Additional file 4 of PilG and PilH antagonistically control flagellum-dependent and pili-dependent motility in the phytopathogen Xanthomonas campestris pv. campestris

Additional file 4:Figure S4. Bacterial two-hybrid experiment and pull-down assay demonstrated interaction between FliN and PilG or PilH. (A) Bacterial two-hybrid experiment showed that PilG and PilH interacted with FliN protein. The reporter strain XL1-Blue MRF′ with different plasmid pairs was grown on no selective plates and double-selective indicator plates containing 5 mM 3-AT and 12.5 μg ml− 1 streptomycin. Protein-protein interactions activate the expression of addA and HIS3 genes within the reporter gene cassette of the reporter strain, resulting in resistance to streptomycin and 3-AT. (B) The pull-down assay demonstrated interaction between FliN and PilG or PilH in vitro. Lanes: 1, crude extract of BL21/pET30a after induction with IPTG; 2, crude extract of BL21/pET30a-PilG after induction with IPTG; 3, affinity-purified His6- PilG protein; 4, crude extract of BL21/pET30a- PilH; 5, affinity-purified His6- PilH protein; 6, crude extract of M15/pQE30 after induction with IPTG; 7, crude extract of M15/pQE30- FliN after induction with IPTG; 8, affinity-purified His6- FliN protein; 9, pull-down of protein His6- PilG by FliN; 10, pull-down of protein His6- PilH by FliN; 11, pull-down of protein His6- HpaR1 by FliN. M, molecular mass marker.