Abstract

Nitric oxide (NO) regulates the deployment of a phalanx of immune responses, chief among which is the activation of a constellation of defence-related genes. However, the underlying molecular mechanisms remain largely unknown. The Arabidopsis thaliana zinc finger transcription factor (ZF-TF), S-nitrosothiol (SNO) Regulated 1 (SRG1), is a central target of NO bioactivity during plant immunity. Here we characterize the remaining members of the SRG gene family. Both SRG2 and, especially, SRG3 were positive regulators of salicylic acid-dependent plant immunity. Analysis of SRG single, double and triple mutants implied that SRG family members have additive functions in plant immunity and, surprisingly, are under reciprocal regulation. SRG2 and SRG3 localized to the nucleus and functioned as ethylene-responsive element binding factor-associated amphiphilic repression (EAR) domain-dependent transcriptional repressors: NO abolished this activity for SRG3 but not for SRG2. Consistently, loss of GSNOR function, resulting in increased (S)NO concentrations, fully suppressed the disease resistance phenotype established from SRG3 but not SRG2 overexpression. Remarkably, SRG3 but not SRG2 was S-nitrosylated in vitro and in vivo. Our findings suggest that the SRG family has separable functions in plant immunity, and, surprisingly, these ZF-TFs exhibit reciprocal regulation. It is remarkable that, through neofunctionalization, the SRG family has evolved to become differentially regulated by the key immune-related redox cue, NO.

Highlights

  • A key feature upon attempted pathogen infection is the rapid production of the small, redox-active molecules nitric oxide (NO) and reactive oxygen species (ROS) (Grant & Loake, 2000; Gupta et al, 2011; Yu et al, 2014)

  • The absence of GSNOR function, leading to increased (S)NO concentrations, fully suppressed the disease resistance phenotype established from SRG3 overexpression but this was not found to be the case for overexpression of SRG2

  • Phylogenetic analysis showed that four C2H2-type zinc finger transcription factor (ZF-TF) were classified into a small group: SNO Regulated Gene 1 (SRG1), SRG2 (At3g46090), SRG3 (At5g59820) and SRG4 (At3g46070) (Fig. S1a)

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Summary

Introduction

A key feature upon attempted pathogen infection is the rapid production of the small, redox-active molecules nitric oxide (NO) and reactive oxygen species (ROS) (Grant & Loake, 2000; Gupta et al, 2011; Yu et al, 2014). The principal route for NO bioactivity is thought to be S-nitrosylation, the addition of a NO moiety to a cysteine (Cys) thiol to form an S-nitrosothiol (SNO) (Spadaro et al, 2010; Astier et al, 2011; Corpas & Barroso, 2014). S-nitrosoglutathione reductase (GSNOR) can turn over the natural NO donor, S-nitrosoglutathione (GSNO) (Feechan et al, 2005; Lee et al, 2008; Chen et al, 2009), formed by the reaction of NO with glutathione (GSH), with GSNO acting as a reservoir of NO bioactivity (Corpas & Barroso, 2014). GSNOR RNA interference lines show similar phenotypes in tomato (Hussain et al, 2019), suggesting the function of GSNOR is conserved across numerous dicotyledonous species

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