Fig. 6: P. syringae proline utilization A (putA) is required for maximal type III secretion system deployment and bacterial growth during infection of Arabidopsis.
a Reactions catalyzed by PutA. Pro=proline, P5C = Δ1-pyrroline-5-carboxylate, GSA = glutamic semialdehyde, Glu=glutamic acid. b Time course of P. syringae DC3000 and DC3000ΔputA growth in M9 medium supplemented with 10 mM proline. Graphed are means ± SE of optical density at λ = 600 nm (OD600) measurements at indicated time points, n = 3. c Growth of P. syringae DC3000 and DC3000ΔputA in M9 medium supplemented with 10 mM of individual amino acids as indicated. Graphed are means ± SE of OD600 measurements at 22 h post-inoculation relative to OD600 at t = 0, n = 3. d GFP fluorescence from P. syringae DC3000 hrpLpromoter:gfp and DC3000ΔputA hrpLpromoter:gfp reporter strains incubated for 4.5 h in minimal medium supplemented with 10 mM fructose and 200 μM of individual metabolites as indicated. CA = citric acid, 4hba = 4-hydroxybenzoic acid, fruc = fructose. Graphed are means ± SE of normalized GFP fluorescence, n = 3. e AvrPto protein abundance in leaf tissue five hours after infiltration of leaves with either P. syringae DC3000 or DC3000ΔputA. Upper panel is immunoblot detection of AvrPto levels in protein extracts from infected leaves, lower panel is Coomassie Brilliant Blue (CBB) staining of the immunoblot to assess equal loading. Data shown are representative of three independent experiments. f Leaf bacteria levels three days after infiltration of Arabidopsis leaves with 1 × 106 cfu/mL of P. syringae DC3000 or DC3000ΔputA. Graphed are log transformed means ± SE of colony-forming units (cfu) of bacteria isolated from infected tissue, n = 14. Data were pooled from three independent experiments, n = 4-5 per experiment. Results in panels b–d are representative of two independent experiments. Asterisks in panels c, d and f denote statistical significance based on two-sided t-test, *p < 0.05. **p < 0.01, ***p < 0.001.
