(A) Wild-type and the T459A mutant version of NFRe localize on plasma membrane of N. benthamiana leaf cells. Co-localisation of YFP-labelled NFRe proteins with the AtPIP2-DsRED plasma membrane marker after plasmolysis (arrows) is shown. (B) NFRe forms homo and heteromeric complexes with NFR1 and NFR5 in N. benthemiana leaves. YFP fluorescence is detected when wild type or T459A mutant version of NFRe tagged with the N-terminal part of YFP protein (n–Y) is co-expressed with NFRe, NFR5 or NFR1 tagged with the C-terminal of the YFP (c–Y). No signal is detected when the same constructs are expressed alone. The left panel shows images collected using the YFP channel, and the right panel shows the corresponding bright field image. (C) Co-expression of NFRe and NFR5 induces cell death in N. benthamiana leaves. This phenotype is dependent on the presence of NFR5 and NFRe (upper panel), and on an active NFRe kinase (lower panel). Co-expression of NFRe and NFR1 does not lead to cell death in tobacco leaves (lower panel). A representative image illustrating the absence of cell death is shown in the lower panel for the following expressions: NFReT485A and NFR5, NFRe and NFR1, NFRe, NFR5, NFR1. (D). NFRe signalling in L. japonicus roots is dependent on NFR5. Unlike NFR5 (upper panel), the NFRe, NFR or NeK receptors expressed from the 35S promoter fail to activate Nin promoter in the nfr5-2-Nin:GUS plants exposed to M. loti bacteria for 5 weeks (representative root is shown in the lower panel). Scale bars, 20 μm (A), 100 μm (B).