CHARACTERIZATION OF A FUSARIUM GRAMINEARUM XYLANASE EXPRESSED DURING WHEAT INFECTION AND KNOCK-OUT OF ITS ENCODING GENE

Fusarium graminearum is the causal agent of Fusarium head blight (FHB) of wheat, barley and other cereal grains. During the infection process, this fungus secretes a large number of hydrolytic enzymes acting on the plant cell wall. In particular, F. graminearum is able to degrade xylans, the main constituent in the cell walls of monocot plants like wheat, through the coordinate action of a group of extracellular enzymes; among them, endo-ß-1,4-xylanases hydrolyze the inner ß-1,4 glycosidic bond and could be important pathogenic weapons for (pathogens) attacking cereal plants. However, xylanases could also exert effects independent from their enzymatic activity: a Botrytis cinerea xylanase, named Xyn11A, has been shown to contribute to virulence on tobacco and tomato leaves with its necrotizing activity and not with the xylan hydrolyzing activity. In the genome of F. graminearum there is a xylanase encoding gene (named Xyl1) whose deduced amino acidic sequence has a 55% identity with the B. cinerea xylanase Xyn11A. Since the transcript of Xyl1 gene has been detected in wheat spikelets infected with F. graminearum, we cloned this gene for heterologous expression in Pichia pastoris. The purified xylanase has been characterized by studying its enzymatic activity in vitro and its necrotizing activity on wheat tissue. Knock-out mutants of the corresponding encoding gene have been also obtained by site-directed homologous recombination. Wheat infection experiments aimed at evaluating the virulence of these mutants are in progress.