Figure 2: Deletion strategies for the ALT7 in the tomato pathotype of A. alternata. (A) A fusion PCR method was used to construct the ALT7 replacement vector. All PCR primers used in this figure are listed in Table 1. The 5 region of ALT7 was amplified by PCR with the primer pair ALT7AF/ ALT7AR, and the 3 region was amplified with the primer pair ALT7BF/ ALT7BR; the sizes of the PCR products were 540 and 600 bp, respectively. The hph gene was amplified with the primer pair fushphF/fushphR. The three PCR products were then used as a template for fusion PCR using the primer pair ALT7AF/ALT7BR, and the resulting PCR product was used for transformation. (B) The ALT7 locus before and after replacement of ALT7 with the marker gene hph. (C) PCR analysis of the gene replacement events in ALT7 in the wild-type strain As-27 (WT) and the three ALT7 disruptants (T1 to T3) and one ectopic transformant (T4) using the primer pairs hphF/hphR (C)-a, ALT7inF/ALT7inR (C)-b, and ALT7homoF/hph homo R (C)-c.