Fig. 2

Targeted disruption of LtGPX3 in L. theobromae using a CRISPR-Cas9-aided split marker approach. a Schematic illustration of the PCR strategy for generating split marker fragments overlapping within the hygromycin phosphotransferase gene (HYG) cassette under the control of the Aspergillus nidulans trpC gene promoter (P) and terminator (T) within LtGPX3 (in shaded bars). ΔLtgpx3/GPX3 expressing LtGPX3 under the control of LtGPX3 native promoter. The arrowheads indicate the primer locations and directions. b Schematic illustration of plasmid pmCas9-LtGPX3 construction. The nucleotides in red indicate the Esp3I enzyme digestion site. The gRNA spacers were synthesized by annealing the sense and antisense oligonucleotides with 5′-ACCT and AAAC-3′ overhangs and inserted into Esp3I-digested pmCas9 empty vector by T4 DNA ligase to generate plasmid pmCas9-LtGPX3. c PCR amplification of DNA fragments from genomic DNA of WT and two ΔLtgpx3 mutants (ΔLtgpx3-2 and -3) with indicated primers. Two pairs of primers (16F/16R and 17F/17R) were used to confirm LtGPX3 deletion transformants. Primers 16F and 17R were used to examine in situ integration of HYG within the LtGPX3 allele. Two LtGPX3-specific primers (15F/15R) amplified an expected 824 bp fragment from the genomic DNA of WT and ΔLtgpx3/GPX3 strains