Fig. 3: Organ-specific efflux pumps are required for full colonization of Arabidopsis thaliana Col-0 and fitness in leaves.

a Locus position in the MF376 genome of statistically significant plant association mutations (black dots, association genes below the gray dots, negative association genes above). Organ-specific efflux pump systems benefit the association with Arabidopsis shoots (green) or roots (purple) but not with other organs and host. Log2(FC) of mutant abundance in association with plant tissue compared to soil is plotted against their location on the genome. b Our data suggest a model in which Arabidopsis produces two different antimicrobial agents, one from the shoot (green) and one from the root (purple), that can inhibit bacterial growth. To reach full colonization MF376 and WCS417 express different organ-specific efflux pumps that remove the plant-derived compound and protect the bacteria. c Reduced fitness of mutant Paraburkholderia bryophila MF376 d90 in an extract of macerated Arabidopsis leaves measured in a competition assay. A Chloramphenicol-resistant (Chl^R) Paraburkholderia bryophila MF376 clone with scarless deletion of the shoot association efflux pump (d90, light orange) was competed against a Tetracycline resistant (Tet^R) wild-type clone (WT, dark orange) overnight in macerated Arabidopsis leaves (top). Bacterial growth was quantified by plating serial five-fold dilutions of 1:1000 diluted overnight culture (left to right) on agar plates containing differentiating antibiotics, demonstrated that, while the wild-type clone grows well in macerated leaves, the mutant bacteria did not grow. A reciprocal experiment (bottom) confirmed these results. d Reduced fitness of Pseudomonas simiae WC417 d90 in macerated Arabidopsis leaf extract. Final OD₆₀₀ of overnight cultures of either wild-type Pseudomonas simiae WCS417 (WCS417-WT, dark cyan, solid line, circles, n = 3 biologically independent cultures) or a mutant in the homologous shoot association efflux pump (WCS417-d90, light cyan, dashed line, triangles, n = 3 biologically independent cultures) in medium supplemented with diluted macerated Arabidopsis leaf extract (shoot dilution). e Expression of the bacterial shoot association efflux pump, ef90, was induced following infiltration into the Arabidopsis apoplast (at 10⁷ cfu/ml Expression of ef90 in either Pseudomonas simiae WCS417 (cyan) or Paraburkholderia bryophila MF376 (orange) 15 and 60 min after infiltration into Arabidopsis leaves (For each n = 8 biologically independent leaves in 2 independent experiments). In both (d, e), boxplots show median (black line), mean (colored x), first and third quartiles (box) and one standard deviation (whiskers) of each treatment.