Molecular toolkit development for gene expression and gene silencing technologies in the homobasidiomycete Fungi Agaricus bisporus and Coprinus cinereus

Resumen de la conferencia presentada al VI Meeting on Genetics and Cellular Biology of Basidiomycetes (GCBB-VI), organizado por y celebrado en la Universidad Pública de Navarra el 3-6 de junio de 2005.We have developed a “Molecular Toolkit” comprising interchangeable promoters and marker genes to facilitate transformation of homobasidiomycete mushrooms and subsequent analysis of gene expression. We will describe the testing of a wide range of promoters in both Agaricus bisporus and Coprinus cinereus when linked to a range of selectable and visual marker genes, along with the parameters required to successfully achieve foreign gene expression within these organisms. It has been previously demonstrated that a prerequisite for GFP expression in A. bisporus and C. cinereus is an intron. We describe the construction of an expression vector containing a multiple cloning site linked to an intron thus allowing different genes to be easily expressed in A. bisporus and C. cinereus. We report on the development of gene silencing technologies within A. bisporus and C. cinereus. In particular the serine protease has been targeted for gene silencing in A. bisporus. Serine protease has been implicated in post-harvest and age-related senescence of sporophores. On harvesting, mushrooms degenerate rapidly to give browned caps and loss of texture in the fruit body, and such problems can dramatically reduce sale ability of the mushrooms. Suppression of genes involved in these pathways could increase mushroom shelf-life and profitability for mushroom growers, or help to further elucidate the complex biochemical pathways involved in post-harvest degradation. Progress will also be reported on gene silencing in C. cinereus

The pOT and pLOB vector systems: Improving ease of transgene expression in Botrytis cinerea

This paper outlines the construction of a novel vector system comprising interchangeable terminators, as well as a multiple cloning site (MCS), to facilitate the transformation of the fungal plant pathogen Botrytis cinerea. Previous molecular studies on B. cinerea have relied upon the pLOB1 based vector system (controlled by the Aspergillus nidulans oliC promoter and a region reported to be the B. cinerea tubA terminator). Investigations, however, have revealed that, rather than the genuine B. cinerea tubA terminator, the pLOB1 terminator fragment is from another gene locus within the genome. Because previous studies have found that terminators aide in transcript stability, the main aims of this study were to develop and evaluate both vector systems, pOT (controlled by the A. nidulans oliC promoter and A. nidulans trpC terminator) and pLOB, with a range of exogenous genes, including enhanced green fluorescent protein (eGFP), monomeric red fluorescent protein (mRFP), luciferase (LUC) and ß-glucuronidase (GUS). Our investigations demonstrate that pLOB and pOT based vectors are capable of expressing all four reporter genes and may be applied to future molecular studies on B. cinerea and other related ascomycetes. Additionally, this is the first reported expression of mRFP and LUC in B. cinerea