Rhizobial infection of legume root hairs requires a rearrangement of the actin cytoskeleton to enable the establishment of plant-made infection structures called infection threads. In the SCAR/WAVE (Suppressor of cAMP receptor defect/WASP family verpolin homologous protein) actin regulatory complex, the conserved N-terminal domains of SCAR proteins interact with other components of the SCAR/WAVE complex. The conserved C-terminal domains of SCAR proteins bind to and activate the actin-related protein 2/3 (ARP2/3) complex, which can bind to actin filaments catalyzing new actin filament formation by nucleating actin branching. We have identified, SCARN (SCAR-Nodulation), a gene required for root hair infection of Lotus japonicus by Mesorhizobium loti. Although the SCARN protein is related to Arabidopsis thaliana SCAR2 and SCAR4, it belongs to a distinct legume-sub clade. We identified other SCARN-like proteins in legumes and phylogeny analyses suggested that SCARN may have arisen from a gene duplication and acquired specialized functions in root nodule symbiosis. Mutation of SCARN reduced formation of infection-threads and their extension into the root cortex and slightly reduced root-hair length. Surprisingly two of the scarn mutants showed constitutive branching of root hairs in uninoculated plants. However we observed no effect of scarn mutations on trichome development or on the early actin cytoskeletal accumulation that is normally seen in root hair tips shortly after M. loti inoculation, distinguishing them from other symbiosis mutations affecting actin nucleation. The C-terminal domain of SCARN binds to ARPC3 and ectopic expression of the N-terminal SCAR-homology domain (but not the full length protein) inhibited nodulation. In addition, we found that SCARN expression is enhanced by M. loti in epidermal cells and that this is directly regulated by the NODULE INCEPTION (NIN) transcription factor. Characterization of Lotus japonicus mutants defective for nodule infection by rhizobia led to the identification of a gene we named SCARN. Two of the five alleles caused formation of branched root-hairs in uninoculated seedlings, suggesting SCARN plays a role in the microtubule and actin-regulated polar growth of root hairs. SCARN is one of three L. japonicus proteins containing the conserved N and C terminal domains predicted to be required for rearrangement of the actin cytoskeleton. SCARN expression is induced in response to rhizobial nodulation factors by the NIN (NODULE INCEPTION) transcription factor and appears to be adapted to promoting rhizobial infection, possibly arising from a gene duplication event. SCARN binds to ARPC3, one of the predicted components in the actin-related protein complex involved in the activation of actin nucleation.

Nodule Inception (NIN) is one of the most important root nodule symbiotic genes as it is required for both infection and nodule organogenesis in legumes. Unlike most legumes with a sole NIN gene, there are four putative orthologous NIN genes in soybean (Glycine max). Whether and how these NIN genes contribute to soybean–rhizobia symbiotic interaction remain unknown. In this study, we found that all four GmNIN genes are induced by rhizobia and that conserved CE and CYC binding motifs in their promoter regions are required for their expression in the nodule formation process. By generation of multiplex Gmnin mutants, we found that the Gmnin1a nin2a nin2b triple mutant and Gmnin1a nin1b nin2a nin2b quadruple mutant displayed similar defects in rhizobia infection and root nodule formation, Gmnin2a nin2b produced fewer nodules but displayed a hyper infection phenotype compared to wild type (WT), while the Gmnin1a nin1b double mutant nodulated similar to WT. Overexpression of GmNIN1a, GmNIN1b, GmNIN2a, and GmNIN2b reduced nodule numbers after rhizobia inoculation, with GmNIN1b overexpression having the weakest effect. In addition, overexpression of GmNIN1a, GmNIN2a, or GmNIN2b, but not GmNIN1b, produced malformed pseudo-nodule-like structures without rhizobia inoculation. In conclusion, GmNIN1a, GmNIN2a, and GmNIN2b play functionally redundant yet complicated roles in soybean nodulation. GmNIN1b, although expressed at a comparable level with the other homologs, plays a minor role in root nodule symbiosis. Our work provides insight into the understanding of the asymmetrically redundant function of GmNIN genes in soybean.

Legumes acquire fixed nitrogen (N) from the soil and through endosymbiotic association with diazotrophic bacteria. However, establishing and maintaining N₂-fixing nodules are expensive for the host plant, relative to taking up N from the soil. Therefore, plants suppress symbiosis when N is plentiful and enhance symbiosis when N is sparse. Here, we show that the nitrate transporter MtNRT2.1 is required for optimal nodule establishment in Medicago truncatula under low-nitrate conditions and the repression of nodulation under high-nitrate conditions. The NIN-like protein (NLP) MtNLP1 is required for MtNRT2.1 expression and regulation of nitrate uptake/transport under low- and high-nitrate conditions. Under low nitrate, the gene encoding the C-terminally encoded peptide (CEP) MtCEP1 was more highly expressed, and the exogenous application of MtCEP1 systemically promoted MtNRT2.1 expression in a compact root architecture 2 (MtCRA2)-dependent manner. The enhancement of nodulation by MtCEP1 and nitrate uptake were both impaired in the Mtnrt2.1 mutant under low nitrate. Our study demonstrates that nitrate uptake by MtNRT2.1 differentially affects nodulation at low- and high-nitrate conditions through the actions of MtCEP1 and MtNLP1.

Most legume plants can associate with diazotrophic soil bacteria called rhizobia, resulting in new root organs called nodules that enable N2 fixation. Nodulation is an energy-consuming process, and nodule number is tightly regulated by independent systemic signaling pathways controlled by CLE/SUNN and CEP/CRA2. Moreover, nitrate inhibits legume nodulation via local and systemic regulatory pathways. In Medicago truncatula, NLP1 plays important roles in nitrate-induced inhibition of nodulation, but the relationship between systemic and local pathways in mediating nodulation inhibition by nitrate is poorly understood. In this study, we found that nitrate induces CLE35 expression in an NLP1-dependent manner and that NLP1 binds directly to the CLE35 promoter to activate its expression. Grafting experiments revealed that the systemic control of nodule number involves negative regulation by SUNN and positive regulation by CRA2 in the shoot, and that NLP1's control of the inhibition of rhizobial infection, nodule development, and nitrogenase activity in response to nitrate is determined by the root. Unexpectedly, grafting experiments showed that loss of CRA2 in the root increases nodule number at inhibitory nitrate levels, probably because of CEP1/2 upregulation in the cra2 mutants, suggesting that CRA2 exerts active negative feedback regulation in the root.