Recognition of the Cladosporium fulvum Ecp2 elicitor in tomato and non-host plants

Resistance against the tomato fungal pathogen Cladosporium fulvum is often conferred by Hcr9 genes iliomologues of the ~. fulvum resistance gene Cf-~) that are located in the Milky Way cluster on the short arm of Chromosome 1. These Hcr9 genes mediate recognition of matching fungal avirulence gene products. In contrast, the resistance gene Cf-Ecp2 mediates recognition of the pathogenicity factor Ecp2 and is located in the Orlon (OR) cluster on the short arm of Chromosome 1. The main part of this thesis concentrates on the cloning of the Cf-Ecp2 Orion (OR) cluster and the identification of the functional CfEcp2 resistance gene that mediates HR-mediated resistance upon Ecp2 recognition.In CHAPTER 2, we report the map- and homology-based cloning of the OR Hcr9 cluster. A method was optimised to generate clone-specific fingerprint data that were subsequently used in the efficient establishment of genomic DNA contigs. Three Hcr9s were identified as candidate Cf-Ecp2 genes. By PCR-based cloning using specific OR sequences, orthologous Hcr9 genes were identified from different Lycopersicon species and haplotypes. The OR Hcr9s are very homologous to each other. However, based on a relative low sequence homology to other Hcr9s, the OR Hcr9s are classified as a new subgroup. As a consequence, the origin and the mode of action of this unique class of Hcr9s may differ from the other Hcr9s. To support allele mining, mapping, cloning and mRNA profiling of tomato Hcr9 genes, a resistance gene analogue (RGA) fingerprint method was developed to generate novel Hcr9-specific markers (CHAPTER 3). The presence of both conserved and variable sequence domains in Hcr9s is exploited using a combination of PCR amplification and subsequent digestion. By the development of a fluorescent end-Iabelling method for restriction fragments, referred to as A/T labelling, high-resolution size-separation and detection of the complex RGA fingerprint pattern with a LlCOR automated sequencer became possible. The RGA fingerprint method was validated by the analysis of near-isogenic lines and the analysis of two Orlon (OR) Hcr9 loci harbouring the Cf-Ecp2 resistance gene or the recessive cf-ecp2 allele. We identified several RGA-markers cosegregating with Cf-Ecp2 resistance that corresponded to the th ree Hcr9s that are located at the OR locus. In addition, results indicate that the Hcr9 RGA fingerprint method facilitates the discrimination of highly homologous genes in the analysis of a mapping population. Finally, the Hcr9 RGA fingerprint method was applied to study the Hcr9 gene expression and showed that two out of the three OR Hcr9s were expressed in planta. The various methods to identify the functional Cf-Ecp2 gene are described in CHAPTER 4. Transient expression in Nicotiana species and complementation analysis in tomato were exploited to test candidate Cf-Ecp2 genes for the ability to mediate Ecp2 recognition. Despite applying all commonly used functional assays, we were not able to identify which of the three OR Hcr9 represents the functional Cf-Ecp2 gene. 8ased on these results we have to conclude that recognition of the C. fulvum Ecp2 elicitor is not solely mediated by an OR-Hcr9 and an additional tomato-derived HR-stimulating factor is required for Cf-Ecp2/Ecp2 mediated resistance.In addition to the three independent chapters on the cloning and identification of Cf-Ecp2, two related research topics were investigated. Cladosporium fulvum is a fungal pathogen of tomato that grows exclusively in the intercellular spaces of leaves. In tomato, recognition of elicitors is followed by a hypersensitive response (HR) resulting in resistance. However, HR-associated recognition of Ecp2 has also been observed in Nicotiana paniculata, N. sylvestris, N. tabacum and N. undulata that are non-host plants of C. fulvum (CHAPTER 5). Absence of Ecp2-recognition did not lead to growth of C. fulvum on Nicotiana plants. We show that HR-associated recognition of Ecp2 is mediated by a single dominant gene in N. paniculata. However, based on PCR- and hybridisation analysis this gene is not homologous to known Cf-genes.DNA sequence analysis of the Avr proteins (so-called race-specific elicitors) Avr2, Avr4, Avr4E and Avr9 have revealed that the change from a virulence to virulence is associated with DNA mutations in Avr coding regions. The high frequency of these mutations are most likely the result of high selection pressure caused by the frequent use of matching Cf resistance genes in commercial tomato lines. However, the Cf-Ecp resistance genes have rarely been employed and previous research showed that no variation was found the Ecp elicitors. In CHAPTER 6 the Ecotilling method was used in strains of C. fulvum that have been collected world-wide to compare the sequence variation in Avr and Ecp elicitor encoding genes while the variation in ribosomal internal transcribed spacers (lTS) was used as evolutionary clock. No polymorphisms in lTS sequences were observed. Silent mutations in Avrs occurred more frequently. However, the very fast majority of the mutations in Avr proteins were associated with virulence and indicate a high selection pressure in C. fulvum Avr elicitor genes. In Ecp elicitor genes, however, mutations occurred rarely and were not associated with virulence. These results show a very high mutation rate in elicitor proteins and confirms the lack of selection pressure on the Ecp genes by Cf-Ecp resistance genes.The thesis is concluded with a general discussion on Cf- and Cf-like proteins involved in disease resistance (CHAPTER 7). Current knowledge on the genetics and evolution of Cf genes, Cf protein characteristics, elicitor perception and signaI transduction in the tomato - C. fulvum pathosystem is discussed. In addition, the current knowledge on Cf-like proteins that are involved in other pathosystems is presented. Finally, this chapter describes some future directions in research on the described pathosystems