Phosphatidic acid accumulation is an early response in the Cf-4/Avr4 interaction

The Cladosporium fulvum (Cf)-4 gene of tomato confers resistance to the fungus C. fulvum, expressing the corresponding avirulence (Avr)4 gene, which codes for an elicitor protein. Little is known about how such mechanisms work, but previous studies have shown that elicitor recognition activates Ca(2+) signalling and protein kinases, such as mitogen-activated protein kinase (MAPK) and calcium-dependent protein kinase (CDPK). Here, we provide evidence that a new signalling component, the lipid second messenger phosphatidic acid (PA), is produced within a few minutes of AVR4/Cf-4 interaction. Using transgenic tobacco cells expressing the tomato Cf-4-resistance gene as a model system, phospholipid signalling pathways were studied by pre-labelling the cells with (32)P(i) and assaying for the formation of lipid signals after challenge with the fungal elicitor AVR4. A dramatic rapid response was an increase in (32)P-PA, together with its metabolic product diacylglycerol pyrophosphate (DGPP). AVR4 increased the levels of PA and DGPP in a Cf-4(+)-, time- and dose-dependent manner, while the non-matching elicitor AVR9 did not trigger any response. In general, PA signalling can be triggered by two different pathways: via phospholipase D (PLD), which generates PA directly by hydrolysing structural phospholipids like phosphatidylcholine (PC), or via PLC, which generates diacylglycerol (DAG) that is subsequently phosphorylated to PA by DAG kinase (DGK). To determine the origin of the AVR4-induced PA formation, a PLD-specific transphosphatidylation assay and a differential (32)P-labelling protocol were used. The results clearly demonstrated that most PA was produced via the phosphorylation of DAG. Neomycin and U73122, inhibitors of PLC activity, inhibited AVR4-induced PA accumulation, suggesting that the increase in DGK activity was because of increased PLC activity producing DAG. Lastly, evidence is provided that PLC signalling and, in particular, PA production could play a role in triggering responses, such as the AVR4-induced oxidative burst. For example, PLC inhibitors inhibited the oxidative burst, and when PA was added to cells, an oxidative burst was induced.