indicated that the gpd::pah1 constructs were unstable

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. Note added in proof
  9. References

Marguerite Picard is gratefully acknowledged for her constant interest in this work and helpful discussions. We are grateful to Professor H. D. Osiewacz for providing the pRP81-1 plasmid. We thank Guillaume Balavoine for his help in sequence analysis of homeodomain proteins; Michèle Chablat for the gift of the cosmid library and the cDNA from P. anserina; Gwenael Ruprich-Robert for the gift of the pUCHYGRO plasmid; Thierry Langin and Marc-Henri Lebrun for the gift of Magnaporthe grisea DNA.

Note added in proof

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. Note added in proof
  9. References

The GenBank accession number of the pah1 sequence is AJ297955.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. Note added in proof
  9. References
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