A guide to binary vectors and strategies for targeted genome modification in fungi using Agrobacterium tumefaciens-mediated transformation

Abstract

Agrobacterium tumefaciens-mediated transformation (ATMT) of fungi has become a common technique for the study of a wide variety of different fungal species over the past 12 years. The discovery that the host range of A. tumefaciens could be extended to include fungi provided an efficient transformation tool for species in which it was previously impossible to conduct molecular genetics experiments. ATMT experiments can be divided into three groups: i) Forward genetics (i.e., random mutagenesis), ii) Reverse genetics (i.e., targeted genome modification and random integration) and iii) the introduction of reporter genes (e.g., GFP, RFP and GUS) that allow in situ monitoring of the fungus. The use of ATMT for forward genetics experiments has primarily included classic random insertional inactivation strategies to obtain loss-of-function mutants. For reverse genetics experiments, ATMT has been used to introduce targeted genome modifications (e.g., disruptions, replacements, overexpression and complementation) and to generate random integrations for complementation, heterologous expression, expression of transcriptional and translational fusion reporters and RNAi-mediated down-regulation of gene expression. This review summarizes the technical advances within the field from 1998 to the summer of 2011, focusing on the development of binary vectors that are compatible with fungal transformation (over 180 general vectors) and methods for constructing binary vectors for targeted integration of T-DNA into fungal genomes.