Independent Regulation of Symbiotic Nodulation by the SUNN Negative and CRA2 Positive Systemic Pathways

Plant systemic signaling pathways allow the integration and coordination of shoot and root organ metabolism and development at the whole-plant level depending on nutrient availability. In legumes, two systemic pathways have been reported in the Medicago truncatula model to regulate root nitrogen-fixing symbiotic nodulation. Both pathways involve leucine-rich repeat receptor-like kinases (LRR-RLKs) acting in shoots and proposed to perceive signaling peptides produced in roots depending on soil nutrient availability. In this study, we characterized in the M. truncatula Jemalong A17 genotype a mutant allelic series affecting the Compact Root Architecture 2 (CRA2) receptor. These analyses revealed that this pathway acts systemically from shoots to positively regulate nodulation and is required for the activity of C-terminally encoded peptides (CEPs). In addition, we generated a double mutant to test genetic interactions of the CRA2 systemic pathway with the CLAVATA3-like peptide (CLE)/Super Numeric Nodule (SUNN) receptor systemic pathway negatively regulating nodule number from shoots, which revealed an intermediate nodule number phenotype close to the wild type. Finally, we showed that the nitrate inhibition of nodule numbers was observed in cra2 mutants but not in sunn and cra2 sunn mutants. Overall, these results suggest that CEP/CRA2 and CLE/SUNN systemic pathways act independently from shoots to regulate nodule numbers

Local and Systemic Regulation of Plant Root System Architecture and Symbiotic Nodulation by a Receptor-Like Kinase

International audienceIn plants, root system architecture is determined by the activity of root apical meristems, which control the root growth rate, and by the formation of lateral roots. In legumes, an additional root lateral organ can develop: the symbiotic nitrogen-fixing nodule. We identified in Medicago truncatula ten allelic mutants showing a compact root architecture phenotype (cra2) independent of any major shoot phenotype, and that consisted of shorter roots, an increased number of lateral roots, and a reduced number of nodules. The CRA2 gene encodes a Leucine-Rich Repeat Receptor-Like Kinase (LRR-RLK) that primarily negatively regulates lateral root formation and positively regulates symbiotic nodulation. Grafting experiments revealed that CRA2 acts through different pathways to regulate these lateral organs originating from the roots, locally controlling the lateral root development and nodule formation systemically from the shoots. The CRA2 LRR-RLK therefore integrates short-and long-distance regulations to control root system architecture under non-symbiotic and symbiotic conditions

Independent Regulation of Symbiotic Nodulation by the SUNN Negative and CRA2 Positive Systemic Pathways

International audiencePlant systemic signaling pathways allow the integration and coordination of shoot and root organ metabolism and development at the whole-plant level depending on nutrient availability. In legumes, two systemic pathways have been reported in the Medicago truncatula model to regulate root nitrogen-fixing symbiotic nodulation. Both pathways involve leucine-rich repeat receptor-like kinases acting in shoots and proposed to perceive signaling peptides produced in roots depending on soil nutrient availability. In this study, we characterized in the M. truncatula Jemalong A17 genotype a mutant allelic series affecting the Compact Root Architecture2 (CRA2) receptor. These analyses revealed that this pathway acts systemically from shoots to positively regulate nodulation and is required for the activity of carboxyl-terminally encoded peptides (CEPs). In addition, we generated a double mutant to test genetic interactions of the CRA2 systemic pathway with the CLAVATA3/EMBRYO SURROUNDING REGION peptide (CLE)/Super Numeric Nodule (SUNN) receptor systemic pathway negatively regulating nodule number from shoots, which revealed an intermediate nodule number phenotype close to the wild type. Finally, we showed that the nitrate inhibition of nodule numbers was observed in cra2 mutants but not in sunn and cra2 sunn mutants. Overall, these results suggest that CEP/CRA2 and CLE/SUNN systemic pathways act independently from shoots to regulate nodule numbers

<i>CRA2</i> expression in the shoot, root and symbiotic nodules.

<p>The spatial expression pattern of <i>CRA2</i> was analyzed using a promoter (1,8 kb)-GUS fusion (<b>A–D</b> and <b>I–N</b>) or by <i>in situ</i> hybridization (<b>E–H</b>). <b>A</b>, A root apex observed in bright field with dichroic illumination (Nomarski). The dotted lines indicate the position of the “cone-shaped” transition zone (TZ) between the cell proliferation zone (CPZ) and the cell elongation zone (CEZ). <b>B</b> and <b>D</b>, Root transversal sections in the CEZ (<b>B</b>) and the CPZ (<b>D</b>). <b>C</b>, Detail of the root meristem transition zone using a z-stack projection in confocal sections. The cell walls are visualized with a Propidium Iodide counterstaining, and GUS staining appears in reflectance as blue dots. <b>E–F</b>, <i>In situ</i> hybridization of the <i>CRA2</i> transcripts in root longitudinal sections. (<b>F</b>) Detail of the purple signal associated with vasculature strands. <b>G–H</b>, <i>In situ</i> hybridization of the <i>CRA2</i> transcripts in stem transversal sections (<b>G</b>, purple signal) or with a sense probe used as a negative control (<b>H</b>). Brackets indicate vascular bundles. <b>I–J</b>, Detail of the stele in the root differentiated region in bright field (<b>I</b>) to visualize the GUS staining (in dark blue) and phloem vascular bundles poles (Phl, in turquoise blue) or under UV illumination (<b>J</b>, same section as I) to visualize the blue autofluorescence of the xylem vascular bundle poles (Xy) and endodermis (End). <b>K–L</b>, Lateral root primordium initiation (<b>K</b>, arrow) and emergence (<b>L</b>) observed in bright field. <b>K</b> is a root transversal section. <b>M–N</b>, Nodule primordium (<b>M</b>, three days post-inoculation [dpi] with <i>S. meliloti</i> 1021) and mature nitrogen-fixing nodule (<b>N</b>, 14 dpi) observed in bright field. Bars  = 100 µm.</p

The <i>cra2</i> lateral root and root apical meristem phenotypes can be disconnected.

<p><b>A</b> and <b>E</b>, Representative examples of the wild-type (WT) and <i>cra2-1</i> Root Apical Meristems (RAM, stained with Propidium Iodide to visualize cell walls) three days post germination (dpg; <b>A</b>) or one dpg (<b>E</b>). The arrowhead indicates the apical position of the “cone-shaped” transition zone (as defined in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004891#pgen.1004891.s002" target="_blank">S2 Fig</a>.) between the cell proliferation zone (CPZ) and the cell elongation zone (CEZ). Bar  = 100 µm. <b>B, F</b>, Quantification in the WT and <i>cra2-1</i> roots of CPZ and CEZ length at three dpg (<b>B</b>) or one dpg (<b>F</b>). <b>C</b>, Quantification in the WT and <i>cra2-1</i> roots of the cells number in the same zones at three dpg. In <b>B</b>, <b>C</b>, and <b>F</b>, the error bars represent standard deviation, and a Mann-Whitney test was used to determine the significant differences between genotypes (*, α<5%, n = 10). <b>D</b>, Transversal sections of three dpg WT and <i>cra2-1</i> roots showing a similar radial organization of cell layers. Bar  = 100 µm. <b>G</b>, Representative examples of the WT and <i>cra2-1</i> root system architecture seven days post excision (dpe) of the RAM. The excision was performed either one day after germination (dpg, upper pictures) or three dpg (lower pictures). Bar  = 1 cm. <b>H</b>, Quantification of the lateral roots at two, four and seven days post excision (dpe) of the RAM in WT or <i>cra2-1</i> plants. As shown in (<b>G</b>), the meristems were excised at either one dpg (upper graph) or three dpg (lower graph). The error bars represent the confidence interval (α = 5%), and a Mann-Whitney test was used to determine significant differences between the genotypes for each time point (*, α<5%; n>25).</p

<i>CRA2</i> locally regulates lateral root formation and systemically regulates symbiotic nodule formation from the shoots.

<p><b>A</b> and <b>C</b>, Representative images of different grafting combinations between the wild-type (WT) and <i>cra2-1</i> plants that were grown in greenhouse with a perlite-sand mixture for eight weeks on an N-rich medium (<b>A</b>) or on an N-deprived medium with <i>Sinorhizobium meliloti</i> 1021 (<b>C</b>). The shoot/root grafting combinations are indicated above each picture. In panel (<b>C</b>), detailed pictures showing nodules (arrows) are included below. Bar  = 1 cm. <b>B</b>, Quantification of the lateral root density (number of emerged lateral roots/centimeter of parental root) in the different grafting combinations that are shown in (<b>A</b>). <b>D</b> and <b>E</b>, Quantification of the nodule numbers (<b>D</b>) and the nodule number related to the root dry weight (<b>E</b>) in the different grafting combinations that are shown in (<b>C</b>). In <b>B</b>, <b>D</b> and <b>E</b>, the error bars represent confidence intervals (α = 5%), and the significant differences were determined using a Kruskal and Wallis test (indicated by letters, α<5%; n>10).</p

The <i>CRA2</i> gene encodes a Leucine-Rich Repeat Receptor-Like Kinase (LRR-RLK).

<p><b>A</b>, Structure of the CRA2 protein indicating the 10 mutant alleles (arrowheads) that were identified by forward and reverse genetic screens (the indicated position is related to the predicted ATG) and functional domains. The vertical black bars indicate the predicted transmembrane domains; in grey are the Leucine-Rich Repeats; and the hatched region represents the kinase domain. The black arrowheads represent alleles that are linked to a <i>Tnt1</i> retro-element insertion; the grey arrowheads represent another insertional element; and the white arrowhead represents a nucleotide deletion causing a translational frameshift. Bar  = 50 residues. <b>B</b>, Phylogenetic tree of selected LRR-RLKs that are related to CRA2 from <i>Arabidopsis</i> (subfamily XI) or are functionally characterized in legumes. The sequences were aligned using Muscle, and the regions that were conserved between all of the sequences were defined with Gblocks. The phylogenetic relationships were determined using a maximum likelihood analysis (PhyML), and statistical support for each node was estimated by approximate likelihood ratio tests. The <i>Chlamydomonas reinhardtii</i> XP001698687 protein was used to root the tree.</p

The “<i>compact root architecture 2</i>” (<i>cra2</i>) mutants have short roots and more lateral roots independently of the growth conditions.

<p><b>A</b> and <b>B</b>, Representative examples of wild-type (WT) and <i>cra2-1</i> plants that were grown in the greenhouse for one month on a perlite-sand mixture (<b>A</b>) or for three months on soil (<b>B</b>). Bar  = 1 cm in A, 10 cm in B. <b>C</b>, Quantification of the stems, pods and seeds dry weight of the WT, <i>cra2-1</i> and <i>cra2-2</i> plants that are shown in (<b>B</b>). The error bars represent confidence intervals (α = 5%). A Kruskal and Wallis test was used to determine the significant differences (indicated by letters, α<5%; n = 10). <b>D</b>, Quantification of the root length (upper graph), lateral root number (middle graph) and lateral root density (lower graph) of the WT, <i>cra2-1</i> and <i>cra2-2</i> plants that were grown <i>in vitro</i> for 10 days post-germination (dpg) on an N-deprived “i” medium <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004891#pgen.1004891-GonzalezRizzo2" target="_blank">[42]</a>. The error bars represent confidence intervals (α = 1%). A Kruskal and Wallis test was used to determine the significant differences (indicated by letters, α<1%; n>25). <b>E</b>, Representative examples of the WT and <i>cra2-1</i> plants that were grown <i>in vitro</i> for three dpg on an N-deprived “i” medium <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004891#pgen.1004891-GonzalezRizzo2" target="_blank">[42]</a> or for 14 dpg on the same medium (- N), on an N-rich medium (+N, Fahraeus with NH₄NO₃ 10 mM; <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004891#pgen.1004891-Truchet1" target="_blank">[43]</a>), or on an N- and C-rich medium (+N+C, “Lateral Root Inducing Medium; <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004891#pgen.1004891-GonzalezRizzo2" target="_blank">[42]</a>). Note that in <i>cra2</i>, the lateral roots were already emerged at three dpg (arrowhead) and that the “compact root system architecture” phenotype was detectable independently of the growth medium. Bars  = 0,5 cm.</p

Two direct targets of cytokinin signaling regulate symbiotic nodulation in Medicago truncatula.

International audienceCytokinin regulates many aspects of plant development, and in legume crops, this phytohormone is necessary and sufficient for symbiotic nodule organogenesis, allowing them to fix atmospheric nitrogen. To identify direct links between cytokinins and nodule organogenesis, we determined a consensus sequence bound in vitro by a transcription factor (TF) acting in cytokinin signaling, the nodule-enhanced Medicago truncatula Mt RR1 response regulator (RR). Among genes rapidly regulated by cytokinins and containing this so-called RR binding site (RRBS) in their promoters, we found the nodulation-related Type-A RR Mt RR4 and the Nodulation Signaling Pathway 2 (NSP2) TF. Site-directed mutagenesis revealed that RRBS cis-elements in the RR4 and NSP2 promoters are essential for expression during nodule development and for cytokinin induction. Furthermore, a microRNA targeting NSP2 (miR171 h) is also rapidly induced by cytokinins and then shows an expression pattern anticorrelated with NSP2. Other primary targets regulated by cytokinins depending on the Cytokinin Response1 (CRE1) receptor were a cytokinin oxidase/dehydrogenase (CKX1) and a basic Helix-Loop-Helix TF (bHLH476). RNA interference constructs as well as insertion of a Tnt1 retrotransposon in the bHLH gene led to reduced nodulation. Hence, we identified two TFs, NSP2 and bHLH476, as direct cytokinin targets acting at the convergence of phytohormonal and symbiotic cues

A dual legume‐rhizobium transcriptome of symbiotic nodule senescence reveals coordinated plant and bacterial responses

International audienceSenescence determines plant organ lifespan depending on aging and environmental cues. During the endosymbiotic interaction with rhizobia, legume plants develop a specific organ, the root nodule, which houses nitrogen (N)-fixing bacteria. Unlike earlier processes of the legume-rhizobium interaction (nodule formation, N fixation), mechanisms controlling nodule senescence remain poorly understood. To identify nodule senescence-associated genes, we performed a dual plant-bacteria RNA sequencing approach on Medicago truncatula-Sinorhizobium meliloti nodules having initiated senescence either naturally (aging) or following an environmental trigger (nitrate treatment or salt stress). The resulting data allowed the identification of hundreds of plant and bacterial genes differentially regulated during nodule senescence, thus providing an unprecedented comprehensive resource of new candidate genes associated with this process. Remarkably, several plant and bacterial genes related to the cell cycle and stress responses were regulated in senescent nodules, including the rhizobial RpoE2-dependent general stress response. Analysis of selected core nodule senescence plant genes allowed showing that MtNAC969 and MtS40, both homologous to leaf senescence-associated genes, negatively regulate the transition between N fixation and senescence. In contrast, overexpression of a gene involved in the biosynthesis of cytokinins, well-known negative regulators of leaf senescence, may promote the transition from N fixation to senescence in nodules