CHARACTERIZATION OF FRAGARIA VESCA – RHIZOCTONIA FRAGARIAE INTERACTION BY GENE EXPRESSION ANALYSIS

Abstract

The present work was carried out to characterize the Fragaria vesca -Rhizoctonia fragariae interaction by analysis of differentially expressed DNA. Four Fragaria vesca genotypes obtained from a specific breeding programme were utilized. Two of them were shown to be resistant against Rhizoctonia (Rhizoctonia -resistant) ; the others were susceptible to Rhizoctonia disease. For each genotype as a whole 45 plants were utilized. In particular 25 of them were artificially inoculated with Rhizoctonia mycelium, the others were used as testers (non-inoculated plants). Five sampling times were applied; 15, 30, 45, 60 and 75 days after inoculation. For each sampling time 5 inoculated and 4 tester plants were collected. Plant roots, crown regions and leaves were separately collected and stored in liquid nitrogen. RNA was purified separately from roots, crowns and leaves. In order to isolate differentially expressed genome fractions, cDNA was obtained. Specific and random primers were applied to isolate differentially expressed sequences among susceptible and resistant genotypes. All the differentially expressed fragments obtained were isolated and sequenced. All sequences were compared to a database to evaluate similarity with genes already isolated. All fragments isolated were used to produce a specific cDNA library. A large percentage of the fragments sequenced showed homologies with ESTs. Considering the fragments showing homologies with genes encoding proteins with known activity, particular attention was directed to the expressed fractions related to beta-galactosidase, chitinase and glucanase genes. The fragments encoding for beta-galactosidase synthesis were isolated only in the resistant genotypes, starting from the first sampling time. Considering the enzymatic activity of this gene, it is possible to assume an indirect control of the pathogen.