Nitrogen fixation in soybean takes place in root nodules that arise from de novo cell divisions in the root cortex. Although several early nodulin genes have been identified, the mechanism behind the stimulation of cortical cell division during nodulation has not been fully resolved. Here we provide evidence that two paralogs of soybean SHORT-ROOT (GmSHR) play vital roles in soybean nodulation. Expression of GmSHR4 and GmSHR5 (GmSHR4/5) is induced in cortical cells at the beginning of nodulation, when the first cell divisions occur. The expression level of GmSHR4/5 is positively associated with cortical cell division and nodulation. Knockdown of GmSHR5 inhibits cell division in outer cortical layers during nodulation. Knockdown of both paralogs disrupts the cell division throughout the cortex, resulting in poorly organized nodule primordia with delayed vascular tissue formation. GmSHR4/5 function by enhancing cytokinin signaling and activating early nodulin genes. Interestingly, D-type cyclins act downstream of GmSHR4/5, and GmSHR4/5 form a feedforward loop regulating D-type cyclins. Overexpression of D-type cyclins in soybean roots also enhanced nodulation. Collectively, we conclude that the GmSHR4/5-mediated pathway represents a vital module that triggers cytokinin signaling and activates D-type cyclins during nodulation in soybean.

Symbiotic nitrogen fixation (SNF) provides sufficient nitrogen (N) to meet most legume nutrition demands. In return, host plants feed symbionts carbohydrates produced in shoots. However, the molecular dialogue between shoots and symbionts remains largely mysterious. Here, we report the map-based cloning and characterization of a natural variation in GmNN1, the ortholog of Arabidopsis thaliana FLOWERING LOCUS T (FT2a) that simultaneously triggers nodulation in soybean and modulates leaf N nutrition. A 43-bp insertion in the promoter region of GmNN1/FT2a significantly decreased its transcription level and yielded N deficiency phenotypes. Manipulating GmNN1/GmFT2a significantly enhanced soybean nodulation, plant growth, and N nutrition. The near-isogenic lines (NILs) carrying low mRNA abundance alleles of GmNN1/FT2a, along with stable transgenic soybeans with CRISPR/Cas9 knockouts of GmNN1/FT2a, had yellower leaves, lower N concentrations, and fewer nodules than wild-type control plants. Grafting together with split-root experiments demonstrated that only shoot GmNN1/FT2a was responsible for regulating nodulation and thereby N nutrition through shoot-to-root translocation, and this process depends on rhizobial infection. After translocating into roots, shoot-derived GmNN1/FT2a was found to interact with GmNFYA-C (nuclear factor-Y subunit A-C) to activate symbiotic signaling through the previously reported GmNFYA-C-ENOD40 module. In short, the description of the critical soybean nodulation regulatory pathway outlined herein sheds novel insights into the shoot-to-root signaling required for communications between host plants and root nodulating symbionts.

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