Identification of vib-1, a Locus Involved in Vegetative Incompatibility Mediated by het-c in Neurospora crassa

Corresponding author: N. Louise Glass, University of California, Berkeley, CA 94720-3102., lglass{at}uclink.berkeley.edu (E-mail)

Communicating editor: M. SACHS

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

A non-self-recognition system called vegetative incompatibility is ubiquitous in filamentous fungi and is genetically regulated by het loci. Different fungal individuals are unable to form viable heterokaryons if they differ in allelic specificity at a het locus. To identify components of vegetative incompatibility mediated by allelic differences at the het-c locus of Neurospora crassa, we isolated mutants that suppressed phenotypic aspects of het-c vegetative incompatibility. Three deletion mutants were identified; the deletions overlapped each other in an ORF named vib-1 (vegetative incompatibility blocked). Mutations in vib-1 fully relieved growth inhibition and repression of conidiation conferred by het-c vegetative incompatibility and significantly reduced hyphal compartmentation and death rates. The vib-1 mutants displayed a profuse conidiation pattern, suggesting that VIB-1 is a regulator of conidiation. VIB-1 shares a region of similarity to PHOG, a possible phosphate nonrepressible acid phosphatase in Aspergillus nidulans. Native gel analysis of wild-type strains and vib-1 mutants indicated that vib-1 is not the structural gene for nonrepressible acid phosphatase, but rather may regulate nonrepressible acid phosphatase activity.

FILAMENTOUS fungi grow by hyphal tip extension and branching. Within the interior of a colony, hyphae undergo fusion to form a network that makes up the fungal individual (BULLER 1933 ; GLASS et al. 2000 ; HICKEY et al. 2002 ). Filamentous fungi can also undergo hyphal fusion between isolates, resulting in the formation of a heterokaryon, in which genetically different nuclei coexist in a common cytoplasm. The viability of such heterokaryons, however, is genetically controlled by a number of loci, termed het (for heterokaryon) or vic (vegetative incompatibility; GLASS and KULDAU 1992 ; LESLIE 1993 ). In heterokaryotic cells, a genetic difference in allelic specificity at any het locus between two fungal strains causes a phenomenon called vegetative incompatibility (GLASS et al. 2000 ; SAUPE 2000 ). In many fungal species, a macroscopic barrage forms when two incompatible individuals meet (ESSER and BLAICH 1994 ), which is caused by abnormal or lethal hyphal fusions in the area of contact. Vegetative incompatibility also can be triggered in forced heterokaryons, partial diploids, or transformants that contain het alleles of alternative specificity (MYLYK 1975 ; PERKINS 1975 ; SAUPE and GLASS 1997 ; WU and GLASS 2001 ). In pseudohomothallic fungal species, self-incompatible progeny can be obtained from crosses between two incompatible parents (SAUPE 2000 ; SAENZ et al. 2001 ). Phenotypic aspects of vegetative incompatibility include hyphal compartmentation and death (HCD), lack of conidiation (in species that produce conidia), and growth inhibition (GARNJOBST and WILSON 1956 ; BOUCHERIE and BERNET 1978 ; JACOBSON et al. 1998 ; WU and GLASS 2001 ).

Vegetative incompatibility has been studied extensively in two ascomycete species, Neurospora crassa and Podospora anserina (for reviews, see GLASS et al. 2000 ; SAUPE 2000 ). A total of 9 het loci in P. anserina and 11 het loci in N. crassa have been described. Genes at 4 het loci in N. crassa and 3 het loci in P. anserina have been cloned. The predicted protein products of these het loci are diverse. N. crassa het-c and het-6 and P. anserina het-s and het-e are not essential for cellular processes other than vegetative incompatibility. They encode a plasma membrane protein, a putative protein, a prion analog, a protein with a GTP-binding site, and a C-terminal WD repeat domain, respectively (TURCQ et al. 1990 ; SAUPE et al. 1995 , SAUPE et al. 1996 ; SMITH et al. 2000). The other three het loci encode proteins involved in biological processes in addition to vegetative incompatibility. The N. crassa mating-type genes, mat A-1 and mat a-1, regulate mating and vegetative incompatibility and encode putative transcriptional factors with predicted DNA-binding domains (GLASS et al. 1990 ; STABEN and YANOFSKY 1990 ). The un-24 locus encodes a polypeptide with high identity to the large subunit of type I ribonucleotide reductase (SMITH et al. 2000A , SMITH et al. 2000B ), which catalyzes the synthesis of deoxyribonucleotides that are required for DNA replication and repair (JORDAN and REICHARD 1998 ). P. anserina het-c encodes a putative glycolipid transfer protein required for ascospore maturation (SAUPE et al. 1994 ).

Mutations that relieve vegetative incompatibility have also been identified in these two species. Mutations at the tol locus suppress mating-type vegetative incompatibility in N. crassa (NEWMEYER 1970 ; VELLANI et al. 1994 ), and tol mutations do not affect vegetative incompatibility mediated by other het loci (LESLIE and YAMASHIRO 1997 ). tol encodes a putative protein (SHIU and GLASS 1999 ) that shares three conserved amino acid regions with HET-6 in N. crassa and HET-E in P. anserina (SMITH et al. 2000). In P. anserina, mod (for modifier) mutants that suppress some phenotypic aspects of vegetative incompatibility have been described (SAUPE 2000 ). Mutations in mod-A relieve growth inhibition caused by interactions between nonallelic het loci, but cannot fully relieve HCD (BELCOUR and BERNET 1969 ). Complete suppression of vegetative incompatibility in a mod-A mutant requires a second mutation at the mod-B locus. mod-A has been cloned and encodes a novel protein (BARREAU et al. 1998 ). Mutations at another mod locus, mod-C, suppress nonallelic vegetative incompatibility between het-R and het-V, but not other nonallelic incompatibility interactions. mod mutants also show morphological or developmental defects. Extragenic mutations (mod-D through mod-G) that suppress these morphological or developmental defects have been identified. Two of these genes, mod-D and mod-E-1, have been characterized at the molecular level. They encode an -subunit of trimeric G protein and HSP90, respectively (LOUBRADOU et al. 1997 , LOUBRADOU et al. 1999 ).

The het-c locus in N. crassa has been used as a model system to understand molecular mechanisms of non-self-recognition (SAUPE et al. 1996 ; SAUPE and GLASS 1997 ; WU and GLASS 2001 ) and to assess selection mechanisms for polymorphisms at het loci (WU et al. 1998 ; MUIRHEAD et al. 2002 ). The het-c locus encodes three allelic specificities, termed het-c^OR, het-c^PA, and het-c^GR (nomenclature is based on het-c allelic specificity of laboratory strains). The polypeptides encoded by the three het-c allelic specificities are similar except for a variable domain of 34–48 amino acids. This polymorphic region is necessary and sufficient to confer het-c allelic specificity (SAUPE and GLASS 1997 ; WU and GLASS 2001 ). HET-C is a plasma membrane protein; non-self-recognition is correlated with the formation of a HET-C heterocomplex composed of HET-C polypeptides of alternative specificity (S. SARKAR, G. IYER and N. L. GLASS, unpublished data).

In an effort to identify components of vegetative incompatibility in addition to het-c, we identified a number of mutants that suppressed het-c vegetative incompatibility. Previously, we reported on the isolation of a mutant (ahc) identified from a strain that had "escaped" from het-c vegetative incompatibility (XIANG et al. 2002 ). "Escape" is a process whereby strains that contain het alleles of alternative specificity (and display slow, aconidial growth) recover to wild-type-like growth (faster growth and conidiation). The escape process has been associated with deletions and point mutations in genes that relieve vegetative incompatibility, such as within the het locus itself, or in suppressor loci (NEWMEYER 1970 ; DELANGE and GRIFFITHS 1975 ; SMITH et al. 1996 ; COUSTOU et al. 1999 ). The ahc mutant carries a large deletion (26 kbp) covering a number of predicted open reading frames (ORFs), including a locus, ham-2, which is required for hyphal fusion (XIANG et al. 2002 ). The introduction of ham-2 into the ahc mutant complemented morphological defects, such as the lack of aerial hyphae formation and hyphal fusion, but did not complement het-c vegetative incompatibility, indicating that a different ORF in the deletion region of the ahc mutant was required for this process. In this study, we isolated two additional mutants, vc1 and vc2 (for vegetative incompatibility and conidiation), from other escape strains that suppressed het-c-mediated vegetative incompatibility. The mutations in vc1 and vc2 were also deletions in the same chromosomal region as the ahc deletion; the three deletions overlapped in a region covering an ORF. Mutations in this ORF (named vib-1, for vegetative incompatibility blocked) restored growth and conidiation in het-c incompatible heterokaryons, but only partially suppressed HCD.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

N. crassa strains and heterokaryon tests:
The strains used in this study are listed in Table 1. Strains were cultured on Vogel's medium (VOGEL 1964 ) with supplements as required. Strains were cultured at 25° in 30- or 50-cm-long race tubes to measure growth rates. Crosses were performed on Westergaard's medium (WESTERGAARD and MITCHELL 1947 ). Most strains constructed in this study carry auxotrophic markers. To improve the fertility of these strains, 5–10% of recommended amounts of corresponding supplements for vegetative growth was added to the crossing medium or, alternatively, the helper strain FGSC 4564 (PERKINS 1984 ) was used to form heterokaryons, which were subsequently used as female strains in crosses. Heterokaryon tests were performed by co-inoculating 1–2 µl each from conidial suspensions (10⁵ conidia/µl) of two different auxotrophic strains onto plates or race tubes containing Vogel's medium (VOGEL 1964 ).

Nucleic acid isolation, Southern hybridization, PCR, and DNA sequence analysis:
Genomic DNA was isolated as described (LEE and TAYLOR 1990 ). Southern hybridization was performed as described (SAMBROOK et al. 1989 ). Primers used to amplify an internal fragment (800 bp) of het-c from the escape transformants were 5'-GGAGACATGGCGATATCG-3' and 5'-CTCACCCAACACGGAGTG-3'. The het-c^OR and het-c^PA PCR products were distinguishable by ApaI digestion. The het-c^OR PCR fragment has an ApaI site, while an ApaI site is absent in the het-c^PA PCR fragment. Primers used to amplify mutated regions from vib-1^rip mutants were 5'-AATCCGGTGCAGATGAATACTG-3' at position 54–75 bp downstream of the start codon in the vib-1 ORF and 5'-ATCTGCTTCGCAGACGTGAACGT-3' at position 1249–1272 bp downstream of the start codon in the vib-1 ORF. DNA sequence determinations were performed using the ABI automated DNA sequencing procedure at DNA Sequencing Facility, Berkeley, California (http://idrive.berkeley.edu/dnaseq/web).

Gene cloning:
DNA sequences of cosmid H57:G1 (XIANG et al. 2002 ) were from the Munich Information Centre for Protein Sequences (MIPS; http://www.mips.biochem.mpg.de/proj/neurospora/). DNA fragments carrying the vib-1 ORF were subcloned from cosmid H57:G1 into plasmid pCB1004, which confers hygromycin resistance (CARROLL et al. 1994 ). Spheroplast isolation and transformation were performed as described (SCHWEIZER et al. 1981 ).

Hyphal compartmentation and death assay:
Sterile pieces of cellophane (Fisher Scientific) were spread onto the surface of solid medium. Heterokaryons were forced by co-inoculating conidia of two strains (10⁵ conidia from each strain) onto the cellophane. At different time points after inoculation, the cellophane containing hyphae was peeled off from the surface of the medium and stained with 1% Evan's Blue (GAFF and OKONG'O-OGOLA 1971 ; JACOBSON et al. 1998 ). Stained hyphae were examined under bright field using a Zeiss Axioskop II microscope.

Native PAGE analysis of phosphatases:
All strains were cultured in liquid Vogel's medium (phosphate rich; VOGEL 1964 ) with corresponding supplements at 30° for 2 days. Cultures were subsequently transferred to either high-phosphate or low-phosphate conditions with constant shaking (100 rpm) at 30° overnight. For high-phosphate conditions, the liquid medium was replaced with fresh phosphate-rich Vogel's medium (VOGEL 1964 ). For low-phosphate conditions, the hyphae were washed thoroughly with distilled water and subsequently transferred to phosphate-depleted Vogel's medium, plus 0.05 mM KH₂PO₄. Harvesting of mycelia, protein extraction, and 8% polyacrylamide gel electrophoresis were performed as described (HOCHBERG and SARGENT 1973 ). Phosphatase activity was examined as described (DORN 1965 ) with minor modifications: the gel was flooded with 0.6 M acetate buffer (pH 4.8) containing 0.05% sodium -naphthyl acid phosphate (Sigma, St. Louis) plus 0.5% fast garnet G. B. C salt (Aldrich Chemical, Milwaukee) for 1 hr at room temperature. During staining, sodium -naphthyl acid phosphate is converted into -naphthol and phosphate by phosphatases. -Naphthol forms an insoluble dark-brown compound with fast garnet G. B. C salt, which is deposited where phosphatases are located in the gel. The reaction was terminated by washing the gel thoroughly with distilled water.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Identifying suppressors of het-c vegetative incompatibility:
Our strategy to identify additional components of vegetative incompatibility was to isolate mutants that suppressed the phenotypic aspects of het-c vegetative incompatibility, namely growth inhibition, repression of conidiation, and HCD. Incompatible partial diploids, heterokaryons, or transformants that contain het alleles of alternative specificity commonly escape from vegetative incompatibility after being maintained in culture for 2 weeks. The escape process is associated with a sudden increase in conidiation and growth rate of the cultures. Escape has been associated with mutations either in one of the het alleles or at a locus required to mediate vegetative incompatibility (NEWMEYER 1970 ; DELANGE and GRIFFITHS 1975 ; VELLANI et al. 1994 ; SMITH et al. 1996 ). In our study, the het-c^OR allele in plasmid pCB1004 (which confers hygromycin resistance; CARROLL et al. 1994 ) was transformed into C9-2 (het-c^PA thr-2 a; Table 1). Approximately 90 hygromycin-resistant incompatible transformants were transferred to slants. Sixty escape transformants were analyzed. Heterokaryon tests and PCR analysis of these escape transformants showed that 24 isolates contained only one het-c allele, either het-c^OR or het-c^PA. The other 36 escape transformants maintained both het-c^OR and het-c^PA alleles (data not shown). The coexistence of both het-c alleles in these escape transformants suggested that extragenic mutations had occurred that suppressed the phenotypic aspects of het-c vegetative incompatibility.

To identify the genetic basis of the escape phenotype in transformants retaining both het-c^OR and het-c^PA, 14 escape transformants were crossed with RLM 57-30 (het-c^OR pyr-4 A; Table 1). All of the escape transformants were fertile as males except for b-11-1, which showed greatly reduced fertility (XIANG et al. 2002 ). We selected for pyr-4 progeny (and thus het-c^OR, to which pyr-4 is closely linked). Heterokaryons were forced between pyr-4 a progeny from each cross and the parental escape transformant [genotype: het-c^PA (pCB1004::het-c^OR) thr-2 a]. If a suppressor mutation was not linked to het-c, the ratio of pyr-4 progeny forming compatible vs. incompatible heterokaryons with the parental escape transformant should be 1:1. In 11 crosses, all pyr-4 progeny (20 progeny from each cross) were incompatible with their parental escape transformants. These mutants presumably contained mutations linked to het-c on LGII (which was selected against in this cross) that suppressed vegetative incompatibility or vegetative incompatibility was suppressed by epigenetic mechanisms in the original escape transformants and was not inherited by the progeny. The twelfth cross (b-11-1 x RLM 57-30) led to the identification of the ahc mutant (XIANG et al. 2002 ). In the remaining two crosses (b-19-5 x RLM 57-30) and (c3-1 x RLM 57-30), half of the pyr-4 a progeny formed compatible heterokaryons with their parental transformants, b-19-5 or c3-1, respectively. These results suggested that b-19-5 and c3-1 carried mutations unlinked to het-c that suppressed het-c vegetative incompatibility.

Phenotypic characterization of the suppressors:
The pyr-4 progeny from the above crosses that formed compatible heterokaryons with b-19-5 or c3-1 showed a similar phenotype of profuse conidiation (Fig 1A; 9-39-10 is descended from b-19-5 and 24-24-9 is descended from c3-1). Approximately one-half of the thr-2 progeny (from both b-19-5 and c3-1 crosses with RLM 57-30) also showed a profuse conidiation phenotype, suggesting that the mutation that resulted in the profuse conidiation phenotype in these progeny was unlinked to pyr-4 or thr-2 (left arm of LGII, linked to het-c).

View larger version (68K): In this window In a new window Download PPT slide   Figure 1. Morphological phenotype of the ahc, vc, and vib-1 mutants as compared to a wild-type strain. (A) The phenotype of the 8-88 (ahc), 9-39-10 (vc1), 24-24-9 (vc2), and 80-32 (vib-1(1)) mutant strains as compared to wild type when grown on petri plates. Conidiation in a wild-type strain occurs around the perimeter of the plate, while the ahc, vc, and vib-1 mutants produce profuse conidia across the plates. (B) The growth rates of ahc, vc, and vib-1(1) mutants as compared to a wild-type strain.

To determine whether the profuse conidiation phenotype segregated with suppression of vegetative incompatibility, a thr-2 het-c^PA progeny from b-19-5 x RLM 57-30 showing the profuse conidiation pattern was crossed with RLM 57-30; 74 progeny were analyzed. Thirty-five progeny from the cross showed the profuse conidiation pattern, while the rest of progeny were wild type in phenotype. Progeny carrying the thr-2 (and thus het-c^PA) or the pyr-4 (and thus het-c^OR) marker were recovered. The pyr-4 het-c^OR progeny with the profuse conidiation phenotype were forced in heterokaryons with thr-2 het-c^PA progeny with the profuse conidiation phenotype (same mating-type pairing). The conidiation pattern of the resulting heterokaryons and their growth rates were indistinguishable from the mutants by themselves (Fig 2, A and B, heterokaryon (9-39-10 + 9-39-7); Fig 1A and Fig B). Analysis of crosses with c3-1 yielded similar results. We named the mutants carrying these mutations as vc1 (mutants derived from escape transformant b-19-5) and vc2 (for mutants derived from escape transformant c3-1).

View larger version (219K): In this window In a new window Download PPT slide   Figure 2. vib-1 mutants relieve repression of conidiation and growth inhibition associated with het-c vegetative incompatibility. (A) A compatible heterokaryon (9-1-5 + FGSC 4564) and a het-c incompatible heterokaryon (FGSC 4564 + C9-2). A heterokaryon between a het-cOR; vib-1(1) (X80-32) and a het-cPA; vib-1(1) (X80-49) strain shows profuse conidiation. Similarly, a vc1 heterokaryon [het-cOR; vc1 (9-39-10) + het-cPA; vc1 (9-39-7)] also shows profuse conidiation. (B) The vib-1 and vc1 mutations suppress growth rate inhibition mediated by allelic specificity differences at het-c and have growth rates similar to the mutants themselves (Fig 1B). The numbers below the columns indicate the heterokaryons with the same numbers shown in A. Standard error bars are shown. (C) The semirecessive nature of vc1 suppression of het-c vegetative incompatibility. The wild-type het-c incompatible heterokaryon is (I-1-51 + Xa-2). The heterokaryon developed conidia in a small section of the plate on the ninth day, which was possibly due to mutational processes resulting in escape. A compatible heterokaryon (I-1-51 + FGSC 4564) is also shown. The middle panel shows a heterokaryon between a het-cOR; vc1 strain and a het-cPA strain (9-39-10 + Xa-2). Conidiation was observed on the fourth day and spread across the plate by the ninth day.

In race tubes under normal laboratory conditions, the vc1 and vc2 mutants formed dense patches of conidia along the length of 50-cm race tubes that later filled in to form a continuous conidial mat. By contrast, a wild-type strain forms dense conidial patches only at the two ends of the race tube; conidiation in the middle of the race tube is suppressed by a high concentration of CO₂ (SARGENT et al. 1972 ). The vc1 and vc2 mutants have a slightly slower growth rate, 4–5 cm/day as compared to 6–7 cm/day for a wild-type strain (Fig 1B).

To determine whether the vc1 and vc2 mutations were recessive or dominant, heterokaryons were forced between the vc1 and vc2 mutants and wild-type strains with het-c alleles of the same or alternative het-c allelic specificity. Heterokaryons between a wild-type strain (FGSC 4564; Table 1) and the vc1 or vc2 mutants (9-39-10 or 24-24-9) of identical het-c specificity displayed wild-type growth rates and a normal conidiation pattern, indicating that the morphological phenotype of the vc1 and vc2 mutants was recessive (data not shown). Heterokaryons between het-c^OR; vc1 or het-c^OR; vc2 mutants with a wild-type het-c^PA strain (Xa-2; Table 1) displayed typical het-c vegetative incompatibility during the first 3 days. However, after 4 days an increase in growth rate was observed in the heterokaryons and conidiation began in the middle of the plate (Fig 2C). By contrast, an increase in growth rate and conidiation was not observed in wild-type het-c incompatible heterokaryons. These results indicate that the suppression of het-c vegetative incompatibility by the vc1 and vc2 mutations was not completely recessive.

Complementation between suppressor mutants:
The ahc, vc1, and vc2 mutants all show suppression of het-c vegetative incompatibility and have a similar phenotype, although the ahc mutant has additional morphological defects. The ahc mutant is female sterile, shows ascus-dominant developmental defects, and is severely restricted in its capacity to undergo hyphal fusion (XIANG et al. 2002 ). Heterokaryon tests were performed to determine if the three mutants can complement each other's morphological defects. A heterokaryon between an ahc mutant (X39-12) and vc1 (9-39-10) or vc2 (24-24-9) mutants (using a modified heterokaryon test; XIANG et al. 2002 ) of identical het-c specificity showed the morphology of a vc1 or vc2 mutant. Although the slow mycelial growth of the ahc mutant was complemented in a heterokaryon with the vc1 or vc2 mutant, the profuse conidiation pattern was not. A heterokaryon between vc1 mutant (X43-12) and vc2 mutant (24-24-9) displayed a similar growth rate and conidiation pattern of vc1 or vc2 mutants themselves. The above results suggest that the mutations resulting in the conidiation defect in ahc, vc1, and vc2 mutants were allelic. We previously mapped the mutation in the ahc mutant to chromosome V between lys-2 and ilv-2 (XIANG et al. 2002 ).

All three suppressor mutants carry deletions that overlap in an ORF:
The ahc mutant carries a deletion covering at least eight predicted ORFs, including ham-2, a locus involved in hyphal fusion (XIANG et al. 2002 ). Southern hybridization was performed to determine whether vc1 and vc2 mutants also carried deletions in this region. Probes were generated from cosmid H57:G1, which was previously shown to span most of the deletion in the ahc mutant (XIANG et al. 2002 ). Fig 3 shows that the 6280-bp HindIII fragment carrying ham-2 was present in both vc1 and vc2 mutants. However, a 5278-bp HindIII fragment is completely absent from the vc1 mutant. Most of this fragment is also absent from vc2. In the ahc mutant, both HindIII fragments are missing.

View larger version (45K): In this window In a new window Download PPT slide   Figure 3. Southern blots showing the vib-1 region in the ahc, vc1, and vc2 deletion mutants. (A) Restriction enzyme sites in the region covering ham-2 and vib-1. The sizes of HindIII DNA fragments are indicated. H, HindIII; B, BamHI. (B) Southern blots of genomic DNA probed with either HindIII5.2-6 or BamHI9-4. Genomic DNA was digested by HindIII. Lane 1, C9-2 (WT); lane 2, 8-88 (ahc); lane 3, 9-39-32 (vc1); lane 4, 24-24-9 (vc2).

The three deletions in ahc, vc1, and vc2 mutants overlap each other in a region (the 5278-bp HindIII fragment in Fig 3) that spans a predicted ORF. The ORF starts from position 61,496 bp (start codon) and ends at position 59,292 bp (stop codon) in contig 9a36 (http://www.mips.biochem.mpg.de/proj/neurospora/). An 4-kbp SacI-HindIII DNA fragment covering the ORF, SAH4-8 (Fig 4), was transformed into two vc1 mutants, 9-39-7 (het-c^PA thr-2; vc1 a) and 9-39-10 (het-c^OR pyr-4; vc1 a; Table 1). The introduction of SAH4-8 into 9-39-7 and 9-39-10 did not fully complement the profuse conidiation phenotype of the vc1 mutants. To determine whether the introduction of SAH4-8 restored vegetative incompatibility, heterokaryons were forced between 9-39-7 (SAH4-8) and 9-39-10 (SAH4-8) transformants. These heterokaryons displayed het-c vegetative incompatibility. We name the ORF required to restore het-c vegetative incompatibility in the vc1 mutant, vib-1.

View larger version (79K): In this window In a new window Download PPT slide   Figure 4. DNA sequence of the 4-kbp SacI-HindIII fragment, the predicted amino acid sequence of the vib-1 ORF, and the GC-to-AT transitions in the vib-1(1) and vib-1(2) mutants. The CAAT box, consensus sequence around the start codon CAGTATGGCA, and the polyadenylation site AATAAA are underlined. The NLS sequence is underlined and in boldface type. The KpnI and XhoI sites used for mutational analysis and the HindIII and SacI sites in the 3' UTR region used for deletion constructs are in boldface type. The six GC-to-AT transitions in vib-1(1) (X80-32 and X80-49) are marked by a boldface "A" (the mutant nucleotide) above "G" (the WT nucleotide). The 26 GC-to-AT transition mutations in vib-1(2) (X80-33) are marked by boldface "a" or "t" (the mutant nucleotide) above "G" or "C" (the WT nucleotide; accession number for vib-1: BK000540).

The predicted vib-1 ORF has two introns and three exons and encodes a predicted polypeptide of 670 amino acids (Fig 4). A consensus sequence for translation initiation sites in Neurospora, CAGTATGGCA (EDELMANN and STABEN 1994 ), is present around the predicted start codon. A CAAT box is located 54 bp upstream from the start codon and a polyadenylation signal, AATAAA, is 669 bp downstream from the predicted stop codon. The 3' untranslated region (3' UTR) is apparently important for vib-1 function. The introduction of deletion constructs bearing 302 bp (HindIII site) or 413 bp (SacI) of the 3' UTR (Fig 4) into the vc1 mutant does not restore the capacity for vegetative incompatibility.

vib-1^rip mutants:
Since the deletions in the ahc, vc1, and vc2 mutants could possibly cover additional ORFs besides vib-1, it was necessary to generate vib-1 mutants. Repeat-induced point (RIP) mutation is a naturally mutagenic mechanism in N. crassa (SELKER 1997 ) that acts on duplicated sequences in the genome, such as those introduced by transformation. An 1-kbp KpnI-XhoI DNA fragment from the 5' end of the vib-1 ORF (Fig 4) was transformed into C9-2 (Table 1). A C9-2 (KpnI-XhoI) transformant was crossed with a wild-type strain 9-1-5 (Table 1). Out of 60 progeny, 3 progeny showing a profuse conidiation pattern were recovered. The conidiation pattern of these progeny was similar to vc1 and vc2 mutants in plates, race tubes, and slants and their growth rates were also very similar to vc1 and vc2 mutants (Fig 1).

The 1-kbp region between KpnI and XhoI in the vib-1 ORF of all three RIP mutants, X80-32, X80-49, and X80-33, was amplified by PCR and cloned. DNA sequencing of the vib-1 fragment in the X80-32 and X80-49 mutants revealed 6 GC-to-AT transitions, which are typical for sequences that have undergone RIP (SELKER 1997 ). The mutations occurred in the same sites in the two mutants, suggesting that they came from the same mutagenic event (Fig 4). Three GC-to-AT transition mutations changed Met (55) to Ile, Val (227) to Met, and Trp (260) to a stop codon. The other 3 GC-to-AT transition mutations occurred in the middle of the first intron (after the stop codon). The X80-33 strain contained 26 GC-to-AT transition mutations, which caused 15 amino acid changes and three stop codons. The first stop codon is at Glu (250). Seven amino acid alterations—His (141) to Tyr, Cys (178) to Tyr, Cys (191) to Tyr, Met (200) to Ile, Ala (218) to Val, Val (227) to Ile, and Asp (249) to Asn—occurred in the vib-1 ORF before the first stop codon at amino acid (aa) 250 (Fig 4). The vib-1 alleles were named vib-1(1) (X80-32 and X80-49) and vib-1(2) (X80-33).

The ability of the vib-1 mutants to suppress het-c-mediated vegetative incompatibility was examined by forcing heterokaryons between X80-32 [het-c^OR pyr-4; vib-1(1) A] and X80-49 [het-c^PA thr-2; vib-1(1) A]. The (X80-32 + X80-49) heterokaryons displayed a phenotype that was similar to X80-32 or X80-49 mutants by themselves (Fig 1; Fig 2A and Fig B). Thus, mutations in vib-1 fully relieve growth inhibition and conidiation repression mediated by het-c vegetative incompatibility.

The vc1 and vc2 mutations were not completely recessive in heterokaryons with wild-type strains of alternative het-c specificity. Similar to the heterokaryon between a vc1 or vc2 mutant and a wild-type strain of alternative het-c specificity (Fig 2C), heterokaryons between a wild-type strain and a vib-1(1) mutant of alternative het-c specificity (X80-32 + Xa-3; Table 1) showed more conidiation and less growth inhibition after 3 days than did wild-type het-c incompatible heterokaryons. Thus, the vib-1(1) mutant phenotype was indistinguishable from the vc1 and vc2 deletion mutants.

vib-1 mutations alter the pattern of HCD mediated by het-c:
In a het-c incompatible heterokaryon, partial diploid or transformant, 20–30% of the hyphal compartments have plugged septa and are dead (JACOBSON et al. 1998 ; WU and GLASS 2001 ). The percentage of dead hyphal compartments is fairly uniform across the colony and has not been associated with any obvious developmental or morphological feature (such as hyphal fusion junctions). To assess whether HCD was also suppressed in the vib-1 mutant, we forced a heterokaryon between X80-32 [pyr-4 het-c^OR; vib-1(1) a] and X80-49 [thr-2 het-c^PA; vib-1(1) a] (Table 1) and stained the hyphae with the vital dye, Evan's Blue (GAFF and OKONG'O-OGOLA 1971 ). Fig 5B–D, shows the HCD pattern from a 2-day-old mycelium of the (X80-32 + X80-49) heterokaryon as compared to a wild-type het-c incompatible heterokaryon (Xa-3 + FGSC 4564, Fig 5A). HCD was not observed in the growth front of the mycelium of the (X80-32 + X80-49) heterokaryon (Fig 5B) in contrast to a wild-type het-c incompatible heterokaryon in which 20% dead hyphal compartments were observed across the colony (Fig 5A). However, from the growth front toward the inoculation point, HCD rates in the (X80-32 + X80-49) heterokaryon increased to 5–10% (Fig 5C and Fig D). The above results indicate that vib-1(1) mutation does not fully suppress HCD and that HCD in the vib-1(1) heterokaryons is dependent upon the age of the mycelium. HCD was also examined in vc1 heterokaryons with alternative het-c alleles (9-39-10 + 9-39-7; Table 1). The rate and pattern of HCD in the vc1 heterokaryons (which contained a deletion covering vib-1) was identical to that of the vib-1(1) (X80-32 + X80-49) heterokaryons (data not shown).

View larger version (139K): In this window In a new window Download PPT slide   Figure 5. The HCD pattern in heterokaryon [X80-32 (pyr-4 het-cOR; vib-1(1) A) + X80-49 (thr-2 het-cPA; vib-1(1) A)] is altered compared to a het-c incompatible heterokaryon. (A) Typical HCD in a 2-day-old het-c incompatible heterokaryon (Xa-3 + FGSC 4564), as observed by staining with the vital dye Evan's blue (GAFF and OKONG'O-OGOLA 1971 ). Arrows indicate dead hyphal compartments. Approximately 20% of the hyphal compartments are dead and were distributed randomly across the colony, consistent with previous observations (JACOBSON et al. 1998 ; WU and GLASS 2001 ). (B–D) HCD in a vib-1(1) heterokaryon (X80-32 + X80-49; Table 1). The 2-day-old vib-1(1) heterokaryon growth distance is 7 cm. (B) A region 0.5 cm away from the growth front; no HCD was observed. (C) A region 3 cm away from the growth front. (D) A region 6 cm away from the growth front; 5–10% HCD was observed. Bars, 20 µm.

VIB-1 has a predicted nuclear localization sequence and shows similarity to PHOG from Aspergillus nidulans:
Database searches revealed that an internal region of VIB-1 (from 157 to 415 aa) was similar to a putative phosphate-nonrepressible acid phosphatase (An PHOG) from A. nidulans (MACRAE et al. 1993 ; 41% identity). This region also has a high similarity to a predicted PHOG in Penicillium chrysogenum (Pc PHOG; MARX et al. 1995 ) and to a hypothetical protein, NCU0429.1, in N. crassa (Fig 6). NCU0429.1 is a predicted polypeptide of 719 amino acids (http://www-genome.wi.mit.edu/ annotation/fungi/neurospora/). VIB-1, An PHOG, and Pc PHOG did not show significant similarity to any other predicted proteins in public databases, including known phosphatases. A short region of similarity was identified between VIB-1, An PHOG, and Pc PHOG and a transcription factor from Saccharomyces cerevisiae, Ntd80p (23% identity over 143 aa, E value 0.003). Ntd80p is required for linking meiosis and sporulation (XU et al. 1995 ; CHU and HERSKOWITZ 1998 ). The N terminus (100 amino acids) and the C terminus (200 amino acids) of VIB-1 do not show high similarity to any other known or hypothetical proteins. Computational analysis (http://psort.ims.u-tokyo.ac.jp) showed that VIB-1 has a predicted bipartite nuclear localization sequence (NLS), RRLQFRIATANNGRRKE, from amino acid position 280 to 297. This type of NLS consists of two basic domains separated by 10 intervening amino acids (ROBBINS et al. 1991 ). The two basic domains in VIB-1 NLS are RR and RRK. This putative NLS was also conserved in the PHOG proteins (Fig 6).

View larger version (113K): In this window In a new window Download PPT slide   Figure 6. Alignment of VIB-1 with An PHOG, Pc PHOG, and NCU04729.1. An PHOG is a 330-aa predicted polypeptide from A. nidulans. NCU04729.1 is a predicted 719-aa-long polypeptide in N. crassa (http://www-genome.wi.mit.edu/annotation/fungi/neurospora/) and Pc PHOG is a predicted 593-amino-acid polypeptide from P. chrysogenum. The shaded boxes represent identical amino acids. The predicted NLS is demarcated by a thick line above the conserved sequence.

vib-1 mutants show reduced nonrepressible acid phosphatase activity:
To determine whether vib-1 is a structural gene for nonrepressible acid phosphatase in N. crassa, native PAGE analysis was used to detect phosphatase activity in the vib-1 mutants. The staining method employed in this study can detect the activities of both alkaline and acid phosphatases (DORN 1965 ). As shown in Fig 7, under low-phosphate conditions, four phosphatases were observed from the two parental wild-type strains (C9-2 and 9-1-5, lanes 1 and 2) and vib-1(1) (X80-32) and vc1 mutants (9-39-10, lanes 3 and 4). Under high-phosphate conditions, only nonrepressible phosphatases were detectable (Fig 7). The activity of the nonrepressible acid phosphatase (B in Fig 7) is lower in vib-1(1) and vc1 mutants compared to that in wild-type strains under both low- and high-phosphate conditions. There were no obvious differences in A, C, and D in Fig 7 phosphatases between the wild-type strains and the vib-1(1) and vc1 mutants, except that 9-1-5 had lower nonrepressible alkaline phosphatase activity under high- phosphate conditions. These data indicate that vib-1 does not encode the structural gene for phosphate nonrepressible acid phosphatase, but may instead encode a positive regulator of nonrepressible acid phosphatase activity.

View larger version (74K): In this window In a new window Download PPT slide   Figure 7. Native gel of phosphatase activity in N. crassa. Fifty micrograms of total protein was loaded per lane. Cultures were grown under low-phosphate or high-phosphate conditions, as described in MATERIALS AND METHODS, prior to protein extraction. The strains used were: lane 1, C9-2 (WT); lane 2, 9-1-5 (WT); lane 3, X80-32 [vib-1(1)]; and lane 4, 9-39-10 (vc1). The predicted phosphatases are A, repressible alkaline phosphatase; B, nonrepressible acid phosphatase; C, nonrepressible alkaline phosphatase; and D, repressible acid phosphatase (HOCHBERG and SARGENT 1973 ).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

In this study and previous work (XIANG et al. 2002 ), we identified mutants that suppressed vegetative incompatibility from transformants that escaped from vegetative incompatibility mediated by the het-c locus. By contrast to partial diploids or heterokaryons that escaped from vegetative incompatibility mediated by mating type or het-6 (DELANGE and GRIFFITHS 1975 ; SMITH et al. 1996 ), the majority of het-c escape transformants (60%) maintained both het-c alleles. The compatibility of these escape transformants presumably was caused by mutations in genes required to mediate vegetative incompatibility or by epigenetic mechanisms. The mutations in these strains could be genetically characterized because they were fertile, in contrast to the near-sterility of escaped partial diploids used to identify mutations in tol and het-6 (VELLANI et al. 1994 ; SMITH et al. 1996 ). Crosses between 14 escape transformants and a wild-type strain revealed three suppressor mutations, ahc, vc1, and vc2. The compatibility of the other 11 escape transformants might have been caused by mutations closely linked to the het-c locus or by epigenetic mechanisms that silenced het-c during vegetative growth but which were not transmitted to progeny (COGONI and MACINO 1999 ).

The three suppressor mutants all carried independent deletions in the same region of chromosome V, between lys-2 and ilv-2. The ahc deletion is 26 kbp and the deletions in vc1 and vc2 are 19 and 8 kbp, respectively (our unpublished data). It is unclear how these deletions occurred, but the removal of vib-1, a locus responsible for mediating het-c vegetative incompatibility, is probably a major factor involved in their appearance. Hyphae containing a nucleus with a deletion or mutation in vib-1 would have a selective advantage for growth and conidiation in an otherwise het-c incompatible colony.

Since the ahc, vc1, and vc2 deletions possibly covered genes in addition to vib-1, we generated vib-1 mutants. The phenotype of the vib-1 mutants was identical to that of the vc1 and vc2 mutants. The distinguishable character of the vc and vib-1 mutants is the profuse conidiation phenotype, suggesting that vib-1 negatively regulates conidiation. A characteristic phenotypic consequence of vegetative incompatibility in N. crassa is the suppression of conidiation (MYLYK 1975 ; PERKINS 1975 ). These data suggest that VIB-1-mediated suppression of conidiation is activated or maintained during het-c vegetative incompatibility.

The profuse conidiation pattern of the vib-1 mutants is similar to cpd-1 and cpd-2 (HASUNUMA and SHINOHARA 1985 , HASUNUMA and SHINOHARA 1986 ), cr-1 (see PERKINS et al. 2001 ), and gna-3 (KAYS et al. 2000 ) mutants, all of which have reduced cAMP levels. We speculated that cAMP signaling might be involved in het-c vegetative incompatibility. The profuse conidiation of a cr-1 mutant (defective in adenylyl cyclase), however, was fully suppressed in cr-1 het-c^OR/het-c^PA partial diploids (our unpublished data). These data suggest that het-c vegetative incompatibility is independent of cAMP signaling. This result is consistent with data showing that the cAMP pathway is not involved in vegetative incompatibility in P. anserina (LOUBRADOU et al. 1999 ).

In addition to the suppression of conidiation, vegetative incompatibility also results in growth inhibition and HCD. Mutations in vib-1 fully relieve het-c-mediated growth inhibition in heterokaryon tests. They cannot, however, fully relieve HCD caused by het-c vegetative incompatibility. In vib-1 or vc1 heterokaryons with alternative het-c alleles, HCD was age dependent and occurred mainly in older hyphae. This pattern is in contrast with the HCD pattern in a wild-type het-c incompatible colony in which 20% HCD takes place in both young and old mycelia (JACOBSON et al. 1998 ; WU and GLASS 2001 ). The HCD pattern caused by vib-1 mutations is similar to that caused by mod-A mutations in P. anserina (BERNET et al. 1973 ). It is possible that the remaining level of HCD observed in vib-1 heterokaryons with alternative het-c alleles is caused by NCU0429.1, a protein sharing high similarity with VIB-1. We are currently mutating NCU0429.1 to see whether a full relief of HCD in (het-c^PA; vib-1 + het-c^OR; vib-1) heterokaryons will occur.

VIB-1 is a predicted polypeptide of 670 amino acids. It has been annotated to be related to acid phosphatases (Swissprot accession no. Q05534; MIPS: http://www.mips.biochem.mpg.de/proj/neurospora). The internal 240-amino-acid region of VIB-1 has a high similarity to An PHOG and Pc PHOG, possible phosphate nonrepressible acid phosphatases (nrAPase) in A. nidulans and P. chrysogenum, respectively, but is not similar to any other known phosphatases. The introduction of An phoG into A. nidulans enhanced acid phosphatase activity in a pacG mutant under high-phosphate conditions (MACRAE et al. 1993 ). The pacG mutant reduced activity of nonrepressible acid phosphatase (CADDICK and ARST 1986 ). An PHOG was thus suggested to be either a nonrepressible acid phosphatase or a regulator of acid phosphatase activity. The Pc phoG gene was cloned by hybridization to An phoG (MARX et al. 1995 ); neither an An phoG nor a Pc phoG mutant has been described in the literature. Our native gel analysis showed that vib-1 mutants possess all four phosphatases described in N. crassa (KUO and BLUMENTHAL 1961A , KUO and BLUMENTHAL 1961B ; NYC et al. 1966 ; JACOBS et al. 1971 ), including nonrepressible acid phosphatase. Thus, vib-1 is not a structural gene for a nonrepressible acid phosphatase. However, VIB-1 may be a positive regulator of nrAPase because the activity of nrAPase was significantly reduced in vib-1(1) and vc1 mutants. VIB-1 has a predicted NLS and could be a nuclear protein and thus may regulate multiple cellular processes at a transcriptional level. Interestingly, VIB-1 also displays limited similarity to Ntd80p in S. cerevisiae, a transcriptional factor involved in gametogenesis (XU et al. 1995 ; CHU and HERSKOWITZ 1998 ). However, the domain of Ntd80p required for DNA binding to transcriptional targets has not been well defined (E. R. JOLLY, and I. HERSKOWITZ, personal communication).

The relationship between HET-C and VIB-1 is unclear. het-c null mutants have no phenotype, with the exception that they are fully compatible in heterokaryons with strains with alternative het-c specificity (SAUPE et al. 1996 ). Non-self-recognition during het-c vegetative incompatibility has been associated with the formation of a heterocomplex composed of het-c proteins of alternative specificity (S. SARKAR, G. IYER and N. L. GLASS, unpublished data). It is possible that VIB-1 is required for formation of a HET-C heterocomplex and thus that the vib-1 mutant suppresses het-c vegetative incompatibility because the HET-C heterocomplex fails to form. Alternatively, the HET-C heterocomplex may form in the vib-1 mutant, but vegetative incompatibility is not triggered. Experiments are currently underway to determine the effect of the vib-1 mutation on HET-C heterocomplex formation.

We conclude that vib-1 is a gene involved in multiple cellular processes. In addition to vegetative incompatibility, it is also implicated in the regulation of conidiation and nrAPase. What is the relationship between het-c vegetative incompatibility and nrAPase? The first possibility is that VIB-1 regulates multiple cellular processes, such as nrAPase production, conidiation, and vegetative incompatibility, and thus there is no causal connection between vegetative incompatibility and nrAPase. The second possibility is that VIB-1 regulates nrAPase, which is required to mediate vegetative incompatibility. In N. crassa, it has been speculated that nrAPase participates in metabolic control systems rather than in phosphate uptake (KUO and HERSKOWITZ 1961b). Thus, nrAPase may be involved in the regulation of certain cellular processes in N. crassa, including vegetative incompatibility. We are currently conducting experiments to distinguish these two possibilities.

	ACKNOWLEDGMENTS

We thank Drs. Robert Metzenberg, Patrick Shiu, and Jennifer Wu for technical help and Dr. George Haughn for helpful suggestions. We thank members of the Glass laboratory for critical reading of this manuscript. This work was funded by a National Institutes of Health grant (GM-60468-01) to N.L.G.

Manuscript received April 8, 2002; Accepted for publication June 10, 2002.

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