Table of Contents
The pHg/pSILBAã-Vector System
The Use of pHg/pSILBAã for RNA Silencing of MiSSP7
Minna J. Kemppainen
Laboratorio de Micología Molecular, Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes, and Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Roque Sáenz Peña, Bernal, Provincia de Buenos Aires, Argentina
Alejandro G. Pardo
Consejo Nacional de Investigaciones, Científicas y Técnicas (CONICET), Provincia de Buenos Aires, Argentina
Series: Genetics – Research and Issues
Mycorrhizas, fungal-root associations, are the most prevalent mutualistic symbiotic relationships on earth. In nature the plant nutrient uptake from the soil takes place via the extraradical mycelia of the symbiotic fungi as an exchange for photosyntates. While most herbaceous plants and tropical trees form endomycorrhiza-type interactions, trees of boreal and temperate ecosystems are typically ectomycorrhizal (ECM). These species include the majority of ecologically and economically important trees and the fungal symbionts are mainly homobasidiomycetes. The symbiotic phase in the life cycle of ECM homobasidiomycetes is the dikaryon.
Therefore, studies on symbiosis regulated gene functions in these organisms require the inactivation of both gene copies. RNA silencing is a sequence homology-dependent degradation of target RNAs in the Eukaryota domain. In different such organisms, like fungi, the RNA silencing pathway can be artificially triggered to target and degrade gene transcripts of interest, resulting in gene knock-down. Most importantly, RNA silencing can act at the cytosolic level affecting mRNAs originating from several gene copies and different nuclei thus offering an efficient means of altering gene expression in dikaryotic, diploid and polyploid organisms. The pHg/pSILBAÃ£ silencing vector was constructed for RNA silencing triggering in homobasidiomycetes using the ECM fungus Laccaria bicolor as a model. This cloning vector harbors the Agaricus bisporus gpdII-promoter, two multiple cloning sites separated by a L. bicolor intron and the Aspergillus nidulans trpC terminator.
The pSILBAÃ£ plasmid allows a two-step PCR-cloning of hairpin sequences for expression in homobasidiomycetes. Further cloning into pHg, a pCAMBIA1300-based binary vector carrying a hygromycin resistance marker for fungal selection, makes the pHg/pSILBAÃ£ system compatible with Agrobacterium-mediated transformation. The pHg/pSILBAÃ£ vector usually produces single integrations of T-DNAs in the fungal genome. Moreover, the integration sites can be determined by plasmid rescue in E. coli as pHg/pSILBAÃ£ carries an ampicilline selection marker and an ORI from pBluescript in its T-DNA. Besides the optimized use in L. bicolor for RNA silencing and overexpression, this vector system is compatible with other homobasidiomycetes and different transformation techniques.
(Imprint: Nova Biomedical )