Plants have developed elaborate defence mechanisms to protect themselves against pathogens. Recently, the ubiquitin-proteasome pathway has been proven to play important roles in regulating plant disease resistance. Previously, the tobacco (Nicotiana tabacum) ACRE276 and its Arabidopsis homolog AtPUB17 have been identified as E3 ligases that are positive regulators of the Cf-9/Avr9- and N/p50-elicited hypersensitive response (HR) in tobacco. In addition, AtPUB17 is required for the RPM1- and RPS4-mediated resistance responses in Arabidopsis. The identification of AtPUB17 signalling partners would allow us to understand the mode of action of AtPUB17 during plant defence. AtPOB1, a BTB/POZ-domain protein was isolated as an AtPUB17 interactor in a yeast-two-hybrid screen. The aim of this study was to confirm this interaction and to investigate the potential involvement of AtPOB1 in mediating disease resistance responses. The analysis of the Atpob1 knock out plants revealed a novel BTB/POZ protein implicated in plant defence. Atpob1 plants rapidly accumulated reactive oxygen species (ROS), induced the expression of pathogenesis related (PR) genes and developed spontaneous necrotic lesions upon infection with a virulent pathogen. AtPOB1 transcript and protein levels were induced by virulent Pseudomonas syringae. And transient overexpression of AtPOB1 in Cf-9 tobacco compromised the Avr9-triggered HR. In addition, Atpob1 plants showed signs of premature senescence. These results indicate that AtPOB1 is a negative regulator of plant defence- and senescence-associated pathways. The Nicotiana benthamiana AtPOB1 homolog was also identified and its cDNA sequence was used to investigate the role of NbPOB1 and its close relative NtPOB1 in disease resistance signalling. Transient overexpression of NbPOB1 and RNA interference (RNAi)-based silencing of NtPOB1 in Cf-9 tobacco compromised and accelerated the Avr9-triggered HR, respectively. Moreover, virus induced gene silencing (VIGS) of NbPOB1 accelerated the dark-induced senescence in N. benthamiana leaves. These observations identify NbPOB1 and NtPOB1 as the orthologs of AtPOB1. The subcellular localization of AtPOB1 and NbPOB1 was analyzed by transiently overexpressing GFP-AtPOB1 and GFP-NbPOB1 fusion proteins in tobacco leaf tissue. Analysis by confocal microscopy revealed that GFP fluorescence was localized in the nucleus of leaf tissue tested. The overexpression of AtPOB1 fused with a nuclear export signal (NES) failed to compromise the Avr9-triggered HR in Cf-9 tobacco, indicating the nuclear localization of AtPOB1 is crucial for its function. The BTB/POZ domain is a highly conserved protein-protein interaction interface that mediates homo- and/or hetero-dimerization of BTB/POZ proteins. The D146A and D141A mutation in the BTB/POZ domain of AtPOB1 and NbPOB1, respectively reduces their dimerization efficiency. These mutants fail to negatively regulate the Cf-9/Avr9-mediated HR, supporting the importance of an intact BTB/POZ interface for the function of AtPOB1 and NbPOB1. Finally, yeast-two-hybrid and immunoprecipitation assays indicate that AtPOB1 interacts with AtCUL3A, a component of E3 ligase complexes, in which AtPOB1 would confer substrate-specificity. We propose that AtPOB1 (and Nicotiana POB1) negatively regulate cell death and senescence possibly through ubiquitin-mediated pathways.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:495388 |
| Date | January 2009 |
| Creators | Mesmar, Joelle |
| Publisher | University of Glasgow |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://theses.gla.ac.uk/659/ |
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