Construction of a polar mutant of phtD

A PCR-derived amplicon containing the phtD gene from the phaseolotoxin gene cluster, from strain NPS3121, was cloned in the pUC19 vector; gene phtD was then disrupted by digestion with the restriction enzyme MscI and the insertion, in the opposite orientation to the transcription of the gene, of a 3.8 kb SmaI fragment containing the uidA-aph cassette from pWM6 [34]. The construct was confirmed by restriction digestion and introduced by electroporation into P. syringae pv. phaseolicola NPS3121; consequently, a polar mutant of phtD was obtained by the replacement of the wild type allele in the P. syringae pv. phaseolicola chromosome by a double recombination event. Kanamycin resistance was used to select for double-recombination events. The fidelity of the double recombination was confirmed by PCR analyses.