S-Nitrosylation of AtSABP3 Antagonizes the Expression of Plant Immunity

Yi-Qin Wang,Angela Feechan,Byung-Wook Yun,Reza Shafiei,Andreas Hofmann,Paul Taylor,Peng Xue,Fu-Quan Yang,Zhen-Sheng Xie,Jacqueline A Pallas,Cheng-Cai Chu,Gary J Loake

doi:10.1074/jbc.m806782200

Abstract

Changes in cellular redox status are a well established response across phyla following pathogen challenge. In this context, the synthesis of nitric oxide (NO) is a conspicuous feature of plants responding to attempted microbial infection and this redox-based regulator underpins the development of plant immunity. However, the associated molecular mechanism(s) have not been defined. Here we show that NO accretion during the nitrosative burst promotes increasing S-nitrosylation of the Arabidopsis thaliana salicylic acid-binding protein 3 (AtSABP3) at cysteine (Cys) 280, suppressing both binding of the immune activator, salicylic acid (SA), and the carbonic anhydrase (CA) activity of this protein. The CA function of AtSABP3 is required for the expression of resistance in the host against attempted pathogen infection. Therefore, inhibition of AtSBAP3 CA function by S-nitrosylation could contribute to a negative feedback loop that modulates the plant defense response. Thus, AtSABP3 is one of the first targets for S-nitrosylation in plants for which the biological function of this redox-based post-translational modification has been uncovered. These data provide a molecular connection between the changes in NO levels triggered by attempted pathogen infection and the expression of disease resistance.

Highlights

Plants have evolved a complex series of integrated defense systems in response to microbial colonization [1, 2]
We found that exposure of AtSABP3 to glutathione forms S-nitrosoglutathione (GSNO) but not GSH resulted in a dramatic, concentration-dependent decrease in carbonic anhydrase (CA) activity (Fig. 3A)
Attempted pathogen infection is a prominent feature of the amino acid residues required for salicylic acid (SA) binding have not yet been plant defense response [6, 7, 26]

Summary

EXPERIMENTAL PROCEDURES

Detection of Protein S-Nitrosylation—PstDC3000(avrB) challenged Arabidopsis leaf tissue was frozen in liquid nitrogen at the stated times post-inoculation. Donors were removed using Micro Bio-Spin P6 columns and the resulting protein was subjected to the biotin switch technique [17]. Site-directed Mutagenesis and Analysis of Protein Structure— Site-directed mutagenesis of AtSABP3 was undertaken using QuikChange௡ II Site-directed Mutagenesis Kits (Stratagene Corporate). The reaction with 300 nM 14C-labeled SA (20.5 Ci mmol; 1 Ci ϭ 37 GBq; New England Biolabs) with or without unlabeled SA (10,000-fold molar excess) was carried out in 100 ␮l of assay buffer (30 mM sodium citrate, pH 6.3, 1 mM EDTA) for 1 h on ice. Unbound ligand was removed with a 1-ml Bio-Spin 6 (BioRad) column by centrifugation for 4 min at 1,000 ϫ g. To monitor bacterial growth in planta given Arabidopsis lines were inoculated with avirulent PstDC3000(avrB) at 105 colony forming units mlϪ1

RESULTS

To determine a possible role for

DISCUSSION