Mycorrhizas: Gene to Function

Substantial progress has been made toward development of molecular tools for identification and quantification of mycorrhizal fungi in roots and evaluation of the diversity of ectomycorrhizal (ECM) fungi and the phylogeny and genetic structure of arbuscular mycorrhizal (AM) fungi. rDNA analysis confirms high diversity of ECM fungi on their hosts, and for AM fungi has revealed considerable genetic variation within and among morphologically similar AM fungal species. The fungal and plant genes, regulation of their expression, and biochemical pathways for nutrient exchange between symbiotic partners are now coming under intense study and will eventually be used to define the ecological nutritional role of the fungi. While molecular biological approaches have increased understanding of the mycorrhizal symbiosis, such knowledge about these lower-scale processes has yet to influence our understanding of larger-scale responses to any great extent.     

Production of recombinant proteins by filamentous fungi

The initial focus of recombinant protein production by filamentous fungi related to exploiting the extraordinary extracellular enzyme synthesis and secretion machinery of industrial strains, including Aspergillus, Trichoderma, Penicillium and Rhizopus species, was to produce single recombinant protein products. An early recognized disadvantage of filamentous fungi as hosts of recombinant proteins was their common ability to produce homologous proteases which could degrade the heterologous protein product and strategies to prevent proteolysis have met with some limited success. It was also recognized that the protein glycosylation patterns in filamentous fungi and in mammals were quite different, such that filamentous fungi are likely not to be the most suitable microbial hosts for production of recombinant human glycoproteins for therapeutic use. By combining the experience gained from production of single recombinant proteins with new scientific information being generated through genomics and proteomics research, biotechnologists are now poised to extend the biomanufacturing capabilities of recombinant filamentous fungi by enabling them to express genes encoding multiple proteins, including, for example, new biosynthetic pathways for production of new primary or secondary metabolites. It is recognized that filamentous fungi, most species of which have not yet been isolated, represent an enormously diverse source of novel biosynthetic pathways, and that the natural fungal host harboring a valuable biosynthesis pathway may often not be the most suitable organism for biomanufacture purposes. Hence it is expected that substantial effort will be directed to transforming other fungal hosts, non-fungal microbial hosts and indeed non microbial hosts to express some of these novel biosynthetic pathways. But future applications of recombinant expression of proteins will not be confined to biomanufacturing. Opportunities to exploit recombinant technology to unravel the causes of the deleterious impacts of fungi, for example as human, mammalian and plant pathogens, and then to bring forward solutions, is expected to represent a very important future focus of fungal recombinant protein technology.     

Genetic transformation of filamentous fungi

Many filamentous fungi of all taxa can now be subject to DNA-mediated transformation. Many dominant selectable markers are available and the range available is increasing as new genes are cloned. Transformation is especially valuable in cloning genes defined by mutations with selectable phenotypes and is allowing investigation of many problems in fungi with good genetic systems. Increasingly sophisticated techniques for inactivating genes, targetingin vitro generated mutations to specific loci, and altering gene expression and its regulation are being developed. These approaches are being used to investigate the wealth of basic and applied biological problems available in filamentous fungi.     

Gibberellin biosynthesis in fungi: genes, enzymes, evolution, and impact on biotechnology

Gibberellins (GAs) constitute a large family of tetracyclic diterpenoid carboxylic acids, some members of which function as growth hormones in higher plants. As well as being phytohormones, GAs are also present in some fungi and bacteria. In recent years, GA biosynthetic genes from Fusarium fujikuroi and Arabidopsis thaliana have been cloned and well characterised. Although higher plants and the fungus both produce structurally identical GAs, there are important differences indicating that GA biosynthetic pathways have evolved independently in higher plants and fungi. The fact that horizontal gene transfer of GA genes from the plant to the fungus can be excluded, and that GA genes are obviously missing in closely related Fusarium species, raises the question of the origin of fungal GA biosynthetic genes. Besides characterisation of F. fujikuroi GA pathway genes, much progress has been made in the molecular analysis of regulatory mechanisms, especially the nitrogen metabolite repression controlling fungal GA biosynthesis. Basic research in this field has been shown to have an impact on biotechnology. Cloning of genes, construction of knock-out mutants, gene amplification, and regulation studies at the molecular level are powerful tools for improvement of production strains. Besides increased yields of the final product, GA₃, it is now possible to produce intermediates of the GA biosynthetic pathway, such as ent-kaurene, ent-kaurenoic acid, and GA₁₄, in high amounts using different knock-out mutants. This review concentrates mainly on the fungal biosynthetic pathway, the genes and enzymes involved, the regulation network, the biotechnological relevance of recent studies, and on evolutionary aspects of GA biosynthetic genes.     

The development of gene expression systems for filamentous fungi

Filamentous fungi have been used for decades in the commercial production of enzymes, antibiotics, and specialty chemicals. Traditionally, improving the yields of these products has involved either mutagenesis and screening or modification of fermentation conditions. Generally, selective breeding of strains has not been successful, because most of the commercially important fungal species lack a sexual cycle. For a few species, strain improvements have been made possible by employing the parasexual cycle for genetic crosses (30). The recent development of DNA-mediated transformation systems for several industrially important fungal species has spawned a flurry of research activity directed toward the development of gene expression systems for these microorganisms. This technology is now a viable means for novel and more directed approaches to improving existing fungal strains which produce enzymes or antibiotics. In addition, fungal expression systems are now being tested for the production of heterologous gene products such as mammalian pharmaceutical proteins. The goal of this review is to present a summary of the gene expression systems which have recently been developed for some filamentous fungi of commercial importance. To insure that the most recent developments are presented we have included data from not only scientific papers, but also from personal communications, abstracts, symposia, and our own laboratory.     

真菌DNA条形码研究进展

DNA条形码（DNA barcode）是通过一段短的标准DNA片段实现物种的快速、准确和标准化鉴定。线粒体细胞色素C氧化酶亚基I（COI）基因作为动物的DNA条形码已广泛应用于物种鉴定中,在植物上已选定叶绿体rbcL和matK基因作为基本的DNA条形码。目前世界各国真菌学家正对不同的真菌类群进行不同基因片段的筛选与评价,并在第四届国际生命条形码大会上正式推荐了ITS作为真菌的首选DNA条形码。对国内外真菌DNA条形码的研究进展进行总结与分析,并展望真菌DNA条形码的应用前景。     

Genetic transformation in Aspergilli: tools of the trade

DNA-mediated transformation is a powerful tool that allows the introduction of specific genetic changes in an organism. Transformation of Aspergilli, acclaimed for their wide use in the industry, has been possible for about two decades now. Several basic and applied problems related to fungal biology have been addressed using this technique. Nonetheless, new markers and strategies for transformation are still being developed for these filamentous fungi. Different methods and markers that are currently available for the transformation of Aspergilli are summarized here. The review also brings out the importance of these transformation systems in analyzing fungal gene function. Aspects of Aspergillus niger transformation are selectively emphasized.     

Exploiting the full potential of disease-resistance genes for agricultural use

Effective and sustained control of fungal pathogens and nematodes is an important issue for all agricultural systems. Global losses caused by pathogens are estimated to be 12% of the potential crop production [1], despite the continued release of new resistant cultivars and pesticides. Furthermore, fungi are continually becoming resistant to existing resistance genes and fungicides, and a few of the pesticides are being withdrawn from the market for environmental reasons. In addition to reducing crop yield, fungal diseases often lower crop quality by producing toxins that affect humans and human health. Additional methods of disease control are therefore highly desirable. Breeding programs based on plant disease-resistance genes are being optimized by incorporating molecular marker techniques and biotechnology. These efforts can be expected to result in the first launches of new disease-resistant crops within the next five years.     

Comparison of the Yeast Proteome to Other Fungal Genomes to Find Core Fungal Genes

The purpose of this research was to search for evolutionarily conserved fungal sequences to test the hypothesis that fungi have a set of core genes that are not found in other organisms, as these genes may indicate what makes fungi different from other organisms. By comparing 6355 predicted or known yeast (Saccharomyces cerevisiae) genes to the genomes of 13 other fungi using Standalone TBLASTN at an e-value <1E-5, a list of 3340 yeast genes was obtained with homologs present in at least 12 of 14 fungal genomes. By comparing these common fungal genes to complete genomes of animals (Fugu rubripes, Caenorhabditis elegans), plants (Arabidopsis thaliana, Oryza sativa), and bacteria (Agrobacterium tumefaciens, Xylella fastidiosa), a list of common fungal genes with homologs in these plants, animals, and bacteria was produced (938 genes), as well as a list of exclusively fungal genes without homologs in these other genomes (60 genes). To ensure that the 60 genes were exclusively fungal, these were compared using TBLASTN to the major sequence databases at GenBank: NR (nonredundant), EST (expressed sequence tags), GSS (genome survey sequences), and HTGS (unfinished high-throughput genome sequences). This resulted in 17 yeast genes with homologs in other fungal genomes, but without known homologs in other organisms. These 17 core, fungal genes were not found to differ from other yeast genes in GC content or codon usage patterns. More intensive study is required of these 17 genes and other common fungal genes to discover unique features of fungi compared to other organisms.Reviewing Editor: Prof. David Gottman     

Mycoviruses and virocontrol

Viruses are widespread in all major groups of fungi. The transmission of fungal viruses occurs intracellularly during cell division, sporogenesis, and cell fusion. They apparently lack an extracellular route for infection. Recent searches of the collections of field fungal isolates have detected an increasing number of novel viruses and lead to discoveries of novel genome organizations, expression strategies and virion structures. Those findings enhanced our understanding of virus diversity and evolution. The majority of fungal viruses have dsRNA genomes packaged in spherical particles, while ssRNA mycoviruses, possessing or lacking the ability to form particles, have increasingly been reported. This review article discusses the current status of mycovirus studies and virocontrol (biocontrol) of phytopathogenic fungi using viruses that infect them and reduce their virulence. Selected examples of virocontrol-associated systems include the chestnut/chestnut blight/hypovirus and fruit trees/white root rot fungus/mycoviruses. Natural dissemination and artificial introduction of hypovirulent fungal strains efficiently contributed to virocontrol of chestnut blight in European forests. Attempts to control white root rot with hypovirulence-conferring mycoviruses are now being made in Japan.     

Fungal pathogenicity genes in the age of 'omics'

The identification of the fungal genes essential for disease underpins the development of disease control strategies. Improved technologies for gene identification and functional analyses, as well as a plethora of sequenced fungal genomes, have led to the characterization of hundreds of genes, denoted as pathogenicity genes, which are required by fungi to cause disease. We describe recent technologies applied to characterize the fungal genes involved in disease and focus on some genes that are likely to attract continuing research activity.     

Genetic manipulation in vesicular-arbuscular mycorrhizal fungi

The symbiosis between vesicular-arbuscular mycorrhizal (VAM) fungi and host plants develops after successful interactions between both partners. These interactions probably involve signal molecules produced by the host plant, by the fungi, or by both. So far the biotrophic status of VAM fungi has hampered the understanding of the processes regulating their physiology. However, among different methods for co-cultivating VAM fungi, root organ cultures (ROC) appear to be a useful technique for studying VAM development. This system has been useful in defining the nutritional requirements of VAM fungi in the precolonization stage and in obtaining axenic fungal material in various developmental stages. The work discussed here focuses on the application of Polymerase Chain Reaction (PCR) technology and the potential of promoting hyphal growth in the absence of the plant. These techniques are being used to study VAM fungi in two main areas. The first concerns the determination of the DNA sequences coding for the SSU ribosomal RNA of two VAM fungi. This approach has allowed the design of specific primers for the rapid identification and quantification of VAM fungi. The second area of research concerns the potential use of PCR technology to study selective expression of specific genes during fungal spore development in defined in vitro conditions. The achievement of this future prospect depends on the ability to prepare PCR-based cDNA libraries from small amounts of fungal material after stimulation of hyphal growth with CO₂ and plant flavonols.     

Chromosome-length polymorphism in fungi.

The examination of fungal chromosomes by pulsed-field gel electrophoresis has revealed that length polymorphism is widespread in both sexual and asexual species. This review summarizes characteristics of fungal chromosome-length polymorphism and possible mitotic and meiotic mechanisms of chromosome length change. Most fungal chromosome-length polymorphisms are currently uncharacterized with respect to content and origin. However, it is clear that long tandem repeats, such as tracts of rRNA genes, are frequently variable in length and that other chromosomal rearrangements are suppressed during normal mitotic growth. Dispensable chromosomes and dispensable chromosome regions, which have been well documented for some fungi, also contribute to the variability of the fungal karyotype. For sexual species, meiotic recombination increases the overall karyotypic variability in a population while suppressing genetic translocations. The range of karyotypes observed in fungi indicates that many karyotypic changes may be genetically neutral, at least under some conditions. In addition, new linkage combinations of genes may also be advantageous in allowing adaptation of fungi to new environments.     

Evolution of beta-lactam biosynthesis genes and recruitment of trans-acting factors

Penicillins and cephalosporins belong chemically to the group of beta-lactam antibiotics. The formation of hydrophobic penicillins has been reported in fungi only, notably Penicillium chrysogenum and Emericella nidulans, whereas the hydrophilic cephalosporins are produced by both fungi, e.g., Acremonium chrysogenum (cephalosporin C), and bacteria. The producing bacteria include Gram-negatives and Gram-positives, e.g. Lysobacter lactamdurans (cephabacins) and Streptomyces clavuligerus (cephamycin C), respectively. For a long time the evolutionary origin of beta-lactam biosynthesis genes in fungi has been discussed. As often, there are arguments for both hypotheses, i.e., horizontal gene transfer from bacteria to fungi versus vertical descent. There were strong arguments in favour of horizontal gene transfer, e.g., fungal genes were clustered or some genes lack introns. The recent identification and characterisation of cis-/trans-elements involved in the regulation of the beta-lactam biosynthesis genes has provided new arguments in favour of horizontal gene transfer. In contrast to the bacterium S. clavuligerus, all regulators of fungal beta-lactam biosynthesis genes represent wide-domain regulators which were recruited to also regulate the beta-lactam biosynthesis genes. Moreover, the fungal regulatory genes are not part of the gene cluster. If bacterial regulators were co-transferred with the gene cluster from bacteria to fungi, most likely they would have been non-functional in eukaryotes and lost during evolution. Alternatively, it is conceivable that only a part of the beta-lactam biosynthesis gene cluster was transferred to some fungi, e.g., the acvA and ipnA gene without a regulatory gene.