Molecular tools for functional genomics in filamentous fungi: recent advances and new strategies

Advances in genetic transformation techniques have made important contributions to molecular genetics. Various molecular tools and strategies have been developed for functional genomic analysis of filamentous fungi since the first DNA transformation was successfully achieved in Neurospora crassa in 1973. Increasing amounts of genomic data regarding filamentous fungi are continuously reported and large-scale functional studies have become common in a wide range of fungal species. In this review, various molecular tools used in filamentous fungi are compared and discussed, including methods for genetic transformation (e.g., protoplast transformation, electroporation, and microinjection), the construction of random mutant libraries (e.g., restriction enzyme mediated integration, transposon arrayed gene knockout, and Agrobacterium tumefaciens mediated transformation), and the analysis of gene function (e.g., RNA interference and transcription activator-like effector nucleases). We also focused on practical strategies that could enhance the efficiency of genetic manipulation in filamentous fungi, such as choosing a proper screening system and marker genes, assembling target-cassettes or vectors effectively, and transforming into strains that are deficient in the nonhomologous end joining pathway. In summary, we present an up-to-date review on the different molecular tools and latest strategies that have been successfully used in functional genomics in filamentous fungi.