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 strain containing a plasmid with NFR1/NFR5 genes. Dashed red lines indicate leaf discs where different proteins were expressed. (A, B) NopT (A), but not NopTC93S (B), a protease-inactive version of NopT, suppressed NFR1NFR5-induced cell death. (C) Expression of NopM-induced cell death in N. benthamiana. (D) Expression of NopT could not suppress the cell death response triggered by expression of Avr3a/R3a, NopM, BAX, INF1 or AtCERK1. Numbers in (D) represent the number of leaf discs showing cell death and total leaf discs tested. Cell death in leaf disc results in the formation of necrotic plaques, which restrains pathogens within deceased cells. These plaques commonly manifest as leaf dehydration, frequently accompanied by a translucent appearance. Brown and shriveled leaf discs serve as indicators of cell death. The pictures shown in this figure are representative of at least three independent biological replicates.

NopT interacts with NFR1 and NFR5

Interactions between NopT and NFR1 or NFR5 were detected using BiFC (Split-YFP) (A, B), Split-LUC complementation (C) and co-IP (D) assays in N. benthamiana leaves. (A, B) For BIFC analysis, nYFP and cYFP tags were fused at the C-terminus of NopT, NFR1, NFR5, and the flagellin receptor FLS2 (negative control). YFP fluorescence signals represent protein-protein interactions. Scale bar=25 μm. (C) For the Split-LUC complementation assay, the NLuc and CLuc tags were fused at the C-terminus of NopT, NFR1, NFR5 and FLS2 (negative control). Luminescence signals represent protein-protein interactions. (D) For the co-IP assay, HA-tagged NFR1 or NFR5 and FLAG-tagged NopT were expressed in N. benthamiana cells followed by immunoprecipitation using an anti-FLAG antibody. Immunoblot analysis was performed using anti-HA and anti-FLAG antibodies (NopTC denotes the truncated version of NopT after autocleavage). The images and immunoblots shown in this figure are representative of three biological replicates

NopT proteolyzes NFR5 at its 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 NopT) cleaves NFR5-GFP protein expressed in N. benthamiana cells by releasing NFR5CD-GFP (NopTC denotes autocleaved NopT). The cleavage efficiency was marked under the lane. (B) NopT but not NopTC93S cleaves NFR5-Myc expressed in hairy roots of L. japonicus by releasing NFR5CD-Myc. The cleavage efficiency was marked under the lane. The asterisk indicates the HA-tagged NFR5 cleavage product containing the kinase domain and a C-terminal tail region (C) Analysis of the CDs of NFR1 and NFR5 co-expressed with NopT or NopTC93S in E. coli cells. Cleavage of NFR5CD was observed for NopT but not NopTC93S, while NFR1CD was not proteolyzed by NopT. In the presence of NFR1CD, a slower migrating band was observed, possibly representing phosphorylated NopT (NopTP). The cleavage efficiency was marked under the lane. (D) A repeat experiment confirmed that NopT is able to cleave NFR5CD (the asterisk indicates the HA-tagged NFR5 cleavage product containing the kinase domain and a C-terminal tail region). The cleavage efficiency was marked under the lane. (E) NopT cleaves His-SUMO-NFR5JM-GFP (His-SUMO and GFP linked by the JM domain of NFR5) in vitro. F.T. indicates proteins in flow through samples after purification with Ni-beads. (F) NopT expressed in E. coli cells was unable to cleave co-expressed NFR5CD-5m-HA, a modified version of NFR5CD-HA in which five amino acids of the JM were substituted by other residues (S283Y, G294Q, Y303S, A310I, T311Y). (G) NopT expressed in E. coli cells was unable to cleave co-expressed M. truncatula NFPCD-HA and the NFR5JM-NFPKC-HA fusion protein, while NFPJM-NFR5KC was proteolyzed (KC stands for the kinase domain and a C-terminal tail region, modified nod factor receptors without the JM).

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 inoculation with rhizobia (Mesorhizobium loti MAFF303099) and was largely dependent on NFR1. (B) The relative protein amount of each lane, as shown in (A), was quantified with ImageJ software (three biological replicates). The value of the control band in each gel was set to 1 for comparison. Values are means ± SEM. (C) NFR1CD but not NFR1CD-K351E phosphorylates NopT in E. coli cells. The phosphorylated full-length form of NopT could be dephosphorylated by CIAP (gel band shift). Abbreviations: NFR1CD-P, autophosphorylated NFR1; NopTP, phosphorylated NopT; NopT, non-phosphorylated NopT; NopTC, autocleaved NopT. (D) The phosphorylation sites of NopT identified by LC-MS were either substituted to alanine (A) or aspartate (D). The indicated NopT variants were subsequently tested for autocleavage and NFR5CD proteolysis in E. coli cells. Wild-type NopT (WT) and protease-dead NopTC93S were included into the analysis.

NopT regulates rhizobial infection in L. japonicus

(A) Analysis of rhizobial infection in L. japonicus roots inoculated with GUS-labelled S. fredii NGR234 (wild-type; WT) or a nopT knockout mutant (NGR234ΔnopT; abbreviated as ΔnopT in other panels). Scale bar=100 µm. Infection represents both infection focus and infection thread. The infection threads in the root hairs were shown in the left image in the upper pannel, while the infection foci were shown in the left image in the middle pannel. (B) GUS staining pictures showing roots of L. japonicus plants expressing GUS expression under control of the NIN promoter (pNIN:GUS). Plants were inoculated with NGR234 or NGR234ΔnopT and analyzed at 7 dpi. Scale bar=1 mm. (C) Infection data (ITs, infection threads) for roots shown in (A) at 7 dpi (n=10, Student’s t-test; * indicates P < 0.01). (D) Quantification of GUS staining sites for roots shown in (B) (n=10, Student’s t-test, P < 0.01). (E) Nodule primordia formation in L. japonicus wild-type roots inoculated with NGR234 (WT), NGR234ΔnopT or NGR234 over-expressing nopT (pT7:NopT). Roots were analyzed at 14 dpi (n=16, Student’s t-test: P < 0.01). (F) Infection data for wild-type roots inoculated with NGR234 (WT), NGR234ΔnopT and NGR234ΔnopT expressing indicated NopT variants at 7 dpi (n=5, Student’s t-test: P < 0.01). (G) Analysis of rhizobial infection in hairy roots of L. japonicus (wild-type) expressing GFP (EV, empty vector control) or NopT. Plants were inoculated with DsRed-labeled M. loti MAFF303099 and analyzed at 5 dpi (n=8, Student’s t–test: P < 0.01). (H) Expression of NFR5 and NFR5-NFPKC in hairy roots of nfr5-3 mutant plants. Plants were inoculated with GFP-labelled NGR234 (WT) or NGR234ΔnopT and analyzed at 8 dpi. Roots expressing NFR5-NFPKC showed high numbers of infection foci for both strains whereas significant differences were observed for NFR5 expressing roots (n=10, Student’s t-test: P < 0.01).

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. diazoefficiens USDA110; NopT257, NopT of S. fredii USDA257; NopT103, NopT of S. fredii HH103). Immunoblot analysis indicated NFR5 cleavage by NopT proteins from S. fredii strains. (B) Expression of NopT257 in N. benthamiana could not inhibit the cell death triggered by co-expressed NFR1 and NFR5. (C) L. japonicus Gifu pNIN:GUS plants were inoculated with S. fredii NGR234, NGR234ΔnopT and NGR234ΔnopT expressing NopT257 (ΔnopT+NopT257). Roots were subjected to GUS staining at 7 dpi. Scale bar=2.5 mm. (D) Quantitative analysis of GUS-stained roots shown in panel C (n=19, Student’s t–test: P < 0.01). (E) A proposed model for NopT of NGR234 interacting with NFRs. NopT and NopTC (autocleaved NopT) proteolytically cleave NFR5 at the JM domain to release the intracellular domain of NFR5 (cleaved NFR5). NFR1 phosphorylates full-length NopT to block its proteinase activity. NopTC cannot be phosphorylated by NFR1.