The AVR9 elicitor peptide of the tomato pathogen Cladosporium fulvum : molecular aspects of recognition

The interaction between the fungal pathogen <em>Cladosporium fulvum</em> and tomato has been used as a model system to study the molecular basis of gene-for-gene relationships. <em>C. fulvum</em> 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 <em>C. fulvum</em> and tomato (De Wit and Spikman, 1982). Injection of these intercellular washing fluids in tomato plants resistant to the <em>C. fulvum</em> 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 <em>C. fulvum</em> 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 reversiblebinding 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.As described in chapters 3, 4, and 5, experiments were performed to prove or reject one of these two hypotheses. To determine whether the high-affinity binding site for AVR9 is indeed a functional receptor, we studied the correlation between binding affinity and necrosis-inducing activity of mutant AVR9 peptides. We determined structure-activity relationships of the AVR9 peptide by independently substituting each amino acid of AVR9 by alanine, using a site-directed mutagenesis approach. In addition, surfaceexposed amino acid residues of AVR9 were substituted by other amino acids. Activity of mutant Avr9 constructs was studied by expressing the constructs in MM-Cf9 tomato plants using the potato virus X (PVX) expression system, and assessing the severity of necrosis induced by each PVX::Avr9 construct. This allowed direct identification of amino acid residues of AVR9 that are essential for elicitor activity. We identified amino acid substitutions resulting in AVR9 mutants with higher, similar or lower elicitor activity compared to the wild-type AVR9 peptide. Mutants of the amino acid residues Phe21 and Leu24 had completely lost elicitor activity. Necrosis-inducing activity of isolated AVR9 peptides correlated well with the necrosis induced by the corresponding PVX::Avr9 constructs. It was concluded the PVX expression system is ideally suited to analyze necrosis-inducing activity of AVR9 peptides. We analyzed whether there is a correlation between elicitor activity of the mutant AVR9 peptides and their affinity to the binding site in membranes of tomato. Therefore, <em>Nicotiana clevelandii</em> plants were inoculated with a selection of PVX::Avr9 constructs and mutant AVR9 peptides were purified from these plants. In addition, some AVR9 peptides were chemically synthesized. Characterization by Electrospray Mass Spectrometry, Circular Dichroism-, and 'H-NMR- spectroscopy revealed that both the <em>in planta</em> produced and the synthetic mutant peptides were correctly folded. AVR9 peptides purified from PVX::Avr9-infected <em>N. clevelandii</em> contained one N-acetylglucosamine, although small amounts of non- glycosylated AVR9 peptides were also detected. The glycosylated AVR9 peptides showed lower affinity to the binding site than the non-glycosylated AVR9 peptides, whereas they did not differ significantly in necrosis-inducing activity. For both the non- glycosylated and glycosylated mutant AVR9 peptides, a positive correlation between their affinity to the membranelocalized binding site and their necrosis-inducing activity in MM-Cf9 tomato was found, i.e. peptides with higher affinity to the binding site showed higher necrosis-inducing activity. This correlation suggested that the characterized high-affinity binding site for AVR9 is indeed a functional receptor that initiates the AVR9- CF-9-dependent HR in MM-Cf9 plants.In <em>chapter</em> 5, we studied whether the Cf-9 resistance gene or (one of) its homologues code for an AVR9 binding site. We tested binding of AVR9 to microsomal membranes of a variety of solanaceous and non-solanaceous plant species and analyzed these species for the presence of Cf-9-homologues. All solanaceous species tested contain homologues of the Cf-9 resistance gene and membranes of these plants contain a highaffinity binding site for AVR9. However, a high affinity binding site for AVR9 is also present on membranes of the non-solanaceous plant species cucumber, barley and oat, which do not contain homologues of the Cf-9 resistance gene. Membranes of tobacco, transgenic for the Cf-9 resistance gene, showed no change in the number of AVR9 binding sites. Arabidopsis does not have a binding site for AVR9 and membranes of Arabidopsis, transgenic: for the Cf-9 resistance gene, also showed no AVR9 binding. From this we concluded that not only the Cf-9 resistance gene, but also its homologues are not required for high-affinity binding of AVR9. Based on the presented data, we have developed a model, explaining recognition of AVR9 in MM-Cf-9 tomato <em>(chapter</em> 6). This model predicts that the high-affinity binding protein either 'presents' the AVR9 elicitor to the Cf-9-encoded protein or that binding of AVR9 induces a conformational change of the high-affinity binding protein. The latter results in recruitment of Cf-9 into the AVR9- receptor complex. Subsequently, signal cascade(s) resulting in HR will be initiated.