Quantitative co-culture assays. A. Prey cells (MFN1032 carrying pSMC2.1 gfp) were cultured alone or mixed with P. fluorescens MFE01 or various MFE01 T6SS mutants in a 1:5 ratio. After incubation for 4 h at 28°C, the MFN1032 colonies were counted (n = 6, the error bars represent the standard error of the mean). ** indicates a significant difference in the number of MFN1032 cfu (p-value < 0.01) relative to MFN1032 co-cultured with MFE01; * indicates a significant difference in the number of MFN1032 cfu (p-value < 0.05) relative to MFN1032 co-cultured with MFE01; ns indicates no significant difference. ⧫ indicates a significant difference in the number of MFN1032 cfu (p-value < 0.05) relative to the MFN1032-alone control assay. EV means empty pPSV35 (plasmid control). B. Prey cells (P. aeruginosa H103 carrying pSMC2.1 gfp) were cultured alone or mixed with P. fluorescens MFE01 or various MFE01 T6SS mutants in a 1:5 ratio. After 4 h at 28°C, H103 colonies were counted (n = 6, the error bars represent the standard error of the mean). ** indicates a significant difference in the number of H103 cfu (p-value < 0.01) relative to H103 co-cultured with MFE01; * indicates a significant difference in the number of H103 cfu (p-value < 0.05) relative to H103 co-cultured with MFE01; ns indicates no significant difference. ⧫ indicates a significant difference in the number of H103 cfu (p-value < 0.05) relative to the H103-alone control assay. EV means empty pPSV35 (plasmid control). C. Prey cells (MFP05 carrying pSMC2.1 gfp) were cultured alone or mixed with P. fluorescens MFE01 or various MFE01 T6SS mutants in a 1:5 ratio. EV indicates empty vector for the pPSV35 control. After 4 h at 28°C, MFP05 colonies were counted (n = 6, the error bars represent the standard error of the mean). ** indicates a significant difference in the number of MFP05 cfu (p-value < 0.01) relative to MFE05 co-cultured with MFE01; * indicates a significant difference in the number of MFE05 cfu (p-value < 0.05) relative to MFE05 co-cultured with MFE01; ns indicates no significant difference. ⧫ indicates a significant difference in the number of MFE05 cfu (p-value < 0.05) relative to the MFE05-alone control assay.