In a previous study, we constructed a Tn5-tagged PXO99A mutant library, consisting of 24,192 Xoo transformants (clones), with a six times coverage of the PXO99A genome . In an attempt to identify major virulence genes in PXO99A, we screened the library and isolated mutants with reduced virulence. Here, we reported the isolation and characterization of a hrcQ-Tn5-insertion mutant PXM69 with
no virulence in host rice and no ability to elicit HR in non-host tobacco (Nicotiana benthamiana). We found that reintroduction of the hrcQ gene could only partially complement the loss of pathogenic function in PXM69. Xoo strains used in this study were PXO99A (wild-type) and its
mutants such as PXM69. Escherichia coli strain DH5α was used Vemurafenib mw in constructing plasmids for marker exchange mutagenesis. Xoo strains were grown at 28 °C on TSA Idelalisib medium (tryptone, 10 g L− 1; sucrose, 10 g L− 1; glutamic acid, 1 g L− 1; and agar, 15 g L− 1; pH 6.8–7.0) or NB medium (peptone, 5 g L− 1; yeast extract, 1 g L− 1; sucrose, 10 g L− 1; and beef extract, 3 g L− 1; pH 6.8 − 7.0). The E. coli strain was grown at 37 °C in Lutia − Bertani medium (tryptone, 10 g L− 1; yeast extract, 5 g L− 1; and sodium chloride, 10 g L− 1; pH 6.8–7.0). The broad host-range vector pHM1 was used to produce complementary constructs. Antibiotics used in the study were ampicillin (Amp) 100 μg mL− 1, kanamycin (Km) 50 μg mL− 1, spectinomycin (Sp) 100 μg mL− 1, and rifampicin (Rf) 50 μg mL− 1. The indica rice cultivar JG30, highly susceptible to PXO99A, was planted
in the field or in a greenhouse reaching 28–32 °C in daylight hours. Inoculations were performed on the plants at the maximum tillering stage (40 to 50 days old) by the leaf-clipping method . N. benthamiana Urocanase plants were grown in a growth cabinet under standard conditions (day and night temperatures of 25 °C and 20 °C, respectively), with 16 h light (30 to 40 μmol s− 1 m− 2) and 50%–60% humidity. Expanded leaves of 5- to 7-week-old plants were inoculated using a needleless syringe . The pathogenicity of all Xoo strains was evaluated using the leaf-cutting method . In the first round of screening for Tn5-insertion mutants, saturated cultures of Xoo strains grown in TSA medium were pelleted down and re-suspended in sterile distilled water (SDW) at an optical density of 1.0 at 600 nm (OD600 1.0). Scissors dipped in the inocula were used to clip fully expanded leaves of JG30. Disease symptoms were recorded two weeks after inoculation.