TY - JOUR

T1 - A gene locus for targeted ectopic gene integration in Zymoseptoria tritici

AU - Kilaru, S

AU - Schuster, M

AU - Latz, Meike Anna Christine

AU - Das Gupta, S

AU - Steinberg, N

AU - Fones, H

AU - Gurr, S J

AU - Talbot, N J

AU - Steinberg, G

N1 - Septoria tritici blotch disease of wheat: Tools and techniques to study the pathogen Zymoseptoria tritici

PY - 2015

Y1 - 2015

N2 - 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.

AB - 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.

KW - Ascomycota

KW - Gene Expression

KW - Genetic Loci

KW - Genetics, Microbial

KW - Molecular Biology

KW - Mutagenesis, Insertional

KW - Recombination, Genetic

KW - Succinate Dehydrogenase

U2 - 10.1016/j.fgb.2015.03.018

DO - 10.1016/j.fgb.2015.03.018

M3 - Journal article

C2 - 26092798

VL - 79

SP - 118

EP - 124

JO - Fungal Genetics and Biology

JF - Fungal Genetics and Biology

SN - 1087-1845

ER -