Recruitment of novel calcium-binding proteins for root nodule symbiosis in Medicago truncatula

Abstract

Legume rhizobia symbiotic nitrogen (N2) fixation plays a critical role in sustainable nitrogen management in agriculture and in the Earth's nitrogen cycle. Signaling between rhizobia and legumes initiates development of a unique plant organ, the root nodule, where bacteria undergo endocytosis and become surrounded by a plant membrane to form a symbiosome. Between this membrane and the encased bacteria exists a matrix-filled space (the symbiosome space) that is thought to contain a mixture of plant- and bacteria-derived proteins. Maintenance of the symbiosis state requires continuous communication between the plant and bacterial partners. Here, we show in the model legume Medicago truncatula that a novel family of six calmodulin-like proteins (CaMLs), expressed specifically in root nodules, are localized within the symbiosome space. All six nodule-specific CaML genes are clustered in the M. truncatula genome, along with two other nodule-specific genes, nodulin-22 and nodulin-25. Sequence comparisons and phylogenetic analysis suggest that an unequal recombination event occurred between nodulin-25 and a nearby calmodulin, which gave rise to the first CaML, and the gene family evolved by tandem duplication and divergence. The data provide striking evidence for the recruitment of a ubiquitous Ca(2+)-binding gene for symbiotic purposes.

Figure 1.

Analysis of CaML1 expression in M. truncatula. A, RNA gel-blot analysis in developing nodules and in various tissues. Each lane in the top image contains 15 μg of total RNA from root (R), effective nodules 8 DAI (8), 10 DAI (10), and 14 DAI (14), ineffective nodules induced by S. meliloti strain T202 (IN), stem (S), leaf (L), flower (F), and pod (P). The blot was hybridized with a cDNA insert specific for the CaML1 transcript. The numbers at the right indicate the size of the hybridizing band in kilobases. The bottom image shows the ethidium bromide-stained rRNA on the gel prior to blotting to the membrane. B, Semiquantitative RT-PCR analysis of CaMLs during nodule initiation. Total RNA was isolated from uninoculated roots (0), inoculated roots at 4 DAI (4), root segments with nodules at 6 and 8 DAI (6, 8), and nodules 10 DAI (10) and used for RT and subsequent PCR to detect transcript accumulation. Typical CaM transcript (CaM1) was also amplified as a positive control. Thirty cycles were used for the amplification of CaM1 and 26 to 36 cycles for CaML1 to 6.

Figure 2.

Localization of CaML1 transcripts and protein within Medicago root nodules. Nodule nomenclature is classified according to Vasse et al. (1990): meristem (I), invasion zone (II), interzone (*), nitrogen-fixing zone (III), and senescent zone (IV). Un, Uninfected cell; in, infected cell; ba, bacteroid; SymM, symbiosome membrane. A to C, Longitudinal cross section of mature effective and ineffective M. truncatula root nodules hybridized with antisense CaML1 probe. High expression of CaML1 is found in the infected cells throughout effective M. truncatula root nodules in A and B. B, Magnification of the boxed area in A. Mature ineffective root nodules induced by S. meliloti strain T202 can be seen in C. Note CaML1 transcripts are detected only in infected cells of the late interzone (*) and early zone III of ineffective nodules. Scale bars in A and C are 0.5 mm; in B, 0.02 mm. D, CaML1:GUS reporter gene activity in effective alfalfa nodules at 8 DAI (scale bar = 0.3 mm). E to I, Confocal images of CaML1:GFP localization in effective alfalfa nodules. Nodules stained with SYTO63 (E) nucleic acid stain showing location of nucleic acids in red. GFP localization in nodule in E visualized by scanning with 488-nm laser line (F). Overlay of E and F resulted in G, showing GFP is exclusively localized in infected cells. Scale bars in E to G are 0.25 mm. Magnification (60×) of cells in zone III (H) shows GFP reporter gene activity localized in Syms of infected cells (scale bar = 0.02 mm). CaML1:GFP reporter gene activity (I) is localized in Syms released from infected cells (scale bar = 0.01 mm). J, Immunogold localization of CaML1 protein in alfalfa root nodules. Arrowhead points to gold particles (scale bar = 0.5 μm). K to M, Immunolocalization of CaML1 in M. truncatula root nodule Syms using CaML1 primary antibodies and secondary antibody conjugated to ALEXA546. CaML1 protein appears red (K) and is localized to SymS (white arrows). Bacteroid nucleic acids in K stained with SYTO13 appear green (L). Overlay of K and L resulted in M. CaML1 protein surrounds the bacteroid nuclear material (scale bar = 3 μm for K and M). Light scattering from the fluorochrome can contribute to the SymS appearing broader than expected.

Figure 3.

CaML protein is abundant in the SymS. A, Immunoblot detection of CaML protein using specific antibodies on cell-free extracts from roots (lane1), nodules 8 DAI (lane 2), 10 DAI (lane 3), and 14 DAI (lane 4), purified SymS protein (lane 5, 40 μg), and His-CaML protein (lane 6, 90 ng). Lanes 1 through 4 each contained 100 μg protein. The numbers at the right of each image indicate M_r markers in kilodaltons. B, Electrophoretic mobility shift assays on minigels in the presence (Ca²⁺) or absence (EGTA) of calcium carried out on His-CaML protein (His-CaML) and on a typical bovine CaM (B-CaM) as a control. C, Analysis of the ability of the purified fusion proteins G10-CaML protein (G10-CAML) and His-CaML protein and a typical bovine CaM to bind ⁴⁵Ca on a nitrocellulose miniblot after SDS-PAGE.

Figure 4.

All six M. truncatula CaML genes, nodulin-25, and nodulin-22 cluster within 112 kb of the M. truncatula genome. BLASTN analysis identified BAC-end sequence matches (shaded gray) to CaML2, CaML3, CaML5, CaML6, nodulin-22, and nodulin-25. PCR amplification was performed on all seven BACs using primers designed from CaML1 (○), CaML2 (▴), CaML3 (▪), CaML4 (▵), CaML5 (•), CaML6 (□), nodulin-25 (⋄), and nodulin-22 (▾). The results of PCR amplification were used to order the genes on the contig. For final verification, PCR products from the amplification of all eight genes using BAC Mth2-124L21 as template were cloned and sequenced.

Figure 5.

Multiple sequence alignment of conserved nodule-specific signal peptides in eight unique genes or gene families. Designations at left are GenBank accession numbers, TIGR tentative consensus identifiers, or MsCaM and MtCaML numbers.