Hypovirus-responsive transcription factor gene pro1 of the chestnut blight fungus Cryphonectria parasitica is required for female fertility, asexual spore development, and stable maintenance of hypovirus infection

Abstract

We report characterization of the gene encoding putative transcription factor PRO1, identified in transcriptional profiling studies as being downregulated in the chestnut blight fungus Cryphonectria parasitica in response to infection by virulence-attenuating hypoviruses. Sequence analysis confirmed that pro1 encodes a Zn(II)(2)Cys(6) binuclear cluster DNA binding protein with significant sequence similarity to the pro1 gene product that controls fruiting body development in Sordaria macrospora. Targeted disruption of the C. parasitica pro1 gene resulted in two phenotypic changes that also accompany hypovirus infection, a significant reduction in asexual sporulation that could be reversed by exposure to high light intensity, and loss of female fertility. The pro1 disruption mutant, however, retained full virulence. Although hypovirus CHV1-EP713 infection was established in the pro1 disruption mutant, infected colonies continually produced virus-free sectors, suggesting that PRO1 is required for stable maintenance of hypovirus infection. These results complement the recent characterization of the hypovirus-responsive homologue of the Saccharomyces cerevisiae Ste12 C(2)H(2) zinc finger transcription factor gene, cpst12, which was shown to be required for C. parasitica female fertility and virulence.

FIG. 1.

Comparative sequence analysis of the predicted amino acid sequences of the C. parasitica pro1 gene and homologous genes reported to function in fungal sexual development or from closely related plant pathogenic fungi. (A) Phylogram of predicted amino acid sequences of pro1 gene orthologues. The tree was generated by CLUSTALW (http://align.genome.jp/) with the default parameter setting, using the full-length amino acid sequence. FL Nc, N. crassa fluffy protein (GenBank accession no. AAB80932); NosA An, A. nidulans NosA protein (GenBank accession no. CAJ76908); RosA An, A. nidulans RosA protein (GenBank accession no. CAD58393); MGG_00494 Mg, M. grisea hypothetical protein MGG_00494 (GenBank accession no. XP_368750); PRO1 Cp, C. parasitica PRO1 protein (GenBank accession no. FJ348264); PRO1 Nc, N. crassa PRO1 protein (GenBank accession no. CAB89819); PRO1 Sm, S. macrospora PRO1 protein (GenBank accession no. CAB52588); FG07067.1 Gz, G. zeae hypothetical protein FG07067.1 (GenBank accession no. XP_387243). The 0.1 scale bar shows 10% sequence aberration. (B) Alignment of the Zn(II)₂Cys₆ fungal binuclear cluster motif for five clustered PRO1 orthologues. The alignment was performed with MegAlign in Lasergene, using the default setting. Cp, C. parasitica PRO1 protein; Gz, G. zeae hypothetical protein FG07067.1; Mg, M. grisea hypothetical protein MGG_00494; Nc, N. crassa PRO1 protein; Sm, S. macrospora PRO1 protein. The cysteine residues involved in the coordination of the Zn²⁺ atoms are highlighted with an asterisk. The position of the conserved intron is indicated by an arrowhead. The two predicted bipartite nuclear localization signals are indicated with a line above the sequence.

FIG. 2.

Growth and tissue-specific accumulation of pro1 gene transcripts. Real-time RT-PCR analysis was performed as described in Materials and Methods for RNA isolated from wild-type C. parasitica strain EP155 cultures harvested from PDA-cellophane plates at weekly intervals and from perithecia collected from chestnut tree twigs. The values were normalized to the pro1 transcript accumulation level measured for 1-week-old PDA-cellophane culture (value set to 1), with the standard deviations, based on three independent measurements of two independent RNA preparations, indicated by the error bars.

FIG. 3.

Disruption of the C. parasitica pro1 gene. (A) Cartoon of pro1 gene organization and disruption construct. A pro1 gene disruption construct was generated by replacing a 200-bp fragment that encodes most of the putative DNA activation domain with a neomycin resistance gene cassette, using a PCR-based strategy as described in Materials and Methods. (B) PCR analysis of pro1 disruption mutants. Templates used for PCR analysis included genomic DNA isolated from wild-type strain EP155 (lanes 1 and 4) and pro1 disruption mutants Δpro1T3 (lanes 2 and 5) and Δpro1T6 (lanes 3 and 6). (C) Southern blot analysis of the EP155 (lane 1), Δpro1T3 (lane 2), and Δpro1T6 (lane 3) strains. Genomic DNA was digested with SacI and hybridized with a pro1-specific probe as shown in panel A.

FIG. 4.

Colony morphology of the pro1 disruption mutant strain. Colonies of wild-type strain EP155 (left) and pro1 disruption mutant Δpro1T6 (right) after 7 and 14 days of culturing are shown in panels A and B, respectively. Photographs were taken of cultures incubated on PDA at 22 to 24°C under standard light conditions (12-h/12-h light/dark cycle with a light intensity of 1,300 to 1,600 lx). The pro1 disruption mutant formed small putative proto-pycnidia (C, right) rather than mature conidium-producing pycnidia as produced by strain EP155 (C, left). The defect in conidium production in the pro1 disruption mutant was restored to wild-type levels in the complemented Δpro1T6/pG1 strain (Table 2). Panel D shows the colonies formed by the hypovirus CHV1-EP713-infected EP155 (left) and Δpro1T6 (right) strains after 7 days of culturing under the conditions described above.

FIG. 5.

The pro1 disruption mutants are female sterile on American chestnut tree twigs. Mating experiments were performed with pro1 disruption mutant or EP155 strains as the female and strain EP146, of the opposite mating type, as the male. Photographs were captured at 6 months after initiation of the mating, using a Pentax K100D digital SLR camera equipped with a macro lens at a magnification of ×4. Mature perithecia with protruding ostiolar necks (arrows) were present in abundance for the control EP146(♂) × EP155(♀) crosses (A) but were completely absent in the EP146(♂) × Δpro1T6(♀) crosses (B).

FIG. 6.

Semiquantitative real-time RT-PCR analysis of pro41 expression in the EP155, EP713, Δpro1T6, and Δcpst12 strains. Relative transcript levels were measured using cDNA of 18S rRNA generated in the same RT reaction for normalization, with standard deviations, based on three independent measurements of two independent RNA preparations, indicated by the error bars.

FIG. 7.

Requirement of the pro1 gene for CHV1-EP713 virus maintenance. (A and B) Anastomosis-mediated transfer of hypovirus CHV1-EP713 from strain EP713 (left side of each panel) to wild-type strain EP155 (A) and pro1 disruption mutant Δpro1T6 (B). (C) Subcultures taken from the converted, CHV1-EP713-infected pro1 disruption mutant culture at a point near the initial contact between the paired colonies (position 1) exhibited virus infection symptoms that included loss of orange pigment production and altered colony morphology (Fig. 4D). (D) Subcultures taken from rapidly growing sectors originating from the converted pro1 disruption mutant (position 2) exhibited a virus-free colony phenotype. (E) With successive subculturing (transfer every 7 days), the CHV1-EP713-infected Δpro1T6 mycelia produced rapidly growing sectors at a high frequency and the colonies derived from those sectors always exhibited a virus-free phenotype (F). (G) Total RNA was extracted from colonies originating from mycelia taken from positions 1 and 2 (C and D, respectively) and analyzed on an agarose gel. The slowly migrating bands present in lane 1 but absent in lane 2 are full-length and defective CHV1-EP713 replicative double-stranded RNAs. (H) RT-PCR analysis detected a virus-specific product (lane 1) from colonies originating from position 1 (C) but not from position 2 (D). Primers specific for CHV1-EP713 genome RNA and pro1 mRNA were used along with primers specific for β-tubulin mRNA as reaction and loading controls.