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

Authors

  • Camiel F. De Jong,

    1. Laboratory of Phytopathology, Wageningen University, Marijkeweg 22, NL-6709 DG Wageningen, the Netherlands, and
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  • Ana M. Laxalt,

    1. Department of Plant Physiology, Swammerdam Institute for Life Sciences, University of Amsterdam, Kruislaan 318, NL-1098 SM Amsterdam, the Netherlands
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  • Bastiaan O. R. Bargmann,

    1. Department of Plant Physiology, Swammerdam Institute for Life Sciences, University of Amsterdam, Kruislaan 318, NL-1098 SM Amsterdam, the Netherlands
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  • Pierre J. G. M. De Wit,

    1. Laboratory of Phytopathology, Wageningen University, Marijkeweg 22, NL-6709 DG Wageningen, the Netherlands, and
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  • Matthieu H. A. J. Joosten,

    1. Laboratory of Phytopathology, Wageningen University, Marijkeweg 22, NL-6709 DG Wageningen, the Netherlands, and
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  • Teun Munnik

    Corresponding author
    1. Department of Plant Physiology, Swammerdam Institute for Life Sciences, University of Amsterdam, Kruislaan 318, NL-1098 SM Amsterdam, the Netherlands
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For correspondence (fax +31 20 5257934; e-mail munnik@science.uva.nl).

Current address: Department of Analytical Chemistry and Applied Spectroscopy, Vrije Universiteit, De Boelelaan 1083, 1081 HV Amsterdam, the Netherlands.

Current address: Instituto de Investigaciones Biologicas, Facultad de Ciencias Exactas y Naturales, Universidad Nacional de Mar del Plata, CC 1245, 7600 Mar del Plata, Argentina.

Both authors contributed equally to the work.

Summary

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 Ca2+ 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 32Pi and assaying for the formation of lipid signals after challenge with the fungal elicitor AVR4. A dramatic rapid response was an increase in 32P-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 32P-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.

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