Targeted Mutagenesis and Complementation of Effector Genes in R. solanacearum

The mutagenesis of effectors in HA4-1 strain was performed as previously described (Cheng et al., 2021 (link)). To generate a mutant of the R. solanacearum target effector gene, the effector gene was replaced with a cassette harboring spectinomycin resistance. The genome fragment containing the target gene and its two flanking regions was amplified from the strain HA4-1 genome and then was cloned into pCE2-TA-Blunt-Zero vector (Vazyme 5min™ TA/Blunt-Zero Cloning Kit C601-01/02). A reverse amplification of the above recombinant vector was performed to delete the target gene and generate a linear vector only with the flanking region of the effector. A spectinomycin resistance gene (Spe) was connected with the linear to replace the target gene. The mutant plasmid was successfully constructed. Then the spectinomycin cassette with the effector flanking regions was generated, which was used to be transformed into R. solanacearum competent cells to replace the target effector gene.
To generate the effector complementation strain, the coding sequences with their native promoters and a Km^r cassette were cloned into the plasmid pH7C and then the fragments containing the coding sequences with the effector promoters and the antibiotic gene were amplified and inserted into a permissive chromosome site by transferred into the mutant competent cell, as described previously (Monteiro et al., 2012 (link)).