Tomatinase from Fusarium oxysporum f. sp lycopersici is required for full virulence on tomato plants

Saponin detoxification enzymes from pathogenic fungi are involved in the infection process of their host plants. Fusarium oxysporum L sp lycopersici, a tomato pathogen, produces the tomatinase enzyme Tom1, which degrades cc-tomatine to less toxic derivates. To study the role of the tom1 gene in the virulence of E oxysporum, we performed targeted disruption and overexpression of the gene. The infection process of tomato plants inoculated with transformants constitutively producing Tom1 resulted in an increase of symptom development. By contrast, tomato plants infected with the knockout mutants showed a delay in the disease process, indicating that Tom1, although not essential for pathogenicity, is required for the full virulence of E oxysporum. Total tomatinase activity in the disrupted strains was reduced only 25%, leading to beta(2)-tomatine as the main hydrolysis product of the saponin in vitro. In silico analysis of the E oxysporum genome revealed the existence of four additional putative tomatinase genes with identities to tomatinases from family 3 of glycosyl hydrolases. These might be responsible for the remaining tomatinase activity in the Delta tom1 mutants. Our results indicate that detoxification of alpha-tomatine in F. oxysporum is carried out by several tomatinase activities, suggesting the importance of these enzymes during the infection process.