Fig. 3: EV-localized PsTET1 and PsTET3 proteins are required for the full virulence of P. sojae.

a Transcript levels of PsTET genes at different stages (My, non-sporulating mycelium grown in V8 broth; SP, sporangium; ZO, zoospores; IF20min = 20 min post-inoculation). Transcript levels measured by RT-qPCR were normalized to levels in mycelium using the PsActin gene as an internal reference. The data are presented as fold changes in a heat map. b, c P. sojae PsTET1 and PsTET3 double knockout mutants exhibit reduced virulence. b Disease symptoms photographed at 2 days post-inoculation. c Relative Phytophthora biomass in inoculated etiolated hypocotyls measured as the ratio between the amounts of P. sojae DNA and soybean DNA assayed at 2 dpi by qPCR; levels in P6497-inoculated soybean were set to 1.0. Data are presented as mean values (±SD). Statistical analyses were performed using Two-tailed Student’s t test. d NTA analysis of EV levels released by double knockout mutants and wild type. Data are presented as mean values (±SD). Statistical analyses were performed using Two-tailed Student’s t test. e Immunoblots of culture fluid from P. sojae transformant over-expressing eGFP-PsTET3. The P100 (pellet) fraction and S100 (supernatant) fractions from the culture fluid were analyzed alongside a whole mycelium protein lysate. CBB staining was used as a loading control. f Trypsin digestion of P. sojae EVs. The red star indicates the size expected for the N-terminal eGFP-containing fragment protected from trypsin. g EV sucrose gradient centrifugation and protein content analyzed by western blot. h Localization of eGFP-PsTET3 during infection of soybean hypocotyl epidermal cells by an eGFP-PsTET3 overexpressing P. sojae transformants. White arrows indicate putative EVs. The control transformant expresses eGFP in the P. sojae. Scale bars, 10 μm. The brightness of all images was increased identically to enhance visualization of EVs. i Confocal microscopy and fluorescence analysis of EVs isolated from P. sojae expressing eGFP-PsTET3 with or without FM4-64. Scale bars were indicated. All experiments were repeated three times with similar results. Source data are provided as a Source Data file.