Genetic dissection of nitric oxide signalling network In plant defence response

Abstract

Following pathogen recognition, nitric oxide (NO) is rapidly produced in plants, this small molecule has emerged as a key signal in plant defence responses. S-nitrosylation is the major route of NO signal transduction in plants, a redox-based modification by addition of an NO moiety on cysteine thiol to form an S-nitrosothiol (SNO). S-nitrosoglutathione reductase (GSNOR) regulates cellular levels of S-nitrosylation and displays a key role in regulating the plant defence response. In this context, NO is important to orchestrate both defence gene expression and the hypersensitive response (HR) during attempted microbial infection. However, how the plant immune system recognizes NO and how NO level could elicit plant defence responses are poorly understood. The Arabidopsis thaliana (Arabidopsis) mutant NO overproducing 1 (nox1) was employed to characterize how NO level elicits defence dynamics. In response to microbial infection, resistance (R) gene-mediated defence and basal resistance were found to be compromised in the nox1 mutant relative to wild type Col-0 plants. Interestingly, nox1 mutant exhibit similar levels of HR and pathogen susceptibility to the GSNOR loss-of-function mutant atgsnor1-3. This phenomenon suggests that NO might regulate defence responses via GSNOR-mediated S-nitrosylation. Therefore, the nox1 atgsnor1-3 double mutant was generated and characterized to clarify this hypothesis. Accelerated HR and increased pathogen susceptibility are shown in the double mutant, which implies that increased NO mediated by nox1 and elevated SNOs resulting from atgsnor1-3, are additive with respect to the plant defence response. To identify genes responsible for NO perception, forward genetic screens were developed to identify Arabidopsis mutants with abnormal NO recognition. NO marker genes for genetic screens were identified from both lab and open source microarray data. Two genes, At3g28740 and At1g76600 were selected and experimentally confirmed to be strongly induced by NO. Transgenic Arabidopsis plants were generated carrying a NO reporter cassette, which consist of a luciferase reporter gene (LUC) driven by the promoter of NO marker gene. This forward genetic approach might be a powerful tool to identify genes integral to NO signal transduction. Three C2H2 zinc finger transcription factors (ZnTFs) ZAT7, ZAT8 and ZAT12 were identified as being rapidly and strongly induced by NO donors, which could be modulators of redox/NO-dependent signalling pathway. T-DNA insertion mutants within these ZnTFs have been identified. Basal resistance against Pseudomonas syringae pv tomato (Pst) DC3000 is compromised in all single knockout lines. Therefore, the full characterisation of defence phenotype of these mutants would be necessary to explore the role of these TFs in the plant defence. Furthermore, zat8 mutant is more sensitive to nitrosative stress when compared to wild type Col-0. This suggests that ZAT8 may be involved in protecting plants against nitrosative stress. However, the molecular mechanisms that underpin this function remain to be determined. In conclusion, NO and SNOs might regulate plant disease resistance via distinct pathways. Our work has also established NO-reporter lines to identify genes responsible for NO perception. In addition, three NO-induced ZnTFs have been identified that participate in regulation of basal resistance, which might unveil aspects of NO signalling related to the regulation of transcription.