Characterization of a fungal protein kinase from Cryphonectria parasitica and its transcriptional upregulation by hypovirus

Abstract

SummaryThe chestnut blight fungus Cryphonectria parasitica and its hypovirus comprise useful model system to study the mechanisms of hypoviral infection. We used degenerate primers based on fungal protein kinases to isolate a gene, cppk1, which encodes a novel Ser/Thr protein kinase of C. parasitica. The gene showed highest homology to ptk1, a Ser/Thr protein kinase from Trichoderma reesei. The encoded protein had a predicted mass of 70.5 kDa and a pI of 7.45. Northern blot analyses revealed that the cppk1 transcript was expressed from the beginning of culture, with a slight increase by 5 days of culture. However, its expression was specifically affected by the presence of virus, and it was transcriptionally upregulated in the fungal strain infected with the hypovirus. A kinase assay using Escherichia coliderived CpPK1 revealed CpPK1-specific phosphorylated proteins with estimated masses of 50 kDa and 44 kDa. In addition, the phosphorylation of both proteins was higher in a cell-free extract from the hypovirulent strain. The increased expression of cppk1 by the introduction of an additional copy results in a subset of viral symptoms of reduced pigmentation and conidiation in a virus-free isolate. cppk1 overexpression also causes the downregulation of mating factor genes Mf2/1 and Mf2/2, resulting in female sterility. The present study suggests that the hypovirus disturbs fungal signalling by transcriptional upregulation of cppk1, which results in reduced pigmentation and conidiation and female sterility.

Characterization of the ERK homologue CpMK2 from the chestnut blight fungus Cryphonectria parasitica

“…studies have demonstrated the implementation of a signal transduction pathway during viral symptom development. Genes modulated during this process encode components of various signal transduction pathways, including heterotrimeric G-proteins, a putative regulator of these G-proteins (Choi et al, 1995;Kasahara & Nuss, 1997;Kasahara et al, 2000), a novel kinase (Kim et al, 2002) and many others that are under investigation (Dawe & Nuss, 2001). Recently, we have demonstrated the hypoviral regulation of components of a mitogen-activated protein kinase (MAPK) signalling pathway in C. parasitica (Park et al, 2004).…”

“…To test for autophosphorylation, purified CpMK2 was used in a kinase assay without MBP. A kinase assay of cell-free extracts was conducted as described previously (Kim et al, 2002).…”

“…Anti-CpMK2 antibody was obtained as described previously (Kim et al, 2002). Purified recombinant CpMK2 (100 mg) was injected into an 8-week-old BALB/c mouse, which was boosted with the same amount of the CpMK2 emulsified in incomplete Freund's adjuvant 10 days after the initial injection.…”

“…Transformants were selected from agar plates that were supplemented with 150 mg hygromycin B ml 21 (Calbiochem) or 1?5 mg benomyl ml 21 (DuPont) as appropriate, passaged three or four times on selective media and single-spore-isolated, as described previously (Kim et al, 1995(Kim et al, , 2002. PCR and Southern blot analysis were conducted with genomic DNA from the transformants to confirm the replacement and complementation of the cpmk2 gene in trans.…”

“…Laccase activity of culture filtrate was determined as described previously (Kim et al, 1995). Expression of the virus-regulated cryparin (Crp), laccase (lac1) and mating pheromone (Mf2/1) genes was examined by Northern blot analysis (Kim et al, 1995(Kim et al, , 2002.…”