title: Exploitation of sulfonylurea resistance marker and non-homologous end joining mutants for functional analysis in Zymoseptoria tritici.
abstract: The lack of techniques for rapid assembly of gene deletion vectors, paucity of selectable marker genes available for genetic manipulation and low frequency of homologous recombination are major constraints in construction of gene deletion mutants in Zymoseptoria tritici. To address these issues, we have constructed ternary vectors for Agrobacterium tumefaciens mediated transformation of Z. tritici, which enable the single step assembly of multiple fragments via yeast recombinational cloning. The sulfonylurea resistance gene, which is a mutated allele of the Magnaporthe oryzae ILV2 gene, was established as a new dominant selectable marker for Z. tritici. To increase the frequency of homologous recombination, we have constructed Z. tritici strains deficient in the non-homologous end joining pathway of DNA double stranded break repair by inactivating the KU70 and KU80 genes. Targeted gene deletion frequency increased to more than 85% in both Z. tritici ku70 and ku80 null strains, compared to ⩽10% seen in the wild type parental strain IPO323. The in vitro growth and in planta pathogenicity of the Z. tritici ku70 and ku80 null strains were comparable to strain IPO323. Together these molecular tools add significantly to the platform available for genomic analysis through targeted gene deletion or promoter replacements and will facilitate large-scale functional characterization projects in Z. tritici.
authors.affiliation: Biosciences, University of Exeter, Stocker Road, Exeter EX4 4QD, UK.
authors.forename: Y S
authors.initials: YS
authors.lastname: Sidhu
authors.affiliation: Biosciences, University of Exeter, Stocker Road, Exeter EX4 4QD, UK.
authors.forename: T C
authors.initials: TC
authors.lastname: Cairns
authors.affiliation: Biosciences, University of Exeter, Stocker Road, Exeter EX4 4QD, UK.
authors.forename: Y K
authors.initials: YK
authors.lastname: Chaudhari
authors.affiliation: Biosciences, University of Exeter, Stocker Road, Exeter EX4 4QD, UK.
authors.forename: J
authors.initials: J
authors.lastname: Usher
authors.affiliation: Biosciences, University of Exeter, Stocker Road, Exeter EX4 4QD, UK.
authors.forename: N J
authors.initials: NJ
authors.lastname: Talbot
authors.affiliation: Biosciences, University of Exeter, Stocker Road, Exeter EX4 4QD, UK.
authors.forename: D J
authors.initials: DJ
authors.lastname: Studholme
authors.affiliation: Syngenta, Jealott's Hill International Research Centre, Bracknell, Berkshire RG426EY, UK.
authors.forename: M
authors.initials: M
authors.lastname: Csukai
authors.affiliation: Biosciences, University of Exeter, Stocker Road, Exeter EX4 4QD, UK. Electronic address: k.haynes@exeter.ac.uk.
authors.forename: K
authors.initials: K
authors.lastname: Haynes
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics; D001203:Ascomycota / Q000235:genetics* / Q000502:physiology; D025141:Drug Resistance, Fungal; D017353:Gene Deletion; D018390:Gene Targeting / Q000379:methods*; D005819:Genetic Markers; D005822:Genetic Vectors / Q000302:isolation & purification*; D059765:Homologous Recombination; D012641:Selection, Genetic; D013453:Sulfonylurea Compounds / Q000633:toxicity*; D014170:Transformation, Genetic
chemical_list: D005819:Genetic Markers; D013453:Sulfonylurea Compounds
keywords: Agrobacterium tumefaciens mediated transformation; Gene targeting frequency; Homologous recombination; Ku70; Ku80; Non homologous end joining mutants; Sulfonylurea; Yeast recombinational cloning; Zymoseptoria tritici