The Cladosporium fulvum-tomato interaction : elicitor proteins and their perception

The gene-for-gene concept postulates that for every dominant gene determining resistance in the host plant, there is a corresponding dominant gene conditioning avirulence in the pathogen. The simplest way to explain the biochemical basis of this concept is direct interaction between an elicitor protein, which is encoded by an avirulence ( Avr ) gene of the pathogen, and a receptor protein, which is encoded by the matching resistance ( R ) gene of the host. Perception of the elicitor protein by the host plant subsequently leads to the activation of defence responses, often including a hypersensitive response (HR). The research described in this thesis is focussed on the characterisation of elicitor proteins of the fungus Cladosporium fulvum and the analysis of their perception by resistant tomato plants. A striking feature of all elicitor proteins of C. fulvum is that their mature form contains an even number of cysteine residues. These cysteine residues are thought to be involved in disulfide bridges, which are essential for proper conformation and stability of the elicitors. Mutational analysis of elicitor proteins ECP1, ECP2 and ECP5, however, revealed that the role of (the even number) of cysteine residues is more complex than anticipated, as not all cysteine residues appeared to be critical for the HR-inducing activity of the elicitor proteins (Chapter 2). During colonisation of the apoplastic space of tomato leaves, C. fulvum secretes elicitor proteins into the apoplast. All Cf genes, mediating resistance to particular races of C. fulvum , are predicted to encode extracytoplasmic, membrane-anchored glycoproteins that contain many leucine-rich repeats (LRRs). LRR domains are thought to be involved in protein-protein interactions. The extracellular localisation of the LRR region of the Cf proteins is consistent with a direct, extracellular perception of the corresponding elicitor proteins. To validate this hypothesis, binding studies were performed between avirulence protein AVR9 and the matching resistance protein Cf-9. Although extensive studies were performed in a multidisciplinary collaboration to prove a direct interaction between the two proteins, no specific binding between AVR9 and Cf-9 could be detected (Chapter 3). This implies that the simplest interpretation of the gene-for-gene concept, involving direct interaction of a pathogen-derived elicitor with a matching resistance gene product, does not hold for the Avr9 / Cf-9 gene pair and that at least a third component is involved in the perception of AVR9 by Cf-9. Also for Avr2/Cf-2 -mediated resistance a third component, Rcr3, seems to be involved. To allow dissection of the biochemical mechanism of perception of avirulence protein AVR2, we set out to clone Avr2 (Chapter 4). Avr2 cDNA was cloned based on the specific HR-inducing activity of the encoded protein in Cf2 tomato plants. Like the other Avr genes of C. fulvum , Avr2 encodes a small, secreted protein with an even number of cysteine residues. Analysis of strains of C. fulvum that are virulent on Cf2 tomato lines revealed various mutations in the Avr2 ORF that all result in the production of a truncated AVR2 protein. Interestingly, an additional modification was discovered, involving the insertion of a LINE-like element (a retrotransposable element), Cfl1, in the Avr2 ORF. Cfl1 is the first LINE-like element identified in C. fulvum and provides the first example of loss of avirulence of a plant pathogen due to insertion of a retrotransposable element in an Avr gene. Analysis of two different rcr3-mutant Cf2 tomato plants revealed that their ability to respond to AVR2 with a HR correlates with their degree of resistance to AVR2-producing strains of C. fulvum . These data support a role for Rcr3 in the perception of AVR2 by Cf-2. Direct perception of elicitor proteins by R proteins has been the prevailing working hypothesis to explain the biochemical basis of the gene-for-gene concept for years. The results of the research that is described in this thesis, however, do not support this hypothesis. Also for most other gene-for-gene relationships studied so far, experimental evidence appears to be more consistent with indirect perception of an AVR protein by an R protein (Chapter 5). Indirect perception implies that, beside the AVR and the R protein, at least a third component is required to induce defence responses. For several gene-for-gene relationships the nature of the putative third component is known. Although each of these components are suggested to be involved in basal defence mechanisms, their nature appears to be diverse. Hence, we argue that, although some elicitors might be directly perceived by the matching R protein, for most gene-for-gene relationships elicitor perception will turn out to be more complex.