Post-transcriptional silencing of the tomatinase gene in Fusarium oxysporum f. sp lycopersici

Fusarium oxysporum f. sp. lycopersici produces an extracellular enzyme tomatinase, which has been known to detoxify a tomatine, a preformed inhibitor of fungal growth in tomato plants. To understand the role of the tomatinase in pathogenicity, we performed transformation-mediated disruption of the tomatinase gene in F. oxysporum f. sp. lycopersici. Mutants obtained showed increase in sensitivity to a tomatine and decrease in tomatinase activity in vitro. Inoculation assay showed reduced pathogenicity of the mutants on tomato plants, suggesting an important role of tomatinase in pathogenicity of this fungus. However, Southern blot analysis revealed that the resident tomatinase gene of the mutants was not disrupted but the gene replacement vector was ectopically integrated into the genome of the mutants. To clarify the mechanisms involved, we examined one of the ectopic insertion mutants at both DNA and RNA levels. No methylation of the resident tomatinase gene was detected. Northern blot analysis revealed low level of the tomatinase gene expression and also the presence of antisense RNA in the ectopic mutant. Thus, post-transcriptional gene silencing seems to be involved in the suppression of tomatinase in the ectopic mutant.