An Optimized cDNA-AFLP Protocol for the Identification of TDFs Involved in the Malus-Venturia inaequalis Interaction

The interaction of apple genotypes and Venturia inaequalis, the causal agent of apple scab, is nowadays the most studied plant-pathogen interaction in a non-model woody plant. After the cloning of the apple scab resistance gene HcrVf2, the cascade of reactions induced after pathogen recognition is under investigation. To understand the gene networks that underlie plant defense responses, it is necessary to identify and characterize the genes responding to pathogen infection. Young leaf samples were collected from genetically modified 'Gala' plants carrying the HcrVf2 gene, at different times after inoculation with V. inaequalis. A cDNA-AFLP procedure, successfully applied to study plant-pathogen interactions, has been chosen in order to identify sequences (TDFs, transcript derived fragments) that are differentially expressed after pathogen inoculation. An optimized and highly reproducible cDNA-AFLP protocol was set up on PAGE, starting with an RNA extraction from apple leaves until gel band elution from polyacrylamide gels. The feasibility of this cDNA-AFLP protocol by the dHPLC for fragment separation in order to automatize all band elution steps will be discussed.