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Functional analysis of DIR1, a putative lipid transfer protein that promotes long distance signalling during systemic acquired resistance in Arabidopsis.

Final Activity Report Summary - LIPSIGNAL (Functional analysis of DIR1, a putative lipid transfer protein that promotes long distance signalling during systemic acquired resistance in Arabidopsis.)

Dir1-1 is specifically compromised in SAR, exhibiting wild type gene-for-gene resistance in incompatible interactions and wild type levels of basal resistance in compatible interactions. The aim of this project was to characterise the mechanism of action of DIR1 with the following objectives: localisation of DIR1 LTP and tracking possible relocation of DIR1 during the induction of SAR, investigation of potential ligands for DIR1 and identification and functional characterisation of proteins interacting with DIR1.

Localisation of DIR1 LTP
A. Maldonado began the work by generating DIR1-GFP fusioned plants under 35S promoter in the Ws background. DIR1 was fusioned to GFP by a 10 Alanines linker. The 35Sp-DIR1-GFP transgene was able to complement dir1-1 mutation. But western blotting experiment with either DIR1 or GFP antibodies showed that 2 proteins were detectable, the GFP fusioned to DIR1 and the GFP alone. Analysis of the homozygous lines of DIR1-GFP plants under dexamethasone promoter with a shorter linker (3 Alas) showed different level of expression but we still observed the degradation of the fused DIR1-GFP. Confocal images realised after 24h of induction by dexamethasone showed apoplastic fluorescence in the DIR-GFP or SP-GFP plant .The analysis of the (re)location of DIR1 in response to pathogen challenge is in progress.

Investigation of potential ligands for DIR1
The overexpression of DIR1 in transgenic plants didn't induce SAR therefore the hypothesis in which DIR1 acts in cooperation with a mobile signal is then strong. Therefore the mature DIR1 (8 kDa), has been first cloned into a vector that is driving the synthesis and the secretion of DIR1 in the culture medium by methanol induction. First experiments done in collaboration with Dr Marion (INRA Nantes, FRANCE), showed that DIR1 is able to bind lipids. Some new experiments showed that LTPs can form a covalent complex with lipids hydroperoxides (D. Marion et al, unpublished data). Experiments using petiole exudates of SAR-induced leaves are in progress in order to validate or not this hypothesis.

Identification and functional characterisation of proteins interacting with DIR1
Chaperone or transporter functions might not involve specific high affinity interactions with a small number of discrete proteins. To answer the question of the capacity of DIR1 to interact with other partner we wanted to use first a yeast two-hybrid screen question. We have chosen the CYTOTRAP Two-Hybrid system developed by Stratagene that seems to be convenient for detecting interactions between membrane protein and soluble protein. The generation of a cDNA library of correct size (at least 2 millions clones) is in progress but we are experiencing some problems with the construction of the cDNA library. We are looking at possible receptor(s) on plasmalemma membrane for DIR1 too. Indeed, LTPs show some similarities to elicitins (2) and I showed during my PhD that wheat LTP competes with elicitin for a low abundance, high affinity binding site on tobacco plasma membranes (3). Binding and displacement experiments realised in collaboration with Dr Blein in Dijon pointed out that there is a binding site for DIR1 which is specific and saturable, on the tobacco plasma membrane. Displacements experiments with cold DIR1 showed that this fixation was reversible and identical to elicitins one. We are studying now the possibility of using BIACORE technology to transpose that study to Arabidopsis.