Fig. 3
Deletion mutagenesis of Xss-V2–18 tal genes. a Schematic diagram of suicide plasmids pKMSA1 and pKMSA2. Fragments a (580 bp) and b (350 bp) were amplified on the left and right sides of the CRR, respectively, and cloned as a fused fragment in pKMSA1. Fragments c (580 bp) and d (150 bp) were amplified on the left and right sides of the CRR, respectively, and cloned as a fused fragment in pKMSA2. Constructs pKMSA1 and pKMSA2 were introduced into Xcm strain Xss- V2–18 by electroporation, and deletion of the CRR region was conducted as described in Methods. b Confirmation of 930- and 450- bp inserts in pKMSA1 and pKMSA2, respectively, by digestion with XbaI and SmaI. c PCR analysis of 41 putative mutants with primers pKMSA1-5F and pKMSA1-3R. A 930-bp fragment was amplified in M1, M2, M3, and M4, indicating that these four mutants underwent a homologous recombination and potential deletion of the CRR region. pKMSA1 was included as a control. d Southern hybridization analysis of Xss-V2–18 and mutant strains M1-M4. Plasmid DNA of WT Xss-V2–18 and mutants were isolated and digested with BamHI. The internal SphI fragment of pthXo1 (from Xoo) was used as a hybridization probe to detect tal genes. e PCR screening for putative mutants using primers pKMSA2-5F and pKMSA2-3R. pKMSA2 was included and used as a positive control. f Southern hybridization analysis of mutant M4 (used for second round of mutagenesis), M5 and M6. Plasmid DNA of M4, M5 and M6 were isolated and digested with BamHI, and the internal SphI fragment of pthXo1 was used a hybridization probe to detect tal genes