Fig. 2

Fatty acid modifications of proteins involved in plant immunity. a Arabidopsis protoplasts were transformed with HA epitope-tagged FLS2 wild-type (WT) or an fls2 mutant encoding C830S, C831S. Protoplasts were treated with 10 μM Alk14, incubated for 6 h, and cells collected. Total protein was extracted, FLS2 proteins immunoprecipitated using anti-HA resin, and click chemistry performed. Incorporated Alk14 was visualized by fluorescence imaging and total protein was detected by anti-HA western blotting. b Transgenic Arabidopsis plants conditionally expressing avrPto were treated with 20 μM dexamethasone to induce transgene expression. Leaves were infiltrated twice with 10 μM Alk12, 6 h after induction and 6 h before sampling. Tissue was collected 30 h after induction and total protein extracted. AvrPto was immunoprecipitated using anti-AvrPto resin and a biotin tag added using click chemistry. Streptavidin-HRP western blotting was used to detect incorporation of Alk12. Anti-AvrPto western blotting was used to verify equal amounts of protein in all samples. c Nicotiana benthamiana leaves were infiltrated with Agrobacterium strains carrying avrPto-YFP fusion constructs encoding the WT protein or a G2A mutant. 10 μM Alk12 was infiltrated twice, 24 h after Agrobacterium infiltration and 6 h before sampling. Tissue was collected 48 h after transformation and total protein extracted. AvrPto proteins were immunoprecipitated using anti-GFP resin and a biotin tag attached using click chemistry. Incorporated Alk12 was detected by streptavidin-HRP western blotting. The anti-GFP western blot shows relative protein levels. d Nicotiana benthamiana was used to transiently express Pto-YFP fusions encoding the WT protein or a G2A mutant. 10 μM Alk12 was infiltrated twice, 24 h after Agrobacterium infiltration and 6 h before sampling. Tissue was collected 48 h after transformation, total protein extracted, and Pto proteins immunoprecipitated using anti-GFP resin. A biotin tag was attached using click chemistry and incorporation of Alk12 was detected by streptavidin-HRP western blotting. Protein levels were visualized by anti-GFP western blotting