Fig. 3: DUF4123 proteins physically link cognate effectors to variable C-terminal extensions of VgrG proteins.
A Outcome of intraspecific growth competitions between the indicated P. aeruginosa PA14 donor and recipient strains. The competitive index is calculated as a change (final/initial) in the donor-to-recipient ratio. Error bars represent mean values +/− SEM, n = 3 biological replicates. Asterisks indicate statistically significant differences in the competitive indices between indicated donor and recipient strain (p < 0.05). Differences between groups were calculated using an ordinary one-way ANOVA with multiple comparisons against the parent strain. Against the ∆ptx2∆pti2 recipient, the p-value for the mean difference between parent and ∆tap6 is <0.0001. Against the ∆rhsP2∆rhsI2 recipient, the p-value for the mean difference between parent and ∆tap14 is 0.0004. All other p-values are not significant. B Schematic representation of VgrG6 and VgrG14. The inset represents pairwise alignment from residues 644-680. C AlphaFold3 models of VgrG6 and VgrG14 trimers. Colored regions represent residues 653–680. D Western blot analysis of co-purification experiments between the indicated heterologously expressed tagged (hexahistidine (H) or FLAG (F)) proteins. E Size exclusion chromatograms of indicated combinations of recombinant proteins. F Western blot analysis of immunoprecipitation experiments between the indicated heterologously expressed tagged (VSV-G or V5) proteins. G Western blot analysis of co-purification experiments between the indicated heterologously expressed tagged (hexahistidine (H) or FLAG (F)) proteins. RhsP2 was detected using a polyclonal antibody raised against a truncation of this protein lacking its C-terminal toxin domain. Control lanes represent co-expression of VgrG14CT-His and RhsP2 without Tap14-FLAG. The asterisk represents a non-specific band detected by the RhsP2 polyclonal antibody. Western blots are representative of three independent experiments.
