Figures and data
NopT specifically suppresses cell death triggered by NFR1 & NFR5 in N. benthamiana. Agrobacterium strains harboring genes were NopT or EV infiltrated into N. benthamiana,12 hpi followed by infiltration with agrobacterium containing NFR1 and NFR5. Different leaf discs indicate where different proteins were expressed. NopT (A) but not NopTC93S, the protease–inactive version of NopT (B) suppressed cell death induced by expression of NFR1 and NFR5. (C) Expression of NopM induced cell death in N. benthamiana. (D) Expression of NopT could not suppress the cell death triggered by expression of Avr3a&R3a, NopM, BAX, INF1 or AtCERK1. Digital numbers in (D) represent the number of leaf discs with cell death and total leaf discs tested. All pictures in the Figure are representative of at least three independent biological replicates.
NopT interacts with NFR1 and NFR5.
Interactions between NopT and NFR1 and NFR5 were detected using Split–YFP assay (A) and (B), Split–LUC (C) and co–IP (D) assays in N. benthamiana. (A) and (B). nYFP and cYFP tags were fused at the C– terminus of NopT, NFR1, NFR5, and negative control FLS2. YFP fluorescence signals represent protein–protein interactions. Scar bar=25 um. (C). nLUC and cLUC tags were fused at the C–terminus of NopT, NFR1, NFR5, and negative control FLS2. Luminescence signals represent protein–protein interactions. (D). HA–tagged NFR1 and NFR5 and FLAG–tagged NopT were expressed in plant cells followed by immunoprecipitation using anti–FLAG antibody and determined by immunoblot using anti–HA and anti–FLAG antibodies. All data in Figure are representative of three biological replicates.
NopT proteolyzes NFR5 at its juxtamembrane domain.
(A) NopT but not NopTC93S cleaves NFR5–GFP protein expressed in N. benthamiana cells by releasing NFR5CD–GFP. (B) NopT but not NopTC93S cleaves NFR5-Myc protein expressed in L. japonicus transgenic root by releasing NFR5CD-Myc. Asterisk indicated the cleaved variant of NFR5. (C) A repeated experiment testing the cleavage of NFR5CD but not NFR1CD fused with different tags. Asterisk represents the phosphorylated NopT by inducing a band retardation on SDS–PAGE gel. (D) NopT but not NopTC93S cleaves the CD of NFR5 protein expressed in E. coli cells by releasing the kinase domain of NFR5. Asterisk indicated the cleaved variant of NFR5. (E) NopT cleaves a recombinant protein where His–tagged SUMO and GFP is bridged with the JM domain of NFR5. F.T. represents flow through sample after Ni–beads purification. (F) NopT could not cleave a mutant version of NFR5CD–HA with 5 residues in the JM substituted into other residues (S283Y, G294Q, Y303S, A310I, T311Y). (G) NopT could not cleave Medicago truncatula NFP and from NFR5JM–NFPKD. CD represents cytoplasmic domain, KC represents kinase domain and C-terminal tail region, NopTC indicates the truncated version of NopT after autocleavage by releasing about 50 a.a. at its N–terminus.
Phosphorylation of NopT by NFR1 suppresses its proteolytic activity.
(A) In vivo phosphorylation assay in L. japonicus roots. Phosphorylation of NopTC93S was induced with rhizobia treatment and dependent on nfr1. (B) NFR1CD phosphorylates NopT by inducing a gel band shift determined by immunoblotting. (C) The phoshorylation sites on NopT identified by liquid chromatography–mass spectrometry (LC–MS) were either substituted into alaine (A) or asparatate (D) and subsequently tested for their ability to proteolytically cleave NFR5CD.
NopT regulates rhizobial infection in L. japonicus.
(A) Rhizobial infection in L. japonicus using GFP-labelled WT and nopt mutant strains of S. fredii NGR234. Fluorescent, GFP-expressing bacteria are imaged in Cyan. Scar bars=12.5µm. (B) GUS staining pictures from the roots of L. japonicus transgenically expressing GUS under pNIN promoter inoculated with WT and nopT mutant strains of NGR234. Scar bars=1mm (C) Statistical analysis of rhizobial infection in A (n=10, Student’s t–test: P < 0.01). (D) Statistical analysis of GUS straining sites in B (n=10, Student’s t–test: P < 0.01). (E) Nodule primordia number on the roots 14dpi after inoculation WT or nopT mutant strains (n>20, Student’s t–test: P < 0.01). (F) Infection foci number from the wild type plant roots inoculated with WT strains and nopt mutant strains expressing NopT with different variations (n=5, Student’s t–test: P < 0.01). (G) Statistical analysis of rhizobial infection in L. japonicus transgenic root expressing GFP (EV, empty vector control) or NopT using DsRed-labeled M. loti MAFF303099 (n=8, Student’s t–test: P < 0.01).
Working model of NopT in association with NF receptors.
(A) NopT234, NopT from S. fredii NGR234; NopT1110 and NopT2110, NopT homologs from B. diazoefficiens USDA110; NopT257, NopT from S. fredii USDA257; NopT103, NopT from S. fredii HH103. All NopT homologs were tested in the proteolysis assay using NFR5CD as a target. (B) Rhizobial infection NopT257 could not inhibit the cell death triggered by NFR1 and NFR5 in N. benthamiana. (C)The indicated rhizobial strains induced nin::gus expressing in Lotus root. Scar bars=2.5mm. (D) Statistical analysis of GUS straining in C (n=19, Student’s t–test: P < 0.01). (E) A proposed model of NopT in association with NF receptors. Both NopT and NopTC after autocleavage exert proteolytic activities to cleave NFR5 at the juxtamembrane domain to release the kinase domain of NFR5 (cleaved NFR5). NFR1 phosphorylates intact NopT, but not NopTC, to block its proteinase activity.
Analyses of the functions of all 15 effectors from S. fredii NGR234 in preventing LjNFR1- and LjNFR5-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. LjNFR1 and LjNFR5 from L. japonicus were C-terminally tagged with HA and Myc, respectively, and cloned into binary vectors for expression in N. benthamiana. All plasmids were electroporated into Agrobacterium tumefaciens EH105. (A) Agrobacterium strain harboring each effector gene or control empty vector (EV) were infiltrated into N. benthamiana. 12 hour post infiltration (hpi), agrobacterium strains harboring LjNFR1 and LjNFR5 were mixed and hand-infiltrated in the same leaf discs as shown in (B). 48 hpi, cell death phenotypes were evaluated (C). At least three replicates in the same leaf were performed to test the function of each effector in suppressing cell death-triggered by LjNFR1 and LjNFR5. (D) Results of all 15 effector proteins in suppressing programmed cell death by overexpression of NFR1 and NFR5. (E) Abundance of LjNFR1, LjNFR5, and NopT proteins, as measured by immunoblot with specific antibodies. Leaf discs expressing LjNFR1, LjNFR5, and NopT or LjNFR1 and LjNFR5 and with EV were used for immunoblot analyses. Actin was used as loading control. (F) Another example of suppression of programmed cell death by NopT in the leaf discs coexpressing NFR1 and NFR5. Rep1, Rep2, and Rep3 represent three technical repeats in the same leave.
Split–LUC assay testing the interactions between different NopT mutants and NFRs. (A)The interaction between NopT with mutated acylation site and NFR1/NFR5. NopTAAA (G50A C51A C52A). (B) The protein expressing of indicating genes in the leave of (A). (C) NopC93S interact with NFR1 and NFR5. (D) The interaction between NopTΔN50 and NFR5. NopTΔN50 represents a truncated NopT with deletion of about 50 a.a. from its N–terminus.
NopT cleaves NFR5 at the juxtamembrane domain. (A) NopT cannot cleave NFR1-GFP expressed in N. benthamiana leaves. recombinant Sumo-NFR5KD-HA lacking the juxtamembrane (JM) domain. (B) NopT could not cleave Sumo–NFR5KD–HA, a recombinant protein expressed in E. coli with the juxtamembrane domain of NFR5 was replaced with Sumo tag. (C) Cleavage assay using two mutant versions of NFR5CD in the presence of NopT or AvrPphB. NFR5CD288-294A represents a mutant NFR5CD with residues 288 to 294 replaced with seven alanines; NFR5CD288-294PphB represents a mutant NFR5CD with residues 288 to 294 replaced with AvrPphB recognition sites. (D) Autocleavage assay, using different NopT variants with single amino acid mutations. H205A and D220A indicate His-205 and Asp-220 replaced with alanine, respectively. (E-H) NopT cleavage assay using 19 variants of NFR5CD with three adjacent amino acids replaced with three alanines. The numbers indicate the location of the three residues replaced with alanines. (I) NopT cleavage assay using seven truncated variants of NFR5CD with 10 adjacent amino acids deleted. The numbers indicate the location of the 10 deleted residues. (J) NopT but not NopTC93S could cleave the CDs of AtLYK5 and LjLYS11 expressed in E. coli. NopT could cleave both AtLYK5JM– NFR5KC, LjLYS11JM–NFR5KC and LjNFR1JM–NFR5KC, three recombinant proteins expressed in E. coli with the JM of NFR5 was replaced with the JMs from AtLYK5, LjLYS11 and NFR1, respectively. (K) NopT but not NopTC93S could cleave recombinant protein NFR5268-445-NFP458-595 and NFP270-457-NFR5456-595.
Characterization of cleavage site of LysM receptor. (A) Domain and motif annotation of NFR5. (B) Protein sequence alignment of the cytoplasmic domain of AtLYK5, LjLYS11, LjNFR5 and MtNFP. Yellow boxes delineated highly conserved residues (S283, G294, Y303, A310, T311) and brown box delineated kinase domain. Green frame delineated cleavage site of NFR5 and red frame delineated residues similar to NopT autocleavage region. (C) Mass spectrometry analysis of cleaved NFR5. Blue lines indicated the peptides characterized by MS.
The cytoplasmic domains (CDs) of NFR1, NFR5, and kinase– dead NFR1CDK351E were used for kinase assays in E. coli.
Proteins were detected by immunoblotting using anti–HA and anti–Myc antibodies. Asterisk indicated the band retardation on the gel representing the phosphorylated NFR5CD proteins.
Rhizobial infection in the mutant versions of NFR5. (A) NFR55m failed to form rhizobial infection (n=7, Student’s t–test: P < 0.01). (B)NFR5-NFPKD-CT could recuse the rhizobial infection of wild type NGR234 (n=10, Student’s t–test: P < 0.01). The transgenic roots expressing NFR5 or NFR55m or NFR5-NFPKCT in nfr5-3 background under the control of native promoter were inoculated with GFP-labelled wild type NGR234 and NGR234ΔnopT respectively. The data was collected 9 days post inoculation.
Phylogenetic tree based on the amino acid sequence of NopT homologs from different bacterial species.
Transient expression of NopT triggers cell death in Arabidopsis thaliana and Nicotiana tabacum. (A) Pseudomonas syringae pv tomato DC3000 harboring control plasmids (left) or expressing NopT (right) were infiltrated into Arabidopsis leaves. Note the cell death in the right-side panel. (B) Agrobacterium strains harboring NopT, NopT with an N-terminal 50–amino acid deletion, NopTC93S, or NopTC93S with an N-terminal 50–amino acid deletion were infiltrated into N. tabacum leaves.
Phosphopeptides identified by liquid chromatography– mass spectrometry.
An in vitro kinase assay was performed using the CD of NFR1 and NopT. NopT was separated by SDS-PAGE, and the digested gel slices representing the phosphorylated NopT were used for analysis. The deduced phosphopeptides are listed.
