Figure 4. The IAQVV/LVKIE region of the Pikp-HMA domain determines high-affinity binding to AVR-PikD.
(A) Protein sequence alignment showing the Pikp–ancHMA swap chimeras. The amino acid sequences of ancestral HMA (ancHMA), Pikp-HMA, and chimeras are aligned, with the protein model above corresponding to the Pikp-HMA structure. The colour-coded rectangles correspond to polymorphic regions used for chimeric swaps. (B) Schematic representation of Pikp-HMA (blue) in complex with AVR-PikD (pink) (De la Concepcion et al., 2018), with polymorphic regions between the Pikp-HMA and the ancHMA colour-coded as in (A). The molecular surfaces of the polymorphic residues are also shown. (C) Association between AVR-PikD (N-terminally tagged with FLAG) and Pikp-1, Pikp-1E230R, Pikp-1:ancHMA, and Pikp-1:ancHMA chimeras (N-terminally tagged with HA), labelled above, was tested in planta in co-IP experiment. Wild-type (WT) Pikp-1 and Pikp-1E230R were used as a positive and negative control, respectively. Immunoprecipitates (HA-IP) obtained with anti-HA probe and total protein extracts (input) were immunoblotted with the appropriate antisera, labelled on the left. Rubisco loading control was performed using Pierce staining solution. Arrowheads indicate expected band sizes. Results from three independent replicates of this experiment are shown in Figure 4—figure supplement 1.
Figure 4—figure supplement 1. Replicates of the co-immunoprecipitation (co-IP) experiment between AVR-PikD and the Pikp-1:ancHMA chimeras.
