3-hydroxy-3-methylglutaryl coenzyme a reductase 1 interacts with NORK and is crucial for nodulation in Medicago truncatula

Abstract

NORK in legumes encodes a receptor-like kinase that is required for Nod factor signaling and root nodule development. Using Medicago truncatula NORK as bait in a yeast two-hybrid assay, we identified 3-hydroxy-3-methylglutaryl CoA reductase 1 (Mt HMGR1) as a NORK interacting partner. HMGR1 belongs to a multigene family in M. truncatula, and different HMGR isoforms are key enzymes in the mevalonate biosynthetic pathway leading to the production of a diverse array of isoprenoid compounds. Testing other HMGR members revealed a specific interaction between NORK and HMGR1. Mutagenesis and deletion analysis showed that this interaction requires the cytosolic active kinase domain of NORK and the cytosolic catalytic domain of HMGR1. NORK homologs from Lotus japonicus and Sesbania rostrata also interacted with Mt HMGR1, but homologous nonsymbiotic kinases of M. truncatula did not. Pharmacological inhibition of HMGR activities decreased nodule number and delayed nodulation, supporting the importance of the mevalonate pathway in symbiotic development. Decreasing HMGR1 expression in M. truncatula transgenic roots by RNA interference led to a dramatic decrease in nodulation, confirming that HMGR1 is essential for nodule development. Recruitment of HMGR1 by NORK could be required for production of specific isoprenoid compounds, such as cytokinins, phytosteroids, or isoprenoid moieties involved in modification of signaling proteins.

Figure 1.

Mt HMGR1ΔN Coimmunoprecipitates with Mt NORK-IC. Top panel: Arrowheads indicate c-Myc–tagged (GFP-MYC and HMGR1ΔN-GFP-MYC) and HA-tagged (HA-GFP and HA-NORK-IC) proteins produced in Arabidopsis protoplasts by protein gel blot analysis using anti-MYC and anti-HA antibodies, respectively. In the HA-tagged protein extracts, asterisks indicate an aspecific band that runs slightly faster but was clearly different from HA-NORK-IC. Bottom panel: Detection of c-Myc–tagged proteins in the anti-HA immunoprecipitates. Immunoglobulin G heavy chain (IgG hc) and light chain (IgG lc) of rat anti-HA antibody used for the immunoprecipitation are present in all samples and detected by weak cross-reaction with the sheep anti-mouse antibody. + and − indicate the presence or the absence, respectively, of given proteins in the reactions.

Figure 2.

Alignment of the Full-Length M. truncatula HMGR Proteins. TM1 and TM2 are the hydrophobic transmembrane domains. Lines above the sequence alignments indicate HMGR1ΔN (broken line), HMGR1ΔNΔC1 (dotted line), and HMGR1ΔNΔC2 (full line). Asterisks mark positions that are variable in the NORK interaction domain among the five Mt HMGR proteins.

Figure 3.

Specificity of the NORK–HMGR Interactions. (A) Interaction of the NORK-IC with the different members of the Mt HMGR family in the Y2H system was evaluated by the growth (after 3 and 6 d) and β-galactosidase activity of yeasts. (B) The different constructs of HMGRs and NORK or NORK homologs used for pairwise Y2H interactions. The size of the different derivates and the special motifs in the proteins are indicated. TM, transmembrane domain; SP, signal peptide; LRRs, Leu-rich repeats; AA, amino acids. The asterisk indicates the mutation in NORK-R38. (C) Results of the pairwise Y2H interactions. ++ indicates yeast growth at 3 d (strong interaction), + indicates yeast growth at 6 d (weak interaction), and − indicates no yeast growth (no interaction).

Figure 4.

Expression Pattern and Subcellular Localization of Mt HMGR1. (A) Expression pattern of NORK and the HMGR genes in different plant organs by RT-PCR analysis. The constitutively expressed Mtc27 was used as control for RNA loading in (A) to (C). R, roots; N, nodules; L, leaves; S, stems; F, flowers. (B) Expression pattern of NORK and HMGR1 in roots, Nod factor–treated roots (R+NF), nodule primordia (NP), young nitrogen fixing nodules (YNFN), and mature nitrogen fixing nodules (MNFN). (C) Expression of NORK and HMGR1 in root hairs (RH) and roots devoid of root hairs (R-RH). (D) Localization of NORK (NORK-AS) and HMGR1 (HMGR1-AS) transcripts by in situ hybridization in the nodulation-competent root zone. Dark color (arrows) corresponds to the hybridization signal, which is absent in the control section hybridized with the sense HMGR1 (HMGR1-S) probe. Bars = 50 μm. (E) Subcellular localization of the YFP control and MtHMGR1-YFP in Arabidopsis protoplasts. DIC, differential interference contrast. Bars = 10 μm.

Figure 5.

Effect of the HMGR Inhibitor Lovastatin on Medicago Root Growth and Nodule Development. (A) Primary root growth at different concentrations of lovastatin. Small letters (a to d) in (A), (B), and (D) represent significantly distinct categories by Student's t test at 95% confidence. (B) Lateral root formation at different concentrations of lovastatin. At 5 μM lovastatin, only lateral root primordia were formed that did not develop further. (C) Growth of S. meliloti is unaffected by lovastatin. (D) Nodulation efficiency in the presence of different lovastatin concentrations in three independent experiments.

Figure 6.

Downregulation of HMGR1 in Hairy Roots Inhibits Nodulation. (A) and (B) Transgenic M. truncatula roots expressing pRNAiMtHMGR1. (C) and (D) Transgenic M. truncatula roots expressing pRNAiMtHMGR4. Roots were observed under bright field in (A) and (C) and fluorescence microscope in (B) and (D) to identify hairy roots that were DsRED1 positive (DsRED1⁺) and distinguish them from the DsRED1-negative (DsRED1⁻; non-cotransformed) roots. Arrows indicate nodules. Bars = 0.5 cm. (E) Nodule number at 3 weeks after inoculation on DsRED1⁺ versus DsRED1⁻ roots of pRNAiMtHMGR1, pRNAiMtHMGR4, or pRedRoot:GFP transformation. Nodulation was similar in all plants except the DsRED1⁺ pRNAiMtHMGR1 roots, where no nodules developed. (F) Relative expression levels of HMGR1 and its closest homolog HMGR2 measured by quantitative RT-PCR in DsRED1⁺ roots expressing pRNAiMtHMGR1 compared with DsRED1⁻ ones. (G) Optimized hairy root nodulation allows nodule initiation on DsRED1⁺ pRNAiMtHMGR1 roots but in reduced number and with no or aborted infection. Error bars in (E) to (G) represent sd. (H) Nodules on DsRED1⁺ pRNAiMtHMGR1 roots were mainly uninfected and halted in the primordium state. (I) Occasional aborted surface infections marked by the blue color (arrow) of S. meliloti lacZ activity in a DsRED1⁺ pRNAiMtHMGR1 nodule.

Figure 7.

MVA Pathway in Plant Cells and Putative Roles for HMGR in Nodulation. (A) The steps of the cytoplasmic isoprenoid synthesis with indication of the relevant intermediates and important end products (in italics and underlined). The enzymatic reaction catalyzed by HMGR (boxed) is shown as well as inhibition of HMGR by lovastatin. (B) Model for potential implications of HMGR1 via NORK in nodule development. See text for details.