Plant immunity requires conformational changes [corrected] of NPR1 via S-nitrosylation and thioredoxins

Abstract

Changes in redox status have been observed during immune responses in different organisms, but the associated signaling mechanisms are poorly understood. In plants, these redox changes regulate the conformation of NPR1, a master regulator of salicylic acid (SA)-mediated defense genes. NPR1 is sequestered in the cytoplasm as an oligomer through intermolecular disulfide bonds. We report that S-nitrosylation of NPR1 by S-nitrosoglutathione (GSNO) at cysteine-156 facilitates its oligomerization, which maintains protein homeostasis upon SA induction. Conversely, the SA-induced NPR1 oligomer-to-monomer reaction is catalyzed by thioredoxins (TRXs). Mutations in both NPR1 cysteine-156 and TRX compromised NPR1-mediated disease resistance. Thus, the regulation of NPR1 is through the opposing action of GSNO and TRX. These findings suggest a link between pathogen-triggered redox changes and gene regulation in plant immunity.

Fig. 1

GSNO facilitates the oligomerization of NPR1. (A) SA induces NPR1 monomer release as well as oligomerization. Nonreducing (–DTT) and reducing (+DTT) immuno-blot analysis revealed oligomeric (O), monomeric (M), and total (T) NPR1. (B) Re-oligomerization of monomeric NPR1-GFP in a cell-free assay. (C) NPR1 monomer disappears with an increasing concentra tion of GSNO but not of SNP or H₂O₂, while total protein levels are unaffected. (D) SA induces nuclear localization of the NPR1-GFP monomer in the wild type but not in the atgsnor1-3 mutant. (E) SA-induced PR-1 gene expression is compromised in atgsnor1-3 plants. PR-1 expression was determined by real-time polymerase chain reaction (PCR) and normalized with ubiquitin (UBQ). Error bars represent SD.

Fig. 2

S-nitrosylation of Cys¹⁵⁶ facilitates the assembly of NPR1 oligomer. (A) SA induces S-nitrosylation of endogenous NPR1 and the NPR1-GFP proteins in vivo. Sodium ascorbate (Asc) was used to specifically detect S-nitrosylated (SNO) NPR1. Equal loading was verified with antibodies against NPR1 or NPR1-GFP. (B) GSNO, but not mock (–) or SNP treatment, induces S-nitrosylation of NPR1-GFP in plant extracts. S-nitrosylated NPR1-GFP was detected with the biotin-switch assay. An antibody against NPR1-GFP was used to verify equal loading. (C) GSNO, but not mock (–) or SNP treatment, induces S-nitrosylation and multimerization (black arrows) of recombinant His6-NH (NPR1 residues 1 to 246) monomer (gray arrow). Equal loading was verified with an antibody to NPR1. (D) Cys¹⁵⁶ is the principal site of S-nitrosylation in NPR1. Recombinant His6-NH and His6-NH-C156A proteins were incubated with different GSNO concentrations, and S-nitrosylation was detected by the biotin-swich assay. Equal loading was verified with an antibody to NPR1. (E) The C156A mutation impairs GSNO-induced oligomerization. Recombinant His6-NH and His6-NH-C156A proteins were treated with GSNO and with (+) or without (–) sodium ascorbate. Subsequently, monomers were allowed to re-oligomerize for the indicated times. Monomeric (–DTT) and total (+DTT) proteins were detected with an antibody to NPR1.

Fig. 3

S-nitrosylation of Cys¹⁵⁶ is essential for NPR1 protein homeostasis and SA-induced disease resistance. (A) SA treatment reduces the constitutive nuclear fluorescence of NPR1C156A-GFP. (B) The C156A mutation impairs NPR1 oligomer formation in response to SA. 35S::NPR1-GFP and 35S::NPR1C156A-GFP plants were treated with SA. The relative amount of NPR1 oligomer was determined by calculating densitometric ratios between induced and uninduced samples and normalized against total NPR1 protein. Error bars represent SD (n = 3 measurements). (C) SA-induced resistance is compromised in NPR1C156A plants. Error bars represent 95% confidence limits (n = 8 xxxxx). cfu, colony-forming unit. (D) SA treatment decreases NPR1C156A protein levels. 35S::NPR1-GFP and 35S::NPR1C156A-GFP plants were treated with (+) or without (–) SA for 48 hours. NPR1-GFP protein was detected with an antibody to GFP, and equal loading was verified with an antibody against constitutively expressed Ca²⁺-sensing receptor (CAS).

Fig. 4

TRX is a redox mediator of NPR1. (A) Immobilized His6-NH pulls down TRXs in vitro. TRXs were detected with protein-specific antibodies. (B) 35S::NPR1-TAP transformed and untransformed plants were treated with or without SA. SA treatment enhanced coimmunoprecipitation of TRX-h5 with NPR1-TAP as detected by an antibody to TRX-h5. (C) NPR1-GFP oligomer (O) is reduced to monomer (M) by TRX-h5 in vitro. DTT (0.33 mM) was added to recycle TRX activity. Detection of total (T) NPR1-GFP verified equal loading. (D) In vivo reduction of NPR1 oligomer to monomer in Col-0 and trx-h5 plants 12 hours after SA treatment. Non-reducing (–DTT) and reducing (+DTT) SDS-polyacrylamide gel electrophoresis were performed in parallel, and the endogenous NPR1 was detected with an antibody to NPR1. The asterisk indicates possible NPR1 dimer complexes. (E) SA-induced PR-1 gene expression is compromised in trx mutants. PR-1 expression was determined by real-time PCR and normalized with ubiquitin (UBQ). Error bars represent SD. (F and G) Induction of SAR significantly decreased disease symptoms and Psm ES4326 growth in Col-0 plants but not in the trx, ntra, and npr1-1 mutants. Error bars represent 95% confidence limits (n = 8 samples).