The rhizobial effector NopT targets Nod factor receptors to regulate symbiosis in Lotus japonicus
- National Key Lab of Agricultural Microbiology, Hubei Hongshan Laboratory, Huazhong Agricultural University, China
- State Key Laboratory of Biocontrol and Guangdong Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat-sen University, China
- Divisions of Plant Science and Technology, Christopher S. Bond Life Sciences Center, University of Missouri, United States
Figures
Figure 1 with 1 supplement
NopT specifically suppresses NFR1/NFR5-triggered cell death in N. benthamiana.
Agrobacterium strains harboring plasmid DNA encoding NopT or the empty vector (EV) were infiltrated into N. benthamiana leaves. At 12 hpi, a second infiltration was performed with an Agrobacterium …
Figure 1—figure supplement 1
Analyses of the functions of all 15 effectors from S. fredii NGR234 in preventing NFR1- and NFR5-induced cell death in N. benthamiana leaves.
15 genes from S. fredii NGR234 encoding effector proteins were amplified and cloned into binary vector for expression of Strep-tagged proteins in N. benthamiana. NFR1 and NFR5 from L. japonicus were …
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Figure 1—figure supplement 1—source data 1
Original files for western blot analysis displayed in Figure 1—figure supplement 1E.
- https://cdn.elifesciences.org/articles/97196/elife-97196-fig1-figsupp1-data1-v1.zip
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Figure 1—figure supplement 1—source data 2
PDF file containing original western blots for Figure 1—figure supplement 1E, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/97196/elife-97196-fig1-figsupp1-data2-v1.pdf
Figure 2 with 2 supplements
NopT interacts with NFR1 and NFR5.
Interactions between NopT and NFR1 or NFR5 were detected using bimolecular fluorescence complementation (BiFC) (Split-YFP) (A, B), Split-LUC complementation (C) and co-IP (D) assays in N. benthamiana…
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Figure 2—source data 1
Original files for western blot analysis displayed in Figure 2D.
- https://cdn.elifesciences.org/articles/97196/elife-97196-fig2-data1-v1.zip
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Figure 2—source data 2
PDF file containing original western blots for Figure 2D, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/97196/elife-97196-fig2-data2-v1.pdf
Figure 2—figure supplement 1
NopT interacts with both NFR1 and NFR5.
The full blot of co-IP assay as shown in Figure 2D. The smaller bands are marked with asterisks. Compared to NFR1, NFR5 might be more susceptible to degradation during protein extraction from N. …
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Figure 2—figure supplement 1—source data 1
Original files for western blot analysis displayed in Figure 2—figure supplement 1.
- https://cdn.elifesciences.org/articles/97196/elife-97196-fig2-figsupp1-data1-v1.zip
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Figure 2—figure supplement 1—source data 2
PDF file containing original western blots for Figure 2—figure supplement 1, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/97196/elife-97196-fig2-figsupp1-data2-v1.pdf
Figure 2—figure supplement 2
Split-LUC assay testing the interactions between different NopT mutants and NFRs.
The interaction between NopT with mutated acylation site and NFR1/NFR5. NopTG50A/C51A/C52A represents a modified NopT form lacking acylation sites. (B) The protein expressing of indicating genes in …
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Figure 2—figure supplement 2—source data 1
Original files for western blot analysis displayed in Figure 2—figure supplement 2B.
- https://cdn.elifesciences.org/articles/97196/elife-97196-fig2-figsupp2-data1-v1.zip
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Figure 2—figure supplement 2—source data 2
PDF file containing original western blots for Figure 2—figure supplement 2B, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/97196/elife-97196-fig2-figsupp2-data2-v1.pdf
Figure 3 with 8 supplements
NopT proteolyzes NFR5 at its juxtamembrane (JM) domain.
Proteins with indicated tags were expressed in N. benthamiana (A), L. japonicus (B), or E. coli cells (C–G) and detected by immunoblotting. (A) NopT but not NopTC93S (a protease-dead version of …
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Figure 3—source data 1
Original files for western blot analysis displayed in Figure 3A–G.
- https://cdn.elifesciences.org/articles/97196/elife-97196-fig3-data1-v1.zip
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Figure 3—source data 2
PDF file containing original western blots for Figure 3A–G, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/97196/elife-97196-fig3-data2-v1.zip
Figure 3—figure supplement 1
NopT cleaves NFR5 but not NFR1.
Proteins with indicated tags were expressed in either N. benthamiana or E. coli cells and detected by immunoblotting. (A) NopT did not cleave NFR1-GFP expressed in N. benthamiana leaves. (B) NopT …
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Figure 3—figure supplement 1—source data 1
Original files for western blot analysis displayed in Figure 3—figure supplement 1A and B.
- https://cdn.elifesciences.org/articles/97196/elife-97196-fig3-figsupp1-data1-v1.zip
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Figure 3—figure supplement 1—source data 2
PDF file containing original western blots for Figure 3—figure supplement 1A and B, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/97196/elife-97196-fig3-figsupp1-data2-v1.zip
Figure 3—figure supplement 2
NopT interacts with the juxtamembrane (JM) domain of NFR5.
NopT interacts with NFR5JM in in vitro pull-down assay. IB: immunoblotting, IP: immunoprecipitation. His-SUMO-NFR5JM-GFP: a recombinant protein containing His tag, SUMO tag, the JM domain of NFR5, …
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Figure 3—figure supplement 2—source data 1
Original files for western blot analysis displayed in Figure 3—figure supplement 2.
- https://cdn.elifesciences.org/articles/97196/elife-97196-fig3-figsupp2-data1-v1.zip
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Figure 3—figure supplement 2—source data 2
PDF file containing original western blots for Figure 3—figure supplement 2, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/97196/elife-97196-fig3-figsupp2-data2-v1.pdf
Figure 3—figure supplement 3
Conserved domains and residues of NFR5 and related proteins.
(A) Domain and motif annotation of NFR5. LysM: Lysin-Motif; TM: transmembrane domain; JM: juxtamembrane domain; KD: kinase domain; CT: C-terminal domain. (B) Alignment of amino acid residues of …
Figure 3—figure supplement 4
NopT cleaves NFR5 at the juxtamembrane domain.
(A) Cleavage assay using two mutant versions of NFR5CD in the presence of NopT or AvrPphB. NFR5CD288-294A represents a mutant NFR5CD with residues 288–294 replaced with seven alanines; NFR5CD288-294P…
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Figure 3—figure supplement 4—source data 1
Original files for western blot analysis displayed in Figure 3—figure supplement 4A–G.
- https://cdn.elifesciences.org/articles/97196/elife-97196-fig3-figsupp4-data1-v1.zip
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Figure 3—figure supplement 4—source data 2
PDF file containing original western blots for Figure 3—figure supplement 4A–G, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/97196/elife-97196-fig3-figsupp4-data2-v1.zip
Figure 3—figure supplement 5
Mass spectrometry analysis of cleavage site of NFR5 by NopT.
(A) The sequence of NFR5 juxtamembrane domain (286–320 a.a.). (B) Mass spectrometry analysis of peptides of NFR5CD after proteolysis by NopT. Blue lines indicated the peptides characterized by mass …
Figure 3—figure supplement 6
NopT cleaves the NFR5 homolog proteins at the juxtamembrane (JM) domain.
NopT but not NopTC93S could cleave the CDs of AtLYK5 and LjLYS11 expressed in E. coli cells. NopT could cleave both AtLYK5JM-NFR5KC, LjLYS11JM-NFR5KC, and NFR1JM-NFR5KC, three recombinant proteins …
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Figure 3—figure supplement 6—source data 1
Original files for western blot analysis displayed in Figure 3—figure supplement 6.
- https://cdn.elifesciences.org/articles/97196/elife-97196-fig3-figsupp6-data1-v1.zip
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Figure 3—figure supplement 6—source data 2
PDF file containing original western blots for Figure 3—figure supplement 6, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/97196/elife-97196-fig3-figsupp6-data2-v1.pdf
Figure 3—figure supplement 7
NopT cleaves the juxtamembrane (JM) of MtNFP.
NopT cleaves a recombinant protein where His-tagged SUMO and GFP is bridged with the JM domain of NFP. F.T. represents flow through sample after Ni-beads purification. IB: immunoblotting.
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Figure 3—figure supplement 7—source data 1
Original files for western blot analysis displayed in Figure 3—figure supplement 7.
- https://cdn.elifesciences.org/articles/97196/elife-97196-fig3-figsupp7-data1-v1.zip
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Figure 3—figure supplement 7—source data 2
PDF file containing original western blots for Figure 3—figure supplement 7, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/97196/elife-97196-fig3-figsupp7-data2-v1.pdf
Figure 3—figure supplement 8
NopT cleaves recombinant proteins NFR5268-445-NFP458-595 and NFP270-457-NFR5456-595.
NopT but not NopTC93S could cleave recombinant proteins NFR5268-445-NFP458-595 and NFP270-457-NFR5456-595 detected by immunoblotting (IB).
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Figure 3—figure supplement 8—source data 1
Original files for western blot analysis displayed in Figure 3—figure supplement 8.
- https://cdn.elifesciences.org/articles/97196/elife-97196-fig3-figsupp8-data1-v1.zip
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Figure 3—figure supplement 8—source data 2
PDF file containing original western blots for Figure 3—figure supplement 8, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/97196/elife-97196-fig3-figsupp8-data2-v1.pdf
Figure 4 with 1 supplement
Phosphorylation of NopT by NFR1 suppresses its proteolytic activity.
(A) In vivo phosphorylation assay with proteins expressed in L. japonicus roots (wild-type and nfr1-1 mutant plants) using Zn2+-Phos-tag SDS–PAGE. Phosphorylation of NopTC93S was induced by …
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Figure 4—source data 1
Original files for western blot analysis displayed in Figure 4A, C and D.
- https://cdn.elifesciences.org/articles/97196/elife-97196-fig4-data1-v1.zip
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Figure 4—source data 2
PDF file containing original western blots for Figure 4A, C and D, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/97196/elife-97196-fig4-data2-v1.zip
Figure 4—figure supplement 1
NFR1CD phosphorylates NFR5CD in vitro.
The cytoplasmic domains (CDs) of NFR1, NFR5, and kinase-dead NFR1CD-K351E were used for kinase assays in E. coli cells. Proteins were detected by immunoblotting using anti-HA and anti-Myc …
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Figure 4—figure supplement 1—source data 1
Original files for western blot analysis displayed in Figure 4—figure supplement 1.
- https://cdn.elifesciences.org/articles/97196/elife-97196-fig4-figsupp1-data1-v1.zip
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Figure 4—figure supplement 1—source data 2
PDF file containing original western blots for Figure 4—figure supplement 1, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/97196/elife-97196-fig4-figsupp1-data2-v1.pdf
Figure 5 with 2 supplements
NopT regulates rhizobial infection in L. japonicus.
(A) Analysis of rhizobial infection in L. japonicus roots inoculated with GUS-labeled S. fredii NGR234 (wild-type; WT) or a nopT knockout mutant (NGR234ΔnopT; abbreviated as ΔnopT in other panels). …
Figure 5—figure supplement 1
Cross-section images of nodules at 14 dpi after toluidine blue staining.
NGR234 and NGR234ΔnopT mutants inoculated L. japonicus Gifu at 14 dpi. Scale bars correspond to 100 μm.
Figure 5—figure supplement 2
Rhizobial infection in the mutant versions of NFR5.
NFR55m failed to form rhizobial infection (n = 7, Student’s t-test: p < 0.01). The transgenic roots expressing NFR5 or NFR55m in the nfr5-3 mutant under the control of native promoter were …
Figure 6 with 3 supplements
S. fredii NopT proteins cleave NFR5 and working model for NopT of NGR234.
(A) NopT of S. fredii NGR234 and homologs from other rhizobial strains were co-expressed with NFR5CD in E. coli cells (NopT234, NopT of NGR234; NopT1110 and NopT2110, NopT proteins of B. …
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Figure 6—source data 1
Original files for western blot analysis displayed in Figure 6A.
- https://cdn.elifesciences.org/articles/97196/elife-97196-fig6-data1-v1.zip
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Figure 6—source data 2
PDF file containing original western blots for Figure 6A, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/97196/elife-97196-fig6-data2-v1.pdf
Figure 6—figure supplement 1
Phylogenetic tree based on the amino acid sequence of NopT homologs from different bacterial species.
Figure 6—figure supplement 2
The cleavage efficiency of NFR5 in different system.
Figure 6—figure supplement 3
Transient expression of NopT triggers cell death in Arabidopsis thaliana and Nicotiana tabacum.
Pseudomonas syringae pv. tomato DC3000 harboring control plasmids (left) or expressing NopT (right) were infiltrated into Arabidopsis leaves. Pictures were taken 3 days post inoculation. (B) Agrobact…
Additional files
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Supplementary file 1
Phosphopeptide identification and oligonucleotide used in the study.
(A) Phosphopeptides identified by liquid chromatography–mass spectrometry. (B) Oligonucleotides used in the study.
- https://cdn.elifesciences.org/articles/97196/elife-97196-supp1-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/97196/elife-97196-mdarchecklist1-v1.docx
