Fig. 4

CCR is disrupted in PfMig1⁸⁸. Graphical representation of a Mig1⁸⁸ cassette constructed for homologous recombination and cloned in pCambia1302, and b Truncated Mig1⁸⁸ protein. c PCR amplification products using Mig1 flanking primers. Lane 1 represents DNA ladder, Lane 2 shows amplification of 2248-bp fragment indicating native Mig1 gene and flanking sequence of NCIM1228, and Lane 3 shows amplification of 3035-bp fragment using P1 and P2 primers indicating homologous recombination of PfMig1⁸⁸ cassette leading to loss of native Mig1 sequence. d RACE experiments to confirm the replacement of native gene with Mig1⁸⁸ at RNA level. Lane 1 represents DNA ladder, Lane 2 shows amplification of 1248-bp band representing full-length Mig1 RNA in NCIM1228 and no amplification in Lane 3 shows its absence in PfMig1⁸⁸ transformant, Lanes 4 and 5 represent 5′ RACE where 253-bp fragment from 5′ end of Mig1 RNA was amplified in both NCIM1228 and PfMig1⁸⁸, and Lanes 6 and 7 represent 3′ RACE where NCIM1228 shows amplification of 319-bp fragment representing intact 3′-end of Mig1 RNA, whereas failed 3′ RACE in PfMig1⁸⁸ shows truncated Mig1⁸⁸ RNA. 1-kb Plus DNA ladder (Fermentas) was used as DNA marker. Conidiospores of NCIM1228 and PfMig1⁸⁸ were spotted on e SC media having 2% Avicel in the absence and presence of 0.5% 2-DG, where PfMig1⁸⁸ was found resistant to 2-DG, and on f SC media having 2% glucose in the absence and presence of 1 mM AA, where PfMig1⁸⁸ was found sensitive to 1 mM AA