Cf-dependent early defence responses induced by avirulence proteins of the tomato pathogen : Cladosporium fulvum

The outcome of a plant-pathogen interaction is determined by both the presence of resistance ( R ) genes in the plant and matching avirulence ( Avr ) genes in the pathogen. According to the gene-for-gene concept, for a dominant R gene in the host plant resistant to a specific strain of a pathogen, a corresponding dominant Avr gene exists in that strain of the pathogen. R gene-mediated recognition of an Avr gene product triggers a signal transduction cascade, eventually resulting in a hypersensitive response (HR). This HR consists of a collapse of plant cells at the primary site of infection, resulting in an arrest of growth of the pathogen. As it is impractical to study defense signaling responses in whole plants, often cell suspensions are used for this purpose. Directly after treatment of cell suspensions with elicitors, activation of signal transduction processes, such as ion fluxes over the plasma membrane (detectable as alkalization of the extra cellular medium), phospholipid signaling and protein phosphorylation occur. Also reactive oxygen species, which are thought to play a role both in defense signaling and in direct defense against the pathogen, are produced after AVR perception. The interaction between Cladosporium fulvum and tomato is a well-studied plant-pathogen interaction that obeys the gene-for-gene concept. From this pathosystem several resistance ( Cf ) and Avr genes have been cloned, from which the matching gene pairs Cf-4/Avr4 and Cf-9/Avr9 are the best studied. Although many efforts were undertaken to study defense signaling in cell suspensions derived from Cf- carrying tomato plants, they were not responsive to the matching AVRs. Therefore, Cf4 +-and Cf-9 +- tobacco cell suspensions, which are responsive to the matching AVR protein, were used to study Cf/Avr- mediated defense signaling. In Chapter 2, defense responses in Cf-9 +- tobacco leaves and Cf-9 +- tobacco cell suspensions induced by both wild-type (WT-AVR9) and mutant AVR9 analogues were studied. Upon injection into leaves of both tomato MM-Cf9 and Cf-9 +- tobacco leaves, the mutant AVR9 peptides R08K, F10A and F21A showed higher, lower and no necrosis-inducing activity, respectively, as compared to WT-AVR9. Similar relative activities were found for these 4 peptides when assayed in Cf-9 +- tobacco cell suspensions. R08K showed a stronger, whereas F10A and F21A showed a lower oxidative burst-inducing activity as compared to WT-AVR9. In a medium alkalization assay equal activities were observed for R08K and WT-AVR9, whereas F10A showed a lower activity and no medium alkalization activity at all was observed for F21A. Surprisingly, the oxidative burst was induced at peptide concentrations that were 100 times lower as compared to those inducing medium alkalization. Concentrations inducing a full medium alkalization response are similar to peptide concentrations that induce necrosis in leaves of Cf-9- carrying tomato or tobacco plants. Treatment of Cf-9 +- tobacco cell suspensions with WT-AVR9 resulted in the activation of a MAP kinase, whereas F21A activated the MAP kinase only to a small extent. WT-AVR9 also induced massive cell death at 18 hr after addition to Cf-9 +- tobacco cell suspensions, whereas tobacco cells not expressing Cf-9 remained viable, illustrating the specificity of this response. In Chapter 3 we have shown that, upon AVR4 treatment of Cf-4 +-tobacco cells, levels of the second messenger phosphatidic acid (PA) increased dramatically. This response occurred within 2 min after addition of AVR4 and was highly specific. The PA conversion product diacylglycerol pyrophosphate (DGPP) accumulated between 4-8 minutes after addition of AVR4. Whether DGPP is a second messenger in its own right or serves as a negative regulator for PA signaling is still unclear. A differential labeling strategy showed that AVR4-induced PA accumulation resulted predominantly from the conversion of diacylglycerol (DAG) into PA by diacylglycerol kinase (DGK). DAG can be generated during signaling events by the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP 2 ) by phospholipase C (PLC). Pretreatment of Cf-4 +- tobacco cells with the PLC inhibitors neomycin and U73122 blocked AVR4-induced PA accumulation, indicating that PA is indeed generated via PLC activity. The AVR4-induced oxidative burst was blocked by the NADPH oxidase inhibitor diphenylene iodonium (DPI), whereas it did not block AVR4-induced PA accumulation. Conversely, the PLC inhibitor U73122 blocked both AVR4-induced PA accumulation and oxidative burst in a dose-dependent way. Treatment with a synthetic, water-soluble PA derivative induced a small, and transient oxidative burst in Cf-4 +- tobacco cells. These data demonstrate the importance of phospholipid signaling in the AVR4-induced oxidative burst. Additional studies showed that AVR4-induced medium alkalization and MAP kinase activation were also blocked by PLC inhibitors, suggesting that these responses are also PLC-dependent. During the experiments described in Chapter 2 it became clear that AVR9-induced defense responses are temperature sensitive. The temperature-sensitivity of Cf/Avr- mediated defense responses is studied in Chapter 4. Injection of AVR4 or AVR9 in leaves of tobacco and tomato plants carrying Cf - 4 or Cf-9, respectively, resulted in necrosis in the injected area at 20°C, whereas at 33°C this response was suppressed. At 20°C, tomato seedlings expressing both a Cf gene and matching Avr gene germinate but develop systemic HR after unfolding of the cotyledons and subsequently die. These seedlings could be rescued at 33°C but rapidly died after transfer to 20°C. Gel blot analysis of RNA isolated at different time points after transfer of the rescued Cf/Avr seedlings to 20°C, revealed a controlled induction of expression of various typical defense-related genes. This synchronized onset of HR provides an excellent basis for the identification of novel, HR-related genes by cDNA-AFLP analysis. In cell suspensions we found that both the AVR4- and AVR9-induced medium alkalization response is slowly suppressed at 33°C, but quickly recovers upon transfer to 15°C. For AVR4- and AVR9-induced medium alkalization, differences in the kinetics of the suppression of this response at an elevated temperature were demonstrated. The high affinity binding site for AVR9 is thought to be the AVR9 receptor, which is involved in the initiation of Cf-9/Avr9- mediated defense responses. It was shown that binding of AVR9 to microsomal fractions isolated from cell suspensions incubated at 33°C was decreased by 80%, as compared to microsomal fractions isolated from cell suspensions incubated at 20°C. The decrease of AVR9 binding was caused by a decrease in amount of binding sites rather than by a decrease in the affinity of the binding site for AVR9, providing a molecular basis for the temperature sensitivity of Cf-9/Avr9- mediated defense. In Chapter 5 the results described in this thesis are discussed and some additional unpublished data are included. A model for AVR-induced signaling in the C. fulvum/ tomato interaction is presented. In addition, new techniques, future experiments and perspectives to further unravel Cf/Avr- mediated signaling are discussed