Elsevier

Biotechnology Advances

Volume 31, Issue 8, December 2013, Pages 1562-1574
Biotechnology Advances

Research review paper
Molecular tools for functional genomics in filamentous fungi: Recent advances and new strategies

https://doi.org/10.1016/j.biotechadv.2013.08.005Get rights and content

Highlights

  • Various molecular tools for genetic manipulation in filamentous fungi.

  • Examples of random-mutation library construction in filamentous fungi.

  • Strategies for improving genetic transformation efficiency in filamentous fungi.

  • A brief summary on large-scale screening of mutants and some practical experiences.

  • Application of Omics-analysis in functional genomics research was briefly commented.

Abstract

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.

Introduction

Filamentous fungi encompass a diverse group of simple eukaryotes with compact genomes and are widely distributed in nature. Many species are directly relevant to humans and human activities, with some species playing important roles in industry, agriculture, and medicine. For instance, filamentous fungi are involved in the production of primary and secondary metabolites, including organic acids, enzymes, exopolysaccharides, antibiotics, and biofilms for industrial uses (Gutiérrez-Correa et al., 2012, Meyer, 2008). In addition, several species have become model organisms for understanding basic biological processes. Apart from the species of biotechnical and/or pharmaceutical significance, many filamentous fungi are also pathogenic in plants and animals, including humans (Fisher et al., 2012).

One major area of current research on filamentous fungi focuses on delineating their molecular mechanisms of pathogenesis. Additionally, investigation on the beneficial properties of filamentous fungi highlights another major area of research to improve our understanding of physiological and genetic information of those industrially important fungi through the application of functional genomics. Since initial DNA transformation experiments were carried out in Neurospora crassa (Mishra and Tatum, 1973), several protocols utilizing different methods for genetic transformation and subsequent gene-function assessment have been developed for filamentous fungi. These include the standard methods by PEG/protoplast transformation (Hinnen et al., 1978), electrotransformation (Karube et al., 1985), biolistics (Armaleo et al., 1990), restriction enzyme mediated integration (REMI) (Lu et al., 1994), Agrobacterium tumefaciens mediated transformation (ATMT) (de Groot et al., 1998), and RNA interference (RNAi) techniques (Akashi et al., 2005), highlighting the significant application for these new techniques to functional genomics research on filamentous fungi.

With the rapid development of DNA sequencing techniques, the complete genome sequences over 100 fungi (http://en.wikipedia.org/wiki/List_of_sequenced_fungi_genomes) are available. Several fungi species are significant to industry and agriculture: the mold Aspergillus niger (Pel et al., 2007), the plant pathogen Magnaporthe oryzae (Dean et al., 2005), the entomopathogenic fungus Metarhizium anisopliae (Gao et al., 2011), and the “Chinese mushroom of immortality” Ganoderma lucidum (Chen et al., 2012). These large-scale functional and comparative genome analyses are now possible in a wide range of species (Soanes et al., 2008). Current research on filamentous fungi highlights the significant contribution of the genomics era, and recognizes that new advanced approaches to efficiently analyze fungi through the application of functional genomics will undoubtedly contribute further to the understanding and utilization of filamentous fungi.

Previous reviews have summarized several common approaches and applications (Kück and Hoff, 2010, Meyer, 2008, Weld et al., 2006), but lacked extensive insight on technological progress. As such, a full-scale review is warranted to provide up-to-date information on molecular tools and strategies for performing functional genomics research in filamentous fungi. This paper provides a comprehensive overview of the popular and robust tools for both random mutagenesis and targeted gene disruption. Many successful and practical strategies for improving the genetic manipulation of filamentous fungi are also discussed.

Section snippets

Molecular tools for gene functional research in filamentous fungi

An efficient strategy for investigating gene function in fungus and analysis on the associated phenotypic changes results through the use of targeted gene mutation techniques. The most common targeted gene replacement technique depends on homologous recombination (HR), and has been successfully applied to many species, including bacteria (Kurien and Scofield, 1995), yeasts (Kavanagh et al., 1991), plants (Maas and Werr, 1989) and mammalian cells (Koller and Smithies, 1989). Recently, various

Choosing suitable marker genes and screening system

Effective markers and screening systems are very important in genetic transformation experiments. The common markers used in fungi are divided into three classes. The most frequently used fungal markers are resistant genes against chemical drugs, such as hph (Hygromycin B resistance), ble (Phleomycin resistance), neo (Neomycin, Geneticin and Kanamycin resistance), bar (Glyphosate resistance), and some others that confer resistance to Benomyl, Carboxin, Fludioxonil, Blasticidin S and

Large-scale research of mutants in filamentous fungi

Once mutants are obtained, many types of analyses are required. The first step typically is verification of the mutants through PCR, which is then followed by a series of tests on phenotypic variation including genetic stability of mutation, growth rate, morphological features, sporulation, changes of virulence or pathogenicity, and some nichetargeting physiological–biochemical index (Xie et al., 2013, Zhou et al., 2012). Analyses of molecular characteristics usually follow, such as Southern

Conclusions and perspectives

Filamentous fungi are a huge class of lower eukaryotes, which are closely related with the life and production process of humans. Over the years, their unique physiological features have been shown to serve as both beneficial and pathogenic and consequently have attracted the attentions of researchers. Over the past 40 years, scientists have made great efforts in understanding the function of genes, enabling the development of various molecular tools and strategies suitable for functional

Acknowledgments

We are grateful to Prof. Jianping Xu (McMaster University, Canada), Dr. Ariana Harari (Albert Einstein College of Medicine, USA), and Dr. Ellen Leffler (University of Chicago, USA) for their valuable comments and critical discussions. The research described here is jointly supported by the National Basic Research Program of China (2013CB127500), the National Natural Science Foundation of China (approved nos. 31272093 and 31360019), the West Light Foundation of the Chinese Academy of Sciences

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