Gene knockout in X. citri

Single (∆XAC1768, ∆XAC1769, ∆XAC1770, ∆XAC1777) and double (∆XAC1768-XAC1769) gene knockout mutants were obtained by a two-step allelic exchange procedure. DNA fragments (~1.2 kb) corresponding to regions upstream and downstream to the target genes were amplified by PCR from the X. citri genome (Supplementary Table ²⁶). Each corresponding pair of fragments was ligated and then cloned into the pNPTS138 suicide vector⁹⁵ in the corresponding restriction sites. The plasmids were introduced into X. citri by electroporation (~2.3 kV, ~5 ms), and sucrose-sensitive and kanamycin-resistant colonies were selected (LBON-agar, 100 µg mL⁻¹ ampicillin, 100 µg mL⁻¹ kanamycin with and without 5% sucrose, respectively). This step selected colonies that suffered the first homologous recombination event, when the plasmid is inserted into the bacterial genome. These colonies were grown in LBON, 100 µg mL⁻¹ ampicillin without selection to allow the occurrence of the second recombinant event, when the plasmid is excised from the genome. The cultures were plated, and individual colonies were selected for simultaneous sucrose resistance and kanamycin sensitivity. Deletions were confirmed by PCR and DNA-sequencing (Supplementary Table ²⁷).