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Title: Redox regulation of salicylic acid biosynthesis by S-nitrosylation
Author: Casasola Zamora, Samuel
ISNI:       0000 0004 8497 5525
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2019
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A general outcome of plant-pathogen interactions is the oxidative and nitrosative burst, characterized for the accumulation of reactive oxygen and nitrogen species (ROS and RNA) which coordinate signalling cascades. Among RNS, Nitric oxide (NO) stands as a key signalling molecule in different physiological processes including plant immunity. An established mechanism for the transfer of NO bioactivity is S-nitrosylation (SNO), the reversible binding of a NO molecule to the thiol group of a susceptible cysteine, allowing specific proteins to respond to changes in the cellular REDOX state. The first genetic evidence about the role of NO in plant immunity was noted in Arabidopsis thaliana plants carrying a loss-of-function mutation of the S-nitrosoglutathione (GSNO) reductase 1 (GSNOR1) gene. This mutation resulted in increased total cellular S-nitrosylation and compromised basal, non-host and R-gene mediated immunity, and impaired synthesis and accumulation of the immune activator salicylic acid (SA). SA plays a pivotal role in the regulation of basal and systemic resistance. It is mainly produced by the activity of the enzyme Isochorismate Synthase 1 (ICS1) in response to pathogens. To date, significant progress has been achieved in understanding the mechanisms by which S-nitrosylation regulates SA signalling. However, the molecular mechanisms by which NO regulates SA biosynthesis remains elusive. To investigate if NO mediates transcriptional or posttranscriptional regulation over ICS1 expression we generated the reporter line ICS1::GUS. Our data suggest that ICS1 is subjected to transcriptional repression by S-nitrosylation. In agreement with previous observations about inhibition of the DNA-binding of SARD1 to the ICS1 promoter upon S-nitrosylation of SARD1 at Cysteine 438. We observed a significant reduction in the binding affinity of recombinant wild type (WT) SARD1 but not in SARD1 with a Cystein 438 to Serine mutation. To expand this observations and investigate the biological relevance of S-nitrosylation of Cys438 we generated transgenic lines expressing a C-terminal HA and Nano luciferase SARD1 and SARD1C438S fusion proteins. We showed that SARD1 can be S-nitrosylated in vivo. We did not observe any difference in local immunity against Pseudomonas syringae infection between the WT and C438S lines. Interestingly, the SARD1C438S lines showed impaired activation of systemic acquired resistance (SAR) compared to the WT. In addition, we observed that SARD1 protein level follows a circadian rhythm after SA treatment, which was impaired in the C438S mutant, suggesting that S-nitrosylation of SARD1 is necessary for optimal activation of SAR. It is possible that S-nitrosylation of SARD1 coordinates protein-protein interactions between SARD1 and other SAR activators. We developed a strategy to express and purify recombinant SARD1 for structural studies. Solving the three-dimensional structure of SARD1 can foster our understanding on the molecular interactions behind the regulation of SARD1. Finally, we designed a forward mutant screening to search for second-site mutations that can suppress the gsnor1 phenotype, which we speculate could be related with a novel mechanism for GSNO-turnover or NO metabolism. Collectively, our work can contribute to integrate NO cues in the regulation of SA biosynthesis and suggests a role for S-nitrosylation of SARD1 in systemic immunity.
Supervisor: Loake, Gary ; Nakayama, Naomi Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: salicylic acid ; plant immunity ; SARD1 ; S-nitrosylation