Plasmid Construction and Mutagenesis

Plasmids were constructed as described previously^{16 (link)} using oligonucleotides listed in Supplementary Table 1. Bacterial strains were cultured in Luria-Bertani broth (LB) containing 50 µg ml⁻¹ Carbenicillin (Roth, 6344.3).
For generation of a plasmid encoding triple HA-tagged SseF, p2643 (sscB sseF::HA) was used and sseF::HA on p2643 was replaced by sseF::3HA using Gibson assembly GA. Primers for generation of vector fragment, check primers and sequence of synthetic sseF::3xHA (gBlocks, IDT) are listed in Supplementary Table 1.
Mutagenesis was performed for plasmids p4286, p4295, p4300, and p4305 encoding sseJ::HaloTag, pipB2::HaloTag, sopD2::HaloTag or sifA::HaloTag, respectively, using primers listed in Supplementary Table 1. Deletion of codons 331-336 in sifA removes the previously described prenylation site^{24 (link)} and was expected to reduce membrane interaction. Mutations Y281D Y284D K288D (sopD2), N309D L312D V316D (sseJ), and Y54D L57D M61D (pipB2) affect coiled-coil (CC) domains that were reported to contribute to membrane interactions of translocated T3SS effector proteins^{25 (link)}. Site-directed mutagenesis was performed using the Q5 SDM kit (NEB, E0554S), and resulting plasmids were confirmed by sequencing (SeqLab, Göttingen).