A Lotus japonicus E3 ligase interacts with the Nod Factor Receptor 5 and positively regulates nodulation

Tsikou, Daniela; Ramirez, Estrella E.; Psarrakou, Ioanna S.; Wong, Jaslyn E. M. M.; Jensen, Dorthe Bødker; Isono, Erika; Radutoiu, Simona; Papadopoulou, Kalliope Κ.

doi:10.1186/s12870-018-1425-z

Cited by 10 publications

(6 citation statements)

References 51 publications

(71 reference statements)

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“…The recognition of NF from compatible rhizobia is a crucial step for root nodule symbiosis. Two NF receptor (NFR) genes or their orthologs were identified and characterized in several legumes with root hair infection [18][19][20][21][22][23][24]. These NFR genes were named NFR1 and NFR5 in L. japonicus, both encoding LysM-type serine/threonine receptor kinases [19].…”

Section: Introductionmentioning

confidence: 99%

The application of CRISPR/Cas9 in hairy roots to explore the functions of AhNFR1 and AhNFR5 genes during peanut nodulation

et al. 2020

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Background Peanut is an important legume crop growing worldwide. With the published allotetraploid genomes, further functional studies of the genes in peanut are very critical for crop improvement. CRISPR/Cas9 system is emerging as a robust tool for gene functional study and crop improvement, which haven’t been extensively utilized in peanut yet. Peanut plant forms root nodules to fix nitrogen through a symbiotic relationship with rhizobia. In model legumes, the response of plants to rhizobia is initiated by Nod factor receptors (NFRs). However, information about the function of NFRs in peanut is still limited. In this study, we applied the CRISPR/Cas9 tool in peanut hairy root transformation system to explore the function of NFR genes. Results We firstly identified four AhNFR1 genes and two AhNFR5 genes in cultivated peanut (Tifrunner). The gene expression analysis showed that the two AhNFR1 and two AhNFR5 genes had high expression levels in nodulating (Nod+) line E5 compared with non-nodulating (Nod-) line E4 during the process of nodule formation, suggesting their roles in peanut nodulation. To further explore their functions in peanut nodulation, we applied CRISPR technology to create knock-out mutants of AhNFR1 and AhNFR5 genes using hairy root transformation system. The sequencing of these genes in transgenic hairy roots showed that the selected AhNFR1 and AhNFR5 genes were successfully edited by the CRISPR system, demonstrating its efficacy for targeted mutation in allotetraploid peanut. The mutants with editing in the two AhNFR5 genes showed Nod- phenotype, whereas mutants with editing in the two selected AhNFR1 genes could still form nodules after rhizobia inoculation. Conclusions This study showed that CRISPR-Cas9 could be used in peanut hairy root transformation system for peanut functional genomic studies, specifically on the gene function in roots. By using CRISPR-Cas9 targeting peanut AhNFR genes in hairy root transformation system, we validated the function of AhNFR5 genes in nodule formation in peanut.

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Section: Introductionmentioning

confidence: 99%

The application of CRISPR/Cas9 in hairy roots to explore the functions of AhNFR1 and AhNFR5 genes during peanut nodulation

et al. 2020

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“…LjPUB13 is an E3 ligase that interacts with and ubiquitinates LjNFR5 in vitro (Tsikou et al, 2018). Thus, the biological role of LiPUB13 in promoting rhizobial symbiosis may be similar to that of NopM, as both proteins can ubiquitinate LjNFR5.…”

Section: Discussionmentioning

confidence: 99%

“…Once NF signaling is initiated, protein turnover and signaling activity of NFRs may be critical for maintaining proper symbiotic signaling and subsequent rhizobial infection. Previous research demonstrated that mutation in the LjPUB13 gene suppresses nodulation in L. japonicus (Tsikou et al, 2018). LjPUB13 is an E3 ligase that interacts with and ubiquitinates LjNFR5 in vitro (Tsikou et al, 2018).…”

Section: Discussionmentioning

confidence: 99%

Rhizobial effector NopM ubiquitinates Nod factor receptor NFR5 and promotes rhizobial infection inLotus japonicus

Wang,

Bao,

Lei

et al. 2024

Preprint

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Bacterial pathogens and nitrogen-fixing rhizobia employ type III protein secretion system (T3SS) effectors as potent tools to manipulate plant signaling pathways, thereby facilitating infection and host colonization. However, the molecular mechanisms underlying effects of rhizobial effectors on legume infection remain largely elusive. In this study, we investigated the symbiotic role of T3SS effectors in the interaction between Sinorhizobium fredii NGR234 and Lotus japonicus. Mutants deficient in the T3SS genes Tts1 or NopA showed enhanced rhizobial infection of L. japonicus roots. Further mutant analysis showed that the NopT effector negatively affects infection, while the NopM effector in the absence of NopT promotes infection. Notably, NopM interacts with the Nod factor receptors LjNFR1 and LjNFR5. NopM ubiquitinates LjNFR5 with ten ubiquitinated lysine residues in LjNFR5 identified using mass spectrometry. Expression of NopM in L. japonicus resulted in an approximately twofold increase in LjNFR5 protein levels and enhanced rhizobial infection. Our findings indicate that NopM directly interferes with the symbiotic signaling pathway through interaction and ubiquitination of Nod factor receptors, which likely benefits rhizobial infection of L. japonicus. Our research contributes to the intricate interplay between the Nod factor signaling pathway and rhizobial T3SS effectors delivered into host cells.

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“…Host plants secrete flavonoids, inducing rhizobia to synthesize and secrete Nod factors (NFs), a type of lipo-chitooligosaccharide molecules [ 1 , 2 ]. Two plant LysM-type serine/threonine receptor kinases, NFR1 and NFR5 in Lotus japonicus (Nod Factor Receptor 1 and 5) [ 3 , 4 , 5 ], cooperating with the leucine-rich repeat (LRR) receptor-like kinase SymRK (Symbiosis Receptor-like Kinase) [ 6 ], recognize NFs and initiate the NF signaling pathway [ 7 , 8 , 9 , 10 ]. The core component of the NF signaling pathway is a calcium oscillation which is decoded by the nucleus-localized CCaMK (calcium/calmodulin-dependent protein kinase) [ 11 , 12 ].…”

Section: Introductionmentioning

confidence: 99%

Dephosphorylation of LjMPK6 by Phosphatase LjPP2C is Involved in Regulating Nodule Organogenesis in Lotus japonicus

Yan

Cao

Fan

et al. 2020

IJMS

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The mitogen-activated protein kinase (MAPK) LjMPK6 is a phosphorylation target of SIP2, a MAPK kinase that interacts with SymRK (symbiosis receptor-like kinase) for regulation of legume-rhizobia symbiosis. Both LjMPK6 and SIP2 are required for nodulation in Lotus japonicus. However, the dephosphorylation of LjMPK6 and its regulatory components in nodule development remains unexplored. By yeast two-hybrid screening, we identified a type 2C protein phosphatase, LjPP2C, that specifically interacts with and dephosphorylates LjMPK6 in vitro. Physiological and biochemical assays further suggested that LjPP2C phosphatase is required for dephosphorylation of LjMPK6 in vivo and for fine-tuning nodule development after rhizobial inoculation. A non-phosphorylatable mutant variant LjMPK6 (T224A Y226F) could mimic LjPP2C functioning in MAPK dephosphorylation required for nodule development in hairy root transformed plants. Collectively, our study demonstrates that interaction with LjPP2C phosphatase is required for dephosphorylation of LjMPK6 to fine tune nodule development in L. japonicus.

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A Lotus japonicus E3 ligase interacts with the Nod Factor Receptor 5 and positively regulates nodulation

Cited by 10 publications

References 51 publications

The application of CRISPR/Cas9 in hairy roots to explore the functions of AhNFR1 and AhNFR5 genes during peanut nodulation

The application of CRISPR/Cas9 in hairy roots to explore the functions of AhNFR1 and AhNFR5 genes during peanut nodulation

Rhizobial effector NopM ubiquitinates Nod factor receptor NFR5 and promotes rhizobial infection inLotus japonicus

Dephosphorylation of LjMPK6 by Phosphatase LjPP2C is Involved in Regulating Nodule Organogenesis in Lotus japonicus

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