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. 2018 Jun 29;7:e33506. doi: 10.7554/eLife.33506

Figure 4. NFRe signalling is dependent of NFR5.

(A) The NFRe kinase phosphorylates NFR5 kinase, whereas the NFReT459A shows no phosphorylation activity. The kinase of NeKA1 receptor in which the activation segment of NFRe was swapped with the corresponding region of NFR1 also phosphorylates NFR5 kinase. NFR1 kinase serves as positive control for NFR5 kinase transphosphorylation. Bovine serum albumin and NFR5 kinase domain are negative controls. (B) Bimolecular fluorescence complementation (BiFC) of YFP signal indicates protein-protein interactions in tobacco leaves. NFRe forms homomers (NFRe-nY +NFRe cY), and heteromers with NFR1 (NFRe-nY +NFR1 cY), or NFR5 (NFRe-cY +NFR5 nY). Formation of heteromeric complexes with NFR5 is not dependent on an active NFRe kinase (NFRe T459A -nY + NFR5 cY). (C) Working model of Nod factor signalling (green line) in the susceptible zone ensuring an efficient nodulation on the expanding root system. NFRe (dark grey line) has a constant expression in the epidermal cells of the susceptible zone. NFR1 (light grey line) dominates the uninoculated root in terms of expression level and spatial distribution (1). Once the symbiotic process is initiated by the Nod factor (NF), the expression of NFR1 is rapidly downscaled (2). A sustained expression of NFRe in the epidermal cells ensures an idling signalling in the susceptible zone, keeping the expanding root system tuned in to rhizobia (3). NFR1 acts as a master switch triggering recurrent symbiotic events in a fast and efficient manner from NFRe-attuned epidermal cells (4).

Figure 4.

Figure 4—figure supplement 1. NFRe is localized on plasma membrane and signals together with NFR5.

Figure 4—figure supplement 1.

(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).