Conserved cis-elements enable NODULES WITH ACTIVATED DEFENSE1 regulation by NODULE INCEPTION during nodulation.

Root nodule symbiosis within nitrogen-fixing clade (NFC) plants is thought to have arisen from a single gain followed by massive losses in the genomes of ancestral non-nodulating plants. However, molecular evidence supporting this model is limited. Here, we confirm through bioinformatic analysis that NODULES WITH ACTIVATED DEFENSE1 (NAD1) is present only in NFC plants and is thus an NFC-specific gene. Moreover, NAD1 was specifically expressed in nodules. We identified three conserved nodulation-associated cis-regulatory elements (NACE1-3) in the promoter of LjNAD1 from Lotus japonicus that are required for its nodule specific expression. A survey of NFC plants revealed that NACE1 and NACE2 are specific to the Fabales and Papilionoideae, respectively, while NACE3 is present in all NFC plants. Moreover, we found that Nodule inception (NIN) directly binds to all three NACEs to activate NAD1 expression. Mutation of L. japonicus LjNAD1 resulted in the formation of abnormal symbiosomes with enlarged symbiosome space and frequent breakdown of bacteroids in nodules, resembling phenotypes reported for Medicago truncatula Mtnad1 and Mtnin mutants. These data point to NIN-NAD1 as an important module regulating rhizobial accommodation in nodules. The regulation of NAD1 by NIN in the NFC ancestor represent an important evolutionary adaptation for nodulation.

Soybean ethylene response factors GmENS1 and GmENS2 promote nodule senescence.

The final phase in root nodule development is nodule senescence. The mechanism underlying the initiation of nodule senescence requires further elucidation. Here, we investigated the intrinsic signals governing soybean (Glycine max L. Merr.) nodule senescence, uncovering ethylene as a key signal in this intricate mechanism. Two AP2/ERF transcription factor genes, GmENS1 and GmENS2 (Ethylene-responsive transcription factors required for Nodule Senescence), exhibit heightened expression levels in both aged nodules and nodules treated with ethylene. Overexpression of either GmENS1 or GmENS2 accelerated senescence in soybean nodules, whereas the knockout or knockdown of both genes delayed senescence and enhanced nitrogenase activity. Furthermore, our findings indicated that GmENS1 and GmENS2 directly bind to the promoters of GmNAC039, GmNAC018, and GmNAC030, encoding three NAC transcription factors essential for activating soybean nodule senescence. Notably, the nodule senescence process mediated by GmENS1 or GmENS2 overexpression was suppressed in the soybean nac039/018/030 triple mutant compared with the wild-type control. These data indicate GmENS1 and GmENS2 as pivotal transcription factors mediating ethylene-induced nodule senescence through the direct activation of GmNAC039/GmNAC018/GmNAC030 expression in soybean.

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 NIRE1 at a conserved residue, Tyr-109, inducing a functional switch in NIRE1, which enables NIRE1 to mediate K63-linked polyubiquitination, thereby stabilizing NFR1/NFR5 in infected root cells. The introduction of phospho-dead NIRE1Y109F leads to delayed nodule development, underscoring the significance of phosphorylation at Tyr-109 in orchestrating symbiotic processes. Conversely, expression of the phospho-mimic NIRE1Y109E 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 phosphorylation-dependent functional switch to dynamically and precisely regulate NF receptor protein levels.

A soybean cyst nematode suppresses microbial plant symbionts using a lipochitooligosaccharide-hydrolysing enzyme.

Cyst nematodes are the most damaging species of plant-parasitic nematodes. They antagonize the colonization of beneficial microbial symbionts that are important for nutrient acquisition of plants. The molecular mechanism of the antagonism, however, remains elusive. Here, through biochemical combined with structural analysis, we reveal that Heterodera glycines, the most notorious soybean cyst nematode, suppresses symbiosis by secreting an enzyme named HgCht2 to hydrolyse the key symbiotic signalling molecules, lipochitooligosaccharides (LCOs). We solved the three-dimensional structures of apo HgCht2, as well as its chitooligosaccharide-bound and LCO-bound forms. These structures elucidated the substrate binding and hydrolysing mechanism of the enzyme. We designed an HgCht2 inhibitor, 1516b, which successfully suppresses the antagonism of cyst nematodes towards nitrogen-fixing rhizobia and phosphorus-absorbing arbuscular mycorrhizal symbioses. As HgCht2 is phylogenetically conserved across all cyst nematodes, our study revealed a molecular mechanism by which parasitic cyst nematodes antagonize the establishment of microbial symbiosis and provided a small-molecule solution.

NODULATION TRIO in Medicago truncatula: Unveiling the redundant roles of MtLYK2, MtLYK3, and MtLYK2bis.

Three Medicago truncatula LysM domain receptor kinases have redundant functions in nodulation, with multiple specificities mediating both entry and signaling responses and with distinct contributions to nodulation likely resulting from differing transcription patterns.

Wild species rice OsCERK1DY-mediated arbuscular mycorrhiza symbiosis boosts yield and nutrient use efficiency in rice breeding.

Meeting the ever-increasing food demands of a growing global population while ensuring resource and environmental sustainability presents significant challenges for agriculture worldwide. Arbuscular mycorrhizal symbiosis (AMS) has emerged as a potential solution by increasing the surface area of a plant's root system and enhancing the absorption of phosphorus, nitrogen nutrients, and water. Consequently, there is a longstanding hypothesis that rice varieties exhibiting more efficient AMS could yield higher outputs at reduced input costs, paving the way for the development of Green Super Rice (GSR). Our prior research study identified a variant, OsCERK1DY, derived from Dongxiang wild-type rice, which notably enhanced AMS efficiency in the rice cultivar "ZZ35." This variant represents a promising gene for enhancing yield and nutrient use efficiency in rice breeding. In this study, we conducted a comparative analysis of biomass, crop growth characteristics, yield attributes, and nutrient absorption at varying soil nitrogen levels in the rice cultivar "ZZ35" and its chromosome single-segment substitution line, "GJDN1." In the field, GJDN1 exhibited a higher AM colonization level in its roots compared with ZZ35. Notably, GJDN1 displayed significantly higher effective panicle numbers and seed-setting rates than ZZ35. Moreover, the yield of GJDN1 with 75% nitrogen was 14.27% greater than the maximum yield achieved using ZZ35. At equivalent nitrogen levels, GJDN1 consistently outperformed ZZ35 in chlorophyll (Chl) content, dry matter accumulation, major nutrient element accumulation, N agronomic efficiency (NAE), N recovery efficiency (NRE), and N partial factor productivity (NPFP). The performance of OsCERK1DY overexpression lines corroborated these findings. These results support a model wherein the heightened level of AMS mediated by OsCERK1DY contributes to increased nitrogen, phosphorus, and potassium accumulation. This enhancement in nutrient utilization promotes higher fertilizer efficiency, dry matter accumulation, and ultimately, rice yield. Consequently, the OsCERK1DY gene emerges as a robust candidate for improving yield, reducing fertilizer usage, and facilitating a transition towards greener, lower-carbon agriculture. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-024-01459-8.

A pathogenesis-related protein, PRP1, negatively regulates root nodule symbiosis in Lotus japonicus.

The legume-rhizobium symbiosis represents a unique model within the realm of plant-microbe interactions. Unlike typical cases of pathogenic invasion, the infection of rhizobia and their residence within symbiotic cells do not elicit a noticeable immune response in plants. Nevertheless, there is still much to uncover regarding the mechanisms through which plant immunity influences rhizobial symbiosis. In this study, we identify an important player in this intricate interplay: Lotus japonicus PRP1, which serves as a positive regulator of plant immunity but also exhibits the capacity to decrease rhizobial colonization and nitrogen fixation within nodules. The PRP1 gene encodes an uncharacterized protein and is named Pathogenesis-Related Protein1, owing to its orthologue in Arabidopsis thaliana, a pathogenesis-related family protein (At1g78780). The PRP1 gene displays high expression levels in nodules compared to other tissues. We observed an increase in rhizobium infection in the L. japonicus prp1 mutants, whereas PRP1-overexpressing plants exhibited a reduction in rhizobium infection compared to control plants. Intriguingly, L. japonicus prp1 mutants produced nodules with a pinker colour compared to wild-type controls, accompanied by elevated levels of leghaemoglobin and an increased proportion of infected cells within the prp1 nodules. The transcription factor Nodule Inception (NIN) can directly bind to the PRP1 promoter, activating PRP1 gene expression. Furthermore, we found that PRP1 is a positive mediator of innate immunity in plants. In summary, our study provides clear evidence of the intricate relationship between plant immunity and symbiosis. PRP1, acting as a positive regulator of plant immunity, simultaneously exerts suppressive effects on rhizobial infection and colonization within nodules.

Mitogen-activated protein kinases MPK3 and MPK6 phosphorylate receptor-like cytoplasmic kinase CDL1 to regulate soybean basal immunity.

Soybean cyst nematode (SCN; Heterodera glycines Ichinohe), one of the most devastating soybean (Glycine max) pathogens, causes significant yield loss in soybean production. Nematode infection triggers plant defense responses; however, the components involved in the upstream signaling cascade remain largely unknown. In this study, we established that a mitogen-activated protein kinase (MAPK) signaling module, activated by nematode infection or wounding, is crucial for soybeans to establish SCN resistance. GmMPK3 and GmMPK6 directly interact with CDG1-LIKE1 (GmCDL1), a member of the receptor-like cytoplasmic kinase (RLCK) subfamily VII. These kinases phosphorylate GmCDL1 at Thr-372 to prevent its proteasome-mediated degradation. Functional analysis demonstrated that GmCDL1 positively regulates immune responses and promotes SCN resistance in soybeans. GmMPK3-mediated and GmMPK6-mediated phosphorylation of GmCDL1 enhances GmMPK3 and GmMPK6 activation and soybean disease resistance, representing a positive feedback mechanism. Additionally, 2 L-type lectin receptor kinases, GmLecRK02g and GmLecRK08g, associate with GmCDL1 to initiate downstream immune signaling. Notably, our study also unveils the potential involvement of GmLecRKs and GmCDL1 in countering other soybean pathogens beyond nematodes. Taken together, our findings reveal the pivotal role of the GmLecRKs-GmCDL1-MAPK regulatory module in triggering soybean basal immune responses.

Dual RNA-Seq Analysis Pinpoints a Balanced Regulation between Symbiosis and Immunity in Medicago truncatula-Sinorhizobium meliloti Symbiotic Nodules.

Legume-rhizobial symbiosis initiates the formation of root nodules, within which rhizobia reside and differentiate into bacteroids to convert nitrogen into ammonium, facilitating plant growth. This process raises a fundamental question: how is plant immunity modulated within nodules when exposed to a substantial number of foreign bacteria? In Medicago truncatula, a mutation in the NAD1 (Nodules with Activated Defense 1) gene exclusively results in the formation of necrotic nodules combined with activated immunity, underscoring the critical role of NAD1 in suppressing immunity within nodules. In this study, we employed a dual RNA-seq transcriptomic technology to comprehensively analyze gene expression from both hosts and symbionts in the nad1-1 mutant nodules at different developmental stages (6 dpi and 10 dpi). We identified 89 differentially expressed genes (DEGs) related to symbiotic nitrogen fixation and 89 DEGs from M. truncatula associated with immunity in the nad1-1 nodules. Concurrently, we identified 27 rhizobial DEGs in the fix and nif genes of Sinorhizobium meliloti. Furthermore, we identified 56 DEGs from S. meliloti that are related to stress responses to ROS and NO. Our analyses of nitrogen fixation-defective plant nad1-1 mutants with overactivated defenses suggest that the host employs plant immunity to regulate the substantial bacterial colonization in nodules. These findings shed light on the role of NAD1 in inhibiting the plant's immune response to maintain numerous rhizobial endosymbiosis in nodules.

GmNAC039 and GmNAC018 activate the expression of cysteine protease genes to promote soybean nodule senescence.

Root nodules are major sources of nitrogen for soybean (Glycine max (L.) Merr.) growth, development, production, and seed quality. Symbiotic nitrogen fixation is time-limited, as the root nodule senesces during the reproductive stage of plant development, specifically during seed development. Nodule senescence is characterized by the induction of senescence-related genes, such as papain-like cysteine proteases (CYPs), which ultimately leads to the degradation of both bacteroids and plant cells. However, how nodule senescence-related genes are activated in soybean is unknown. Here, we identified 2 paralogous NAC transcription factors, GmNAC039 and GmNAC018, as master regulators of nodule senescence. Overexpression of either gene induced soybean nodule senescence with increased cell death as detected using a TUNEL assay, whereas their knockout delayed senescence and increased nitrogenase activity. Transcriptome analysis and nCUT&Tag-qPCR assays revealed that GmNAC039 directly binds to the core motif CAC(A)A and activates the expression of 4 GmCYP genes (GmCYP35, GmCYP37, GmCYP39, and GmCYP45). Similar to GmNAC039 and GmNAC018, overexpression or knockout of GmCYP genes in nodules resulted in precocious or delayed senescence, respectively. These data provide essential insights into the regulatory mechanisms of nodule senescence, in which GmNAC039 and GmNAC018 directly activate the expression of GmCYP genes to promote nodule senescence.

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 GmNISP112D on the roots could significantly increase nodule numbers compared with the treatment with phosphorylation-inactive GmNISP112A . 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.

The Perspective of Arbuscular Mycorrhizal Symbiosis in Rice Domestication and Breeding.

In nature, symbiosis with arbuscular mycorrhizal (AM) fungi contributes to sustainable acquisition of phosphorus and other elements in over 80% of plant species; improving interactions with AM symbionts may mitigate some of the environmental problems associated with fertilizer application in grain crops such as rice. Recent developments of high-throughput genome sequencing projects of thousands of rice cultivars and the discovery of the molecular mechanisms underlying AM symbiosis suggest that interactions with AM fungi might have been an overlooked critical trait in rice domestication and breeding. In this review, we discuss genetic variation in the ability of rice to form AM symbioses and how this might have affected rice domestication. Finally, we discuss potential applications of AM symbiosis in rice breeding for more sustainable agriculture.

Suppression of LjBAK1-mediated immunity by SymRK promotes rhizobial infection in Lotus japonicus.

An important question in biology is how organisms can associate with different microbes that pose no threat (commensals), pose a severe threat (pathogens), and those that are beneficial (symbionts). The root nodule symbiosis serves as an important model system for addressing such questions in the context of plant-microbe interactions. It is now generally accepted that rhizobia can actively suppress host immune responses during the infection process, analogous to the way in which plant pathogens can evade immune recognition. However, much remains to be learned about the mechanisms by which the host recognizes the rhizobia as pathogens and how, subsequently, these pathways are suppressed to allow establishment of the nitrogen-fixing symbiosis. In this study, we found that SymRK (Symbiosis Receptor-like Kinase) is required for rhizobial suppression of plant innate immunity in Lotus japonicus. SymRK associates with LjBAK1 (BRASSINOSTEROID INSENSITIVE 1-Associated receptor Kinase 1), a well-characterized positive regulator of plant innate immunity, and directly inhibits LjBAK1 kinase activity. Rhizobial inoculation enhances the association between SymRK and LjBAK1 in planta. LjBAK1 is required for the regulation of plant innate immunity and plays a negative role in rhizobial infection in L. japonicus. The data indicate that the SymRK-LjBAK1 protein complex serves as an intersection point between rhizobial symbiotic signaling pathways and innate immunity pathways, and support that rhizobia may actively suppress the host's ability to mount a defense response during the legume-rhizobium symbiosis.

Critical role of cysteine-266 of SIE3 in regulating the ubiquitination and degradation of SIP1 transcription factor in Lotus japonicus.

MAIN CONCLUSION: A conserved cysteine residue (C266)-mediated homo-dimerization of SIE3 is required for the ubiquitination and degradation of SIP1 transcription factor in Lotus japonicas CTLH/CRA/RING-containing proteins have been shown to possess E3-ligase activities and are crucial for the regulation of numerous cellular signaling pathways. In our previous studies, SIE3 (SymRK-Interacting E3 ubiquitin ligase), a CTLH/CRA/RING-containing protein from Lotus japonicus, has been shown to associate with both Symbiosis Receptor Kinase (SymRK) and SIP1 (SymRK interacting protein 1) transcription factor, and ubiquitinate SymRK (Yuan et al. Plant Physiol 160 (1):106-117, 2012; Feng et al. Front Plant Sci 11: 795, 2020). Besides, we previously also demonstrated that the residue, cysteine-266 in the CRA (CT11-RanBPM) domain is required for homodimerization of SIE3 and cysteine-266 residue-mediated homodimerization is important for the symbiosic function of SIE3 (Feng et al. 2020). In this report, SIE3 was shown to induce the ubiquitination and degradation of SIP1. The cysteine-266 residue is essential for the E3-ligase activity and is highly conserved in the SIE3-like proteins. Our works refined the working model that homodimerization of SIE3 is required for ubiquitin-related degradation of SIP1 and found a conserved cysteine residue plays a key role in the activity of a plant dimeric E3 ligase.

New insights into the function of the proteins IsiC and IsiD from Synechocystis sp. PCC 6803 under iron limitation.

Iron is a common cofactor in biological processes such as respiration, photosynthesis, and nitrogen fixation. The genes isiC and isiD encode unknown proteins, and the growth of ΔisiC and ΔisiD mutants is inhibited under iron-deficient conditions. To study the regulatory mechanisms of IsiC and IsiD during iron starvation, we carried out transcriptome and metabolome sequencing. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the photosynthesis, nitrogen metabolism, and ABC transporter pathways play a vital role in regulating iron deficiency. Upon iron repletion, IsiC and IsiD also have a regulatory effect on these pathways. Additionally, KEGG analysis of the differential metabolites of wild type (WT) and mutants showed that they were all enriched in starch and sucrose metabolism after iron limitation. Weighted gene co-expression network analysis (WGCNA) constructed a co-expression network of differentially expressed genes with phenotypes and metabolites, and finally identified five modules. The turquoise module was positively correlated with iron deficiency. In contrast, the WT and blue module exhibited a negative correlation, and the mutants ΔisiC and ΔisiD were positively correlated with the gray and brown modules, respectively. WGCNA also analyzed the relationship between metabolites and phenotypes, and the green module was related to iron starvation. The co-expression network determined the hub genes and metabolites of each module. This study lays a foundation for a better understanding of cyanobacteria in response to iron deficiency. KEY POINTS: • Nitrogen metabolism and ABC transporters are involved in iron regulation. • Starch and sucrose metabolism is related to the regulation of iron deficiency. • WGCNA analyzes the correlation between genes and metabolites.

Phosphorylation of ATG18a by BAK1 suppresses autophagy and attenuates plant resistance against necrotrophic pathogens.

Autophagy is critical for plant defense against necrotrophic pathogens, which causes serious yield loss on crops. However, the post-translational regulatory mechanisms of autophagy pathway in plant resistance against necrotrophs remain poorly understood. In this study, we report that phosphorylation modification on ATG18a, a key regulator of autophagosome formation in Arabidopsis thaliana, constitutes a post-translation regulation of autophagy, which attenuates plant resistance against necrotrophic pathogens. We found that phosphorylation of ATG18a suppresses autophagosome formation and its subsequent delivery into the vacuole, which results in reduced autophagy activity and compromised plant resistance against Botrytis cinerea. In contrast, overexpression of ATG18a dephosphorylation-mimic form increases the accumulation of autophagosomes and complements the plant resistance of atg18a mutant against B. cinerea. Moreover, BAK1, a key regulator in plant resistance, was identified to physically interact with and phosphorylate ATG18a. Mutation of BAK1 blocks ATG18a phosphorylation at four of the five detected phosphorylation sites after B. cinerea infection and strongly activates autophagy, leading to enhanced resistance against B. cinerea. Collectively, the identification of functional phosphorylation sites on ATG18a and the corresponding kinase BAK1 unveiled how plant regulates autophagy during resistance against necrotrophic pathogens.Abbreviations:35s: the cauliflower mosaic virus 35s promoter; A. thaliana: Arabidopsis thaliana; A. brassicicola: Alternaria brassicicola; ABA: abscisic acid; ATG: autophagy-related; ATG18a: autophagy-related protein 18a in A. thaliana; ATG8a: autophagy-related protein 8a in A. thaliana; ATG8-PE: ATG8 conjugated with PE; B. cinerea: Botrytis cinerea; BAK1: Brassinosteroid insensitive 1-associated receptor kinase1 in A. thaliana; BiFC: biomolecular fluorescence complementation; BIK1: Botrytis-insensitive kinase 1 in A. thaliana; BKK1: BAK1-like 1 in A. thaliana; BR: brassinosteroid; Co-IP: coimmunoprecipitation; dai: days after inoculation; DAMPs: damage-associated molecular patterns; E. coli: Escherochia coli; ER: endoplasmic reticulum; ETI: effector-triggered immunity; GFP: green fluorescent protein; HA: hemagglutinin; IP: immunoprecipitation; LC-MS/MS: liquid chromatography-tandem mass spectrometry; LCI: luciferase complementation imaging; MPK3: mitogen-activated protein kinase 3 in A. thaliana; MPK4: mitogen-activated protein kinase 4 in A. thaliana; MPK6: mitogen-activated protein kinase 6 in A. thaliana; N. benthamiana: Nicotiana benthamiana; NES: nuclear export sequence; PAMP: pathogen-associated molecular pattern; PCR: polymerase chain reaction; PE: phosphatidylethanolamine; PRR: pattern recognition receptor; PtdIns(3,5)P2: phosphatidylinositol (3,5)-biphosphate; PtdIns3P: phosphatidylinositol 3-biphosphate; PTI: PAMP-triggered immunity; qRT-PCR: quantitative reverse transcription PCR; SnRK2.6: SNF1-related protein kinase 2.6 in A. thaliana; TORC1: the rapamycin-sensitive Tor complex1; TRAF: tumor necrosis factor receptor-associated factor; WT: wild type plant; Yc: C-terminal fragment of YFP; YFP: yellow fluorescent protein; Yn: N-terminal fragment of YFP.

Arabidopsis CPK5 Phosphorylates the Chitin Receptor LYK5 to Regulate Plant Innate Immunity.

Chitin, a major component of the fungal cell wall, triggers plant innate immunity in Arabidopsis via a receptor complex including two major lysin motif receptor-like kinases, AtLYK5, and AtCERK1. Although AtLYK5 has been proposed to be a major chitin-binding receptor, the pseudokinase domain of AtLYK5 is required to mediate chitin-triggered immune responses in plants. In this study, 48 AtLYK5-interacting proteins were identified using immunoprecipitation and mass spectrometry assay. Among them, Arabidopsis CALCIUM-DEPENDENT PROTEIN KINASE 5 (AtCPK5) is a protein kinase interacting with both AtLYK5 and AtCERK1. Chitin-induced immune responses are inhibited in both Arabidopsis atcpk5 and atcpk5/6 mutant plants. AtLYK5 and AtLYK4 but not AtCERK1 are phosphorylated by AtCPK5 and AtCPK6 in vitro. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis and in vitro kinase assay identified that Ser-323 and Ser-542 of AtLYK5 are important phosphorylation residues by AtCPK5. Transgenic Arabidopsis expressing either AtLYK5-S323A or AtLYK5-S542A in the atlyk5-2 mutant only partially rescue the defects in chitin-triggered MPK3/MPK6 phosphorylation. Overexpression of AtCPK5 could increase AtCERK1 protein level after chitin treatment. These data proposed a model in which AtCPK5 directly phosphorylates AtLYK5 and regulates chitin-induced defense responses in Arabidopsis.

The Lotus japonicus Ubiquitin Ligase SIE3 Interacts With the Transcription Factor SIP1 and Forms a Homodimer.

The symbiosis receptor kinase SymRK plays an essential role in symbiotic signal transduction and nodule organogenesis. Several proteins bind to SymRK, but how the symbiosis signals are transduced from SymRK to downstream components remains elusive. We previously demonstrated that both SymRK interacting protein 1 (SIP1, an ARID-type DNA-binding protein) and SymRK interacting E3 ligase [SIE3, a RING (Really Interesting New Gene)-containing E3 ligase] interact with SymRK to regulate downstream cellular responses in Lotus japonicus during the legume-rhizobia symbiosis. Here, we show that SIE3 interacts with SIP1 in both yeast cells and Nicotiana benthamiana. SIE3 associated with itself and formed a homodimer. The cysteine 266 residue was found to be essential for SIE3 dimerization and for promoting nodulation in transgenic hairy roots of L. japonicus. Our findings provide a foundation for further investigating the regulatory mechanisms of the SymRK-mediated signaling pathway, as well as the biological function of E3 ligase dimerization in nodule organogenesis.

A High-Quality Genome Sequence of Model Legume Lotus japonicus (MG-20) Provides Insights into the Evolution of Root Nodule Symbiosis.

Lotus japonicus is an important model legume for studying symbiotic nitrogen fixation as well as plant development. A genomic sequence of L. japonicus (MG20) has been available for more than ten years. However, the low quality of the genome limits its application in functional genomic studies. Therefore, it is necessary to assemble high-quality chromosome sequences of L. japonicus using new sequencing technology to facilitate the study of functional genomics. In this report, we used the third-generation sequencing combined with the Illumina HiSeq platform to sequence the genome of L. japonicus (MG20). We obtained 544 Mb of genomic sequence using third-generation assembly. Based on sequence analysis, 357 Mb of repeats, 28,251 genes, 626 tRNAs, 1409 rRNAs, and 1233 pseudogenes were predicted in the genome. A total of 27,991 genes were annotated into databases. Compared to the previously published data, the new genome database contains complete L. japonicus sequences in the proper order and orientation with a contig N50 2.81Mb and an excellent genome coverage, which provides more accurate genome information and more precise assembly for functional genomic study.