Soluble phospholipase A2 activity is induced before oxylipin accumulation in tobacco mosaic virus‐infected tobacco leaves and is contributed by patatin‐like enzymes

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Abstract
Recent evidence suggests that oxidized lipid‐derived molecules play significant roles in inducible plant defence responses against microbial pathogens, either by directly deterring parasite multiplication, or as signals involved in the induction of sets of defence genes. The synthesis of these oxylipins was hypothesized to be initiated by the phospholipase A2‐mediated release of unsaturated fatty acids from membrane lipids. Here, we demonstrate that, in tobacco leaves reacting hypersensitively to tobacco mosaic virus, a strong increase in soluble phospholipase A2 (PLA2) activity occurs at the onset of necrotic lesion appearance. This rapid PLA2 activation occurred before the accumulation of 12‐oxophytodienoic and jasmonic acids, two fatty acid‐derived defence signals. Three PLA2 isoforms were separated and the most active enzyme was partially purified, its N‐terminal sequence displaying similarity with patatin, the major storage protein in potato tubers. Three related tobacco patatin‐like cDNAs, called NtPat1, NtPat2 and NtPat3, were cloned, with NtPat2 encoding the PLA2 isolated from infected leaves. RT–PCR experiments showed a rapid transcriptional activation of the three NtPat genes in virus‐infected leaves, preceding the increase in PLA2 activity. Recombinant NtPat1 and NtPat3 enzymes were active in an assay using labelled bacterial membranes, and also displayed high bona fide PLA2 activity on phosphatidylcholine substrate. These results point to a possible new role of patatin‐like phospholipases in inducible plant defence responses. The induction kinetics together with the enzymatic activity data indicate that the NtPat proteins may provide precursors for oxylipin synthesis during the hypersensitive response to pathogens.