Deletion of the cpku80 gene in the chestnut blight fungus, Cryphonectria parasitica, enhances gene disruption efficiency

Abstract

The chestnut blight fungus, Cryphonectria parasitica, and associated virulence-attenuating hypoviruses have emerged as an important model system for studying molecular mechanisms underlying fungal-plant pathogenic interactions. As more gene sequence information becomes available as a result of C. parasitica express sequence tags (ESTs) and ongoing whole genome sequencing projects, the development of an efficient gene disruption system has become an urgent need for functional genomics studies of this important forestry pathogen. Here, we report the cloning of the C. parasitica gene cpku80 that encodes a key component of the nonhomologous end joining DNA repair pathway and the construction of a corresponding deletion mutant strain. The cpku80 mutant was indistinguishable from the parental wild-type strain EP155 in colony morphology, ability to support hypovirus replication, conidiation and virulence. As predicted, the Deltacpku80 strain did exhibit an increased sensitivity to the mutagen methyl methanesulfonate. A test with three selected genes resulted in a gene disruption efficiency of about 80% for the Deltacpku80 strain, a significant increase over the 2-5% levels of homologous recombination generally observed for the wild-type strain EP155. This efficient homologous recombination system provides a powerful tool for large-scale analysis of gene functions in C. parasitica.

Engineering super mycovirus donor strains of chestnut blight fungus by systematic disruption of multilocus vic genes

“…1, gene disruptions were performed using strain DK80, a mutant of C. parasitica genomic reference strain EP155, vic genotype 2211-22, in which the nonhomologous end-joining DNA repair pathway ku80 gene homolog was disrupted to promote homologous recombination (26). The vic1a-2 gene was disrupted using a loxP-Pro gpd -neo-loxPbased disruption cassette as previously described (23,25) and followed by Pro gpd -neo excision by anastomosis with Cre-expressing strain EP155-cre (Materials and Methods).…”

“…Wild-type and genome reference strain EP155 (ATCC 38755) was originally isolated by Sandra Anagnostakis (Connecticut Agricultural Experiment Station, New Haven, CT) in 1977 near Bethany, CT. Strain EP146 (ATCC64671) was isolated by William MacDonald (West Virginia University) in 1976 near Franklin, WV. Strain DK80, a mutant of strain EP155 that contains a disruption of the nonhomologous end-joining DNA repair pathway ku80 gene homolog to promote homologous recombination (26), was used as the founder strain for vic gene disruptions to construct the super donor strains. The 64 vic genotyped EU tester strains (21) were provided by Michael Milgroom (Cornell University).…”

“…An additional consideration in developing this strategy was the presence of the benomyl resistance SMG associated with the ku80 gene disruption in the DK80 strain introduced to promote homologous recombination (26). This SMG lacked the flanking loxP sites and could not be excised by Cre-mediated recombination.…”

“…Multiple rounds of anastomosis may be required to achieve excision of multiple SMGs in a single strain. The benomyl resistance SMG present in strain DK80 lacked loxP flanking elements (26) and was eliminated from the SD strains by replacement with wild-type ku80 gene from parental strain EP146 in a sexual cross between the hph/neo-SMG-free quadruple vic mutant strain (2211-22, Δku80 ben ) and strain EP146 (2211-22, ku80) performed as described by Chen et al (27). Oligonucleotides used in this study are indicated in Table S1.…”

Vegetative Incompatibility Loci with Dedicated Roles in Allorecognition Restrict Mycovirus Transmission in Chestnut Blight Fungus

“…C. parasitica strain DK80, a mutant of EP155 containing a disruption of the nonhomologous end-joining DNA repair pathway ku80 gene homolog to promote homologous recombination (Lan et al 2008), was used for most gene disruption analyses. C. parasitica field isolates collected from Japan (Liu and Milgroom 2007) and Maryland (Milgroom and Cortesi 1999) were kindly provided by Michael Milgroom (Cornell University).…”