Sulfonylurea resistance reconstitution as a novel strategy for ILV2-specific integration in Magnaporthe oryzae.
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.
Temasek Life Sciences Laboratory, 1 Research Link, Singapore. Electronic address: yangfan@tll.org.sg.
Fan
F
Yang
Temasek Life Sciences Laboratory, 1 Research Link, Singapore; Department of Biological Sciences, National University of Singapore, Singapore; School of Biological Sciences, Nanyang Technological University, Singapore. Electronic address: naweed@tll.org.sg.
Naweed I
NI
Naqvi
D000095:Acetolactate Synthase / Q000235:genetics*; D005800:Genes, Fungal; D005822:Genetic Vectors; D020082:Magnaporthe / Q000187:drug effects / Q000235:genetics*; D013453:Sulfonylurea Compounds / Q000494:pharmacology*; D014170:Transformation, Genetic
D013453:Sulfonylurea Compounds; D000095:Acetolactate Synthase
Acetolactate synthase; Homologous recombination; ILV2; Locus-specific integration; Magnaporthe; Sulfonylurea resistance