AP2-ERF Transcription Factors Mediate Nod Factor–Dependent MtENOD11Activation in Root Hairs via a Novelcis-Regulatory Motif

Abstract

Rhizobium Nod factors (NFs) are specific lipochitooligosaccharides that activate host legume signaling pathways essential for initiating the nitrogen-fixing symbiotic association. This study describes the characterization of cis-regulatory elements and trans-interacting factors that regulate NF-dependent and epidermis-specific gene transcription in Medicago truncatula. Detailed analysis of the Mt ENOD11 promoter using deletion, mutation, and gain-of-function constructs has led to the identification of an NF-responsive regulatory unit (the NF box) sufficient to direct NF-elicited expression in root hairs. NF boxmediated expression requires a major GCC-like motif, which is also essential for the binding of root hair-specific nuclear factors. Yeast one-hybrid screening has identified three closely related AP2/ERF transcription factors (ERN1 to ERN3) that are able to bind specifically to the NF box. ERN1 is identical to an ERF-like factor identified recently. Expression analysis has revealed that ERN1 and ERN2 genes are upregulated in root hairs following NF treatment and that this activation requires a functional NFP gene. Transient expression assays in Nicotiana benthamiana have further shown that nucleus-targeted ERN1 and ERN2 factors activate NF box-containing reporters, whereas ERN3 represses ERN1/ERN2-dependent transcription activation. A model is proposed for the fine-tuning of NF-elicited gene transcription in root hairs involving the interplay between repressor and activator ERN factors.

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

“…4A). Their rapid up-regulation reflects their importance in effecting subsequent symbiotic gene expression and their absolute requirement for nodulation (Kaló et al, 2005;Andriankaja et al, 2007;Marsh et al, 2007). Groups G1, G2, and G3 contained an additional 20 TFs that are coinduced along with NIN, ERN1, and NSP2 by 3 hpi (Fig.…”

A Small GTPase of the Rab Family Is Required for Root Hair Formation and Preinfection Stages of the Common Bean–RhizobiumSymbiotic Association

“…Perception of Nod factors occurs primarily in the epidermal cells, where initial induced responses in the plant lead to ENOD induction in the zone of actively growing root hairs (Journet et al, 1994(Journet et al, , 2001Scheres et al, 1990). Expression of ENODs is under the control of several transcription factors (Kalo et al, 2005;Smit et al, 2005;Andriankaja et al, 2007;Middleton et al, 2007). Mutation of the gene encoding the M. truncatula transcription factor ERN blocked the initiation and development of ITs and the Nod factor-induced gene expression (Middleton et al, 2007).…”

“…Bars = 100 mm. infection were selected: ERN1 (ERF required for nodulation) is a transcription factor of the ERF family that is part of the Nod signal transduction pathway downstream of the dmi3 gene and upregulated in root hairs of M. truncatula upon Nod factor treatment (Andriankaja et al, 2007;Middleton et al, 2007); Hap5 was identified in the same suppressive subtractive hybridization screening as RabA2 (Peltzer Meschini et al, 2008), and it encodes the C subunit of the CCAAT transcription factor, a regulator of nodule development (Combier et al, 2006); and Enod40 encodes an RNA containing two small conserved ORFs that is induced by Nod factor in the pericycle and in dividing cells of the cortex (Crespi et al, 1994). Nucleotide sequences of common bean ESTs with high similarity to M. truncatula ERN and Enod40 were obtained by a BLAST search on the Dana-Farber Cancer Institute (DFCI) Bean Gene Index database and used to design gene-specific primers for qRT-PCR experiments.…”

The Small GTPase ROP10 of Medicago truncatula Is Required for Both Tip Growth of Root Hairs and Nod Factor-Induced Root Hair Deformation

“…The GUS gene uidA driven by the promoter of the early nodulin gene ENOD11 (ENOD11pro:GUS) is a widely used molecular marker to visualize the activation of NF signaling in the root epidermis of M. truncatula (Andriankaja et al, 2007;Middleton et al, 2007). To substantiate the involvement of ROP10 in the NF signaling pathway, we used stably transformed ENOD11pro: GUS plants (Journet et al, 2001) and generated hairy roots overexpressing ROP10 or ROP10CA under the control of the Ljubq1 promoter.…”

“…Downstream components of the NF signal transduction pathway in M. truncatula include DMI2, a leucine-rich repeat receptor kinase (Esseling et al, 2004), DMI1, a potassium ion channel (Ané et al, 2004;Riely et al, 2007), NENA, a nuclear pore component (Groth et al, 2010), CCaMK/DMI3, a Ca 2+ and calmodulin-dependent protein kinase (Lévy et al, 2004;Mitra et al, 2004), the CCaMK-regulated transcriptional regulator CY-CLOPS/IPD3 (Messinese et al, 2007;Yano et al, 2008;Singh et al, 2014), and various transcriptional regulators such as NSP1, NSP2, ERN1, and NIN (Kaló et al, 2005;Smit et al, 2005;Marsh et al, 2007, Middleton et al, 2007. As a consequence of NF signaling, the expression of many symbiotic genes such as the early nodulin gene ENOD11 is induced (Andriankaja et al, 2007;Hirsch et al, 2009).…”