Crystallization of the rice immune receptor RGA5A_S with the rice blast fungus effector AVR1-CO39 prepared via mixture and tandem strategies.

RGA5 is a component of the Pia resistance-protein pair (RGA4/RGA5) from Oryza sativa L. japonica. It acts as an immune receptor that directly recognizes the effector AVR1-CO39 from Magnaporthe oryzae via a C-terminal non-LRR domain (RGA5A_S). The interaction between RGA5A_S and AVR1-CO39 relieves the repression of RGA4, leading to effector-independent cell death. To determine the structure of the complex of RGA5A_S and AVR1-CO39 and to understand the details of this interaction, the complex was prepared by fusing the proteins together, by mixing them in vitro or by co-expressing them in one host cell. Samples purified via the first two strategies were crystallized under two different conditions. A mixture of AVR1-CO39 and RGA5A_S (complex I) crystallized in 1.1 M ammonium tartrate dibasic, 0.1 M sodium acetate-HCl pH 4.6, while crystals of the fusion complex RGA5A_S-TEV-AVR1-CO39 (complex II) were grown in 2 M NaCl. The crystal of complex I belonged to space group P321, with unit-cell parameters a = b = 66.2, c = 108.8 Å, α = β = 90, γ = 120°. The crystals diffracted to a Bragg spacing of 2.4 Å, and one molecule each of RGA5A_S and AVR1-CO39 were present in the asymmetric unit of the initial model. The crystal of complex II belonged to space group I4, with unit-cell parameters a = b = 137.4, c = 66.2 Å, α = β = γ = 90°. The crystals diffracted to a Bragg spacing of 2.72 Å, and there were two molecules of RGA5A_S and two molecules of AVR1-CO39 in the asymmetric unit of the initial model. Further structural characterization of the interaction between RGA5A_S and AVR1-CO39 will lead to a better understanding of the mechanism underlying effector recognition by R proteins.