FIGURE 7.

Ice nucleation activity and infection of Brachypodium distachyon with Pseudomonas syringae pv. syringae B728A. (a) Portions of leaves from non‐acclimated (NA) and cold‐acclimated (CA) plants sprayed with C.A. P. syringae pathovar cultures at 24°C standard conditions (the pair of left images) and −3°C (the pair of right images) showing disease incidence, and in the case of the NA leaves at −3°C, freeze damage. (b) Disease incidence measured as a percentage of leaves sprayed with the C.A. P. syringae pathovar showing disease symptoms with leaves from plants that were either NA and CA and then infected as whole plants and kept at the two temperatures shown. (c) Excised Brachypodium distachyon leaves infected with cold activated Pseudomonas syringae pv. syringae B728A, with the five images (left to right) representing pre‐infection, following infection and incubation for 12 h at −3°C, and during recovery at 4°C at 24 h post infection, 48 h post infection, and 1 week post infection showing infected cold‐acclimated (CA) Bd21 wild type, (d) infected CA induced antifreeze protein (AFP) knockdown line prOmiRBdIRI‐1e, and (e) uninfected CA Bd21 wild type controls. As indicted in the Section 2, the bacterial strain was cultured at 28°C to OD₆₀₀ = 0.6–1.0 and placed at 4°C for 2 days before resuspending in 10‐mM MgCl₂ and diluted to OD₆₀₀ = 0.2 to an approximate concentration of 1 × 10⁸ colony forming units ml⁻¹ prior to infection by dipping wounded ends of leaves in cultures. Assay was performed in triplicate with similar results and also performed with leaves from non‐acclimated plants (not shown).