Cloning And Functional Analysis Of Genes Involved In Signal Transduction In The Tomato Cf-4/Avr4 Pathosystem

To feed the expanding world population, food production needs to increase and be protected against devastating pathogens. Crop protection can be achieved by exploiting natural resistance of plants. The hypersensitive response (HR) is an efficient, active defence response of plants against a broad range of pathogens. The HR involves resistance (R) gene-mediated recognition of avirulence (Avr) gene-encoded elicitors of a pathogen at the site of penetration, after which a few plant cells quickly die, preventing further outgrowth of the pathogen and it is still obscure how this process is initiated and executed. The interaction between the pathogenic fungus Cladosporiumfulvum and tomato has served as a model system for studying the mechanism of HR. In this way key signalling genes governing HR and plant resistance can be identified, thereby generating both markers and tools to create durable, broad resistance of plants against pathogens.After synchronizing Cf-4/AVR4-induced systemic HR in intact tomato plants, 426 early-induced genes were identified by cDNA-AFLP analysis by Frank Takken. One hundred and ninety two differentially expressed genes in proceeding of HR were analysed by Virus-Induced Gene Silencing (VIGS) and 20 of them were found to be involved in hypersensitive response were obtained. Among them, silencing of five genes resulted in a complete suppression of both in Avr4 and Infl-induced HR. Six full-length cDNAs of 20 genes were isolated using PCR. They showed high homology to a resistance gene Bs2 from pepper, two L19 ribosomal proteins, GTP-binding protein, a tetratricopeptide repeat (TPR)-containing protein from Arabidopsis and leucine-rich repeat receptor kinase from sweet potato, respectively. Besides GTP-binding protein and leucine-rich repeat receptor kinase, the other four genes were found for the first time to be involved in HR.RGL (Resistance Gene Like), which related to HR in plant and showed high homology to a resistance gene Bs2 from pepper after sequence analysis, was identified from Cf-4 tomato plant against Cladosporium fulvum. Using RGL protein as bait and yeast two-hybrid system, two interacting proteins, RGLIP-1 and RGLIP-2 (RGL Interacting Protein), were identified from tomato cDNA library. RGLIP-1 is a protein of 291 amino acids with significant homology with thylakoid lumen protein, whereas RGLEP-2 is a protein of 248 amino acids with significant homology with transducin protein. VIGS of these two genes resulted in a partial and complete suppression of Avr4-induced HR, indicating that both genes are involved in hypersensitive response.