Geosmin synthase ges1 knock-down by siRNA in the dikaryotic fungus Tricholoma vaccinum

Genetic manipulation for generating knock-out experiments is essential in deciphering the precise function of a gene. However, dikaryotic fungi pose the inherent challenge of having two allelic versions of each gene, one in each nucleus. In addition, they often are slow-growing and do not withstand protoplasting, which is why Agrobacterium tumefaciens-mediated transformation has been adapted. To obtain knock-out strains, however, is not feasible with a mere deletion construct transformation and screening for deletions in both nuclear copies. Hence, a convenient method using chemically synthesized dicer substrate interfering RNA (DsiRNA) for posttranscriptional interference of targeted mRNA was developed, based on the fungal dicer/argonaute system inherent in fungi for sequence recognition and degradation. A proof-of-principle using this newly established method for knock-down of the volatile geosmin is presented in the dikaryotic fungus Tricholoma vaccinum that is forming ectomycorrhizal symbiosis with spruce trees. The gene ges1, a terpene synthase, was transcribed with a 50-fold reduction in transcript levels in the knockdown strain. The volatile geosmin was slightly reduced, but not absent in the fungus carrying the knockdown construct pointing at low specificity in other terpene synthases known for that class of enzymes.