|Abstract in English:|
Many fungal plant pathogens secrete a complex of extracellular cell wall-degrading enzymes (CWDEs) capable of hydrolysing lignocellulosic components of the host cell wall. CWDEs activity is involved in fungus penetration and after host colonization, they also participate in nutrient acquisition from the hydrolysis of plant wall polysaccharides. The principal component of plant cell wall is cellulose, a polysaccharide organized in crystalline microfibrils, whose digestion requires the synergic activity of three major classes of enzymes: endoglucanase (endo-?-1,4-glucanase), exoglucanase or cellobiohydrolase (exo-?-1,4-glucanase), and ?-glucosidase (?-D-glucoside glucohydrolase). Among these enzymes, the endoglucanases produced by various microorganisms (bacteria, yeasts, and fungi), nematodes, plants and protozoans have been well studied. Several investigations demonstrated that during the infection process there is a synergic role of some endoglucanases with other enzymes of the cellulase complex; however, targeted mutational studies have found that individual enzyme genes could only play a partial role in pathogenesis. As an example, here we report about the endo-?-1,4-glucanase PlEGL1, belonging to glycosyl hydrolase (GH) 61 family and cloned from Pyrenochaeta lycopersici, the causal agent of the Corky Root Rot (CRR) of tomato. Plegl1 expression levels are positively correlated, in planta, with the progress of the disease, being undetectable during the initial infection of tomato, in which cellulose hydrolytic activity has not started, and strongly expressed in the later phase, corresponding with root collapse. The recombinant PLEGL1 protein, analysed by polysaccharide affinity precipitation assays, shows specificity for cellulose-based substrates. In vitro, Plegl1 transcription is negatively regulated by the presence of glucose in culture medium (catabolite repression), this probably reflects the strategy, in planta, to reach an efficient use of energy, producing most of CWDEs only when the fungus needs to use host wall polysaccharides as carbon source. Proteins belonging to GH61 family were formerly known as glycosyl hydrolases, but they were recently classified as polysaccharide monooxygenases (PMO) with high affinity for copper. The proposed chemical mechanism for this new intriguing group of enzymes is based on the electron transfer from cellobiose dehydrogenase (CDH) to GH61 proteins which, in turn, promote the insertion of molecular oxygen, cleaving off the glycosidic bonds of cellulose. Determination of the detailed mechanisms by which these enzymes function will significantly contribute to our understanding of cellulose digestion in nature and to the application of cellulose degrading enzymes in the processing of cellulosic biomasses for biomaterials and bioenergy.