<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