Isolation of differentially expressed transcripts after cerato-platanin treatments in Platanus acerifolia

It is well known that fungal and bacterial pathogens activate a series of genes necessary for the establishment of infection in the plant that, in his turn, activates defence genes. For this reason it is important to know the temporal and spatial control of these gene expression events. Ceratocystis fimbriata (Ell. and Halst.) Davidson f.sp. platani Walter (Cfp) is an Ascomycete responsible for the canker stain of the plane tree, a severe disease showing a high incidence in the European Platanus acerifolia (Ait.) Willd populations. Actually, the lack of curative treatments and an effective prophylaxy make difficult to contrast successfully the spread of the pathogen. Cfp produces in culture an acidic protein called cerato-platanin (CP). CP, a member of the new “cerato-platanin protein family”, is also located in the cell walls of ascospores, conidia and hyphae of Cfp and has been proposed to be one of the fungal proteins involved in the first events of the plane canker stain pathogenesis because of its ability to elicit defense-related host responses. We have applied the suppression subtractive hybridisation method (SSH) for the generation of subtracted cDNA libraries and for the identification and isolation of differentially expressed transcripts after PC treatments. The technique is based on the construction of forward and reverse cDNA libraries that allow the identification of the genes that are switched on/off after the treatments. This method allows to obtain cDNA library enriched of transcript only present in one of the two compared samples (in our case treated sample and control sample). The main feature of this technique is the peculiar effectiveness in isolating rarely expressed transcripts. Platanus acerifolia leaves, after wash with sterile distilled water, were shaped in two part: one was treated with 5 drops of sterile distilled water (control sample) and another one with 5 drops containing 1,5 nmoles of CP for centimetre square of leaves (treated sample). Poly (A)+ RNAs were isolated from control and from treated leaves. Generation of subtracted cDNA libraries by SSH method were performed. After evaluation of the subtraction efficiency the cDNA of the subtracted libraries were cloned. We demonstrated the effectiveness of the SSH method by generating specific cDNA libraries and characterizing selected cDNA clones which are sequenced and analysed using the FASTA, BLASTX, BLASTN ProDom and BLOCK programmes. At present we have isolated some interesting clones containing coding sequences implicated in defence response such as: i) a b-ketoacyl-CoA synthase, ii) a tubby-like proteins, iii) a growth-on protein GRO10. The expression of some of the most interesting isolated clones has been analysed.