Two Direct Targets of Cytokinin Signaling Regulate Symbiotic Nodulation in Medicago truncatula    

Abstract

Cytokinin regulates many aspects of plant development, and in legume crops, this phytohormone is necessary and sufficient for symbiotic nodule organogenesis, allowing them to fix atmospheric nitrogen. To identify direct links between cytokinins and nodule organogenesis, we determined a consensus sequence bound in vitro by a transcription factor (TF) acting in cytokinin signaling, the nodule-enhanced Medicago truncatula Mt RR1 response regulator (RR). Among genes rapidly regulated by cytokinins and containing this so-called RR binding site (RRBS) in their promoters, we found the nodulationrelated Type-A RR Mt RR4 and the Nodulation Signaling Pathway 2 (NSP2) TF. Site-directed mutagenesis revealed that RRBS cis-elements in the RR4 and NSP2 promoters are essential for expression during nodule development and for cytokinin induction. Furthermore, a microRNA targeting NSP2 (miR171 h) is also rapidly induced by cytokinins and then shows an expression pattern anticorrelated with NSP2. Other primary targets regulated by cytokinins depending on the Cytokinin Response1 (CRE1) receptor were a cytokinin oxidase/dehydrogenase (CKX1) and a basic Helix-Loop-Helix TF (bHLH476). RNA interference constructs as well as insertion of a Tnt1 retrotransposon in the bHLH gene led to reduced nodulation. Hence, we identified two TFs, NSP2 and bHLH476, as direct cytokinin targets acting at the convergence of phytohormonal and symbiotic cues.

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.…”

“…Ariel et al (2012) noted that treating M. truncatula roots with CKs induces the expression of GA biosynthetic genes, suggesting that the Nod factor-induced biosynthesis of CK may indirectly stimulate GA pathways. Our sampling of multiple early time points reveals nuanced aspects of GA signaling during the legume-rhizobium interaction.…”

Flavonoids and Auxin Transport Inhibitors Rescue Symbiotic Nodulation in the Medicago truncatula Cytokinin Perception Mutant cre1

“…flavonoid-metabolic genes constituted the most significantly enriched functional category (Ariel et al, 2012;Supplemental Table 2). Previous results showing that a local BAP treatment of white clover roots led to cortical cell divisions, CHS induction, and flavonoid accumulation in the dividing cells also support this model (Mathesius et al, 2000).…”

“…This suggests that the cre1 mutant is compromised in its ability to regulate flavonoid accumulation in response to rhizobia either through cytokininmediated changes in biosynthesis or conversion of flavonoids during nodule initiation. A previous study showed that a large number of flavonoid metabolic genes were transcriptionally regulated by cytokinin in M. truncatula roots (Ariel et al, 2012; genes listed in Supplemental Table 2). …”

“…In M. truncatula, the cytokinin receptor CRE1 (CYTOKININ RESPONSE1) is necessary for perceiving exogenous cytokinin in roots, and either mutation or local RNAi-induced silencing of CRE1 in roots leads to the inhibition of nodule formation (Gonzalez-Rizzo et al, 2006;Plet et al, 2011). In response to rhizobia and cytokinins, the cre1 mutant fails to induce several cytokinin primary response genes, such as RESPONSE REGULATOR4 and NSP2 (NODULATION SIGNALING PATHWAY2) (Ariel et al, 2012). A recent transcriptome comparison of gene expression changes in wild-type and cre1 mutant roots in response to a 3-h Nod factor treatment in M. truncatula demonstrated that cytokinin signaling is necessary for the majority (;75% or ;600 transcripts) of Nod factor-induced transcriptional changes (van Zeijl et al, 2015).…”