Efficiency of different strategies for gene silencing in Botrytis cinerea.

摘要: The generation of knock-out mutants in fungal pathogens by gene replacement and insertional mutagenesis is the classical method to validate virulence factors. An alternative strategy consists silencing candidate making use phenomenon RNA interference (RNAi), adding features such as possibility generating knock-down with variable expression levels target or ability simultaneously multiple genes. Two different approaches have been assayed generate RNAi phytopathogenic fungus Botrytis cinerea. In first one, single nitrate reductase B. cinerea genome, niaD, was silenced transformation a construct containing 400-bp niaD fragment between two opposing promoters, so that dsRNA generated. As an approach, mgfp4 coding for green fluorescent protein (GFP) transforming GFP-expressing strains hairpin (hpRNA)-expressing vector, inverted copies 300-bp separated spacer DNA. While dual-promoter produced about half transformants assayed, efficiency hpRNA-expressing vector higher, inducing decrease GFP more than 90 % transformants. degree achieved high both methods, but hpRNA resulted higher proportion strongly