Rapid Phosphoproteomic and Transcriptomic Changes in the Rhizobia-legume Symbiosis

Abstract

Symbiotic associations between legumes and rhizobia usually commence with the perception of bacterial lipochitooligosaccharides, known as Nod factors (NF), which triggers rapid cellular and molecular responses in host plants. We report here deep untargeted tandem mass spectrometry-based measurements of rapid NF-induced changes in the phosphorylation status of 13,506 phosphosites in 7739 proteins from the model legume Medicago truncatula. To place these phosphorylation changes within a biological context, quantitative phosphoproteomic and RNA measurements in wild-type plants were compared with those observed in mutants, one defective in NF perception (nfp) and one defective in downstream signal transduction events (dmi3). Our study quantified the early phosphorylation and transcription dynamics that are specifically associated with NF-signaling, confirmed a dmi3-mediated feedback loop in the pathway, and suggested "cryptic" NF-signaling pathways, some of them being also involved in the response to symbiotic arbuscular mycorrhizal fungi. Legumes have the ability to form a very efficient symbiotic association with rhizobia to meet their nitrogen demand. This results in root nodule formation, inside which the rhizobia can fix atmospheric nitrogen efficiently and transfer it to the plants, in exchange for a carbon source. This interaction is often characterized by a high level of host specificity and generally requires an exchange of diffusible signals between legumes and rhizobia. Flavonoids and isoflavonoids present in the legume root exudates induce the expression of nod genes in rhizobia, and are responsible for the production and secretion of bacterial lipochitooligosaccharides (LCOs), known as Nod factors (NF) 1 (1, 2). NF are generally required for rhizobial infection and nodule development. Mere application of purified NF at low concentrations (10 Ϫ8 to 10 Ϫ12 M) is sufficient to trigger responses in host plants similar to those elicited by the rhizobia themselves (2, 3). Responses are elicited in root cells within a few seconds to minutes after NF application and include changes in ion fluxes across the plasma membrane, as well as accumulation of reactive oxygen species (4, 5). After 15-20 min, oscillations of the nuclear and perinuclear calcium concentration (calcium spiking) are initiated and trigger the expression of early nodulin genes (3). Within the first hour, legume root hairs undergo a cytoplasmic disorganization ,which leads to a transient swelling (6), followed by root hair deformations (7).Forward and reverse genetic approaches, in the model legumes Medicago truncatula (Medicago) and Lotus japonicus (Lotus), have identified several components controlling these events (Fig. 1). NF are perceived with high specificity by LysM receptor-like kinases residing on the plasma membrane. These kinases include nod factor perception (NFP), an essential component of a signaling receptor necessary for early responses to NF and rhizobial infection (8, 9). Mutants in NFP are affected in all known cellular respons...

A Laser Dissection-RNAseq Analysis Highlights the Activation of Cytokinin Pathways by Nod Factors in the Medicago truncatula Root Epidermis

“…Only 14.7% (33 genes) of the NF down-regulated genes at 4 h were still scored downregulated at 24 h, indicating a very high proportion of transient regulation, while only seven genes were found in common among the LCM, RH, and all root tissues 24-h down-regulated genes (Supplemental Table I. Detection of known NF-induced genes in this study and in recent transcriptomic analyses Values correspond to the regulation levels (fold change) following NF treatment for the indicated times or S. meliloti inoculation at 5 d post inoculation (RH-5dpi; skl mutant background), determined in this study versus previous studies: a, Rose et al (2012);b, van Zeijl et al (2015); c, Czaja et al (2012);and d, Breakspear et al (2014). For entries in parentheses, differences were not statistically significant (adjusted P .…”

“…NFinduced gene expression reprogramming was analyzed recently in M. truncatula using whole roots or root segments (Czaja et al, 2012;Rose et al, 2012;van Zeijl et al, 2015) as well as isolated RHs (Breakspear et al, 2014). Two of these studies used Affymetrix microarrays representing approximately 70% of the genome (Czaja et al, 2012;Breakspear et al, 2014), while the two others used RNA sequencing (RNAseq), with sequencing reads mapped on the partial (Mt3.5) or the full (Mt4.0) M. truncatula genome sequence (Rose et al, 2012;van Zeijl et al, 2015). In addition, genes whose transcriptional activation by Rhizobium is NF dependent were identified by root RNAseq analysis (Larrainzar et al, 2015).…”

Battle through Signaling between Wheat and the Fungal Pathogen Septoria tritici Revealed by Proteomics and Phosphoproteomics

“…We identified the differential phosphorylation of a mitogenactivated protein kinase kinase in Stakado and a DENN (AEX-3) domain-containing protein in Sevin, both of which are involved in the regulation of MAPK signaling pathways. A sucrose non-fermenting-related kinase (SNF), the master regulator of energy metabolism and one of the major components in sugar signaling (47), was found to be phosphorylated in Sevin at 3 dai, which was correlated with increased sugar levels in response to infection. SNF1 itself is activated by phosphorylation and can phosphorylate transcription repressors for the de-repression of genes involved in sugar metabolism (3).…”

“…SNF1 itself is activated by phosphorylation and can phosphorylate transcription repressors for the de-repression of genes involved in sugar metabolism (3). The alteration of the phosphorylation level of SNF1 has recently been identified in Arabidopsis in response to abscisic acid (6) and in Rhizobium-legume symbiosis (47). In addition, a protein kinase NPH1 and an RPT2-like protein showed altered phosphorylation levels in Sevin.…”

“…Moreover, a vesicle-associated membrane protein involved in vesicle trafficking was phosphorylated in both cultivars. Vesicle trafficking has been shown to play an important role in the cellular processes of plant defense against pathogens and is possibly mediated by phosphorylation (47,59).…”