(A) The structure of Nfre gene (4663 bp) and predicted protein domains (600 aminoacids). The boxes indicate coding regions, lines are introns, and the location of mutations in the three alleles is indicated. The underlines indicate domains in NFRe; LysM domains, TM: transmembrane, JX: juxtamembrane, kinase. (B), (C) and (D) are binding curves obtained from the biolayer interferometry measurements of NFRe ectodomain, NFR1 ectodomain and AtCERK1 ectodomain interaction with two different ligands, R7A Nod factor and GlcNAc5. Both NFRe and NFR1 ectodomain do not bind to GlcNAc5 but show binding to R7A Nod factor. AtCERK1 ectodomain does not bind R7A Nod factor but binds GlcNAc5. (E) Binding constants of NFRe, NFR1 and AtCERK1 ectodomain to GlcNAc5 and R7A Nod factor obtained from biolayer interferometry steady state-analysis. (F) Nfre encodes an active kinase domain. Autophosphorylation and protein kinase activities of wild-type NFRe, T459A, D436A, D418A NFRe mutant versions, and bovine serum albumin as control are shown. Myelin basic protein was used as substrate for kinase activities. Autoradiogram (top), and SDS-PAGE gels (bottom) are shown. KD, Std. Error and n represent the dissociation constant, standard deviation and number of biological replicates used for the analysis. N.D. represents not detectable.