Fig. 2

Construction of a vector for targeted integration into the Mosdi1 locus in M. oryzae. a The effect of carboxin against mycelial growth of M. oryzae strains on complete media. M. oryzae wild type Guy11 and its derivative mutants were cultured for 3 days on complete media supplemented with various concentrations of carboxin. Mosdi1 ^R is a Mosdi1 point mutation strain; M1, M2 are two strains integrated with vector pMoC-eGFP (Mosdi1 ^ReGFP). b Strategy for generating Mosdi1 point mutation mutant. c Schematic drawing showing the organization of vector pMoC-eGFP. The fluorescent protein eGFP is expressed under the M. oryzae Morak1 promoter. URA3 and 2 micro2_origin cassettes enable yeast recombination-based cloning in S. cerevisiae. After integration into the Mosdi1 locus, a point mutation in the succinate dehydrogenase encoding gene Mosdi1, which resulted in substitution of histidine to leucine (H245L), could confer carboxin resistance for the transformants. Note that fragments are not drawn to scale. For more accurate information on fragment sizes see main text. d Image illustrates the integration of pMoC-eGFP into the native Mosdi1 locus of M. oryzae. In this integration, the carboxin-resistant Mosdi1 ^R allele and cytoplasmic eGFP were simultaneously inserted into the genome of M. oryzae. e The Mosdi1 ^ReGFP mutants (M1–M9) were validated by dialogistic PCR. Genomic DNA from Mosdi1 ^ReGFP strains and wild-type strain Guy11 were extracted, respectively, and used for screening of the integration into Mosdi1 locus by PCR. f The Mosdi1 ^ReGFP mutants (M1–M9) were validated by southern blot. Single integration into the expected locus was found in all nine Mosdi1 ^ReGFP mutants, and expected size bands were obtained in both Mosdi1 ^ReGFP mutants and wild type strain Guy11. The size markers in the corresponding agarose gel are shown at the left