NopD of Bradyrhizobium sp. XS1150 Possesses SUMO Protease Activity

Abstract

Effectors secreted by the type III protein secretion system (T3SS) of rhizobia are host-specific determinants of the nodule symbiosis. Here, we have characterized NopD, a putative type III effector of Bradyrhizobium sp. XS1150. NopD was found to possess a functional N-terminal secretion signal sequence that could replace that of the NopL effector secreted by Sinorhizobium sp. NGR234. Recombinant NopD and the C-terminal domain of NopD alone can process small ubiquitin-related modifier (SUMO) proteins and cleave SUMO-conjugated proteins. Activity was abolished in a NopD variant with a cysteine-to-alanine substitution in the catalytic core (NopD-C₉₇₂A). NopD recognizes specific plant SUMO proteins (AtSUMO1 and AtSUMO2 of Arabidopsis thaliana; GmSUMO of Glycine max; PvSUMO of Phaseolus vulgaris). Subcellular localization analysis with A. thaliana protoplasts showed that NopD accumulates in nuclear bodies. NopD, but not NopD-C₉₇₂A, induces cell death when expressed in Nicotiana tabacum. Likewise, inoculation tests with constructed mutant strains of XS1150 indicated that nodulation of Tephrosia vogelii is negatively affected by the protease activity of NopD. In conclusion, our findings show that NopD is a symbiosis-related protein that can process specific SUMO proteins and desumoylate SUMO-conjugated proteins.

Keywords: effector; legume; nitrogen fixation; nodulation; protease; small ubiquitin-related modifier; symbiosis; type III protein secretion system.

FIGURE 1

NopD of Bradyrhizobium sp. XS1150. (A) Amino acid sequence of NopD, a modular protein that consists of an N-terminal domain (N), a tandem repeat domain (TR) and a C-terminal protease domain (C) with histidine, aspartic acid and cysteine residues (predicted catalytic triad). (B) Phylogenetic analysis of NopD family proteins based on a conserved C-terminal region. The residues 835–982 of NopD were aligned with a selection of related rhizobial proteins (B. elkanii USDA61, B. japonicum Is-34, B. japonicum USDA110, M. loti MAFF303099 and Sinorhizobium fredii HH103) and with XopD of X. campestris pv. campestris 8004. The alignment and accession numbers of the proteins are shown in Supplementary Figure S4. The tree was constructed with MEGA5 software. Bootstrap values are indicated next to branches. The scale bar represents 0.05 substitutions per site. (C) T3SS-dependent secretion of a chimeric NopD-NopL protein. The DNA construct of plasmid pFAJ-NopD:NopL is shown on the top of the panel. The plasmid was mobilized into the NGRΩnopL and NGRΩrhcN mutants of Sinorhizobium sp. NGR234. Equal amounts of secreted proteins from culture supernatants were analyzed by SDS-PAGE and Ponceau staining (loading control of Western blot). NopD-NopL was immunodetected by an antibody against NopL. Lane 1, NGRΩnopL; lane 2, NGRΩrhcN; lane 3: NGRΩnopL carrying pFAJ-NopD:NopL; lane 4, NGRΩrhcN carrying pFAJ-NopD:NopL.

FIGURE 2

Peptidase activity of 6 × His-tagged NopD and NopD-C. (A) Schematic view of the test system that is based on release of the C-terminal 3HA tag from a given GST-SUMO protein by NopD and NopD-C (C-terminal protease domain of NopD). Reaction mixtures were incubated at 30°C for 1 h. 6 × His-tagged NopD-C₉₇₂A (enzymatically inactive variant) and buffer (no enzyme) were used as negative controls. (B) Analysis of reaction mixtures on Western blots probed with an anti-GST antibody. Incubation of GST-AtSUMO1-3HA, GST-AtSUMO2-3HA, GST-GmSUMO1-3HA and GST-PvSUMO1-3HA with 6 × His-tagged NopD or NopD-C resulted in band shifts, indicating release of the C-terminal 3HA tag. Incubation with NopD-C₉₇₂A or buffer alone did not result in a band shift. (C) Results for reactions with GST-SUMO-3HA proteins that were not cleaved by NopD or variants (no obvious band shift).

FIGURE 3

Isopeptidase activity of 6 × His-tagged NopD and NopD-C. SUMO-RanGAP conjugates were prepared to analyze the desumoylation activity of NopD and NopD-C. (A) Preparation of an AtSUMO1-RanGAP conjugate in an in vitro sumoylation system with indicated 6 × His-tagged Arabidopsis proteins and human RanGAP (with 6 × His and Myc tags). The obtained AtSUMO1-RanGAP conjugate was not formed in the presence of 6 × His-tagged XopD. Western blot analysis of sumoylated RanGAP and RanGAP was performed with an anti-Myc antibody. Similar SUMO-RanGAP conjugates were obtained for other SUMO proteins (see Supplementary Figure S6). (B) NopD and NopD-C show SUMO isopeptidase activity for indicated SUMO-RanGAP conjugates. Incubation with 6 × His tagged NopD and NopD-C resulted in desumoylation of the conjugate, whereas NopD-C₉₇₂A (enzymatically inactive) or buffer (no enzyme) showed no effects. Reaction mixtures were analyzed on Western blots probed with an anti-Myc antibody. (C) Western blot results of reactions with indicated SUMO-RanGAP conjugates that were not desumoylated by NopD or NopD-C under the same test conditions.

FIGURE 4

Expression of NopD and NopD-C₉₇₂A in plant cells. (A) NopD induces cell death in tobacco. Expression of NopD and NopD-C₉₇₂A in different sections of a tobacco leaf was performed by infiltration of A. tumefaciens carrying appropriate plasmids (pCAMBIA1302 derivatives). NopT (a T3 effector of Sinorhizobium sp. NGR234) was used for comparison. The photograph was taken 48 h after bacterial infiltration. Cell death (necrotic tissue) was observed for NopD and NopT, whereas expression of NopD-C₉₇₂A showed no visible effects. (B,C) Subcellular localization of YFP-tagged NopD and NopD-C₉₇₂A in Arabidopsis protoplasts. The nuclear marker ARF4 fused to RFP was co-expressed. Cells were analyzed with a confocal microscope for red fluorescence (RFP) emission, yellow fluorescence (YFP) emission and under bright-field illumination (18 h after transformation). YFP fluorescence was evenly distributed throughout the nucleus (type I cells) or preferentially in nuclear bodies (type II cells).

FIGURE 5

Symbiotic phenotype of Bradyrhizobium sp. XS1150 and constructed mutants on the host plant T. vogelii. Data shown are the results of a representative nodulation test. Plants were inoculated with indicated strains and harvested 36 days later. Data indicate means ± SE (8 jars; n = 8). Different letters indicate significant differences (Kruskal–Wallis tests, P < 0.02). (A) Nodule number (number of pink nodules and white nodules per plant). (B) Nodule biomass (fresh weight of pink and white nodules per plant). Abbreviations: XS1150, Bradyrhizobium sp. XS1150 (wild-type); ΔnopD, XS1150ΔnopD (nopD knockout mutant); ΔnopD+nopD, XS1150ΔnopD+nopD (rescued nopD knockout mutant); ΔnopD+nopD-C₉₇₂A, XS1150ΔnopD+nopD-C₉₇₂A (nopD knockout mutant expressing NopD-C₉₇₂A); ΩrhcST, XS1150ΩrhcST (mutant lacking a functional T3SS).