Co-expression with CesT chaperone increases accumulation of recombinant chloroplast-targeted Tir:GFP. Relative GFP fluorescence in intact leaf disks expressing proteins of interest (EspA:GFP, EspD:GFP, NleA:GFP, Tir-ep:GFP, Tir:GFP) with or without T3SS chaperones (CesD, CesAB, CesD2, CesT) was measured by fluorescence microtiter plate spectroscopy. Recombinant proteins and chaperones were targeted to the plant chloroplasts using the transit peptide from the small subunit of tobacco RuBisCO, and all infiltrated samples included the p19 suppressor of gene silencing. Infiltrated samples with and without chaperones were included on the same leaf and paired for statistical analysis. P-values from paired t-tests: (A) EspA:GFP vs. EspA:GFP+CesD, 0.1518; EspA:GFP vs. EspA:GFP+CesAB, 0.7618; EspA:GFP vs. EspA:GFP+CesD+CesAB, 0.9788; (B) EspD:GFP vs. EspD:GFP+CesD, 0.7955; EspD:GFP vs. EspD:GFP+CesD2, 0.0853; EspD:GFP vs. EspD:GFP+CesD+CesD2, 0.4343; (C) NleA:GFP vs. NleA:GFP+CesT, 0.0866; (D) Tir-ep:GFP vs. Tir-ep:GFP+CesT, 0.8414; (E) Tir:GFP vs. Tir:GFP+CesT, 0.0070. Leaf disks were harvested at 4 dpi. The X-axis indicates different plants as biological replicates.