The Medicago truncatula Lysine Motif-Receptor-Like Kinase Gene Family Includes NFP and New Nodule-Expressed Genes

Localization of NFP gene expression throughout nodulation in M. truncatula. A to H, Expression of a Pro_NFP-GUS fusion in transgenic roots, inoculated or not with S. meliloti. A, Whole, noninoculated lateral root showing constitutive GUS activity (blue) in root hairs. B to F, Root segments showing GUS activity (magenta) in the epidermis 1 d postinoculation (dpi; B), in the inner and middle cortex where cell divisions are occurring under a curled root hair (arrow) at 2 dpi (C), in the inner and middle cortex 2 dpi, and in outer cortical cells (D), in outer cortical cells through which an infection thread (arrow) is passing (E), and in an emerging nodule 5 dpi (F). G and H, Longitudinal section of an 18-d-old nodule showing GUS activity (blue) in the apical region and bacteria (magenta) in infection threads and the nitrogen-fixing zones. H, Close-up of the box in G. I, GUS activity (blue) in an 18-d-old whole nodule, with bacteria not stained. J and K, In situ hybridization of NFP gene expression on a sectioned 14-d-old nodule using a ³⁵S-UTP labeled antisense NFP probe. Bright-field image of nodule section with silver grains visible in black (J); epipolarization image of I (K). Arrows indicate points of infection in root hair curls (C and D) and an infection thread (E). Bars = 100 μm.

Analysis of the role of NFP in controlling infection thread formation. Roots of wild-type M. truncatula plants were transformed with either the vector alone (A) or an RNAi construct for NFP (B and C). Confocal images of root hairs in which GFP-expressing S. meliloti are seen in infection threads, which are tubular for the control and sac-like in NFPi root hairs. Root hairs in A and C were counterstained by propidium iodide (0.2 μg/mL). Bars = 10 μm.

Autophosphorylation tests on the intracellular kinase domains of NFP and LYK3. Purified GST-tagged intracellular domains of NFP, LYK3, and BAK1 (GST-NFP, GST-LYK3, and GST-BAK1) were assayed for autophosphorylation activity using [γ-³²P]ATP. Reactions were subjected to SDS-PAGE and proteins visualized with Coomassie Blue (A). Phosphorylated proteins were visualized by phosphor imaging (B). Molecular mass markers of 55 and 72 kD are shown.

Schematic representation of the domain structure of 16 M. truncatula LysM-RLKs, based on the amino acid alignment presented in Supplemental Figure 1 and protein domain predictions explained in the text. SP, Signal peptide; LysM1, LysM2, and LysM3, three LysM domains; CXC, Cys-X-Cys motif; TM, transmembrane segment; PL, P-loop; AL, activation loop; Ser-Thr kinase, predicted intracellular kinase domain.

Phylogenetic tree of kinase domains of LysM-RLK proteins of M. truncatula (16), rice (six), Arabidopsis (five), and NFR1 and NFR5 of L. japonicus, showing bootstrap values. NFP and LYK3 are underlined and the three groups are labeled I, II, and III.

Structural predictions for LysM domains of NFP. 1 and 3, HCA plots showing homologous hydrophobic residues (circled orange) and secondary structural features (boxed; 1) and electropositively (circled blue) and electronegatively (circled red) charged residues (3). 2, Ribbon diagrams showing the N- and C-terminal ends of the LysM domain polypeptides. 4, 3-D models with electrostatic potentials displayed on the molecular surface of LysM domains at −5 kT and +5 kT levels; red (electronegative), blue (electropositive), and white (neutral). A, LysM domain of E. coli murein hydrolase. B to D, Structural predictions for NFP LysM domains built from the atomic coordinates of this E. coli LysM domain: LysM1 (B); LysM2 (C); LysM3 (D). Note the strongly electropositive cavity in LysM2 of NFP (4C).