A Host Factor Involved in Hypovirus Symptom Expression in the Chestnut Blight Fungus, Cryphonectria parasitica

Virus strains.Table 1 shows the virus strains used: the prototype hypovirus CHV1-EP713 (28); its deletion mutants Δp29, Δp40b, and Δp69b (36); and the site-directed mutant Cys(72) (35). The type member of the genus Mycoreovirus, MyRV1-Cp9B21, was described previously (19, 39).

Fungal strains and culturing.The field fungal isolate EP155 (ATCC 38755) and transformants with the EP155 background were infected or uninfected with viruses (Table 1). Fungal colonies were cultured at 25°C to 27°C for 5 to 7 days in potato dextrose broth (PDB) (Difco) for RNA/DNA extraction and spheroplast preparations or on potato dextrose agar (PDA) (Difco) for phenotypic observation. Fungal strains were cultured on regeneration plates (9) for maintenance and stored at 4°C in a refrigerator until use.

Transfection and transformation of C. parasitica spheroplasts.Spheroplasts were prepared from fungal strains with the EP155 backgrounds that were cultured in PDB as described previously by Churchill et al. (9) and transformed with full-length cDNA clones of Cys(72) or genomic DNA fragments as described previously by Choi and Nuss (8). Transfection of EP155 with in vitro-synthesized RNA from cDNA of CHV1-EP713 and its mutant strains or purified virus particles of MyRV1 was carried out according to methods described previously by Suzuki et al. (35) and Hillman et al. (18), respectively.

Phenotypic measurements of fungal colonies.Virulence was measured in apples (cv. Jonathan Gold) as areas of lesions induced by fungal strains as described previously by Fulbright (15) and Hillman et al. (17). Sporulation and pigmentation were evaluated using methods described previously by Hillman et al. (17, 18). Fungal strains were cultured for 14 days on PDA plates that were 60 mm in diameter: the first 4 days on the bench top and the last 10 days under moderate light of approximately 3,000 lx using a 16-h photoperiod. Produced spores were liberated in 6 ml of 0.15% Tween 20 and filtered through a double layer of Miracloth (Calbiochem). Conidia were counted with the aid of a hemocytometer. To measure pigmentation, strains were cultured in 20 ml PDB for 12 days and harvested onto Miracloth. The mycelia (300 mg) were homogenized using a mortar and pestle under liquid nitrogen. After the addition of 3 ml ethanol, the absorbance at 454 nm was determined using an Ultrospec 2000 spectrophotometer (Pharmacia Biotech) (18). Alternatively, pigment production on PDA cultures was visually estimated.

Mutagenesis by random plasmid integration of a strain transformed with the virus cDNA clone.A full-length cDNA clone of CHV1-EP713 mutant strain Cys(72) that had a Cys-to-Gly substitution in p29 at Cys⁷² and that caused phenotypic alterations similar to those induced by Δp29 in EP155 when transfected was described previously by Suzuki et al. (35). After being liberated from the pRFL4-based plasmid by NotI, Cys(72) cDNA was transferred into a transformation vector, pCPXBn1 (14), with the benomyl-resistant gene as the selectable marker to form pBn1-Cys(72). Recombinant DNA procedures were performed according to methods described previously by Sambrook and Russell (30).

One of the transformant strains with pBn1-Cys(72), TCys(72)-1, was mutagenized by random insertion with a plasmid (pHygR) that contained the hygromycin B phosphotransferase gene (hph) controlled by the Aspergillus nidulans trpC promoter (Ptrp) and terminator (Ttrp) (10) at the EcoRV site of pBluescript SK(+) (Stratagene) (sequence is available upon request). Spheroplasts of TCys(72)-1 were prepared as described above, retransformed with pHygR, and subjected to single conidial isolation to obtain homokaryons with insertion mutations. The resulting transformants were screened for mutants with phenotypes that were different from that of the parental strain TCys(72)-1. This screening procedure was based on the visual estimation of fungal phenotypes and comparison with TCys(72)-1 cultured on PDA plates in parallel.

The genetic stabilities of the phenotypes of TCys(72)-1 and mutated fungal strains were examined by comparing the colony morphologies and drug resistances of approximately 50 single conidial isolates and isolates passaged four times to those of the original isolates.

DNA isolation and Southern blot analysis.Total nucleic acids were prepared from mycelia of fungal strains cultured in 20 ml PDB as described previously by Suzuki et al. (37). Chromosomal DNA was enriched by digestion with RNase A overnight at 37°C, followed by phenol-chloroform extraction and ethanol precipitation. Ten micrograms of DNA was digested with the appropriate restriction enzymes overnight at 37°C. The digested DNA was treated with phenol-chloroform, ethyl alcohol precipitated, and suspended in water. The DNA was applied to a well in 0.7% agarose gel, electrophoresed, and blotted onto a Hybond-N+ nylon membrane (Amersham Biosciences, Buckingham, England). The membrane was washed twice in 5× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate [pH 7.0]), baked at 80°C for 10 min, and then probed with digoxigenin (DIG)-11-dUTP-labeled DNA fragments amplified from genomic DNA or cDNA according to methods recommended by the manufacturer (Roche Diagnostics GmbH, Mannheim, Germany). Prehybridizations and hybridizations were carried out at 42°C using the DIG Easy-Hyb Granules kit according to the manufacturer's instructions (Roche). Hybridized bands were detected with a DIG detection kit and a CDP Star kit (Roche). Chemiluminescent signals were visualized on film.

RNA preparation and Northern blot analysis.Total nucleic acids were prepared from mycelia of fungal strains cultured in PDB as described above. Total RNA was enriched by eliminating fungal chromosomal DNA with two-round digestion of the extracted nucleic acids with RQ1 DNase I (Promega, Madison, Wis.) in the presence of an RNase inhibitor (40 units) (Toyobo, Tokyo, Japan) for 1 h at 37°C. After phenol, phenol-chloroform, and chloroform extractions and ethanol precipitation, total RNA was suspended in sterile distilled water at a final optical density at 260 nm of 25.

For Northern blot analysis, single-stranded nucleic acids were precipitated from total nucleic acids with LiCl (2 M) and treated twice with RQ1 DNase I. Total single-stranded RNA (ssRNA) was extracted twice by phenol-chloroform and suspended in sterile distilled water at an optical density at 260 nm of 25. When needed, poly(A) fractions were prepared from the total ssRNA isolated from fungal mycelia using an mRNA isolation kit (Roche Diagnostics). Total ssRNA (25 μg) or poly(A) RNA (10 μg) was denatured, electrophoresed, and capillary transferred onto a Hybond-N+ nylon membrane (Amersham Biosciences, Buckingham, England) as described previously by Suzuki et al. (37). Procedures for prehybridization, hybridization, and detection of hybridization signals were described previously (37).

Plasmid rescue and I-PCR.Genomic DNA (10 μg) obtained from fungal strains was cut with an appropriate restriction enzyme, which digested the inserted plasmid at one site. After extraction by phenol-chloroform, the digested products were self-linked by T4 DNA ligase (3 units) at 4°C overnight. For I-PCR, the ligated DNA (200 ng) was mixed with the primer set HG15 and HG18 (10 pmol) (see Table S1 in the supplemental material for sequences) and the long and accurate (LA) Taq polymerase (2.5 units) (Takara) in a 50-μl reaction solution (10 mM Tris-HCl [pH 8.0], 50 mM KCl, 2.5 mM deoxynucleoside triphosphate). Reactions consisted of 4 min of initial denaturation at 94°C followed by 35 thermal cycles: a denaturation step at 94°C for 30 s, an annealing step at 52°C for 30 s, an extension step at 72°C for 10 min, and a final extension step at 72°C for 10 min. When needed, nested PCR was performed on I-PCR products in which the primer set consisting of HG21 and HG12 was used under the same reaction conditions as those used for I-PCR.

Nucleotide sequencing of genomic DNA and cDNA of nam-1.The sequence of genomic DNA containing a 8.5-kb region of the nam-1 gene was determined using a primer-walking method. First, I-PCR products were sequenced with hph-specific primers to obtain the sequences of the junction regions of pHygR integrated into chromosomes and the restriction enzyme sites used in I-PCR. The 6.5-kb DNA fragment that encoded the nam-1 gene was amplified by genomic PCR with the primer set Nam5 and Nam25, whose sequences were obtained by sequencing of the I-PCR products, and purified by a Wizard SV gel and PCR clean-up system kit (Promega) according to the manufacturer's protocol. The PCR fragments or fragments cloned into the pGEM-T Easy vector (Promega) were sent to Macrogen, Inc. (Seoul, South Korea) to be sequenced with a series of Nam primers listed in Table S1 in the supplemental material.

An almost-full-length cDNA of nam-1 was synthesized by using SuperScript II reverse transcriptase (Invitrogen) and an oligo(dT) primer and amplified by two rounds of PCR with the primer set NamC1 and NamC15 and subsequently with the primer set NamC2 and NamC16. This fragment was sequenced using a series of Nam and NamC primers (see Table S1 in the supplemental material). Both terminal sequences of nam-1 mRNA were determined by using an RLM-RACE kit (Ambion). For 3′ rapid amplification of cDNA ends (RACE), cDNA was synthesized from mRNA isolated as described above using the 3′ RACE adapter containing a dT tail as a primer; amplified by PCR with a primer set, the 3′ RACE outer primer and a gene-specific primer, Nam59; and subsequently amplified with the 3′ inner primer and Nam60. The protocol for 5′ RACE was described previously (39). The deoxyoligonucleotide supplied in the kit was ligated into mRNA treated with calf intestinal phosphatase and tobacco acid pyrophosphatase (decapping enzyme). The resulting RNA was subjected to cDNA synthesis using random decamers and SuperScript reverse transcriptase and PCR amplification with primer sets consisting of the 5′ RACE outer primer and Nam81 as well as the 5′ inner primer and Nam82.

Both genomic and complementary DNAs were sequenced at least twice from two directions. The determined sequences were examined for PCR misincorporation.

Complementation assay.A few plasmid clones were prepared for functional complementation of the namA mutant. After determining the sequences of both flanking regions of the pHygR insertion sites, genomic DNA fragments of the wild-type allele were amplified on EP155 chromosomal DNA using LA PCR with oligonucleotide primer sets Nam5 and Nam25, Nam5 and Nam30-C, and Nam5 and Nam42 (see Table S1 in the supplemental material for sequences). The resulting fragments of approximately 6.5 kb, 7.5 kb, and 8.5 kb, respectively, were cloned into the pGEM-T Easy vector (Promega). After digestion with NotI, the DNA fragments were moved to vector pBSD1 to obtain the complementation constructs pBSDnamC1, pBSDnamC2, and pBSDnam3. pBSD1 contained a coding domain for blasticidin S-deaminase that confers blasticidin S resistance at the EcoRV site of pBluescript S(+) (Stratagene). Spheroplasts of the namA mutant were transformed with the complementing constructs as well as the empty vector pBSD1. The resulting transformants were selected on PDA containing 150 μg/ml blasticidin S (Nacalai Tesque, Kyoto, Japan) or a 1/10,000 (vol/vol) dilution of the blasticidin S-based fungicide Bla-S (Nihon Tokushu Nouyaku, Nihonmatsu, Japan).

Construction of a replacement vector and targeted gene disruption.Targeted gene disruption was achieved by homologous recombination-based gene replacement previously established for this fungus (16, 21). A disruption plasmid clone was constructed using pHygR with the pBluescript S(+) background. A genomic DNA fragment of approximately 1.7 kb (map positions −698 to 977) was amplified by PCR using chromosomal DNA of EP155 as the template and the primer set Nam5 and Nam10 (see Table S1 in the supplemental material) and cloned between the two NotI sites of the pGEM T-Easy vector (Promega). After liberation with NotI, the fragment was subcloned into the NotI site of pHygR. The 3′ 1.3-kb region of nam-1 (map positions 3851 to 5116) was amplified by PCR using primers Nam2-C and Nam25. This fragment was inserted into the HindIII and KpnI sites of the intermediate plasmid. The resulting replacement vector was then used to transform TCys(72)-1, the parental strain for mutagenesis.

Nucleotide sequence accession number.The sequence reported in this study was deposited in the GenBank/EMBL/DDBJ database under accession number AB300388.

Transformation of EP155 with a full-length cDNA clone of Cys(72).Several mutants of prototype hypovirus strain CHV1-EP713 were characterized previously (35, 36). Surmising that different host factors could be isolated using different viral mutants, we chose a site-directed mutant, Cys(72), that induced mild symptoms, allowing the host to sporulate relatively well (35). This moderate level of conidiation was important for the screening procedure that required single conidial isolation to obtain homokaryons. Full-length cDNA of Cys(72) was moved into pCPXBn1 to obtain pBn1Cys(72) and was used to transform EP155. More than 10 transformants with pBn1Cys(72) were selected on benomyl-containing PDA and examined for copy numbers of the viral cDNA integrated into the host chromosomes, phenotypic characteristics, and virus replication. Most benomyl-resistant strains showed symptoms typical of strains transfected with the in vitro-synthesized RNA of Cys(72) (35). However, they have different copy numbers of pBn1Cys(72), ranging from 1 to 3, and relative genetic stability when examined for their colony morphologies and abilities to grow on benomyl-PDA. Considering copy number and genetic stability, one of the transformants, TCys(72)-1, was selected for mutagenesis by plasmid insertion. In Southern blot analyses (Fig. 1), a probe hybridizing the p29 coding sequence detected only a single major band when the chromosomal DNA of TCys(72)-1 was digested by XbaI, KpnI, MfeI, and AfeI (Fig. 1). These enzymes digest the cDNA sequence of Cys(72) at one site (KpnI or XbaI) or more than one site (the rest of enzymes) downstream of the p29 coding sequence, while the foundation vector pCPXBn1 has no (AflI or MfeI), one (XbaI), or more than one (KpnI) cut site. Southern analysis indicated that TCys(72)-1 contained one copy of pBn1Cys(72) per genome. Integrated cDNA was maintained through the next generations in which virus infection could be launched by the transmitted Cys(72) or initially from transcripts of the chromosomally integrated Cys(72) cDNA and, subsequently, by autonomous replication by the viral RNA polymerase complex (27).

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FIG. 1.

Southern blot analysis of the parental strain TCys(72)-1. (A) Ten micrograms of genomic DNA prepared from virus-free EP155 and TCys(72)-1 was digested with four restriction enzymes (shown above the lanes). The restriction enzymes used were XbaI, KpnI, MfeI, and AfeI. Digested DNA was separated on a 0.7% agarose gel, transferred onto a Hybond N+ nylon membrane, and probed with a 0.7-kb, DIG-11-dUTP-labeled p29 coding sequence. M refers to the 1-kb DNA ladder size marker (Fermentas, Life Science). (B) Physical map of the transforming plasmid pBn1-Cys(72). A circular form of pBn1-Cys(72) was used for the transformation of EP155 to prepare TCys(72)-1. Nevertheless, its linear form is shown to scale schematically. The digestion sites of restriction endonucleases and the probe sequence used in Southern blot analysis (A) are shown schematically. pBn1-Cys(72) is based on pCPXBn1, in which the cDNA to the genome of the Cys(72) mutant virus is flanked by the C. parasitica glyceraldehyde-phosphate dehydrogenase gene promoter (Pgpd) and terminator (Tgpd). The shaded bar represents the sequence domains, derived from the foundation vector pCPXBn1 (14), which include the benomyl resistance gene, ampicillin resistance gene, and ColE1 origin.

Screening of the mutant collection for phenotypes different from that of TCys(72)-1.Spheroplasts of TCys(72)-1 were transformed with pHygR for mutagenesis by random plasmid insertion and selected on hygromycin-containing PDA. A mutant population of homokaryonic single conidial isolates was created and observed for phenotypes differing from those of parental strain TCys(72)-1 showing symptoms caused by Cys(72). Of the 1,052 transformants containing pHygR, 109 showed altered phenotypes, while many of the remaining transformants manifested a phenotype that was indistinguishable from that of TCys(72)-1. One of the altered strains, designated namA, was subjected to further characterization in this study. EP155 infected by CHV1-EP713 was severely reduced in sporulation and pigmentation relative to virus-free EP155, while the parental strain TCys(72)-1 was restored in pigmentation like EP155 infected by a p29 null mutant virus, Δp29, as previously reported (35). However, mutant namA had irregular margins with a number of “protrusions” and was reduced in pigment production and asexual sporulation. The namA phenotype resembled that of the EP67 strain containing a virus of Michigan origin reported previously by Anagnostakis (see Fig. 6a, bottom row, in reference 2). The numbers and sizes of the protrusions of namA on PDA varied between cultures (e.g., compare namA cultures in Fig. 2A and 7C), possibly due to slight environmental differences or unknown factors. EP155 anastomosed with namA showed a phenotype similar to that of TCys(72)-1, indicating that Cys(72) cDNA integrated into mutant chromosomes was unaltered during mutagenesis and launched infection by Cys(72).

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FIG. 2.

Characterization of the namA mutant. (A) Colony morphologies of TCys(72)-1, namA, and their virus-cured strains. The namA strain and its parental strain, TCys(72)-1, were cured of the Cys(72) mutant virus to obtain VC-namA and VC-TCys(72)-1, respectively, by a series of single conidial isolations. The strains were cultured on PDA medium for 10 days. VC-TCys(72)-1 and VC-namA showed indistinguishable phenotypes compared to virus-free EP155. (B) Southern blot analysis for namA and VC-namA. Ten micrograms of chromosomal DNA of Cys(72)-containing namA and virus-free VC-namA mutant strains was digested with restriction enzymes (HindIII, SacI, SpeI, EcoRI, and PstI) (shown above the lanes). The enzymes HindIII, SacI, and SpeI cut at one site in the pHygR plasmid sequence outside the hph gene, while EcoRI and PstI cleaved at two sites within and outside the hph gene. The number of hybridizing bands confirmed the single insertion of pHygR into the namA mutant. Chromosomal DNA of nontransformant strain EP155 was also digested by HindIII. After transfer to a Hybond N+ nylon membrane, digested DNA was hybridized to a DIG-11-dUTP-labeled hph probe. The estimated sizes of the hybridizing bands are indicated at the right. M refers to the 1-kb DNA ladder size marker (Fermentas). (C) Colony morphology of VC-TCys(72)-1 and VC-namA strains infected with MyRV1. The photographs were taken at day 10 after inoculation of PDA. (D) Phenotypic properties of the VC-TCys(72)-1 and VC-namA strains infected with hypovirus strains. The hypovirus variants Δp29, Δp40b, and Δp69b and wild-type CHV1-EP713 were introduced into the fungal strains VC-TCys(72)-1 and VC-namA (see Materials and Methods for virus introduction). Phenotype alterations similar to those of the namA mutant are shown in VC-namA infected by Δp29- and Δp40b- but not in Δp69b-infected VC-namA. Strain VC-TCys(72)-1 induced phenotypic changes indistinguishable from those of EP155 upon virus infection irrespective of virus strains. Back views of VC-namA strains infected with CHV1-EP713 and Δp29, showing the characteristic white corrugated edge of namA, are shown. VC-TCys(72)-1 either uninfected or infected with the individual virus was cultured in parallel for comparison.

The phenotype of the mutant namA is altered in a virus infection-specific way.To determine whether the phenotype of the mutant namA was altered in the absence of virus infection or in the presence of other virus strains, we attempted to cure the mutant namA strains of the Cys(72) virus. During the course of the genetic stability assay, pBn1-Cys(72) in namA was found to be relatively stably maintained through asexual spores. However, of 36 single conidial isolates derived from namA, 2 lost benomyl resistance and/or signs of virus infection, possibly due to the deletion of the integrated pBn1-Cys(72). This phenomenon for transformants with cDNA of wild-type CHV1-EP713 was previously reported by Chen et al. (4). Taking advantage of this property, the namA strain was cured of Cys(72) by single conidial isolation. As expected, a few germlings (3 of 60) manifested the typical virus-free phenotype characterized by orange pigment production and high sporulation levels (Fig. 2A). These virus-free (virus-cured) strains were termed VC-namA. Likewise, Cys(72) was cleared from TCys(72)-1 to obtain a virus-free strain, VC-TCys(72)-1. These virus-cured strains, VC-namA and VC-TCys(72)-1, did not harbor viral genomic double-stranded RNA (data not shown) and showed colony morphology attributes indistinguishable from those of wild-type EP155 (Fig. 2A and Table 2). VC-namA receiving Cys(72) back after being fused with Cys(72)-infected EP155 restored the namA phenotype (data not shown). This showed that the mutant phenotype found in namA was virus infection specific.

Southern blot analysis of the namA chromosomal DNA with SalI and XhoI suggested the integration of one copy of pHygR in the chromosomes of namA (data not shown). Utilizing different restriction enzymes, we confirmed it and determined whether the integrated pHygR was retained in VC-namA. When chromosomal DNA was digested by HindIII, SacI, or SpeI, Southern blot analysis revealed that a probe containing the hph sequence was detected in single bands at the same migration positions corresponding to approximately 8 kb in both namA and VC-namA (Fig. 2B). These three restriction enzymes cut the pHygR plasmid at one single digestion site outside the hph sequence. Somehow, digestion with HindIII and SacI provided the hybridization signals with different intensities for namA and VC-namA. Digestion with EcoRI or PstI by cleaving the pHygR sequence at two sites, i.e., within and outside the hph sequence, resulted in the detection of two signals of over 10 kb and 0.6 kb (for EcoRI) and of 4.5 kb and 0.7 kb (for PstI) in namA and VC-namA (Fig. 2B).

These combined results indicated that mutant namA contained a single copy of the mutagenic plasmid pHygR, which was maintained stably in VC-namA.

Phenotypic changes in VC-namA is virus species and virus strain specific.As shown in Fig. 2, the namA mutant reverted to the wild-type (parental) phenotype upon virus elimination, and infection of VC-namA with certain strains of CHV1-EP713 was required to regain the phenotype. We next determined whether the phenotype alteration manifested by VC-namA was virus species specific. A relatively well-characterized mycoreovirus of C. parasitica, MyRV1, was chosen because a transfection protocol was available for this virus (18). Interestingly, unlike the VC-namA strain infected with Cys(72), no significant difference was observed in colony morphologies of VC-namA and EP155 or VC-TCys(72)-1 that were infected with MyRV1 (Fig. 2C). Thus, the mutated gene in VC-namA was considered to be involved in symptom induction by wild-type CHV1 but not by MyRV1.

Furthermore, several CHV1-EP713 mutants (Δp29, Δp40b, and Δp69b) were introduced into VC-namA and VC-TCys(72)-1 by fusing them with the donor strain EP155 infected with the respective virus strains. Upon infection with the wild type, CHV1-EP713, Δp29, or Δp40b, VC-namA showed a reduced growth rate and repressed pigmentation with irregular mycelial extensions (Fig. 2D). As observed with namA, levels of growth suppression and numbers and lengths of hyphal extensions were different between cultures of single strains infected with each virus. These phenotypic characteristics were very similar to those exhibited by namA, being distinguishable from VC-TCys(72)-1 or EP155 infected with the respective viruses that showed regular symptoms as previously reported (36). Irregular colony morphology of virus-infected VC-namA was concurrent with the firm colony edge (Fig. 2D). Interestingly, no significant differences in colony morphology were, however, detected between VC-namA and EP155 or VC-TCys(72)-1 strains when infected with Δp69b (Fig. 2D).

Comparative measures of other biologic properties of VC-namA and EP155 infected with different viral strains are summarized in Table 2. Conidiation levels are usually decreased upon infection by CHV1-EP713 derivatives, although different viral strains have different magnitudes of impact (36). With abnormality of colony morphology, a further decrease in conidiation was found in virus-infected VC-namA. When infected with wild-type CHV1-EP713, Δp29, Δp40b, or Cys(72), VC-namA manifested a 1- to 2-log reduction in conidiation levels relative to those of EP155 or VC-TCys(72)-1 infected with the respective viruses. For example, VC-namA infected with wild-type CHV1-EP713 produced conidia at 2.2 × 10⁴ conidia/ml, a reduction of over 2 magnitudes relative to EP155 infected by CHV1-EP713 (2.8 × 10⁶ conidia/ml). However, no significant difference in conidiation was found in strains VC-namA and EP155 infected with either Δp69b or MyRV1. Uninfected VC-namA and EP155 also sporulated at similar levels (1.9 × 10⁸ versus 1.6 × 10⁸ conidia).

Pigment production was also reduced in VC-namA infected with Δp29 or Δp40b compared to EP155 infected with the respective viruses, while VC-namA showed a level of pigment production similar to those of EP155 and VC-TCys(72)-1 (Fig. 2A and D). Therefore, repression in pigment production and asexual sporulation were observed only in strains that showed altered colony morphologies upon virus infection (Fig. 2A and D and Table 2).

In contrast, no significant difference in virulence was observed between the VC-namA and EP155 backgrounds, regardless of virus infection, when assayed with apples (Table 2). Lesions of similar sizes (ranging from 7.46 to 9.47 cm²) were formed on apples inoculated by the fungal strains EP155 or VC-namA that carried wild-type, Δp29, and Δp40b virus strains. Uninfected EP155 or VC-namA induced much larger lesions (17.15 and 17.43 cm²), while Δp69b and MyRV1 caused a severe reduction in the size of the lesions formed by those fungal strains (2.49 to 2.85 cm²).

Transgenic supply of either p29 or p40 confers the namA phenotype to VC-namA infected by Δp69.The observation that VC-namA shows an irregular phenotype similar to that of namA upon infection by Δp29 or Δp40, but not by Δp69, raises the possibility that the manifestation of the namA phenotype in VC-namA requires either p29 or p40. To test this, effects of transgenic expression of the proteins on the colony morphology of Δp69-infected VC-namA were investigated. Interestingly, transformants of VC-namA with the p29 coding domain (VC-namA/p29) were similar in phenotype to namA when infected with Δp69 (Fig. 3). Likewise, a transgenic supply of p40 (VC-namA/p40) (Fig. 3) or p69 (VC-namA/p69) (data not shown) altered the phenotype of Δp69-infected VC-namA into the namA type. Δp69-infected VC-namA/p29 and VC-namA/p40 were similar to each other while slightly different in the shapes of mycelial extensions. In contrast, Δp69-infected VC-namA that had been transformed with the empty vector pCPXBSD1 showed symptoms that were identical to those of Δp69-infected EP155. These results indicate that either p29 or p40 is required for VC-namA to show the namA phenotype when infected with Δp69.

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FIG. 3.

Effects of transgenic expression of the ORF A-encoded proteins on the colony morphology of VC-namA. Spheroplasts of VC-namA were transformed with pCPXBSD1 containing the coding domains for p29 (VC-namA/p29) or p40 (VC-namA/p40). To introduce Δp69b, independent transformants were fused with EP155 infected with the mutant viral strain. Virus-free transformed fungal colonies and corresponding Δp69b-infected fungal colonies were cultured on PDA for 10 days. A fungal strain transformed with the empty vector was grown in parallel (pCPXBSD).

Determining the insertion site of the mutagenic plasmid HygR and the sequence of its flanking region.The mutant namA was expected to have two different plasmid clones, pBn1Cys(72) and the mutagenic plasmid pHygR. Based on the assumption that a copy of the hph sequence detected by Southern blotting (Fig. 2B) contributed to the phenotype of namA, we used I-PCR to amplify DNA fragments adjacent to the insertion site. As shown in Fig. 4A, I -PCR on HindIII-, XhoI-, and SacI-digested chromosomal DNA of namA and VC-namA with the primer set HG15 and HG18 allowed the amplification of a large DNA fragment. A series of hygromycin resistance gene-specific (HG) primers are specific to the hph sequence (see Table S1 in the supplemental material). The sizes of I-PCR-amplified fragments were approximately 9 kb and 8 kb for HindIII and SacI digests, respectively, of chromosomal DNA from namA or VC-namA (Fig. 4A). I-PCR on XhoI-digested DNA of namA and VC-namA amplified two major fragments of approximately 6 kb and 3.5 kb and one minor 4.5-kb fragment. DNA of the expected size of 6 kb was produced from mutagenic plasmid DNA by I-PCR (pHygR) (Fig. 4A), while no amplification was observed from genomic DNA from EP155. Nested PCR was performed on I-PCR products as shown in Fig. 4B. The nested PCR led to the amplification of large amounts of specific single DNA fragments (9 kb for HindIII, 4.5 kb for XhoI, and 8 kb for SacI) (Fig. 4B). The sizes of the nested-PCR-amplified fragments were in accordance with the sizes of the DNA fragment observed in the Southern blot analysis (Fig. 2B and 4B). The lack of differences in I-PCR and nested PCR profiles between namA and VC-namA provided additional evidence that pHygR was retained stably in VC-namA.

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FIG. 4.

Amplification of pHygR plasmid insertion regions on the namA chromosomal DNA. (A) I-PCR for amplification of upstream and downstream DNA regions flanking the pHygR insertion site. I-PCR with the primer set HG15 and HG18, specific for the hph sequence, was performed on the genomic DNA of namA and VC-namA (approximately 200 ng) digested with HindIII, XhoI, or SacI (indicated above the lanes) and self-ligated. HindIII-digested, self-ligated DNA of virus-free EP155 was used as a negative control, while pHygR plasmid DNA (10 ng) was used as a positive control. Purified pHygR DNA was also electrophoresed (lane plasmid). An aliquot of each I-PCR product was analyzed by 0.7% agarose gel electrophoresis using the 0.5× TAE buffer system (20 mM Tris-acetate, 0.5 mM EDTA [pH 7.8]), stained in ethidium bromide for 30 min, and photographed under a UV transilluminator. M refers to 1-kb DNA ladder size markers in this and subsequent figures (B and C) (Fermentas, Life Science). (B) Nested PCR amplification on the I-PCR products. One microliter of I-PCR products shown in Fig. 4A was subjected to nested PCR with the hph-specific primer set HG21 and HG12. I-PCR products from plasmid DNA (pHygR) and EP155 were also used as positive and negative controls, respectively. Amplified DNA fragments were electrophoresed as described in the legend of Fig. 4A. (C) LA PCR analysis of namA and VC-namA. Genomic PCR was performed using primers designed from the endogenous DNA sequence (nam-1 gene) (Nam2 and Nam5) and the mutagenic plasmid (HG21). The primer set Nam2 and Nam5 allowed the amplification of approximately 5-kb fragments in all the fungal strains, EP155, namA, and VC-namA, while the primer set of hph-specific HG21 and nam-1-specific Nam5 gave products of 6-kb DNA fragments in namA and VC-namA but not in EP155.

These inverse and nested PCR fragments were sequenced directly or after being cloned into the pGEM-T Easy plasmid. A chromosomal DNA fragment of approximately 8.5 kb spanning both sides of the integrated plasmid was sequenced. I-PCR and nested PCR on SacI-cleaved/self-ligated DNA provided the flanking region on the opposite side of that obtained by HindIII and XhoI DNA. The sequence of the 8.5-kb region, excluding the pHygR sequence, and the plasmid insertion site are shown in Fig. S1 in the supplemental material. The corresponding region in the wild-type allele was also sequenced. The sequence of the inserted mutagenic plasmid is available upon request. Linearization of plasmid pHygR at the AmpR coding domain and deletion of a small region of the plasmid occurred during the insertion event.

To ensure insertion site and sequence integrity, genomic LA PCR was performed (Fig. 4C). When PCRs were conducted with the primer set Nam2 and Nam5 (see Fig. 5A for primer map positions), each specific to the endogenous sequence, all three fungal strains tested, EP155, namA, and VC-namA, gave products of the expected size of 5 kb (Fig. 4C). The primer set consisting of Nam5 and hph-specific primer HG21 amplified a 6-kb fragment in namA and VC-namA but not in EP155 that did not harbor pHygR.

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FIG. 5.

Complementation test of the namA mutant. (A) Illustration of plasmid vector constructs for complementation assay. Three nam-1 gene-containing fragments were amplified by LA PCR from genomic DNA of EP155 with the wild-type nam-1 allele using primer sets Nam5 and Nam25, Nam5 and Nam30-C, and Nam5 and Nam42. The amplified DNA fragments of 6.5 kb, 7.5 kb, and 8.5 kb were cloned into the pBSD1 vector to prepare the complementation constructs pBSDnamC1, pBSDnamC2, and pBSDnamC3. pBSD1 contains a coding domain for the blasticidin S resistance gene (blasticidin S-deaminase). Eight exons of the nam-1 gene are indicated by green boxes, while the introns are depicted as gray bars. The pHygR insertion site in the eighth exon of nam-1 is indicated by a triangle. Map positions of primers and recognition sites of restriction enzymes used in I-PCR and Southern blot analyses (Fig. 2 and 4) are shown. An exon-intron structure of the gene immediately downstream of nam-1, which encodes a carboxypeptidase (M. I. Faruk and N. Suzuki, unpublished data), is also shown in the same manner. (B) Colony morphology of the complemented strains of namA. Three representative complementation strains, designated namA/namC1, namA/namC2, and namA/namC3, are shown. Those strains were obtained by transforming namA with pBSDnamC1, pBSDnamC2, and pBSDnamC3, respectively. All complemented strains were grown on PDA in parallel with EP155, TCys(72)-1, namA, and namA transformed with vector pBSD1 for 7 days at 25 to 27°C on the bench top.

Complementation assay.To determine whether the insertion mutation identified above was associated with the virus infection-specific phenotypic changes in namA (Fig. 2A), three DNA fragments of approximately 6.5 kb, 7.5 kb, and 8.5 kb, containing the corresponding unaltered region in the insertion site, were LA PCR amplified from chromosomal DNA from wild-type EP155 (wild-type allele). These fragments were cloned into plasmid pBSD1 containing the blasticidin resistance gene as the selectable marker (pBSDnamC1, pBSDnamC2, and pBSDnamC3) (Fig. 5A) and transformed into mutant strain namA to determine their abilities to complement the phenotypic abnormalities. For each transforming DNA clone, several transformants were obtained. Colony morphologies of representative isolates are shown in Fig. 5B. Consequently, all the plasmid clones complemented the altered phenotype of the mutant and conferred a TCys(72)-1 phenotype to namA; that is, namA transformed with the DNA fragments manifested regular mycelial growth without hyphal extensions and restoration of pigment production to the level shown by TCys(72)-1 (namA/namC1, namA/namC2, and namA/namC3) (Fig. 5B). Transformants with pBSDnamC2 showed a slight increase in pigmentation compared to those with the other two complementing DNA clones. Transformation with the empty vector pBSD1 failed to restore the phenotype of TCys(72)-1 (namA/pBSD1) (Fig. 5B). Two independent transformants with pBSD1 were similar in pigmentations and growth rates but different in the irregular margins from each other (Fig. 5). These results indicated that the 6.5-kb DNA fragment was sufficient for the complementation of defects in a gene(s) involved in the namA phenotype.

Characterization of the nam-1 gene.Based on data from the complementation assay (Fig. 5), the 6.5-kb fragment may contain sufficient sequence for a functional gene mutated in namA. cDNA from mRNA derived from the wild-type allele was synthesized, and its nucleotide sequence was determined to reveal the organization of the gene, termed nam-1. The exon-intron structure of nam-1, which is composed of eight exons and seven introns, is shown in Fig. 5A. An ORF starts at the AUG triplet at map positions 19 to 21 and ends with the TAG codon at map positions 4381 to 4383 (the transcription start site on the genomic DNA is numbered 1). Most splicing sites conform to the GT/AG rule, except for the first intron, where it is 5′-GT—————CG-3′ rather than 5′-GT—————AG-3′. A putative CAAT box and a TATA box (see Fig. S1 in the supplemental material) are found at positions −95 to −98 and −155 to −158, respectively, and are more distantly located from the transcription start site than usual. Additional TATA and CAAT sequences are found in further upstream regions (see Fig. S1 in the supplemental material). The nucleotide sequences of the genomic DNA and cDNA of nam-1 mRNA and the deduced amino acid sequence of NAM-1 comprising 1,257 amino acids are shown in Fig. S1 in the supplemental material. To confirm the expression of the nam-1 gene in wild-type strain EP155 and the parental strain TCys(72)-1, Northern analysis was pursued. It was, however, difficult to detect nam-1 transcripts when total RNA fractions were used. Thus, poly(A)-enriched RNA fractions were prepared for Northern analysis. As a result, nam-1 was shown to be expressed in the two C. parasitica strains (Fig. 6A). No significant difference in nam-1 transcription levels between fungal colonies infected and uninfected with the wild-type CHV1 or Cys(72) was found, suggesting that nam-1 was not responsive to hypovirus infection (Fig. 6A).

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FIG. 6.

Expression of nam-1 in fungal colonies and functional domains of NAM-1. (A) Northern blot analysis of nam-1. Transcription levels of nam-1 between hypovirus-infected and uninfected colonies were compared. Ten micrograms of poly(A)-enriched RNA isolated from the mycelia of wild-type strain EP155, CHV1-EP713-infected EP155 (CHV1-EP713), and TCys(72)-1 was electrophoresed in a 1.4% gel under denaturing conditions and transferred onto a nylon membrane for hybridization with a nam-1-specific, DIG-11-dUTP-labeled probe (top). To detect β-tubulin mRNA as a loading marker, 3 μg of RNA from the same poly(A)-enriched fraction as that in the top panel was probed by a DIG-11-dUTP-labeled probe amplified on β-tubulin cDNA (bottom). (B) Schematic diagram showing two domains, DUF1777 and CorA, of NAM-1 from C. parasitica. A PFAM search (http://www.sanger.ac.uk/Software/Pfam/search.shtml ) with NAM-1 revealed a few domains: CorA as a trusted match and other domains as potential matches represented by DUF1777 with the lowest E value. Positions of the CorA Mg²⁺ transporter domain and a less characterized domain, DUF1777, are shown in the NAM-1 sequence. (C) Sequence alignment of NAM-1 and its counterparts from other filamentous fungi and prokaryotes. The sequences of the C. parasitica NAM-1 were aligned with those of corresponding hypothetical proteins from the ascomycetous fungi Neurospora crassa, Gibberella zeae, and Chaetomium globosum and two bacteria, Burkholderia vietnamiensis and Azotobacter vinelandii. For the overall alignment, see Fig. S2 in the supplemental material. All proteins contain the CorA domain, while only the counterpart from G. zeae possesses the DUF1777 domain. Accession numbers for the GenBank database are SM_955371 for N. crassa, SM_381249 for G. zeae, SM_001226467 for C. globosum, Q4BIY7 for B. vietnamiensis, and Q4IVJ8 for A. vinelandii. The two transmembrane domains, TM1 and TM2, within the CorA consensus sequence are boxed. At the C terminus of TM1, the signature sequence GMN is strictly conserved. Asterisks refer to amino acids that are strictly conserved, while colons and dots indicate changes to chemically similar amino acids.

Dawe et al. (13) constructed a publicly available EST library containing 2,200 genes from C. parasitica. nam-1 was not found in the EST collection. However, a BLAST search of the GenBank library with the deduced amino acid sequence of NAM-1 revealed similar hypothetical proteins from other ascomycetous, filamentous fungi including Neurospora crassa, Gibberella zeae, and Chaetomium globosum. The sequence similarities were found over the entire sequences (see Fig. S2 in the supplemental material). The levels of identities found in NAM-1 and its counterpart were 38%, with an E value of 2e-172, for N. crassa; 34%, with an E value of 3e-121, for G. zeae; and 34%, with an E value of 3e-121, for C. globosum. Highly conserved sequence stretches were detected in the middle and C-terminal regions. The equivalent, hypothetical proteins of N. crassa and C. globosum have a C-terminal extension of approximately 250 amino acids (aa) and a deletion of approximately 200 aa in the central region, respectively, thus being different in size from NAM-1 (see Fig. S2 in the supplemental material).

A search of motif databases with the NAM-1 sequence identified the CorA domain at its C-terminal region (aa 792 to 1170), as defined by PFAM family PF01544 (Fig. 6B), in addition to a number of consensus sequences including glycosylation sites, phosphorylation sites, myristoylation sites, and leucine zipper motifs that are frequently found in protein sequences. Interestingly, the CorA motif was conserved in the counterparts of filamentous fungi. The N-terminal portion of NAM-1 contains the PFAM DUF1777 domain at aa 133 to 253 shared by the counterpart of G. zeae but not by that of N. crassa or C. globosum. The CorA consensus sequence is found in prokaryotic and eukaryotic members of the CorA Mg²⁺ transporter family (25), while the DUF1777 domain is less characterized and defined. The three-dimensional structures of one of the CorA members were recently revealed at 2.9-Å resolution and clearly showed two transmembrane domains (24). The PSORT prediction (http://psort.nibb.ac.jp/form.html ) also revealed two transmembrane sequences of NAM-1, at aa 1111 to 1138 (transmembrane domain 1 [TM1]) and 1148 to 1174 (TM2). The NAM-1 transmembrane region within the CorA domain can be aligned with the corresponding regions of other CorA homologues even with prokaryotic members. The NAM-1 counterparts from the three filamentous ascomycetous fungi and those of two bacteria, Burkholderia vietnamiensis and Azotobacter vinelandii, with the highest sequence identities among prokaryotic CorA members are included in the alignment shown in Fig. 6C. It is also noteworthy that TM1 contains the GMN motif at its C terminus (Fig. 6C), which was previously shown to be highly preserved in prokaryotic and eukaryotic CorA homologues (25).

Disruption of nam-1 in TCys(72)-1 results in virus infection-specific alteration of the colony phenotype.To further confirm the involvement of mutations of the nam-1 gene in the phenotypic change found in namA, a nam-1 disruption construct that contained an hph gene cassette inserted between the 5′ and 3′ sequences of nam-1 was prepared (Fig. 7A). The construct was designed to delete six exons in the central region of the nam-1 gene (Fig. 7A) based upon homologous recombination-based gene replacement. A population of approximately 120 transformants of TCys(72)-1 with the construct was screened for disruptants using selection on hygromycin-containing PDA and subsequently by genomic PCR. Several independent disruptant candidates, such as TCys(72)-1/Δnam1-1 and TCys(72)-1/Δnam1-2 were obtained. Targeted disruption was further confirmed by Southern blot analysis (Fig. 7B); that is, probe 1, spanning map positions 191 to 1381, hybridized 1.8-kb DNA fragments in TCys(72)-1/Δnam1-1 and TCys(72)-1/Δnam1-2, while EP155 and TCys(72)-1 gave a signal corresponding to 5.3 kb when genomic DNA was digested by XbaI. No signal was found in the disruptant candidates with probe 2 harboring the nam-1 sequence that was expected to be deleted, while a 5.3-kb band was detected in EP155 and TCys(72)-1. An hph probe (probe 3) detected the expected signals (2.2 kb) only in disruptant candidates [TCys(72)-1/Δnam1-1 and TCys(72)-1/Δnam1-2] but not in EP155 or TCys(72)-1. These data clearly indicated that TCys(72)-1/Δnam1-1 and TCys(72)-1/Δnam1-2 were nam-1 disruptants.

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FIG. 7.

Disruption of the nam-1 gene of TCys(72)-1. (A) Exon/intron organization of the C. parasitica nam-1 gene (top) and organization of the nam-1 gene disruption construct. The 6-kb genomic DNA containing nam-1 is composed of eight exons (green boxes) and seven introns (gray bars). A nam-1 gene disruption construct was generated by replacing a fragment covering the central six exons between primers Nam10 and Nam2 with a 2.3-kb cassette composed of the Escherichia coli hygromycin B phosphotransferase gene (hph) flanked by the A. nidulans trpC promoter (Ptrp) and terminator (Ttrp). The DNA operation was carried out with the aid of PCR using primers Nam5, Nam10, Nam2, and Nam25. The primers' positions are indicated above the gene organization schematic. DNA regions used as probes (probe 1, probe 2, and probe 3) for Southern blot analysis are indicated by a bar below the diagrams. The recognition site of XbaI used in Southern blotting (Fig. 7B) is indicated on the nam-1 gene organization. A 1-kb scale bar is shown. (B) Southern hybridization analysis of the nam-1 disruption mutants. Endonuclease XbaI-digested DNA (10 μg) of EP155, TCys(72)-1, and the disruptant candidates TCys(72)-1/Δnam1-1 and TCys(72)-1/Δnam1-2 was separated in a 1% agarose gel, blotted onto a nylon membrane, and probed with DIG-11-dUTP-labeled probe 1. The same blot was reprobed sequentially by probe 2 and probe 3. (C) Effect of nam-1 disruption on phenotypic properties of TCys(72)-1. Two independent nam-1 disruption candidates, TCys(72)-1/Δnam1-1 and TCys(72)-1/Δnam1-2, were grown on PDA for 10 days on the laboratory bench at 25 to 27°C. TCys(72)-1, namA, EP155, and VC-namA were cultured in parallel.

TCys(72)-1/Δnam1-1 and TCys(72)-1/Δnam1-2 showed similar colony morphologies (Fig. 7C). The disruptants had uneven, dense aerial hyphae and lacked the protrusions possessed by namA (Fig. 7C). The disruptants were also reduced in growth rate and pigmentation compared with TCys(72)-1 (Fig. 7C). The suppression in pigmentation found in the disruptants was pronounced when cultured in PDB (Fig. 8). Levels of pigmentation suppression were exhibited by the disruptants and were similar to those found in namA, which were lower than those in TCys(72)-1 and much lower than those in EP155 or VC-namA (Fig. 8). These showed the involvement of nam-1 in host pigmentation. To determine the phenotype of virus-free nam-1 disruptants, TCys(72)-1/Δnam1-1 and TCys(72)-1/Δnam1-2 were cured of the Cys(72) virus as in the case of namA and TCys(72)-1 [VC-TCys(72)-1/Δnam1-1 and VC-TCys(72)-1/Δnam1-2]. In the absence of virus infection, the disruptants VC-TCys(72)-1/Δnam1-1 and VC-TCys(72)-1/Δnam1-2 were identical to VC-namA and EP155 (data not shown).

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FIG. 8.

Comparison of pigment production of nam-1 disruption mutants with those of other strains. (A) Pigment production of a nam-1 disruptant [TCys(72)-1/Δnam1-1] in PDB medium. The disruption mutant was cultured in 20 ml PDB (Difco) in parallel with its parental strain, TCys(72)-1, Cys(72)-containing and VC-namA strains, and EP155 for 12 days on a laboratory bench at 25 to 27°C and photographed. (B) Relative levels of pigmentation of different fungal strains. Three hundred milligrams of harvested mycelia from each strain shown in Fig. 8A was pulverized under liquid nitrogen and homogenized in 3 ml absolute ethanol (18). An aliquot of the mixture was transferred into a 1.5-ml microcentrifuge tube, incubated at room temperature for 3 h followed by centrifugation at 12.5 krpm for 1 min, and photographed. (C) Graphical representation of relative levels of pigmentation of different fungal strains. Pigmentation was measured as the absorbance at 454 nm by a densitometer. Ten replicates for each isolate of namA, TCys(72)-1/Δnam1-1, TCys(72)-1, EP155, and VC-namA were used for the measurements to calculate averages and standard deviations.

These results showed that the disruption of nam-1 in the TCys(72)-1 backgrounds had a virus infection-specific effect on colony morphology. The data also suggested that nam-1 was involved in the altered phenotypic changes in namA, but the disruption of the gene was not sufficient to reproduce its phenotype.

Altered, longer versions of nam-1 transcripts contribute to the namA phenotype.The observation that the morphologies of TCys(72)-1/Δnam1-1 and namA were not identical (Fig. 7C) suggested that the phenotype of namA may be due to expression of the modified nam-1 transcripts with altered function rather than because of a simple disruption of its function. To examine this possibility, EP155, namA, and TCys(72)-1/Δnam1-1 were subjected to Northern analysis. Consequently, while no signal was observed in TCys(72)-1/Δnam1-1 at the expected size (approximately 4 kb), transcripts of nam-1 were detected in EP155 and namA (Fig. 9A). Nevertheless, transcripts of a slightly large size than that in TCys(72)-1 or EP155 were detected in namA (Fig. 6A and 9A, compare lane 1 to lane 2). This may indicate that the plasmid insertion into the last exon of the nam-1 gene in namA (Fig. 5) leads to the production of extended transcripts containing the partial pHygR sequence. Data from 3′ RACE supported this possibility and showed that the altered nam-1 transcripts in namA contained a deletion of the 3′-terminal region of approximately 150 nucleotides (nt) and an extension of approximately 450 nt derived from the pHygR plasmid sequence (see Fig. S3 in the supplemental material). The integration of pHygR induced an amino acid sequence change at the C terminus from ——RSTEGKPPGGRIKNGNIL [in EP155 or TCys(72)-1] to ——TNTVLLV (in namA). Thus, it seemed likely that the generation of modified transcripts was involved in the namA phenotype.

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FIG. 9.

Contribution of an altered longer version of nam-1 transcripts to the namA phenotype. (A) Northern hybridization analysis to detect nam-1 transcripts. Ten micrograms of poly(A)-enriched RNA isolated from the mycelia of wild-type strain EP155, the namA mutant, and the nam-1 disruptant with the TCys(72)-1 backgrounds, TCys(72)-1/Δnam1, was electrophoresed in a 1.4% gel under denaturing conditions and transferred onto a nylon membrane for hybridization with a nam-1-specific, DIG-11-dUTP-labeled probe (top). β-Tubulin mRNA was used as a loading marker. To detect β-tubulin mRNA as a loading marker, 3 μg of RNA from the same poly(A)-enriched fraction as that in the top panel was probed by a DIG-11-dUTP-labeled probe amplified on β-tubulin cDNA (bottom). (B) Effects of expression of modified nam-1 on the colony morphology of TCys(72)-1/Δnam1-1. A 6-kb genomic DNA fragment carrying the HygR sequence on exon 8 of nam-1 was amplified on the genomic DNA of namA by LA PCR using primer sets Nam5 and HG17 and cloned into the NotI site of pBSD1 (pBSD-nam1-HygR). Exons/introns of nam-1, the pHygR integration site, and primers' map positions are shown in Fig. 5A. Spheroplasts of the TCys(72)-1/Δnam1-1 strain were transformed with pBSD-nam1-HygR to generate TCys(72)-1/Δnam1-1/nam1-HygR1. The resulting transformant strain was cultured on PDA in parallel with the TCys(72)-1, TCys(72)-1/Δnam1-1, and the namA mutant strain for 8 days on the laboratory bench at 25 to 27°C.

To test this possibility, the nam-1-pHygR sequence was amplified from genomic DNA of namA by LA PCR with the primer pair Nam5 and HG17 (see Fig. 5A for primers' map positions) and cloned into the NotI site of pBSD1. The resulting plasmid, pBSD1-nam1-HygR, which covered the extended region of the altered nam-1 transcripts, was used to transform TCys(72)-1/Δnam1-1. Transformation of TCys(72)-1/Δnam1-1 with pBSD1-nam1-Hyg resulted in colony morphology that resembled that of namA (Fig. 9B) and was characterized by an irregular margin with mycelial protrusions and reduced pigmentation. However, unlike in namA, no firm colony edge was observed in TCys(72)-1/Δnam1-1 transformed with pBSD1-nam1-HygR. Fungal colonies of TCys(72)-1/Δnam1-1 cultured in parallel were reduced in pigmentation but increased in aerial hyphae relative to TCys(72)-1 (Fig. 9B). These results showed that the extension of transcripts of the nam-1 gene contributed to the phenotype of namA.