Figure 8.

ACD6/ACD6-1 Form Complexes with FLS2 and Affect the Trafficking of FLS2 and BAK1 to Plasma Membrane after BTH Treatment.

(A) Co-immunoprecipitation of FLS2 with ACD6-HA and ACD6-1-HA, respectively. Numbers below the blots show the relative amount of total protein quantified from the Coomassie blots and the relative amount of FLS2 after immunoprecipitation, normalized to total protein quantified by densitometry.

(B) Two-dimensional blue native gel analysis shows that FLS2 forms large complexes in ACD6-1-HA (A6-1-HA).

(C) The proportion of FLS2 and BAK1 in the plasma membrane (upper phase) is increased after BTH treatment (48 h, 150 μM) in Col. Additionally, the fraction of FLS2 and BAK1, respectively, at the plasma membrane was higher in ACD6-1-HA plants versus Col even without BTH.

(D) Loss of ACD6 (acd6-2) affects the plasma membrane levels (and fractions of proteins at the plasma membrane) of FLS2 and, to a lesser extent, BAK1 after BTH treatment.

(E) In the npr1 mutant plants, trafficking to the plasma membrane of BAK1 is severely compromised after BTH treatment. Microsomal proteins were isolated from leaves of the indicated plants and solubilized with 0.5% Triton X-100. Solubilized microsomal proteins were obtained for immunoprecipitations, separated by two-dimensional BNG/SDS–PAGE or two phase, and analyzed by immunoblotting with the indicated antibodies. All the lanes in panels (A), (C), and (E), respectively, were from one continuous membrane. These experiments were repeated three times, with similar results.