Content area
Abstract
The interaction between the fungal pathogen Cladosporium fulvum and tomato has been used as a model system to study the molecular basis of gene-for-gene relationships. C. fulvum is a specialized, biotrophic pathogen, which causes leaf mold on tomato. Under humid conditions conidia of C. fulvum germinate and form runner hyphae on the lower side of the leaf. If no resistance genes of the plant match any of the avirulence genes of the fungus, the interaction is compatible and infection will proceed. However, when both a resistance gene and its matching avirulence gene are present, the plant recognizes the fungus and the interaction is incompatible. In an incompatible interaction active defense responses, including the hypersensitive response (HR) are initiated, which inhibit fungal growth effectively. Avirulence genes encode lace-specific elicitors, which are present in intercellular washing fluids obtained from compatible interactions of C. fulvum and tomato (De Wit and Spikman, 1982). Injection of these intercellular washing fluids in tomato plants resistant to the C. fulvum strain from which the washing fluids were obtained, results in specific necrosis at the site of injection. The race-specific elicitor AVR9 was isolated and purified (Scholtens-Toma and de Wit, 1988). AVR9 specifically induces necrosis on tomato genotypes carrying the Cf-9 resistance gene. The encoding AVR9 gene was isolated, and it was shown that this gene specifically determines avirulence of C. fulvum on tomato plants carrying the Cf-9 resistance gene (Van den Ackerveken et al., 1992; Marmeisse et al., 1993). The Avr9 gene encodes a 63-amino acid pre-proprotein containing one potential glycosylation site (Van den Ackerveken et al., 1993). Different forms of the AVR9 elicitor were found, of which the mature AVR9 elicitor of 28 amino acids is predominantly present in C. fulvum-infected tomato plants (Van den Ackerveken et al., 1993). The global structure of the AVR9 peptide shows 3 antiparallel β-sheets and 3 disulfide bonds that are arranged in a cystine knot (Vervoort et al., 1997).
In the research project described in this thesis, we studied AVR9 elicitor perception in tomato plants that carry the Cf-9 resistance gene and compared the results to those obtained with tomato plants lacking this gene. Previously, several research groups had shown that elicitors are recognized through plants receptors, which are localized on the plasma membrane (summarized in chapter 1). To find and characterize the receptor for AVR9, the peptide was labeled with iodine-125 and binding to tomato membranes was studied, as presented in chapter 2. 125 I-AVR9 showed specific, saturable, and reversible<br/>binding to plasma membranes isolated from leaves of the tomato cultivar Moneymaker without Cf resistance genes (MM-Cf0) and to membranes from a near-isogenic genotype containing the Cf-9 resistance gene (MM-Cf9). Binding of AVR9 is characterized by high affinity and low receptor concentration, and thus fulfills several criteria expected for functional receptors (Hulme and Birdsall, 1992). The dissociation constant was determined at 0.07 nM, and the receptor concentration was determined at 0.8 pmol/mg microsomal membrane protein. Binding is highly influenced by pH and ionic strength of the binding buffer and by temperature, indicating the involvement of both electrostatic and hydrophobic interactions. Surprisingly, binding kinetics and binding capacity were identical for membranes of the MM-Cf0 and MM-Cf9 tomato genotype, indicating that the Cf-9 resistance gene is not required for binding of AVR9. By that time, the Cf-9 resistance gene was isolated (Jones et al., 1994). Cf-9 belongs to a gene family and homologues of the Cf-9 resistance gene are present in both resistant and susceptible tomato genotypes. Two new hypotheses were developed of which the first predicts that not only the Cf-9 resistance gene, but also homologues of the Cf-9 gene, encode the high-affinity binding site for AVR9. Only the protein encoded by Cf-9 itself, designated CF-9, would subsequently initiate the signal transduction cascade resulting in HR. The second hypothesis predicts that the AVR9 binding site is neither CF-9 nor a homologue of CF-9. The binding site proposed in the second hypothesis would bind AVR9 and subsequently recruit the CF-9 protein to initiate HR.
