Sulfonylurea resistance reconstitution as a novel strategy for ILV2-specific integration in Magnaporthe oryzae

Fungal Genet Biol. 2014 Jul;68:71-6. doi: 10.1016/j.fgb.2014.04.005. Epub 2014 Apr 24.

Abstract

A sulfonylurea-resistant allele of the ILV2 gene encoding an acetolactate synthase from the rice-blast fungus Magnaporthe oryzae has been extensively used in fungal transformation as a dominant selectable marker that confers resistance to chlorimuron ethyl. We devised a novel strategy for site-specific integration of foreign DNA via sulfonylurea resistance reconstitution (SRR) by replacing the native ILV2 with the sulfonylurea-resistant ILV2(SUR) variant. In contrast to random ectopic integration, SRR-based targeted incorporation at a defined locus eliminates position/orientation effects, unnecessary mutations and/or variation in gene expression. Independent transformants derived from the same SRR construct showed consistent and reproducible fluorescent signal in M. oryzae. Furthermore, the high frequency (>95%) of ILV2-specific targeted integration via SRR circumvents the need for a deficiency in non-homologous end joining (NHEJ) pathway in the recipient strain. Unlike the split-marker technique, which is particularly suitable for targeted gene replacement, the SRR strategy should prove useful for promoter analyses, gene tagging and/or complementation analyses in filamentous fungi.

Keywords: Acetolactate synthase; Homologous recombination; ILV2; Locus-specific integration; Magnaporthe; Sulfonylurea resistance.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetolactate Synthase / genetics*
  • Genes, Fungal
  • Genetic Vectors
  • Magnaporthe / drug effects
  • Magnaporthe / genetics*
  • Sulfonylurea Compounds / pharmacology*
  • Transformation, Genetic

Substances

  • Sulfonylurea Compounds
  • Acetolactate Synthase