A gene locus for targeted ectopic gene integration in Zymoseptoria tritici

Research output: Contribution to journalJournal articleResearchpeer-review

  • S Kilaru
  • M Schuster
  • Latz, Meike
  • S Das Gupta
  • N Steinberg
  • H Fones
  • S J Gurr
  • N J Talbot
  • G Steinberg

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.

Original languageEnglish
JournalFungal Genetics and Biology
Volume79
Pages (from-to)118-124
Number of pages7
ISSN1087-1845
DOIs
Publication statusPublished - 2015
Externally publishedYes

Bibliographical note

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

    Research areas

  • Ascomycota, Gene Expression, Genetic Loci, Genetics, Microbial, Molecular Biology, Mutagenesis, Insertional, Recombination, Genetic, Succinate Dehydrogenase

ID: 162342164