Introduction

Rice blast disease, caused by the filamentous fungus Magnaporthe grisea, is a serious and recurrent problem in all rice-growing regions of the world (Talbot, 2003; Valent & Chumley, 1991). It is estimated that each year enough rice is destroyed by rice blast disease to feed 60 million people. Control of this disease is difficult; new host-specific forms develop quickly to overcome host resistance and chemical control is typically not cost effective (Ou, 1987). Infections occur when fungal spores land and attach themselves to leaves using a special adhesive released from the tip of each spore (Hamer et al., 1988). The germinating spore develops an appressorium, a specialized infection cell, which generates enormous turgor pressure – up to 8 MPa – that ruptures the leaf cuticle allowing invasion of the underlying leaf tissue (de Jong et al., 1997; Dean, 1997). Subsequent colonization of the leaf produces disease lesions from which the fungus sporulates and spreads to new plants. When rice blast infects young rice seedlings, whole plants often die, while spread of the disease to the stems, nodes or panicle of older plants results in nearly total loss of the rice grain. Recent reports have further shown that the fungus has the capacity to infect plant roots (Sesma & Osbourn, 2004). Different host-limited forms of Magnaporthe also infect a broad range of grass species including wheat, barley and millet.

Introduction

Yeast and fungi are important for the production of industrial and therapeutic proteins (Tables 8.1 and 8.2). Product titres can reach several g/l, e.g. for human serum albumin expressed in Pichia pastoris (~7 g/l) (Kobayashi et al., 2000) or cellobiohydrolase expressed in Trichoderma reesei (~20 g/l) (Durand, Clanet & Tiraby, 1988; Nakari-Setälä & Penttilä, 1995). However, in most cases product titres are ~1000-fold lower (Archer, Jeenes & Mackenzie, 1994; Penttilä, 1998; Cereghino & Cregg, 2000). Identification and engineering of the bottleneck in these, usually heterologous, protein production processes will increase their cost efficiency and competitiveness.

The first bottlenecks that were identified were gene copy number (Clare et al., 1991; McGrew et al., 1997; Vassileva et al., 2001) and transcription efficiency (Outchkourov, Stiekema & Jongsma, 2002). Both were overcome by increasing gene dosage (Clare et al., 1991; Parekh, Forrester & Wittrup, 1995; McGrew et al., 1997; Vassileva et al., 2001) and optimization of codon usage (Gouka, Punt & van den Hondel, 1997a; Kraševec, van den Hondel & Komel, 2000; Moralejo et al., 2000; Outchkourov et al., 2002; Cardoza et al., 2003). Reports emerged stating that with increased gene copy number protein production decreased (Parekh et al., 1995). Recombinant protein was shown to remain associated with the cell (Hohenblum, Borth & Mattanovich, 2003) and localized to the endoplasmic reticulum (ER) (Kauffman et al., 2002). Thus, protein folding and posttranslational modification in the ER are a major bottleneck for protein secretion.

Introduction

In order to meet an increasing food and feed demand for a rising human population agricultural production must be increased, in which crop protection plays a key role. For many decades this field has been the domain of inorganic and synthetic organic chemistry. Many pests could be effectively controlled but high toxicity for humans and non-target organisms as well as in some cases a prolonged persistence in the environment, have made a search for less toxic and more environmentally safe compounds mandatory. The emergence of resistant pathogens, especially fungi, has added to the problem making an intensive search for agrochemicals with new target sites necessary. Today, the standards to be met by new agrochemicals are very high, especially with regard to efficiency and ecological safety. The price has to be competitive and, in general, much lower compared to pharmaceutical compounds. In addition, the active ingredients have to be produced in very large quantities. In 2004, 85 000 tons of agrochemicals were produced in Germany, among them 37 000 tons of fungicides (Hübenthal, 2005).

Natural products in plant protection

Natural products obtained from terrestrial and marine microorganisms, plants and animals are important sources of new chemical structures with biological activities useful for medicine or agriculture. In human medicine the natural products themselves, or semi-synthetic derivatives, play key roles in antibiotic therapy, organ transplantation, cancer therapy and other important fields. In the field of plant protection, their roles are much less conspicuous.

Traditional crops are under increasing threat from a number of subterranean pests that have been proven particularly difficult to control. In Europe, these include the larvae of Scarabaeidae (examples include Melolontha melolontha, Amphimallon solstitialis, A. majale, Phyllopertha horticola, Hoplia philanthus), a number of different larvae of Elateridae (for example, Agriotes lineatus, A. obscurus, Limonius spp.) and Curculionidae (Bothynoderes punctiventris, Otiorhynchus sulcatus, for example), Daktulosphaira vitifoliae (Phylloxeridae), as well as the new exotic pest Diabrotica virgifera (Chrysomelidae). Such pests are difficult to control not only because of their hidden niches, but also because of the inherent difficulties of penetrating their habitat with appropriate control agents. In most of the agricultural systems affected in Europe, the application of chemical insecticides is undesirable or impossible. Currently, the use of virulent and ecologically competent strains of insect-pathogenic fungi appears to be the best approach. Fungal pathogens are endemic in pest populations, and they also fulfil the key criteria for biological control agents: effectiveness, auto-dissemination and persistence. In this article, examples of successful use of entomopathogenic fungi Beauveria spp. and Metarhizium anisopliae as preventive control approaches in the subterranean pest control in European agriculture are discussed.

Introduction

Scarabaeidae, Elateridae, Curculionidae, Phylloxeridae, Chrysomelidae and other subterranean pests are of increasing importance in Europe. These pests – especially in their larval stages – cause damage of several billion euros annually to a wide range of economically important crops throughout Europe (Keller & Zimmermann, 2005).

Introduction

The word ‘xenobiotic’ comes from the Greek word ‘xenos’, which means ‘foreign’, and describes foreign compounds that are in direct contact with a living environment. Man-made xenobiotics have been dispersed directly into the environment for many years, dumped as waste products, applied as agrochemicals, or as a result of major accidents, or indirectly, in the form of emissions from incineration processes. Xenobiotic structures are not readily recognized by existing degradative biological systems and have accumulated in the environment, and although substantial progress has been made in reducing chronic industrial derived pollution there is a growing bank of contaminated derelict industrial land – so called ‘brownfield sites’ – in towns and cities all over the country. In order that these sites may be repurposed for housing or for building up new commercial areas, powerful and cost-effective decontamination strategies are needed.

The design of a decontamination strategy for a given site depends on the nature and concentration of contaminants, the site characteristics (especially water movement), and the extent of contamination. Directed bioremediation, an activity in which micro- and phyto-biological processes are used to degrade or transform contaminants into less toxic or non-toxic forms holds considerable potential as a strategy for in situ decontamination. It is generally cost-effective and less disruptive to soil and the natural landscape than ex situ techniques.

Introduction

Nematophagous and entomopathogenic fungi (NEF) comprise an important group of fungal parasites of invertebrates (FPI). NEF belong to a wide range of fungal taxa, but most of them are anamorphic fungi and facultative parasites. These fungi can infect, kill and digest nematodes and insects, respectively, which we will call their canonical, or normal, hosts. These hosts have barriers to the environment (eggshells and cuticles) that have common structural features. Therefore, the infection cycles share common strategies (e.g. adhesion to the host) or metabolites (e.g. proteases and chitinases for host penetration). Some species (e.g. Lecanicillium lecanii) can even be isolated from both infected nematodes and insects. The NEF may also infect other organisms (other fungi and plants) apart from their canonical hosts in a similar or different mode. We will use the term multimodal to describe the mode of action of these biological activities (Fig. 17.1). However, to date, the main emphasis in research has covered their mode of action on their canonical hosts (e.g. nematodes for nematophagous fungi). Many of these fungi are used for biological control of plant-parasitic organisms.

In this review we will describe the NEF and their hosts in general terms (both canonical and non-canonical) at biological, ecological and physiological-molecular levels. We will also analyze the reasons for this multitrophic behaviour, trying to use a comparative approach of both types of hosts (canonical and non-canonical) and pathogens (nematophagous and entomopathogenic fungi) under an evolutionary perspective.

Introduction

The filamentous fungi are proficient and copious producers of secondary metabolites. From the perspective of an organic chemist, the range and variety of chemical structures of these compounds is remarkable. Synthetic organic chemists have often used the very high structural complexity of fungal secondary metabolites to test their own abilities to mimic nature. From the perspective of a medicinal chemist, the diversity of compounds and structural types represents a pool of useful compounds often possessing unique biological properties. The range of structural types can, at first, appear baffling. However, most secondary metabolites produced by fungi fall into a relatively small number of classes: the alkaloids, derived from amines and amino acids; the terpenoids, derived from isopentenyl diphosphate; and the polyketides, generally derived from acetate. This system of classification is based on the biosynthetic origin of the compound in question, that is to say, a combination of the type of starting material and the type of chemical reactions used during biosynthesis. However, fungi also often combine different types of biosynthetic pathway during the manufacture of secondary metabolites. In Bristol, we have focused our efforts on understanding the biosynthesis of polyketides in fungi, but the inclusion of amino-acid derived moieties in the compounds we are interested in has also necessitated wider investigations.

Introduction

Polarized growth is the mechanism by which filamentous fungi extend their hyphae. Microtubules (MT) and filamentous actin (F-actin), in combination with their corresponding motor proteins, kinesins, dynein and myosins, play crucial roles in this process. The exact contribution of the MT cytoskeleton, however, is still under debate. In this review we will summarize recent advances in understanding the role of MTs and MT-dependent motor proteins in fungi with special emphasis on Aspergillus nidulans. Genetic, biochemical and cell biological approaches in A. nidulans and other fungi led to a modified view of many aspects within the past few years. There is increasing evidence that MT strings, which are visualized by immunostaining or GFP-tagging, consist of several MTs and their dynamics appears to be different in fast-growing hyphal tips as compared with young germlings. Whereas the spindle pole bodies were considered as the only or the main microtubule organizing centres (MTOCs) in filamentous fungi, it appears that several additional MTOCs are responsible for the generation of the MT array. In addition to new insights into the MT network and its dynamics, the roles of several kinesins have been elucidated recently and their interplay with dynein investigated. It became clear that MT functions are interwoven with those of the actin cytoskeleton and that three main structures are required for polarized growth, the Spitzenkörper (vesicle supply centre), the polarisome and probably cell end markers at the cortex. We propose a model for polarized growth, where the actin cytoskeleton and the polarisome are crucial for hyphal extension and the MT cytoskeleton continuously provides the building material within vesicles to the Spitzenkörper and determines growth directionality by delivery of cell end marker proteins.

Introduction

Genome resources for filamentous fungi have improved dramatically in the past few years. Since the publication of the N. crassa genome (Galagan et al., 2003) the pace has accelerated, with many projects completed or nearing completion (Table 6.1), and more underway. The number of researchers investigating the molecular genetics of filamentous fungi is relatively small, and the number of species very large, such that our efforts are spread rather thinly compared to the S. cerevisiae community, for example. Nevertheless, there are indications that these new resources will be extremely beneficial for mycology, and will attract new researchers into the field. Unlike yeasts, filamentous fungi are known for their ability to produce a wide variety of secondary metabolites, which are often of importance to man as useful drugs or harmful toxins (Keller, Turner & Bennett, 2005). Studies over the past 20 years have led to the characterization of some of the biosynthetic pathways and the gene clusters which encode them, for example, the penicillin/cephalosporin and aflatoxin/sterigmatocystin pathways (Brakhage, 1998; Hicks, Shimuzu & Keller, 2002). Since these gene clusters often span substantial regions of the genome, their isolation and sequencing was a major undertaking (Keller & Hohn, 1997). The genome sequences now emerging provide us with easily recognizable gene clusters as a starting point for further investigations, show us the entire secondary metabolic capacity of any species, and pose new questions about how the secondary metabolic repertoire of genera and species has evolved.

The fungi are a highly diverse kingdom of eukaryotic microbes that have been exploited commercially for decades due to their ability to secrete large quantities of proteins of commercial value, and because they possess complex secondary metabolic pathways producing a diverse range of bioactive compounds that have had a major impact in the pharmaceutical market. For example, penicillin and cephalosporin (antibiotics), cylosporin (immunosuppressant) and more recently the statins (cholesterol reducing agents) are estimated to be worth over $5 billion per annum, while enzymes and proteins produced commercially in fungal hosts are used in a diverse number of commercial markets including baking, brewing, detergent, textile and animal feed industries. In addition to the exploitation of fungal products, the fungi themselves are increasingly being developed as alternatives to conventional chemically based pest control strategies, as biocontrol agents active against commercially damaging insect pathogens and weeds, and as bioremediation agents capable of transforming organic and inorganic pollutants in the soil environment. Recent advances in the molecular genetics of the fungi together with the recent release of whole genome sequences of an increasing number of fungi will facilitate further the exploitation and commercialization of these important and ubiquitous eukaryotic microorganisms. The objective of this symposium volume is to highlight current and future biological, biochemical and molecular exploitation of the fungi in biotechnology, and act as an interface between current research and future commercialization.

Background

In order to decrease dependence on petroleum, the United States Department of Energy (USDOE) Office of the Biomass Program (OBP) is investing in research and development to enable its vision of the biorefinery. The biorefinery will decrease the use of petroleum through conversion of biomass such as crops or agricultural waste into fuels and products.

In 2004, the USDOE OBP asked researchers at the Pacific Northwest National Laboratory (PNNL) and the National Renewable Energy Laboratory (NREL) to prepare a list of the top ten building-block chemicals that can be derived from simple sugars by biological and/or chemical means. The resulting list of twelve building-block chemicals and the accompanying report (www.eere.energy.gov/biomass/pdfs/35523.pdf) form an informational foundation on which future DOE and industry bioproducts research will be built (Table 1.1).

How do fungi fit into the biorefinery? Analysis of the ‘top ten’ study indicates that nine of the top twelve chemical building blocks are currently produced, or may potentially be produced, by fungal fermentation processes. However, a significant barrier to the use of bio-based products is the economic feasibility – fuels and products must be price-competitive with those derived from petroleum. An obvious way to decrease the costs of biobased products from fungi is to make fermentation strains more productive and processes more efficient. Traditional strain improvement programmes typically span a timescale measured in decades and process development done through the use of batch cultures is extremely labour intensive.

Introduction

Aspergillus spp. are filamentous fungi of which several have been studied in relation to the secretion of proteins. This chapter will only discuss those species for which there is currently genome sequence information even though the secretion of proteins is probably an important aspect of the lifestyles of the more than 180 known species of Aspergillus. Therefore, the emphasis is with a common saprophyte and human pathogen (Aspergillus fumigatus), a sexual species and one that has been developed as a convenient laboratory species for basic studies (Aspergillus nidulans), and two species that are widely exploited commercially for their secreted enzymes (Aspergillus niger and Aspergillus oryzae). The genome sequence data have been recently acquired and made available to the scientific community so it is particularly timely that the annotated sequences serve as a framework for this chapter on protein secretion. That will also avoid a substantial amount of repetition because there are several excellent reviews on protein secretion by Aspergillus that are primarily pre-genomic in their outlook (Conesa et al., 2001; Punt et al., 2002; MacKenzie et al., 2004) even though extensive use of gene sequences and expression data has been made in the reviewed literature. More recent reviews have been able to take a more genomic view from the outset (Archer & Dyer, 2004; Archer & Turner, 2005) but, even then, the full annotated genome sequences were not available.