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.Reduced virulence (hypovirulence) of the chestnut blight fungus, Cryphonectria parasitica, caused by viruses in the family Hypoviridae (hypoviruses), is accompanied by reduced conidiation and loss of female fertility (reviewed in references 3, 21, and 28). These deficiencies in the reproductive capacity of hypovirus-infected C. parasitica strains restrict transmission of hypoviruses through fungal populations and thus reduce the efficacy of hypovirus-mediated biological control of chestnut blight (discussed in reference 24). It has been possible to partially uncouple hypovirulence from reduced conidiation by creating chimeric hypoviruses from mild and severe virus isolates (10). However, a basic understanding of the regulatory pathways that govern C. parasitica asexual and sexual reproduction would provide additional tools for enhancing hypovirus-mediated biological control potential.Our laboratory recently reported that the C. parasitica homologue of the Saccharomyces cerevisiae Ste12 C 2 H 2 zinc finger transcription factor gene, cpst12, identified by microarray analysis as downregulated following hypovirus infection (1, 2), was required for female fertility and virulence (16). A gene encoding a second putative transcription factor with sequence similarity to the Zn(II) 2 Cys 6 transcription factor PRO1 of Sordaria macrospora was also found to be downregulated following infection by mild and severe hypovirus isolates CHV1-Euro7 and CHV1-EP713, respectively (2). Interestingly, the S. macrospora pro1 gene is required for female fertility (22, 23), and the Neurospora crassa pro1 homologue was able to rescue the female infertility phenotype when introduced into the S. macrospora pro1 deletion mutant (23).Thus, it was of int...

A single Argonaute gene is required for induction of RNA silencing antiviral defense and promotes viral RNA recombination

“…C. parasitica genomic DNA was isolated as described in Sun et al (31). Total RNA was extracted from virus-free and hypovirus infected C. parasitica strains as described by Parsley et al (36) with the exception that 5 M LiCl was routinely used for precipitation to collect total host and viral single stranded RNAs as well as viral double stranded RNAs.…”

“…Plasmid pGSN2GFP is similar to the hairpin structure producing plasmid pGS-2GFP (described in ref. 16), except that it has a neomycin resistance gene cassette in place of the benomyl resistance cassette (31). Neomycin-resistant transformants were selected on G418-containing PDA plates and single spore isolates were obtained for each transformant and grown on PDA (20 mL/plate) for 5 days.…”

“…Hygromycin (40 g/mL), benomyl (0.4 g/mL), or G418 (20 g/mL), depending on the selectable marker used, was included in the growth medium to provide selection of transformants. Hyphal anastomosis, sporulation assays, virulence assays, and statistical analysis were performed as described in Sun et al (31).…”

“…The four agl gene disruption mutant strains were independently infected with hypovirus CHV1-EP713, either by transfection of spheroplasts with infectious transcripts (30) or by anastomosis with CHV1-EP713 infected strain EP155 (31). Mutant strains ⌬agl1, ⌬agl3, and ⌬agl4 all responded to CHV1-EP713 infection with phenotypic changes that were indistinguishable from that exhibited by CHV1-EP713-infected wildtype strain EP155.…”

“…The C. parasitica strain EP155 expressing EGFP from a nuclear transgene (16) was transformed with a EGFP hairpin-silencing vector plasmid pGSN2GFP and six The colony morphology for a complemented ⌬agl2 strain infected with hypovirus CHV1-EP713 is shown. The ⌬agl2 deletion mutant was complemented with a genomic DNA clone of the agl2 coding region cloned into plasmid pCPXNBn1 to generate the complementation plasmid pCAGL2, which contains the benomyl resistance cassette and the C. parasitica glyceraldehydes-3-phosphate dehydrogenase promoter (31). Cultures were grown for 7 days on PDA.…”