Legume GRAS (GAI, RGA, SCR)-type transcription factors NODULATION SIGNALING PATHWAY1 (NSP1) and NSP2 are essential for rhizobium Nod factor-induced nodulation. Both proteins are considered to be Nod factor response factors regulating gene expression after symbiotic signaling. However, legume NSP1 and NSP2 can be functionally replaced by nonlegume orthologs, including rice (Oryza sativa) NSP1 and NSP2, indicating that both proteins are functionally conserved in higher plants. Here, we show that NSP1 and NSP2 are indispensable for strigolactone (SL) biosynthesis in the legume Medicago truncatula and in rice. Mutant nsp1 plants do not produce SLs, whereas in M. truncatula, NSP2 is essential for conversion of orobanchol into didehydro-orobanchol, which is the main SL produced by this species. The disturbed SL biosynthesis in nsp1 nsp2 mutant backgrounds correlates with reduced expression of DWARF27, a gene essential for SL biosynthesis. Rice and M. truncatula represent distinct phylogenetic lineages that split approximately 150 million years ago. Therefore, we conclude that regulation of SL biosynthesis by NSP1 and NSP2 is an ancestral function conserved in higher plants. NSP1 and NSP2 are single-copy genes in legumes, which implies that both proteins fulfill dual regulatory functions to control downstream targets after rhizobium-induced signaling as well as SL biosynthesis in nonsymbiotic conditions.

Legumes host their Rhizobium spp. symbiont in novel root organs called nodules. Nodules originate from differentiated root cortical cells that dedifferentiate and subsequently form nodule primordia, a process controlled by cytokinin. A whole-genome duplication has occurred at the root of the legume Papilionoideae subfamily. We hypothesize that gene pairs originating from this duplication event and are conserved in distinct Papilionoideae lineages have evolved symbiotic functions. A phylogenetic strategy was applied to search for such gene pairs to identify novel regulators of nodulation, using the cytokinin phosphorelay pathway as a test case. In this way, two paralogous type-A cytokinin response regulators were identified that are involved in root nodule symbiosis. Response Regulator9 (MtRR9) and MtRR11 in medicago (Medicago truncatula) and an ortholog in lotus (Lotus japonicus) are rapidly induced upon Rhizobium spp. Nod factor signaling. Constitutive expression of MtRR9 results in arrested primordia that have emerged from cortical, endodermal, and pericycle cells. In legumes, lateral root primordia are not exclusively formed from pericycle cells but also require the involvement of the root cortical cell layer. Therefore, the MtRR9-induced foci of cell divisions show a strong resemblance to lateral root primordia, suggesting an ancestral function of MtRR9 in this process. Together, these findings provide a proof of principle for the applied phylogenetic strategy to identify genes with a symbiotic function in legumes.

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