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Regulation of S-nitrosothiol function in plant disease resistance

Reference	BB/D011809/1
Principal Investigator / Supervisor	Professor Gary Loake
Co-Investigators / Co-Supervisors
Institution	University of Edinburgh
Department	Inst for Molecular Plant Science
Funding type	Research
Value (£)	342,754
Status	Completed
Type	Research Grant
Start date	01/03/2006
End date	28/02/2009
Duration	36 months

Abstract

S-nitrosylation is emerging as the prototypic, redox-based, post-translational modification in animals. This process has been demonstrated to have pivotal roles in haemoglobin function, bronchodilation, apoptosis and inflammation. Nitric oxide (NO), generated from nitric oxide synthases, is able to react with the S-H group in cysteine amino acids to form S-NO, an S-nitrosothiol. This can induce a change in target protein activity, location or binding. Thus, S-nitrosylation can be considered akin to protein phosphorylation. The Loake laboratory was the first to show a role for S-nitrosylation in plant biology, suggesting this post-translational modification is conserved between plant and animals. Our findings demonstrated that increased levels of S-nitrosylation abolished resistance (R) gene-mediated protection, basal resistance and non-host resistance (NHR) against microbial pathogens. Therefore, S-nitrosylation, the formation of S-nitrosothiols (SNOs), regulates multiple modes of plant disease resistance. These discoveries were underpinned by the identification of loss- and gain-of-function mutations in an Arabidopsis thaliana S-nitrosothiol glutathione reductase (AtGSNOR1). This enzyme is thought to turnover S-nitrosothiol glutathione (GSNO) in bacteria and mice, which is formed following the reaction of NO with the cellular antioxidant glutathione (GSH). GSNOR has been shown to indirectly control the level of global S-nitrosylation in bacteria and mice by regulating the amount of GSNO, which can drive the formation of protein-SNOs. Our data revealed that a loss-of-function mutation in AtGSNOR1 (atgsnor1-3) increased SNOs in Arabidopsis, while gain-of-function mutants, which over-express AtGSNOR1 (atgsnor1-1 and atgsnor1-2), reduced cellular SNOs. Interestingly, elevated SNO concentrations compromised multiple modes of plant disease resistance. Conversely, reduced SNO levels established broad spectrum resistance against ordinarily virulent pathogens. Thus, manipulating AtGSNOR1 activity provides a novel mechanism to control plant disease resistance. A classical strategy to identify additional genes within a given genetic network is to undertake a search for second-site modifier mutations. Therefore, to further dissect GSNO signalling/turnover, we conducted a suppressor screen in the atgsnor1-3 genetic background to search for mutations that are able to restore GSNO resistance. The growth of the atgsnor1-3 line is inhibited by low concentrations of GSNO. These supressors would most likely harbour mutations in genes that function downstream of or parallel to AtGSNOR1. Employing this approach, four independent atgsnor1-3 suppressor mutations were identified. In this project we will characterise these mutations, termed suppressors of atgsnor1-3 (sgr), to determine if in an atgsnor1-3 background they restore: (1) wild-type SNO, NO and ROI levels; (2) R gene-mediated resistance, basal protection and NHR; (3) SA biosynthesis and signalling; and, (4) wild-type kinetics of hypersensitive cell death. We will also determine the transcriptome in both loss-of-function (atgsnor1-3) and gain-of-function (atgsnor1-1) mutant lines to identify a set of SNO regulated genes, which will provide a further tool to aid characterisation of the atgsnor1-3 suppressors. The ability to exploit both loss- and gain-of-function lines makes these transcriptome experiments particularly powerful. Also, one of the sgr mutations will be selected for positional cloning to identify the cognate gene. We anticipate this gene will encode a key regulator of SNO bioactivity. Using proteomics we have identified a series of proteins that are specifically S-nitrosylated during the establishment of disease resistance. As a prerequisite for their future characterisation (beyond the scope of this project), we will determine if T-DNA insertions within their corresponding genes compromise R gene-mediated, basal or non-host resistance.

Summary

A large proportion of crop plants are lost to disease each year. Furthermore, diseases also have a significant negative impact on crop quality. Plants have evolved a complex battery of defence responses to protect themselves from attempted infection by potential pathogens. A key strategy is the production of reactive nitrogen intermediates (RNIs), such as nitric oxide (NO), an important component of defence responses in animals. NO may be both directly poisonous to the invading pathogen and also function as an alarm signal to trigger the deployment of defence responses. To protect themselves from the toxic effects of NO, plants produce a molecule called glutathione that reacts with NO to form S-nitrosoglutathione (GSNO). This molecule can act as a mobile store of NO bioactivity. The NO group within GSNO can be delivered to proteins, where it can react with the S-H group in an amino acid called cysteine, to produce an S-nitrosothiol (S-NO) group. This process is termed S-nitrosylation and this modification may change the structure, location or activity of the target protein. Therefore, S-nitrosylation may provide an important mechanism to regulate protein function. Previous work in our laboratory has demonstrated that an enzyme, GSNO reductase (AtGSNOR1), degrades GSNO and indirectly controls the cellular levels of S-nitrosylation. Mutations in the gene that encodes AtGSNOR1 result in three major defence systems being abolished, consequently these plants exhibit increased susceptibility to a variety of pathogens. Therefore, AtGSNOR1 has a central role in plant disease resistance. In this project we will characterise mutations within additional genes which function in concert with AtGSNOR1 to control GSNO levels and/or plant disease resistance. Furthermore, we will identify in which gene one of these mutations resides using a procedure termed positional cloning. Also, using gene chip technology, to measure the expression of all plant genes simultaneously, we will identify a set of genes that are controlled by S-nitrosothiols. Using a biochemical approach we have identified a series of proteins that are specifically S-nitrosylated during the establishment of disease resistance, a sub-set of these may have signalling functions. As a prerequisite to their future characterisation (beyond the scope of this project), we will determine if the absence of any of these proteins abolishes disease resistance.

Committee	Closed Committee - Plant & Microbial Sciences (PMS)
Research Topics	Crop Science, Plant Science
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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