Molecular approaches for AM fungal community ecology: A primer

Molecular techniques are no longer optional for ecologists interested in arbuscular mycorrhizal (AM) communities. Understanding the role of these soil fungi in natural systems requires knowledge of their abundance and identity but this is impossible to achieve without a molecular approach. Adapting molecular tools to AM fungi can be challenging because of the unique biology of the fungi. Moreover, many recruits in the field of mycorrhizal ecology have little or no experience with molecular biology. Here, we outline a conceptual framework for designing robust ecological experiments with AM fungi using molecular approaches.     

Bioprocessing strategies to improve heterologous protein production in filamentous fungal fermentations

Filamentous fungi have long been used for the production of metabolites and enzymes. With developments in genetic engineering and molecular biology, filamentous fungi have also achieved increased attention as hosts for recombinant DNA. However, the production levels of non-fungal proteins are usually low. Despite the achievements obtained using molecular tools, the heterologous protein loss caused by extracellular fungal protease degradation persists. This review provides an overview of the potential bioprocessing strategies that can be applied to inhibit protease activity thereby enhancing heterologous protein production.     

Fundamentals of fungal molecular population genetic analyses

The last two decades have seen tremendous growth in the development and application of molecular methods in the analyses of fungal species and populations. In this paper, I provide an overview of the molecular techniques and the basic analytical tools used to address various fundamental population and evolutionary genetic questions in fungi. With increasing availability and decreasing cost, DNA sequencing is becoming a mainstream data acquisition method in fungal evolutionary genetic studies. However, other methods, especially those based on the polymerase chain reaction, remain powerful in addressing specific questions for certain groups of taxa. These developments are bringing fungal population and evolutionary genetics into mainstream ecology and evolutionary biology.     

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.     

Host-cell invasion by malaria parasites: insights from Plasmodium and Toxoplasma

Recent years have seen tremendous progress in our understanding of malaria parasite molecular biology. To a large extent, this progress follows significant developments in genetic, molecular and chemical tools available to study the malaria parasites and related Apicomplexa, in particular Toxoplasma gondii. One area of major advancement has been in understanding parasite host-cell invasion, a process that utilizes several essential molecular mechanisms that are conserved across the different lifecycle stages. Here, we summarize some of the most recent experimental data that shed light on the events underlying preparation and execution of malaria parasite invasion and how these insights might relate to the development of new antimalarial drugs.     

The unified framework for biological invasions: a forest fungal pathogen perspective

Biological invasions in forests are growing in number and importance globally. The best studied examples are those caused by plants and animals, including insects. In contrast, forest invasions caused by microbes, including fungi, have received much lower levels of attention, particularly in the invasion biology literature. This can at least to some extent be due to the large number of these organisms involved and the fact that the majority of these have yet to be discovered and described. This is equally true for tree-infecting fungi, many of which are devastating pathogens responsible for dramatic invasions in natural and planted forests. This situation is changing through the application of molecular genetic tools that make it possible to accurately identify fungal tree pathogens, to determine their origins, pathways of movement, their modes of reproduction and change; all of which can influence invasions. The role and relevance of symbioses between tree pathogens and insects in forest invasions is also gaining increased attention. So too is our understanding that trees live in close association with large numbers of microbes that make up their holobiome. This has substantial relevance to invasion biology (Zenni et al. 2017). This commentary highlights four emerging issues that need to be considered regarding the invasions by fungal pathogens of trees and it emphasizes opportunities to better understand their relevance and impacts on natural and planted forests. A call is also made for plant pathologists to work more closely with ecologists such that fungal pathogens become more commonly integrated into invasion biology programmes.     

Gene therapy for peripheral arterial disease

Our understanding of the molecular biology of vascular disease is rapidly expanding, and this scientific growth has brought with it new opportunities for therapeutic intervention at the molecular and genetic levels. Although our tools for genetic manipulation in vivo and our knowledge of potential molecular targets are still crude and incomplete, the early application of these concepts to clinical problems is already underway, both in the pre-clinical and clinical arenas. The treatment of peripheral vascular disease, although greatly improved over recent decades by surgical and minimally-invasive techniques, remains limited by vascular proliferative lesions and by our inability to modulate the progression of native disease. This review explores some of the evolving concepts of therapeutic gene manipulation and their initial application in the peripheral circulation.     

Using new genetic tools to study the pathogenesis of Blastomyces dermatitidis.

Fungal pathogens have emerged as a public health menace owing to the expanding population of vulnerable patients and a heightened exposure to fungi in our environment, particularly for the systemic dimorphic fungi that inhabit soil worldwide. A better understanding of these invaders and their pathogenic mechanisms is badly needed to further research into therapeutic options. Advances in the molecular tools available for genetic manipulation of Blastomyces dermatitidis have enhanced our ability to study this poorly understood dimorphic fungal pathogen. Recent refinements in gene-transfer techniques, new selection markers, reliable reporter fusions and successes in gene targeting have shed light upon the importance of the mycelium-to-yeast transition and the crucial and complex role the BAD1 adhesin plays in pathogenesis.     

Genetics of ectomycorrhizal fungi: progress and prospects

The variability within and among ectomycorrhizal species provides a substantial genetic resource and the potential to increase forest productivity and environmental sustainability. Two parallel and interacting approaches, classical and molecular genetics, are being developed to acquire the genetic information underpinning selection of improved ectomycorrhizal strains. Determining the genetic traits of the fungi which contribute to symbiosis and plant function are being followed using natural variability combined with classical and molecular genetic manipulations. Classical and molecular manipulations for breeding rely on key information including sexual and parasexual reproduction, postmeiotic nuclear behaviour, mating-types and vegetative incompatibility mechanisms. Progress in the manipulation of genomes of ectomycorrhizal fungi will depend on efficient methods for gene cloning and DNA transformation. Gene transfer into fungal cells have been shown to be successful and include treatment of protoplasts and intact mycelium with naked DNA in the presence of polyvalent cations, electroporation, and microbombardment. The merits and limitations of these methods are discussed. Using this technology the expression of foreign DNA, the functional analysis of fungal DNA sequences, as well as molecular exploitation for commercial purposes can be carried out. This review concentrates on these aspects of fungal molecular biology and discusses the applications of the experimental systems that are currently available to ectomycorrhizal fungi. As it is essential to be able to define the traits which a breeder is seeking to improve, availability of genetically defined strains that are isogenic for a character or differ only in one character and a thorough knowledge of the biochemistry of the symbiosis will be necessary before any genetic manipulation be carried out. Genetic variability of ectomycorrhizal strains has been assessed by DNA fingerprinting. This approach allows the evaluation of DNA variability and the exchange of genetic information in natural populations, the identification of species and isolates by DNA polymorphisms, and tracking the environmental fate of the introduced fungi to determine their survival, growth, and dissemination within the soil.     

Dissecting cellular components of the secretory pathway in filamentous fungi: insights into their application for protein production

Studies on protein production using filamentous fungi have mostly focused on improvement of the protein yields by genetic modifications such as overexpression. Recent genome sequencing in several filamentous fungal species now enables more systematic approaches based on reverse genetics and molecular biology of the secretion pathway. In this review, we summarize recent molecular-based advances in our understanding of vesicular trafficking in filamentous fungi, and discuss insights into their high secretion ability and application for protein production.     

The value of applying molecular biology tools in environmental engineering: Academic and industry perspective in the USA

The integration of molecular biology tools in environmental engineering is a challenge. We discuss our views on the following four critical issues: (i) faculty career development, (ii) tool standardization, (iii) teaching, and (iv) the application of molecular biology tools in practice. For (i), we suggest that administrators and faculty need to understand the special challenges inherent to research and teaching within this highly interdisciplinary area. Furthermore, we suggest preparing two white papers aimed at educating administrators in universities and agencies. For (ii), we conclude that, because molecular biology tools are still in a state of rapid development, proposing standards at this time is premature. In the future, standards for widely applied tools should be in an on-line, peer-reviewed format. Concerning (iii), we believe that molecular biology should be taught only to the degree needed to achieve program goals. For example, environmental engineering practitioners only need to know the vocabulary and basic concepts of molecular biology tools, not be experts at doing them hands on. To help engineering students gain the right level and type of information, learning modules should be developed for them. Finally, although engineering successes applying molecular biology tools are available (iv), the biggest value will come when the tools are fully integrated with practice. Therefore, we encourage the creation of a demonstration project to document the value of applying molecular biology tools in environmental engineering. This revised version was published online in June 2006 with corrections to the Cover Date.     

Bridging the regeneration gap: genetic insights from diverse animal models

Significant progress has recently been made in our understanding of animal regenerative biology, spurred on by the use of a wider range of model organisms and an increasing ability to use genetic tools in traditional models of regeneration. This progress has begun to delineate differences and similarities in the regenerative capabilities and mechanisms among diverse animal species, and to address some of the key questions about the molecular and cell biology of regeneration. Our expanding knowledge in these areas not only provides insights into animal biology in general, but also has important implications for regenerative medicine and stem-cell biology.     

Filamentous fungi as cell factories for heterologous protein production

Filamentous fungi have been used as sources of metabolites and enzymes for centuries. For about two decades, molecular genetic tools have enabled us to use these organisms to express extra copies of both endogenous and exogenous genes. This review of current practice reveals that molecular tools have enabled several new developments. But it has been process development that has driven the final breakthrough to achieving commercially relevant quantities of protein. Recent research into gene expression in filamentous fungi has explored their wealth of genetic diversity with a view to exploiting them as expression hosts and as a source of new genes. Inevitably, the progress in the 'genomics' technology will further develop high-throughput technologies for these organisms.     

合成生物学技术在环境保护中的应用

合成生物学是一个基于生物学和工程学原理的科学领域,其目的是重新设计和重组微生物,以优化或创建具有增强功能的新生物系统。该领域利用分子工具、系统生物学和遗传框架的重编程,从而构建合成途径以获得具有替代功能的微生物。传统上,合成生物学方法通常旨在开发具有成本效益的微生物细胞工厂进而从可再生资源中生产化学物质。然而,近年来合成生物学技术开始在环境保护中发挥着更直接的作用。本综述介绍了基因工程中的合成生物学工具,讨论了基于基因工程的微生物修复策略,强调了合成生物学技术可以通过响应特定污染物进行生物修复来保护环境。其中,规律间隔成簇短回文重复序列(Clustered Regularly Interspersed Short Palindromic Repeats, CRISPR)技术在基因工程细菌和古细菌的生物修复中得到了广泛应用,生物修复领域也出现了很多新的先进技术,包括生物膜工程、人工微生物群落的构建、基因驱动、酶和蛋白质工程等。有了这些新的技术和工具,生物修复将成为当今最好和最有效的污染物去除方式之一。     

Using evolutionary genomics,transcriptomics, and systems biology to reveal gene networks underlying fungal development

Fungal model species have contributed to many aspects of modern biology, from biochemistry and cell biology to molecular genetics. Nevertheless, only a few genes associated with morphological development in fungi have been functionally characterized in terms of their genetic or molecular interactions. Evolutionary developmental biology in fungi faces challenges from a lack of fossil records and unresolved species phylogeny, to homoplasy associated with simple morphology. Traditionally, reductive approaches use genetic screens to reveal phenotypes from a large number of mutants; the efficiency of these approaches relies on profound prior knowledge of the genetics and biology of the designated development trait—knowledge which is often not available for even well-studied fungal model species. Reductive approaches become less efficient for the study of developmental traits that are regulated quantitatively by more than one gene via networks. Recent advances in genome-wide analysis performed in representative multicellular fungal models and non-models have greatly improved upon the traditional reductive approaches in fungal evo-devo research by providing clues for focused knockout strategies. In particular, genome-wide gene expression data across developmental processes of interest in multiple species can expedite the advancement of integrative synthetic and systems biology strategies to reveal regulatory networks underlying fungal development.     

Protozomics: trypanosomatid parasite genetics comes of age

Trypanosomatid protozoans cause important diseases of humans and their domestic livestock. Various molecular genetic tools are now allowing rapid progress in understanding many of the unique aspects of the molecular and cell biology of these organisms. Diploidy and the lack or difficulty of sexual crossing has been a challenge for forward genetics, but powerful selections and functional complementation have helped to overcome it in Leishmania. RNA interference has been adapted for forward genetics in trypanosomes, in which it is also a powerful tool for reverse genetics. Interestingly, the efficacy of different genetic tools has steered research into different aspects of the biology of these parasites.     

DNA marker technology for wildlife conservation

Use of molecular markers for identification of protected species offers a greater promise in the field of conservation biology. The information on genetic diversity of wildlife is necessary to ascertain the genetically deteriorated populations so that better management plans can be established for their conservation. Accurate classification of these threatened species allows understanding of the species biology and identification of distinct populations that should be managed with utmost care. Molecular markers are versatile tools for identification of populations with genetic crisis by comparing genetic diversities that in turn helps to resolve taxonomic uncertainties and to establish management units within species. The genetic marker analysis also provides sensitive and useful tools for prevention of illegal hunting and poaching and for more effective implementation of the laws for protection of the endangered species. This review summarizes various tools of DNA markers technology for application in molecular diversity analysis with special emphasis on wildlife conservation.