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

Abstract

The legume-rhizobium symbiosis is initiated through the activation of the Nodulation (Nod) factor-signaling cascade, leading to a rapid reprogramming of host cell developmental pathways. In this work, we combine transcriptome sequencing with molecular genetics and network analysis to quantify and categorize the transcriptional changes occurring in roots of Medicago truncatula from minutes to days after inoculation with Sinorhizobium medicae. To identify the nature of the inductive and regulatory cues, we employed mutants with absent or decreased Nod factor sensitivities (i.e. Nodulation factor perception and Lysine motif domain-containing receptor-like kinase3, respectively) and an ethylene (ET)-insensitive, Nod factor-hypersensitive mutant (sickle). This unique data set encompasses nine time points, allowing observation of the symbiotic regulation of diverse biological processes with high temporal resolution. Among the many outputs of the study is the early Nod factorinduced, ET-regulated expression of ET signaling and biosynthesis genes. Coupled with the observation of massive transcriptional derepression in the ET-insensitive background, these results suggest that Nod factor signaling activates ET production to attenuate its own signal. Promoter:b-glucuronidase fusions report ET biosynthesis both in root hairs responding to rhizobium as well as in meristematic tissue during nodule organogenesis and growth, indicating that ET signaling functions at multiple developmental stages during symbiosis. In addition, we identified thousands of novel candidate genes undergoing Nod factor-dependent, ET-regulated expression. We leveraged the power of this large data set to model Nod factor-and ET-regulated signaling networks using MERLIN, a regulatory network inference algorithm. These analyses predict key nodes regulating the biological process impacted by Nod factor perception. We have made these results available to the research community through a searchable online resource.

Validating Genome-Wide Association Candidates Controlling Quantitative Variation in Nodulation

“…The Arabidopsis gene with greatest similarity to Medtr2g101090 is PEN3 (66% identical amino acid sequence across 97% of the gene length; E-value = 0), which has been shown to be an essential component of salicylic acid-related defense against biotrophic fungi (Stein et al, 2006;Johansson et al, 2014). The formation of nodules requires direct interaction with bacteria, and there is growing evidence that defense responses can negatively affect nodule formation (Ramu et al, 2002;Lopez-Gomez et al, 2012;Larrainzar et al, 2015). Interestingly, ABC transporters have been shown to be significantly and highly up-regulated in several nodulation mutants relative to wild-type controls (Lang and Long, 2015).…”

Different Pathways Act Downstream of the CEP Peptide Receptor CRA2 to Regulate Lateral Root and Nodule Development

“…NODULE INCEPTION and NODULATION SIGNALING PATHWAY1 and 2 [Kaló et al, 2005;Smit et al, 2005] and MtCLV3/ESR-RELATED12 and 13 [MtCLE12 and 13; Mortier et al, 2010;Saur et al, 2011]). Nodulation is also positively and negatively controlled by complex, context-dependent interactions with various hormones and peptides (Penmetsa and Cook, 1997;Gonzalez-Rizzo et al, 2006;Penmetsa et al, 2008;Mortier et al, 2010;Plet et al, 2011;Saur et al, 2011;Mortier et al, 2012;Larrainzar et al, 2015;van Zeijl et al, 2015). Collectively, these signals, along with the NF/SYM pathway, determine how, when, and where nodules form, as well as the overall nodule number on the root system (Penmetsa and Cook, 1997;Oldroyd et al, 2011).…”

“…Conversely, the ethylene precursor, 1-aminocyclopropane-1-carboxylic-acid (ACC), reduces nodule number and, at high concentration, inhibits calcium oscillations and abolishes nodulation (Penmetsa and Cook, 1997;Oldroyd et al, 2001). Recently, a comprehensive transcriptional profiling of the Mtein2/skl mutant revealed a complex MtEIN2-dependent negative control of NF-mediated responses (Larrainzar et al, 2015).…”

“…Ethylene has a well-established negative role in M. truncatula nodulation, which is illustrated by the ethylene-insensitive, MtEIN2-defective mutant, sickle (skl; Penmetsa et al, 2008;Larrainzar et al, 2015). Rhizobia hyperinfect skl and form numerous underdeveloped nodules that are often fused (Penmetsa and Cook, 1997).…”

“…How CEP peptides simultaneously regulate both root nodule and LR formation is unknown. At early developmental stages LRs and nodules share developmental genes Larrainzar et al, 2015) and morphological features, such as reactivation of cortical, pericycle, and endodermal cell divisions Herrbach et al, 2014;Xiao et al, 2014;Mohd-Radzman et al, 2015), and it is therefore possible that overlapping genetic mechanisms may be involved. In addition, these two developmental pathways are initiated in cells in close proximity to the RAM Herrbach et al, 2014;Xiao et al, 2014).…”