Fig. 7.

SpHtp3 is released from vesicles with the help of SpHtp1 from S. parasitica. a RTG-2 cells in direct contact with S. parasitica are shrunk with a condensed nucleus. In these cells, no cytosolic RNA (SytoRNA) can be detected and infected cells contain a high amount of vesicles (membrane stain FM4-64FX, see also Fig. 1b). In contrast, cells in close proximity but no direct contact do not show any morphological abnormalities (*). Scale bar: 20 µm (n = 3). b RTG-2 cells (c) were challenged with S. parasitica (h) after 1 h incubation with SpHtp3-mRFP. A hyphal tip (arrowhead, DIC) is attacking an RTG-2 cell. Magnification of the infected cell (red square) at different time points (bottom) show vesicles disappearing within a minute (arrowheads). See also Supplementary Movie 1. In contrast, cells in close proximity but no direct contact to S. parasitica contain less disappearing vesicles (*). Scale bar: 20 µm (n = 3). c Quantification of SpHtp3-mRFP containing vesicles of RTG-2 cells from b over time. d Vesicle release of SpHtp3-mRFP into the cytosol of RTG-2 cells after pre-incubation with SpHtp1_21–198-His₆ at pH 7.5. SpHtp3 accumulates in vesicles of RTG-2 cells after self-translocation (see also Fig. 2d). However, after co-incubation of SpHtp1 with SpHtp3, the number of vesicles in the periphery of the cells is reduced and the cytosolic fluorescence of RFP increased. Pictures were taken with a Zeiss Imager M2. Scale bar: 20 µm (n = 2). e Fluorescence intensity of SpHtp3-mRFP across the cell as indicated by dashed lines in d. f In vitro complex formation of recombinant SpHtp1-His₆ and SpHtp3-His₆ after cross-link verified by LC-MS/MS (Supplementary Table 2). An additional band, which only appears in the sample with both proteins is highlighted (Complex)