Characterisation of a divinyl ether biosynthetic pathway specifically associated with pathogenesis in Nicotiana tabacum

In tobacco (Nicotiana tabacum L.), an elicitor- and pathogen-induced 9-lipoxygenase (LOX) gene, NtLOX1, is essential for full resistance to pathogens, notably to an incompatible race of Phytophthora parasitica var. nicotianae (Ppn race 0) (Rancé et al., 1998; Mène-Saffrané et al., 2003). In the present work, we aimed to identify those oxylipins induced during attempted infection by Ppn race 0 and down regulated in NtLOX1-antisense plants. Here we show that colneleic and colnelenic acids, which significantly inhibit germination of Ppn zoospores (Prost et al., 2005), are produced in roots of WT plants inoculated with Ppn but are down regulated in NtLOX1-antisense plants. A search for a tobacco gene encoding the enzyme involved in the formation of these divinyl ether fatty acids resulted in the cloning and characterization of a divinyl ether synthase (DES) clone (NtDES1). NtDES1 is a 9-DES, specifically converting fatty acid 9-hydroperoxides into divinyl ether fatty acids. NtDES1 has the potential to act in combination with NtLOX1, since in the presence of the two enzymes linoleic and linolenic acids were converted in vitro into colneleic and colnelenic acids, respectively. In addition the pattern of NtDES1 gene expression was quite similar to that of NtLOX1. Their transcripts were undetected in healthy tissues from different plant organs, and accumulated locally and transiently after elicitation and fungal infection but not after wounding. Visualization of NtDES1-YFP and NtLOX1-CFP fusion proteins in tobacco leaves indicated that both localize in the cytosol and are excluded from plastids, consistent with the presumed location of the 9-LOX pathway in plants and the lack of transit peptides for NtLOX1 and NtDES1, respectively. Our data suggest that, in tobacco, NtDES1 and NtLOX1 act together and form divinyl ethers in response to pathogen attack, and that this class of oxylipins modulates in vivo the outcome of the tobacco-Ppn race 0 interaction.