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Background  Gene silencing triggered by chemically synthesized small interfering RNAs (siRNAs) has become a powerful tool for deciphering gene function in many eukaryotes. However, prediction and validation of a single siRNA duplex specific to a target gene is often ineffective. RNA interference (RNAi) with synthetic siRNA suffers from lower silencing efficacy, off-target effects and is cost-intensive, especially for functional genomic studies. With the explosion of fungal genomic information, there is an increasing need to analyze gene function in a rapid manner. Therefore, studies were performed in order to investigate the efficacy of gene silencing induced by RNase III-diced-siRNAs (d-siRNA) in model filamentous fungus,  Aspergillus nidulans .    Methodology/Principal Findings  Stable expression of heterologous reporter gene in  A. nidulans  eases the examination of a new RNAi-induction route. Hence, we have optimized  Agrobacterium tumefaciens -mediated transformation (AMT) of  A. nidulans  for stable expression of s GFP  gene. This study demonstrates that the reporter  GFP  gene stably introduced into  A. nidulans  can be effectively silenced by treatment of  GFP -d-siRNAs. We have shown the down-regulation of two endogenous genes, An rasA  and An rasB  of  A. nidulans  by d-siRNAs. We have also elucidated the function of an uncharacterized  Ras  homolog,  rasB  gene, which was found to be involved in hyphal growth and development. Further, silencing potency of d-siRNA was higher as compared to synthetic siRNA duplex, targeting An rasA . Silencing was shown to be sequence-specific, since expression profiles of other closely related  Ras  family genes in d-siRNA treated An rasA  and An rasB  silenced lines exhibited no change in gene expression.    Conclusions/Significance  We have developed and applied a fast, specific and efficient gene silencing approach for elucidating gene function in  A. nidulans  using d-siRNAs. We have also optimized an efficient AMT in  A. nidulans , which is useful for stable integration of transgenes.

Targeting Fungal Genes by Diced siRNAs: A Rapid Tool to Decipher Gene Function in Aspergillus nidulans