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Key Message: Symbiotic nitrogen fixation in root nodules of grain legumes is essential for high yielding. Protein phosphorylation/dephosphorylation plays important role in root nodule development. Differences in the phosphoproteomes may either be developmental specific and related to nitrogen fixation activity. An iTRAQ-based quantitative phosphoproteomic analyses during nodule development enables identification of specific phosphorylation signaling in the Lotus-rhizobia symbiosis. During evolution, legumes (Fabaceae) have evolved a symbiotic relationship with rhizobia, which fix atmospheric nitrogen and produce ammonia that host plants can then absorb. Root nodule development depends on the activation of protein phosphorylation-mediated signal transduction cascades. To investigate possible molecular mechanisms of protein modulation during nodule development, we used iTRAQ-based quantitative proteomic analyses to identify root phosphoproteins during rhizobial colonization and infection of Lotus japonicus. 1154 phosphoproteins with 2957 high-confidence phosphorylation sites were identified. Gene ontology enrichment analysis of functional groups of these genes revealed that the biological processes mediated by these proteins included cellular processes, signal transduction, and transporter activity. Quantitative data highlighted the dynamics of protein phosphorylation during nodule development and, based on regulatory trends, seven groups were identified. RNA splicing and brassinosteroid (BR) signaling pathways were extensively affected by phosphorylation, and most Ser/Arg-rich (SR) proteins were multiply phosphorylated. In addition, many proposed kinase-substrate pairs were predicted, and in these MAPK6 substrates were found to be highly enriched. This study offers insights into the regulatory processes underlying nodule development, provides an accessible resource cataloging the phosphorylation status of thousands of Lotus proteins during nodule development, and develops our understanding of post-translational regulatory mechanisms in the Lotus-rhizobia symbiosis.

Roots of leguminous plants perceive Nod factor signals, and then root hair deformation responses such as swelling and curling are activated. However, very little is known about the molecular mechanisms of such root hair deformation. We have previously shown that LjROP6, a member of the Rho family of small GTPases, was identified as an NFR5 (Nod Factor Receptor 5)-interacting protein and participated in symbiotic nodulation in Lotus japonicus. In this study, we identified ten LjROP GTPases including LjROP6, and they were distributed into groups II, III, IV but not group I by phylogenetic analysis. The expression profiles of ten LjROP genes during nodulation were examined. LjROP6 belonged to group IV and interacted with NFR5 in a GTP-dependent manner. Overexpression of either wild-type ROP6 or a constitutively active mutant (ROP6-CA) generated root hair tip growth depolarization, while overexpression of a dominant negative mutant (ROP6-DN) exhibited normal root hair growth. After inoculating with Mesorhizobium loti or adding Nod factors to hairy roots, overexpression of ROP6 and ROP6-CA exhibited extensive root hair deformation, while overexpression of ROP6-DN inhibited root hair deformation. The infection event and nodule number were increased in ROP6 and ROP6-CA overexpressing transgenic plants; but decreased in ROP6-DN overexpressing transgenic plants. These studies provide strong evidence that ROP6 GTPase, which binds NFR5 in a GTP-dependent manner, is involved in root hair development as well as root hair deformation responses induced by NFs in the early stage of symbiotic interaction in L. japonicus., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Transcription factor complex formation is a central step in regulating gene expression. In this report, a novel MYB coiled-coil transcription factor referred to as IPN2, for Interacting Protein of NSP2, is described. The interaction between IPN2 and NSP2 was examined by protein pull-down assays and bimolecular fluorescence complementation (BiFC). Subcellular localization of proteins, gene expression and gene function were assessed in transgenic hairy roots expressing tagged recombinant proteins, promoter-reporter and RNA interference (RNAi) constructs, respectively. The GRAS domain of NSP2 and the coiled-coil domain of IPN2 were found to be responsible for the interaction between the two proteins. IPN2 had strong transcription activation activity, bound directly to the NIN gene promoter, and was localized to the nuclei of Lotus japonicus root cells. The expression of IPN2 was elevated during nodule development, coinciding with increased NSP2 gene expression during nodule organogenesis. RNAi-mediated knockdown expression of IPN2 did not affect arbuscular mycorrhizal development, but had deleterious effects on rhizobial infection and nodule formation in L. japonicus. These results demonstrate an important role of IPN2 in nodule organogenesis and place a new MYB transcription factor in the Nod signaling pathway., (© 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.)

The symbiosis receptor kinase (SymRK) is required for morphological changes of legume root hairs triggered by rhizobial infection. How protein turnover of SymRK is regulated and how the nodulation factor signals are transduced downstream of SymRK are not known. In this report, a SymRK-interacting E3 ubiquitin ligase (SIE3) was shown to bind and ubiquitinate SymRK. The SIE3-SymRK interaction and the ubiquitination of SymRK were shown to occur in vitro and in planta. SIE3 represents a new class of plant-specific E3 ligases that contain a unique pattern of the conserved CTLH (for C-terminal to LisH), CRA (for CT11-RanBPM), and RING (for Really Interesting New Gene) domains. Expression of SIE3 was detected in all tested tissues of Lotus japonicus plants, and its transcript level in roots was enhanced by rhizobial infection. The SIE3 protein was localized to multiple subcellular locations including the nuclei and plasma membrane, where the SIE3-SymRK interaction took place. Overexpression of SIE3 promoted nodulation in transgenic hairy roots, whereas downregulation of SIE3 transcripts by RNA interference inhibited infection thread development and nodule organogenesis. These results suggest that SIE3 represents a new class of E3 ubiquitin ligase, acts as a regulator of SymRK, and is involved in rhizobial infection and nodulation in L. japonicus.

Nod Factor Receptor5 (NFR5) is an atypical receptor-like kinase, having no activation loop in the protein kinase domain. It forms a heterodimer with NFR1 and is required for the early plant responses to Rhizobium infection. A Rho-like small GTPase from Lotus japonicus was identified as an NFR5-interacting protein. The amino acid sequence of this Rho-like GTPase is closest to the Arabidopsis (Arabidopsis thaliana) ROP6 and Medicago truncatula ROP6 and was designated as LjROP6. The interaction between Rop6 and NFR5 occurred both in vitro and in planta. No interaction between Rop6 and NFR1 was observed. Green fluorescent protein-tagged ROP6 was localized at the plasma membrane and cytoplasm. The interaction between ROP6 and NFR5 appeared to take place at the plasma membrane. The expression of the ROP6 gene could be detected in vascular tissues of Lotus roots. After inoculation with Mesorhizobium loti, elevated levels of ROP6 expression were found in the root hairs, root tips, vascular bundles of roots, nodule primordia, and young nodules. In transgenic hairy roots expressing ROP6 RNA interference constructs, Rhizobium entry into the root hairs did not appear to be affected, but infection thread growth through the root cortex were severely inhibited, resulting in the development of fewer nodules per plant. These data demonstrate a role of ROP6 as a positive regulator of infection thread formation and nodulation in L. japonicus.

The symbiosis receptor kinase, SymRK, is required for root nodule development. A SymRK-interacting protein (SIP2) was found to form protein complex with SymRK in vitro and in planta. The interaction between SymRK and SIP2 is conserved in legumes. The SIP2 gene was expressed in all Lotus japonicus tissues examined. SIP2 represents a typical plant mitogen-activated protein kinase kinase (MAPKK) and exhibited autophosphorylation and transphosphorylation activities. Recombinant SIP2 protein could phosphorylate casein and the Arabidopsis thaliana MAP kinase MPK6. SymRK and SIP2 could not use one another as a substrate for phosphorylation. Instead, SymRK acted as an inhibitor of SIP2 kinase when MPK6 was used as a substrate, suggesting that SymRK may serve as a negative regulator of the SIP2 signaling pathway. Knockdown expression of SIP2 via RNA interference (RNAi) resulted in drastic reduction of nodules formed in transgenic hairy roots. A significant portion of SIP2 RNAi hairy roots failed to form a nodule. In these roots, the expression levels of SIP2 and three marker genes for infection thread and nodule primordium formation were downregulated drastically, while the expression of two other MAPKK genes were not altered. These observations demonstrate an essential role of SIP2 in the early symbiosis signaling and nodule organogenesis.