Fig. 1: Pseudomonas type III secretion system when expressed in A. tumefaciens can deliver T3Es to plant cells.

a Schematics of engineering A. tumefaciens to deliver proteins through a T3SS assay. A. tumefaciens strain GV2260 was mobilized with plasmids containing the P. syringae T3SS (pLN18) and T3E AvrPto tagged with PhiLOV (pBBR1MCS5-AvrPto-PhiLOV) to express the T3SS and AvrPto-PhiLOV. Promoter (P_AvrPto) and coding sequences (CDS) of AvrPto without a stop codon were fused to codon-optimized sequences of PhiLOV. CDS of AvrPto includes sequences encoding a type III secretion signal peptide (T3SS_sp). b AvrPto-PhiLOV secreted in the culture medium by the engineered A. tumefaciens were detected by immunoblotting. GV2260 derived A. tumefaciens strains grown in hrp-derepressing medium were separated into cell pellet and supernatant fractions and probed with PhiLOV-specific antibody. c Schematics of engineering A. tumefaciens to deliver proteins through a T3SS for in planta visualization. A. tumefaciens strain GV2260 was mobilized with pLN18 and a plasmid containing AvrPto-GFP₁₁ to express a T3SS and AvrPto-GFP₁₁. d AvrPto-GFP₁₁ delivered through a T3SS of an engineered A. tumefaciens can complement GFP_1-10 expressed in plants. Representative confocal images of N. benthamiana leaves transiently expressing GFP_1-10 individually infiltrated with A. tumefaciens strains GV2260, GV2260 (pLN18), GV2260 (AvrPto-GFP₁₁) and GV2260 (AvrPto-GFP₁₁, pLN18) are shown. Confocal microscopy was used to visualize GFP fluorescence 48 h post-infiltration. GFP signals were pseudo-colored to green and chlorophyll autofluorescence is shown in red. AvrPto-GFP₁₁ translocated to plant cells through a T3SS of engineered A. tumefaciens complemented GFP_1-10 produced in planta to form functional GFP. Scale bars, 10 µm. Experiments were repeated three times with similar results. Source data are provided as a Source Data file.