NODULE INCEPTION Directly Targets NF-Y Subunit Genes to Regulate Essential Processes of Root Nodule Development in Lotus japonicus

Abstract

The interactions of legumes with symbiotic nitrogen-fixing bacteria cause the formation of specialized lateral root organs called root nodules. It has been postulated that this root nodule symbiosis system has recruited factors that act in early signaling pathways (common SYM genes) partly from the ancestral mycorrhizal symbiosis. However, the origins of factors needed for root nodule organogenesis are largely unknown. NODULE INCEPTION (NIN) is a nodulation-specific gene that encodes a putative transcription factor and acts downstream of the common SYM genes. Here, we identified two Nuclear Factor-Y (NF-Y) subunit genes, LjNF-YA1 and LjNF-YB1, as transcriptional targets of NIN in Lotus japonicus. These genes are expressed in root nodule primordia and their translational products interact in plant cells, indicating that they form an NF-Y complex in root nodule primordia. The knockdown of LjNF-YA1 inhibited root nodule organogenesis, as did the loss of function of NIN. Furthermore, we found that NIN overexpression induced root nodule primordium-like structures that originated from cortical cells in the absence of bacterial symbionts. Thus, NIN is a crucial factor responsible for initiating nodulation-specific symbiotic processes. In addition, ectopic expression of either NIN or the NF-Y subunit genes caused abnormal cell division during lateral root development. This indicated that the Lotus NF-Y subunits can function to stimulate cell division. Thus, transcriptional regulation by NIN, including the activation of the NF-Y subunit genes, induces cortical cell division, which is an initial step in root nodule organogenesis. Unlike the legume-specific NIN protein, NF-Y is a major CCAAT box binding protein complex that is widespread among eukaryotes. We propose that the evolution of root nodules in legume plants was associated with changes in the function of NIN. NIN has acquired functions that allow it to divert pathways involved in the regulation of cell division to root nodule organogenesis.

NODULE INCEPTION creates a long-distance negative feedback loop involved in homeostatic regulation of nodule organ production

“…We investigated the spatial expression patterns of CLE-RS1, CLE-RS2, and NIN in L. japonicus using GUS reporter constructs (17). A 5.5-kb fragment from the putative translation initiation codon of either CLE-RS1 or CLE-RS2 was inserted upstream of the GUS gene.…”

“…We next examined whether NIN actively induces the expression of CLE-RS1 and CLE-RS2. NIN fused to the glucocorticoid receptor (NIN-GR) was overexpressed under the CaMV35S promoter in L. japonicus roots (17). Expression of CLE-RS1 and CLE-RS2 was induced within 4 h of treatment with dexamethasone (DEX) ( Fig.…”

“…Ectopic expression of NIN induces nodule primordium-like structures in the absence of rhizobia (17). We postulated that CLE-RS1 and CLE-RS2 expression is associated with NIN activity in L. japonicus, and investigated the activity of NIN in the systemic suppression of nodulation in response to rhizobial infection through activation of AON.…”

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

“…Members of the heterotrimeric, nucleosome-like NF-Y family of TFs are targets of the NIN protein (Soyano et al, 2013) and mediate early infection and nodule development processes (Petroni et al, 2012;Laloum et al, 2013Laloum et al, , 2014. NF-YA subunits bind to conserved CCAAT motifs in promoters, where they recruit the histone 2A/2B-like NF-YC and NF-YB subunits (Nardini et al, 2013), which also bind DNA, albeit nonspecifically.…”

“…NSP1 has been shown to bind to NODULATION-RESPONSIVE ELEMENT cis-elements in the promoters of ENOD11, ERN1, and NIN, while NSP2 likely binds to this region indirectly by way of its interaction with NSP1 (Hirsch et al, 2009). Likewise, NIN binds to the promoters of NF-YA1, CLE-RS1, CLE-RS2, and NPL in L. japonicus, while NF-YA1 binds to the ERN1 promoter in M. truncatula and is believed to coregulate its expression together with NSP1 and NSP2 (Xie et al, 2012;Soyano et al, 2013Laloum et al, 2014). MERLIN predicts 4,393 regulator-target gene relationships, including several of these known associations.…”