A Remote cis-Regulatory Region Is Required for NIN Expression in the Pericycle to Initiate Nodule Primordium Formation in Medicago truncatula

Abstract

The legume-rhizobium symbiosis results in nitrogen-fixing root nodules, and their formation involves both intracellular infection initiated in the epidermis and nodule organogenesis initiated in inner root cell layers. NODULE INCEPTION (NIN) is a nodule-specific transcription factor essential for both processes. These NIN-regulated processes occur at different times and locations in the root, demonstrating a complex pattern of spatiotemporal regulation. We show that regulatory sequences sufficient for the epidermal infection process are located within a 5 kb region directly upstream of the NIN start codon in Medicago truncatula. Furthermore, we identify a remote upstream cis-regulatory region required for the expression of NIN in the pericycle, and we show that this region is essential for nodule organogenesis. This region contains putative cytokinin response elements and is conserved in eight more legume species. Both the cytokinin receptor 1, which is essential for nodule primordium formation, and the B-type response regulator RR1 are expressed in the pericycle in the susceptible zone of the uninoculated root. This, together with the identification of the cytokinin-responsive elements in the NIN promoter, strongly suggests that NIN expression is initially triggered by cytokinin signaling in the pericycle to initiate nodule primordium formation.

Celebrating 20 Years of Genetic Discoveries in Legume Nodulation and Symbiotic Nitrogen Fixation

“…Cytokinin biosynthesis is mediated by isopentenyl transferase (encoded by LjIPT3), and cytokinin homeostasis during nodule development is maintained by cytokinin oxidase/dehydrogenase3 (encoded by LjCKX3) in L. japonicus and the cytokinin-activating enzyme riboside 59-monophosphate phosphoribohydrolase (encoded by the LONELY GUY gene MtLOG1) in M. truncatula Mortier et al, 2014;Reid et al, 2016). The involvement of the key TF NIN in cytokinin signaling during nodule organogenesis was recently demonstrated by Liu et al, 2019c. A careful analysis of the NIN promoter revealed that a 2.2-kb segment can reverse the excessive root hair curling and restore infection pocket formation, a 5-kb segment can restore IT formation, but only the presence of 10 cytokinin-responsive promoter elements 18 kb upstream of the NIN translation site can restore nodule organogenesis.…”

“…The infectosome comprises the common symbiotic protein MtVPY (MtVAPYRIN) and its two interacting partners, MtLIN (LUMPY INFECTION) and the exocyst complex subunit MtEXO70 H4 (EXOCYST subunit H4; (Pumplin et al, 2010;Murray, 2011;Liu et al, 2019a). Mutating any of the three components causes defects in IT elongation (Liu et al, 2019c). Both Mtvpy and Mtlin mutants produce few nodules, which are small, white, and uninfected, while Mtexo70 h4 mutants form normal, functional nodules, possibly reflecting genetic redundancy (Murray, 2011;Liu et al, 2019a).…”

Mutant analysis in the nonlegume Parasponia andersonii identifies NIN and NF‐YA1 transcription factors as a core genetic network in nitrogen‐fixing nodule symbioses

“…S1b-d). In M. truncatula, similar promoter-GUS studies using a 2.2 kb upstream region revealed that MtNF-YA1 is induced in root hairs of the preinfection zone and in the root pericycle upon rhizobium inoculation (Laporte et al, 2014;Liu et al, 2019). We questioned whether this is also the case for P. andersonii.…”

Global analysis of Two Component System (TCS) member of chickpea and other legume genomes implicates its role in enhanced nodulation

“…Release of Nod factors induces manipulation of phytohormones which is responsible for nodulation. Several cytokinin responsive genes were found to be upregulated during nodulation (Liu et al, 2019; Mortier et al, 2014; Chen et al, 2012). Significant nodulation was observed at 2.5×10 −7 6-BA in chickpea even in the absence of rhizobia.…”