pHg/pSILBAã vector system for efficient gene silencing in homobasidiomycetes: optimization of ihpRNA-triggering in the mycorrhizal fungus Laccaria bicolor.

KEMPPAINEN M & PARDO AG

MICROBIAL BIOTECHNOLOGY

Blackwell Publishing

Lugar: Oxford, United Kingdom; Año: 2010 vol. 3 p. 178 - 200

1751-7915

pSILBAã silencing vector was constructed for efficient RNA silencing triggering in the model mycorrhizal fungus Laccaria bicolor. This cloning vector carries the Agaricus bisporus gpdII-promoter, two multiple cloning sites separated by a L. bicolor nitrate reductase intron and the Aspergillus nidulans trpC terminator. pSILBAã allows an easy oriented two-step PCR-cloning of hairpin sequences to be expressed in basidiomycetes. With one further cloning step into pHg, a pCAMBIA1300 based binary vector carrying a hygromycin resistance cassette, the pHg/pSILBAã plasmid is used for Agrobacterium-mediated transformation. The pHg/pSILBAã-system results in predominantly single integrations of RNA silencing triggering T-DNAs in the fungal genome and the integration sites of the transgenes can be resolved by plasmid rescue. pSILBAã construct and two other pSILBA plasmid variants (pSILBA and pSILBAa) were evaluated for their capacity to silence Laccaria nitrate reductase gene. While all pSILBA-variants tested resulted in up to 65-76 % of transformants with reduced growth on nitrate, pSILBAã produced the highest number (65 %) of strongly affected fungal strains. The strongly silenced phenotype was shown to correlate with T-DNA integration in transcriptionally active genomic sites. pHg/pSILBAã was shown to produce T-DNAs with minimum CpG methylation in transgene promoter regions which assures the maximum silencing trigger production in Laccaria. Methylation of the target endogene was only slight in RNA silencing triggered with constructs carrying an intronic spacer hairpin sequence. The silencing capacity of the pHg/pSILBAã was further tested with Laccaria inositol-1,4,5-triphosphate 5-phosphatase gene. Besides its use in silencing triggering, the herein described plasmid system can also be used for transgene expression in Laccaria. pHg/pSILBAã silencing system is optimized for L. bicolor but it should result highly useful also for other homobasidiomycetes, group of fungi currently lacking molecular tools for RNA silencing.