Ste12 transcription factor homologue CpST12 is down-regulated by hypovirus infection and required for virulence and female fertility of the chestnut blight fungus Cryphonectria parasitica

Abstract

A putative homologue of the Saccharomyces cerevisiae Ste12 transcription factor was identified in a series of expressed sequence tag-based microarray analyses as being down-regulated in strains of the chestnut blight fungus, Cryphonectria parasitica, infected by virulence-attenuating hypoviruses. Cloning of the corresponding gene, cpst12, confirmed a high level of similarity to Ste12 homologues of other filamentous fungi. Disruption of cpst12 resulted in no alterations in in vitro growth characteristics or colony morphology and an increase in the production of asexual spores, indicating that CpST12 is dispensable for vegetative growth and conidiation on artificial medium. However, the disruption mutants showed a very substantial reduction in virulence on chestnut tissue and a complete loss of female fertility, two symptoms normally conferred by hypovirus infection. Both virulence and female fertility were restored by complementation with the wild-type cpst12 gene. Analysis of transcriptional changes caused by cpst12 gene disruption with a custom C. parastica cDNA microaray chip identified 152 responsive genes. A significant number of these putative CpST12-regulated genes were also responsive to hypovirus infection. Thus, cpst12 encodes a cellular transcription factor, CpST12, that is down-regulated by hypovirus infection and required for female fertility, virulence and regulated expression of a subset of hypovirus responsive host genes.

FIG. 1.

Phylogram of the Ste12 homologues from C. parasitica (CpST12) and other fungi. S. cerevisiae Ste12 (GenBank accession no. p13574) was used as an outgroup. Notations of the fungal Ste12 homologues and GenBank accession numbers: CpST12 (DQ458788, C. parasitica), MST12(AF432913, M. grisea), CST1 (AB090340, Colletotrichum lagenarium), CLSTE12(AJ459778, Colletotrichum lindemuthianum, Fst12(AF509340, Fusarium graminearum), pp-1 (AY027529, Neurospora crassa), AnST-12 (XM654802, Aspergillus nidulans), AfST12 (EAL91975, Aspergillus fumigatus), and stlA (AF284062, Penicillium marneffei). The bar represents distances scaled as substitutions per amino acid residue.

FIG. 2.

Genomic organization of C. parasitica cpst12 gene and map of the cpst12 gene disruption construct. (A) A 4.5-kb genomic DNA containing the cpst12 coding region composed of four exons is indicated by the boxes. A cpst12 gene replacement construct was generated by insertion of the 2.8-kb hygromycin cassette using a PCR-based strategy (see Materials and Methods). (B) Southern analysis of wild-type EP155, cpst12 disruptant strains Δcpst-E1 (EP155/Δcpst-E1), Δcpst-E7 (EP155/Δcpst-E7), and ectopic transformant A8. All DNA samples were digested with HindIII and XhoI (left) or independently with ClaI (right). Fragment sizes are indicated in the figure margins. The blot was hybridized with the PCR fragments shown in panel A. (C) Real time RT-PCR analysis of cpst12 gene expression in wild-type EP155, cpst12 disruptant strains EP155/Δcpst-E1 and EP155/Δcpst-E7 and hypovirus CHV1-infected strain EP713.

FIG. 3.

(A) Phenotypes of wild-type strain EP155, CHV1-infected strain EP713 and two cpst12 disruptant strains, Δcpst-E1 and Δcpst-E7. Photograph was taken on day 7 of culture on PDA. (B) Cultures of wild-type EP155 and cpst12 disruptant Δcpst-E1 (EP155/Δcpst-E1) to show the pycnidia and conidial spore production after 2 weeks. Compared to wild-type EP155, the cpst12 disruptant strain Δcpst-E1 produced more pycnidia, which sporulate abundantly on PDA.

FIG. 4.

(A) Virulence assay on dormant American chestnut stems. Representative cankers formed by wild-type strain EP155, strain EP155-infected with hypovirus CHV1-EP713 (EP155/CHV1-EP713), and two cpst12 disruptants (EP155/Δcpst-E1 and EP155/Δcpst-E7). Photographs of cankers were taken 3 weeks postinoculation. (B) The cpst12 disruptants (EP155/Δcpst-E1,shown) failed to produce the stromal pustules that are formed by wild-type strain EP155 on the bark of inoculated dormant chestnut stems, even after prolonged incubation, indicating that the cpst12 disruptants are unable to erupt through the bark of the stems. (C) The cpst12 disruptant Δcpst-E1 was able to grow on the surface of unscored autoclaved chestnut twigs but failed to produce stromal pustules like the wild-type strain EP155, similar to results observed for inoculated chestnut stems.

FIG. 5.

Representative cankers formed by wild-type strain EP155, EP155/CHV1-EP713, cpst12 disruptant EP155/Δcpst-E1, ectopic transformant A8, and the two cpst12 disruptant complemented strains EP155/Δcpst-E1/pCPST-C and EP155/Δcpst-E1/pCPST-G2. Photographs were taken 2 weeks after inoculation.

FIG. 6.

Similarity in expression profiles between CHV-1-EP713 hypovirus-infected strain EP155 and Δcpst12 disruptant strain Δcpst-E1. (A) Venn diagram illustrating the total number of differentially expressed genes identified in hybridizations between EP155/CHV1-EP713 versus EP155 and EP155/Δcpst-E1 versus EP155 (the present study). A total of 47 genes were found on both lists of differentially expressed clones, and these genes are described in Table 2. When differential expression was defined as genes with 1.5-fold changes relative to the wild type, EP155, a total of 99 hypovirus CHV1-EP713-responsive genes overlapped with the differentially expressed genes of the disruptant. (B) Bar chart illustrating the relative magnitude and direction of change in transcript levels produced by microarray for the genes differentially expressed in both CHV1-EP713-infected and cpst12 disruptant strains. Blue-shaded bars indicate the magnitude of transcript accumulation change after CHV1-EP713 infection (fold change [y axis]), whereas the magnitude of change for the same genes in the Δcpst12 disruptant is indicated in purple. The order of AEST clones from left to right is as presented in Table 2. That is, values for AEST-01-G-08 are shown at the far left of the figure.