Using a biotin switch method we show that proteins are S-nitrosylated in <i>Arabidopsis </i>following challenge with both virulent <i>Pseudomonas syringae</i> pv.<i> tomato </i>strain <i>Pst </i>DC3000 and avirulent <i>Pst </i>carrying <i>(avrB). </i>Purification and MALDI analysis indicates that one of these S-nitrosylated proteins is a carbonic anhydrase. A single copy <i>Arabidopsis GS-FDH </i>was identified which shows 75% homology in amino acid sequence to mouse <i>GS-FDH. </i>The induction of <i>GS-FDH </i>by GSNO <i>in Arabidopsis </i>was confirmed by northern analysis. <i>GS-FDH </i>expression in <i>Arabidopsis </i>is constitutive at basal levels. During the HR, which was induced by <i>Pst </i>DC3000 <i>(avrB), </i>GS-FDH expression and activity were found to be rapidly suppressed. Whereas no GS-FDH suppression takes place following infection with virulent <i>Pst </i>DC3000. Therefore GS-FDH expression and activity are tightly regulated during the HR. Three <i>GS-FDH </i>T-DNA insertion lines were identified from the SAIL and SIGnAL databases. Two of these lines <i>fdh1-1 </i>and <i>fdh1-2 </i>are allelic and contain a T-DNA insert in the promoter of GS-FDH. Northern analysis and enzyme activity assays found that GS-FDH is over-expressed in these lines. Characterisation of <i>fdhl-1 </i>revealed that it constitutively expresses the defence gene <i>PDF1.2. fdh1-1 </i>exhibits resistance against the virulent biotroph <i>Pst </i>DC3000. Moreover, <i>fdh1-1 </i>has a delayed HR response following <i>Pst </i>DC3000 <i>(avrB) </i>infiltration. In addition, an activation tagged population was screened to isolate mutants resistant to nitrosative stress, from which ten NO resistant mutant candidates were identified.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:650825 |
| Date | January 2004 |
| Creators | Feechan, Angela |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/10893 |
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