Extended Data Fig. 9: ZorAB recruit ZorC and ZorD during phage invasion.
From: Structure and mechanism of the Zorya anti-phage defence system
a, Complementation experiment between E. coli and P. aeruginosa (Pa) Zorya I. Schematic representation of EcZorI, PaZorI and the constructs for PaZorCD or PaZorD complementation of EcZorI gene deletions. b, Anti-phage defence provided by the constructs in (a), as measured using EOP assays for phages Bas49, Bas52 and Bas57. Data represent the mean of at least 3 replicates (data points indicate replicates) and are normalized to the control samples lacking Zorya. c, Strategy of fusing mNeonGreen (mNG) or HaloTag (HT) or both into EcZorI operon. d, The effects of the mNeongreen (mNG) fusions to EcZorI components on anti-phage defence, as measured using EOP assays for phage Bas24. Data represent the mean of at least 3 replicates (data points indicate replicates) and are normalized to the control samples lacking EcZorI. The boxed constructs (ZorB C-terminal mNG fusion: ZorB-mNG; ZorB C-terminal HT fusion: ZorB-HT; ZorC N-terminal mNG fusion: mNG-ZorC; ZorD C-terminal mNG fusion: ZorD-mNG; Dual-tagged constructs, ZorB C-terminal HT fusion and ZorC N-terminal mNG fusion: ZorB-HT + mNG-ZorC; ZorB C-terminal HT fusion and ZorD C-terminal mNG fusion: ZorB-HT + ZorD- mNG) were used for subsequent microscopy experiments. e, Exemplary denoised TIRF and brightfield microscopy pictures of mNG expression driven by the EcZorI native promoter (p-mNG) either untreated or exposed to Bas24 at an MOI of 5 for 30 min. Scale bar 2 µm. f, Exemplary denoised TIRF microscopy pictures of ZorB C-terminal mNG fusion either untreated or exposed to Bas24 at an MOI of 5 for 30 min. Scale bar 2 µm. g, Comparison of detected maxima of the ZorAB complex foci between untreated or exposed to Bas24 at an MOI of 5 for 30 min (n cells > 250 from n = 3 replicates), p-value: 0.030. Means are derived from three independent biological replicates. h, Exemplary denoised TIRF microscopy pictures of ZorD-mNG either untreated or exposed to increasing Bas24 at MOIs of 1, 5, or 50 for 30 min. i, Statistical comparison of ZorD-mNG maxima between untreated and conditions stated in h, p-values: 0.9978., 0.0009, 0.0258 and <0.0001. Means and exemplarily images in e and h derive from at least three independent biological replicates. For g and i data are presented as mean values and Tukey whiskers. Scale bar 2 µm.
