How many proteins interacting with symbiosis receptor-like kinase （SymRK） involved in nodulation？

Nodule formation is a tightly regulated process that integrates specific signal exchange and coordinated activation of developmental mechanisms to synchronize bacte-rial infection and organ development. Symbiosis receptor kinase （SymRK） is indispensable for symbiotic signal transduction of root nodule symbiosis （RNS） upon stimulation of root cells by microbial signaling molecules. But the protein turnover model of SymRK and the way for nodulation factor signals downstream transduction from SymRK are not clear. Over the past years, a number of proteins interacting with SymRK which required for root nodule symbiosis have been identified. Here we summarized structures and functions of these pro-teins, and concluded that major challenge would be revealing relations between them and the regulation mechanisms of SymRK in nodulation.

High efficient broad-spectrum Bradyrhizobium elkanii Y63-1

Soybean(Glycine max),the primary source of high-quality plant protein,plays a crucial role as a grain and oil crop in China.Harnessing the full potential of symbiotic nitrogen fixation in soybean production holds immense significance for agriculture and ecology alike.Zhongdou 63,a newly developed early-maturing summer soybean cultivar in 2021,exhibits remarkable traits such as high yield,superior quality,multi-resistance,and wide adaptability.In this study,eight distinct rhizobia strains from diverse regions were meticulously screened to identify highly effective strains specifically suited for Zhongdou 63.The aboveground biomass,plant height,chlorophyll content,root length,nodule number,and nodule dry weight of Zhongdou 63 were measured and the data were subjected to statistical analysis.The results demonstrated that Y63-1 is a predominant strain of Zhongdou 63.Subsequently,we conducted further investigations on the broad-spectrum nodulation characteristics of Y63-1.Ten representative soybean cultivars were individually inoculated with Y63-1 and subsequently analyzed for nodule numbers and nodule dry weight in their symbiotic systems with rhizobia.The findings revealed that Y63-1 effectively formed nodules with all ten soybean varieties tested.In summary,our current study identified highly efficient broad-spectrum Bradyrhizobium elkanii strain Y63-1 as the predominant strain in Zhongdou 63 and provided a theoretical foundation for enhancing yield potential not only in Zhongdou 63 but also in other varieties through inoculation with highly efficient rhizobia in production.

Dynamic modulation of nodulation factor receptor levels by phosphorylation-mediated functional switch of a RING-type E3 ligase during legume nodulation

The precise control of receptor levels is crucial for initiating cellular signaling transduction in response to specific ligands;however,such mechanisms regulating nodulation factor(NF)receptor(NFR)-mediated perception of NFs to establish symbiosis remain unclear.In this study,we unveil the pivotal role of the NFR-interacting RING-type E3 ligase 1(NIRE1)in regulating NFR1/NFR5 homeostasis to optimize rhizobial infection and nodule development in Lotus japonicus.We demonstrated that NiRE1 has a dual function in this regulatory process.It associates with both NFR1 and NFR5,facilitating their degradation through K48-linked polyubiquitination before rhizobial inoculation.However,following rhizobial inoculation,NFR1 phosphorylates NIRE1ata conserved residue,Tyr-109,inducing a functional switch in NIRE1,which enables NIRE1tomediateK63-linkedpolyubiquitination,thereby stabilizing NFR1/NFR5 in infected root cells.The introduction of phospho-dead NIRE1Y1osF leads to delayed nodule development,underscoring the significance of phosphorylation at Tyr-1o9 in orchestrating symbiotic processes.Conversely,expression of the phospho-mimic NIRE1Y0E results in the formation of spontaneous nodules in L.japonicus,further emphasizing the critical role of the phosphorylation-dependent functional switch in NiRE1.In summary,these findings uncover a fine-tuned symbiotic mechanism that a single E3 ligase could undergo a phosphorylationdependent functional switch to dynamically and precisely regulate NF receptor protein levels.

A novel secreted protein, NISP1, is phosphorylated by soybean Nodulation Receptor Kinase to promote nodule symbiosis

Nodulation Receptor Kinase(NORK) functions as a co-receptor of Nod factor receptors to mediate rhizobial symbiosis in legumes, but its direct phosphorylation substrates that positively mediate root nodulation remain to be fully identified.Here, we identified a GmNORK-Interacting Small Protein(GmNISP1) that functions as a phosphorylation target of GmNORK to promote soybean nodulation. GmNORKα directly interacted with and phosphorylated GmNISP1. Transcription of GmNISP1 was strongly induced after rhizobial infection in soybean roots and nodules. GmNISP1 encodes a peptide containing 90 amino acids with a “DY” consensus motif at its N-terminus.GmNISP1 protein was detected to be present in the apoplastic space. Phosphorylation of GmNISP1 by GmNORKα could enhance its secretion into the apoplast. Pretreatment with either purified GmNISP1 or phosphorylation-mimic GmNISP1~(12D) on the roots could significantly increase nodule numbers compared with the treatment with phosphorylation-inactive GmNISP1~(12A).The data suggested a model that soybean GmNORK phosphorylates GmNISP1 to promote its secretion into the apoplast, which might function as a potential peptide hormone to promote root nodulation.

Duplication and sub-functionalization of flavonoid biosynthesis genes plays important role in Leguminosae root nodule symbiosis evolution

Gene innovation plays an essential role in trait evolution.Rhizobial symbioses,the most important N2-fixing agent in agricultural systems that exists mainly in Leguminosae,is one of the most attractive evolution events.However,the gene innovations underlying Leguminosae root nodule symbiosis(RNS)remain largely unknown.Here,we investigated the gene gain event in Leguminosae RNS evolution through comprehensive phylogenomic analyses.We revealed that Leguminosae-gain genes were acquired by gene duplication and underwent a strong purifying selection.Kyoto Encyclopedia of Genes and Genomes analyses showed that the innovated genes were enriched in flavonoid biosynthesis pathways,particular downstream of chalcone synthase(CHS).Among them,Leguminosae-gain typeⅡchalcone isomerase(CHI)could be further divided into CHI1A and CHI1B clades,which resulted from the products of tandem duplication.Furthermore,the duplicated CHI genes exhibited exon–intron structural divergences evolved through exon/intron gain/loss and insertion/deletion.Knocking down CHI1B significantly reduced nodulation in Glycine max(soybean)and Medicago truncatula;whereas,knocking down its duplication gene CHI1A had no effect on nodulation.Therefore,Leguminosae-gain typeⅡCHI participated in RNS and the duplicated CHI1A and CHI1B genes exhibited RNS functional divergence.This study provides functional insights into Leguminosae-gain genetic innovation and sub-functionalization after gene duplication that contribute to the evolution and adaptation of RNS in Leguminosae.

Split-root systems applied to the study of the legume-rhizobial symbiosis: What have we learned?

Split-root system（SRS） approaches allow the differential treatment of separate and independent root systems, while sharing a common aerial part. As such, SRS is a useful tool for the discrimination of systemic（shoot origin）versus local（root/nodule origin） regulation mechanisms. This type of approach is particularly useful when studying the complex regulatory mechanisms governing the symbiosis established between legumes and Rhizobium bacteria. The current work provides an overview of the main insights gained from the application of SRS approaches to understand how nodule number（nodulation autoregulation） and nitrogen fixation are controlled both under non-stressful conditions and in response to a variety of stresses. Nodule number appears to be mainly controlled at the systemic level through a signal which is produced by nodule/root tissue, translocated to the shoot, and transmitted back to the root system, involving shoot Leu-rich repeat receptor-like kinases. In contrast, both local and systemic mechanisms have been shown to operate for the regulation of nitrogenase activity in nodules. Under drought and heavy metal stress, the regulation is mostly local,whereas the application of exogenous nitrogen seems to exert a regulation of nitrogen fixation both at the local and systemic levels.

Cellular basis of legume-rhizobium symbiosis

The legume-rhizobium symbiosis represents the most important system for terrestrial biological nitrogen fixation on land.Efficient nitrogen fixation during this symbiosis depends on successful rhizobial infection and complete endosymbiosis,which are achieved by complex cellular events including cell-wall remodeling,cytoskeletal reorganizations,and extensive membrane expansion and trafficking.In this review,we explore the dynamic remodeling of the plant-specific cell wall-membrane system-cytoskeleton(WMC)continuum during symbiotic nitrogen fixation.We focus on key processes linked to efficient nitrogen fixation,including rhizobial uptake,infection thread formation and elongation,rhizobial droplet release,cytoplasmic bridge formation,and rhizobial endosymbiosis.Additionally,we discuss the advanced techniques for investigating the cellular basis of root-nodule symbiosis and provide insights into the unsolved mysteries of robust symbiotic nitrogen fixation.

Mechanisms underlying legume-rhizobium symbioses

Legumes,unlike most land plants,can form symbiotic root nodules with nitrogen-fixing bacteria to secure nitrogen for growth.The formation of nitrogen-fixing nodules on legume roots requires the coordination of rhizobial infection at the root epidermis with cell division in the cortex.The nodules house the nitrogen-fixing rhizobia in organelle-like structures known as symbiosomes,which enable nitrogen fixation and facilitate the exchange of metabolites between the host and symbionts.In addition to this beneficial interaction,legumes are continuously exposed to would-be pathogenic microbes;therefore the ability to discriminate pathogens from symbionts is a major determinant of plant survival under natural conditions.Here,we summarize recent advances in the understanding of root nodule symbiosis signaling,transcriptional regulation,and regulation of plant immunity during legume-rhizobium symbiosis.In addition,we propose several important questions to be addressed and provide insights into the potential for engineering the capacity to fix nitrogen in legume and nonlegume plants.

Computational investigation of small RNAs in the establishment of root nodules and arbuscular mycorrhiza in leguminous plants

Many small RNAs have been confirmed to play important roles in the development of root nodules and arbuscular mycorrhiza. In this study, we carried out the identification of certain small RNAs in leguminous plants(Medicago truncatula, soybean, peanut and common bean), such as miRNAs, tRFs and srRNAs, as well as the computational investigation of their regulations. Thirty miRNAs were predicted to be involved in establishing root nodules and mycorrhiza, and 12 of them were novel in common bean and peanut. The generation of tRFs in M. truncatula was not associated with tRNA gene frequencies and codon usage. Six tRFs exhibited different expressions in mycorrhiza and root nodules. Moreover, srRNA^(5.8S) in M. truncatula was generated from the regions with relatively low conservation at the rRNA 3′ terminal. The protein-protein interactions between the proteins encoded by the target genes of miRNAs, tRFs and srRNAs were computed. The regulation of these three types of sRNAs in the symbiosis between leguminous plants and microorganisms is not a single regulation of certain signaling or metabolic pathways but a global regulation for the plants to own growth or specific events in symbiosis.