title: A gene locus for targeted ectopic gene integration in Zymoseptoria tritici.
abstract: Understanding the cellular organization and biology of fungal pathogens requires accurate methods for genomic integration of mutant alleles or fluorescent fusion-protein constructs. In Zymoseptoria tritici, this can be achieved by integrating of plasmid DNA randomly into the genome of this wheat pathogen. However, untargeted ectopic integration carries the risk of unwanted side effects, such as altered gene expression, due to targeting regulatory elements, or gene disruption following integration into protein-coding regions of the genome. Here, we establish the succinate dehydrogenase (sdi1) locus as a single "soft-landing" site for targeted ectopic integration of genetic constructs by using a carboxin-resistant sdi1(R) allele, carrying the point-mutation H267L. We use various green and red fluorescent fusion constructs and show that 97% of all transformants integrate correctly into the sdi1 locus as single copies. We also demonstrate that such integration does not affect the pathogenicity of Z. tritici, and thus the sdi1 locus is a useful tool for virulence analysis in genetically modified Z. tritici strains. Furthermore, we have developed a vector which facilitates yeast recombination cloning and thus allows assembly of multiple overlapping DNA fragments in a single cloning step for high throughput vector and strain generation.
authors.affiliation: Biosciences, University of Exeter, Exeter EX4 4QD, UK. Electronic address: S.Kilaru@exeter.ac.uk.
authors.forename: S
authors.initials: S
authors.lastname: Kilaru
authors.affiliation: Biosciences, University of Exeter, Exeter EX4 4QD, UK.
authors.forename: M
authors.initials: M
authors.lastname: Schuster
authors.affiliation: Biosciences, University of Exeter, Exeter EX4 4QD, UK.
authors.forename: M
authors.initials: M
authors.lastname: Latz
authors.affiliation: Biosciences, University of Exeter, Exeter EX4 4QD, UK.
authors.forename: S
authors.initials: S
authors.lastname: Das Gupta
authors.affiliation: Geography, University of Exeter, Exeter EX4 4RJ, UK.
authors.forename: N
authors.initials: N
authors.lastname: Steinberg
authors.affiliation: Biosciences, University of Exeter, Exeter EX4 4QD, UK.
authors.forename: H
authors.initials: H
authors.lastname: Fones
authors.affiliation: Biosciences, University of Exeter, Exeter EX4 4QD, UK.
authors.forename: S J
authors.initials: SJ
authors.lastname: Gurr
authors.affiliation: Biosciences, University of Exeter, Exeter EX4 4QD, UK.
authors.forename: N J
authors.initials: NJ
authors.lastname: Talbot
authors.affiliation: Biosciences, University of Exeter, Exeter EX4 4QD, UK. Electronic address: G.Steinberg@exeter.ac.uk.
authors.forename: G
authors.initials: G
authors.lastname: Steinberg
mesh_terms: D001203:Ascomycota / Q000235:genetics*; D015870:Gene Expression; D056426:Genetic Loci; D005827:Genetics, Microbial / Q000379:methods*; D008967:Molecular Biology / Q000379:methods*; D016254:Mutagenesis, Insertional / Q000379:methods*; D011995:Recombination, Genetic; D013385:Succinate Dehydrogenase / Q000235:genetics
chemical_list: D013385:Succinate Dehydrogenase
keywords: Dominant selectable marker; Mycosphaerella graminicola; Septoria tritici blotch; Succinate dehydrogenase; Wheat pathogenic fungi