A Phylogenetic Strategy Based on a Legume-Specific Whole Genome Duplication Yields Symbiotic Cytokinin Type-A Response Regulators

Abstract

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.

Deep Sequencing of the Medicago truncatula Root Transcriptome Reveals a Massive and Early Interaction between Nodulation Factor and Ethylene Signals

“…Type A ARR expression is one hallmark of the activation of CK pathways, and the upregulation of several type A ARRs has been observed following treatments with CK, rhizobium, and purified Nod factors (Op den Camp et al, 2011;Plet et al, 2011;Ariel et al, 2012). We observed the induction of several of these ARRs following inoculation with rhizobium, with most genes induced by 12 to 24 hpi, consistent with the up-regulation of IPT (and presumably de novo CK biosynthesis) at 12 hpi (Supplemental Fig.…”

“…In legumes CKs function to initiate nodule organogenesis (Gonzalez-Rizzo et al, 2006;Murray et al, 2007;Tirichine et al, 2007;Op den Camp et al, 2011;Plet et al, 2011) and autoregulation of nodulation (Sasaki et al, 2014). CKs are formed when an isopetenyl moiety is conjugated to adenine nucleotides via ISO-PENTENYLTRANSFERASES (IPTs).…”

ROLE OF PHYTOHORMONES IN THE CONTROL OF SYMBIOTIC NODULE DEVELOPMENT IN LEGUME PLANTS. I. CYTOKININS (review)

“…Meanwhile, an activation of the cytokinin response regulators was revealed in root hair cells of M. truncatula when inoculation and treatment with Nod factor [73], which was confirmed by the transcriptome analysis [74]. Moreover, transcriptome analysis showed an increased expression of MtCRE1 receptor in root hair cells at inoculation [75].…”

“…During symbiotic events in M. truncatula, the cytokinin receptors were shown to be first associated only with dividing cells in pericycle and the inner cortex, then with the developing nodule primordia, and in the mature nodule with a meristem and invasion zone [72,73]. Meanwhile, an activation of the cytokinin response regulators was revealed in root hair cells of M. truncatula when inoculation and treatment with Nod factor [73], which was confirmed by the transcriptome analysis [74].…”

“…It is known that the expression of A-type primary response genes (RR of A-type), activated by cytokinins, increases significantly when inoculation, i.e., cytokinin response is a part of the signal cascade induced by rhizobia [63,71,73]. The expression of cytokinin-regulated genes encoding MtRR1 and MtRR4 in M. truncatula inoculated with Sinorhizobium meliloti was significantly reduced in Nod  mutants dmi1, dmi2, dmi3 and nsp2, deficient in basic components of the Nod factor signal transmission [63].…”