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1 Introduction

Fungicides have become an extremely important means to combat problems caused by fungal pathogens both within the agricultural and medicinal fields. Although a number of very effective fungicides have been developed, the emergence of resistance has brought a constant need for the identification of new fungicide targets and for novel agents which act against these targets. This chapter discusses the current situation with regard to fungicide targets and available fungicides and the outlook for the development of novel fungicides, with special focus on the application of the ‘omics’ technologies in the drug development process. The availability of these genome-wide technologies represents a major new opportunity to identify novel fungicide targets within pathogenic fungi. Even within the best studied pathogenic fungi there is still a great deal to learn about how disease is established and maintained. A major challenge for drug development today is how best to employ the new genome-wide technologies to indentify which metabolic pathways and gene products are critical for disease establishment and progression and thus to increase the probability of finding novel fungicide targets. This chapter therefore reviews genome-wide approaches that have emerged in the post-genomics era, e.g. comparative genomics and gene expression profiling experiments, including technologies such as microarray-based transcriptional profiling, SAGE, and MPSS. It also reviews results of the application of such projects to date in the field.

2 Currently Deployed Fungicides

The frequent occurrence of resistance to commonly deployed anti-fungals necessitates a continued search for novel fungicides. Ideally new compounds should have a novel mode of action, have a minimal risk in terms of resistance development, and show a high degree of specificity towards the target organism. These compounds must also satisfy the current stringent regulations regarding ecotoxicity and user safety prior to registration.

The most successful fungicides on the market today act relatively broadly by targeting fungal vegetative growth and thus the entire fungal life cycle. Examples of a successful mode of action classes interfering with biochemical processes essential in fungi include compounds targeting respiration and sterol biosynthesis. An example of the former mode of action class is given by compounds which target mitochondrial electron transport within the respiration chain. Fungicides of the strobilurin class have this mode of action. These compounds are structurally based on natural products and interfere with the ubiquinol-cytochrom C oxidoreductase of the cytochrome bc1 in complex III (Becker et al. 1981; Anke and Steglich 1999). A number of fungicides whose mode of action is the inhibition of sterol biosynthesis have been developed. The sterol biosynthetic pathway includes several drugable targets, such as a 3-keto reductase of an enzyme complex of the sterol C-4 demethylation, the squalene epoxidase, Δ14-reductase and/or Δ8→Δ7-isomerase, and sterol 14α-demethylation, which is inhibited by azole fungicides (Köller et al. 1992; Debieu et al. 2001). Although both respiration and sterol biosynthesis have proved good targets for which very successful fungicides have been produced, resistance against such fungicides develops rapidly resulting in a high demand for new targets and fungicides. Furthermore, several fungicides with a broad range of activity have side-effects on benign fungal species, animals, or plants which cannot be excluded. For this reason there is a need for new types of fungicides which exhibit novel modes of action.

3 What Are the Ideal Attributes of the Fungicides of the Future?

Novel targets can be located either in biochemical or signalling pathways essential for vegetative growth or for pathogenic development of the fungus. Targets with high specificity can thereby be expected in pathways involved in adhesion, host-recognition/pre-penetration processes, host colonisation, and the final reproductive differentiation processes during pathogenic development. An attractive proposition is the development of fungicides interfering with pathogenic development but not with vegetative growth. Such a strategy could prevent or cure infections by fungal pathogens without affecting neutral or benign species.

Within the melanin biosynthetic pathway enzymes such as tetra- or trihydroxynaphthalene reductase and sytalone dehydratase have been identified as targets for non-fungitoxic fungicides. Fungicides interfering with these targets, such as tricyclazole, phthalide, pyrochilon, and capropamid have been successfully used in plant protection (Nakasako et al. 1998; Thompson et al., 2000). Such inhibitors of melanin biosynthesis prove effective against the rice blast fungus Magnaporthe grisea where penetration of this species is prevented by blocking the formation of the appressorial melanin layer required for building turgor pressure within this cell (Howard and Ferrari 1989). A good degree of specificity for the target organism is achieved in this case because these inhibitors specifically target enzymes involved in the production of melanin from a pentaketide precursor while melanin in other organisms uses alternative biosynthetic pathways not affected by such agents (Thines et al. 2004).

The differentiation process of appressorium formation itself may offer novel targets for modern plant protection strategies. Inhibitors interfering with the formation of infection structures might be found which do not interfere with mycelial growth. One example for such a protective fungicide is quinoxyfen, a compound which prevents appressorium formation in Blumeria graminis. The mode of action of quinoxyfen is still unclear; however it was proposed recently that quinoxyfen interferes with signalling (Wheeler et al. 2003) and studies of quinoxyfen-resistant mutants recently implicated inhibition of a serine esterase (Lee et al. 2008).

The disadvantage of fungicides with high specificity is their low range of application, which makes them an unattractive commercial proposition for companies. Considering the high costs incurred during the development and registration of a novel fungicide, companies must have to opportunity to recover the enormous costs of development; fungicides active only against a narrow range of species are obviously much less likely to bring a profit to the developer.

4 The Role of Traditional Screening Approaches Used in Fungicide Development

In recent decades traditional screening approaches with compounds derived from chemical synthesis or natural products in greenhouses were very successful and led to a generation of very effective fungicides with toxicologically favourable profiles. Therefore screening on living organisms/plants will play an important role in the future of plant protection research. However, the drawback of this traditional approach is that large numbers of compounds must be tested in order to get to a hit. To overcome this, target-based screening platforms and virtual molecular modelling systems have been developed in which large numbers of molecules can be tested.

5 Determining the Mode of Action of an Anti-Fungal Compound

Determining whether a new compound has a novel mode of action is a critical step in the drug development process and this must be achieved at an early stage. To this end in vitro screening systems targeting distinct biochemical reactions/pathways can also be used to identify the mode of action of compounds showing good antifungal activity during in vivo screening experiments. New compounds for which the mode of action is not known can initially be assessed in simple biochemical tests, such as inhibition of respiration or sterol biosynthesis. Furthermore fungicide-resistant mutants have successfully been used to identify which protein is likely targeted by the agent. When the mode of action of fungicidal compounds cannot be identified by such approaches, novel methods such as metabolome, proteome, or transcriptome analysis can provide signature patterns which can be compared to patterns obtained from experiments with known compounds and may indicate whether or not it is likely that the drug has a novel mode of action.

6 Genome-Wide Approaches

Genome-wide technologies, e.g. gene expression profiling and comparative genomics, lead to the identification of a large number of potential candidate genes encoding factors essential either for invasive growth and development or for vegetative growth. However, these potential targets have to be validated by gene deletion or gene-silencing experiments. Even if deletion of the gene is lethal or the mutant shows a non-pathogenic phenotype, it must be further established whether the target is drugable in vivo.

Once a target has been validated, target-based ultra-high throughput biochemical screening systems can be established in which hundreds of thousands of compounds can be tested daily. The challenge for modern agrochemical research is to convert the in vitro activity of compounds identified in such screening systems to products active in vivo.

The post-genomics era is now truly upon us. More than 50 different fungal genome sequences are now publicly available and more fungal sequencing projects are either planned or in progress. More than half of the finished genomes are of pathogenic species and cover the most important fungal pathogens of humans as well as many of the most devastating fungal pathogens of commercially important crops (Table 11.1). These resources open the door to genome-wide analysis within these species. Recently developed technologies allow the analysis of mRNA (transcriptomics), protein (proteomics), and metabolite (metabolomics) profiles. We focus here largely on transcriptomics and proteomics.

Table 11.1. Fungal species whose genome sequence has been determined or for which a sequence release is imminent. The latest status for ongoing projects and links to finished genomes are available at http://fungalgenomes.org/wiki/Fungal_Genome_Links
Full size table

6.1 Transcriptomics: Microarrays

Microarrays were first employed within fungi more than ten years ago (DeRisi et al. 1997) and microarray technology has since been widely used to study a broad array of processes within fungi (reviewed by Breakspear and Momany 2007). The technology itself is a logical development from Southern/Northern blotting and, very basically, the microarray itself is a series of spots of nucleic acids (cDNAs or oligonucleotides) which are known as features or probes and which are attached to a solid support. cDNA or cRNA samples (somewhat confusingly these are known as targets in this context) can then be hybridised to the microarray and, following high stringency washes, the degree of probe to target annealing at each spot can be quantified and, by comparison of the signal under different conditions, the relative abundance of the target within two different samples can be assessed. Microarray technology is described in detail elsewhere (e.g. see Heller 2002). Several applications of microarray technology have emerged in recent years; however here we confine our discussion largely to the application of microarrays to the study of transcript abundance and how microarray analysis might be exploited to assist in the identification of novel fungicide targets. A number of different examples to illustrate the different potential applications of microarrays are given below.

6.2 Studying Genome-Wide Transcriptional Changes Which Accompany Differentiation

Within plant pathogenic fungi, early studies used sub-genome-wide technologies, often derived from the results of EST sequencing projects, to study development (See Breakspear and Momany 2007 and references therein). In investigations of the rice blast fungus Magnaporthe grisea, for example, the first microarray experiments used cDNA-based technology to investigate the relative transcript abundance of 3500 features (cDNAs with some redundancy) during infection-related morphogenesis to investigate appressorium formation (Takano et al. 2003).

A whole genome microarray of M. grisea is now commercially available from Agilent Biotechnologies and has been used to identify genes important for appressorium formation (Oh et al. 2008). Genes responding to nitrogen starvation, a situation believed to mimic the nutritional state of the fungus post-penetration and which is purported to induce infection-related development, have also been studied using these commercially available microarrays (Donofrio et al. 2006). One gene which responds to nitrogen starvation, SPM1, is predicted to encode a vacuolar serine protease. It was deleted and shown to be required for normal levels of virulence (Donofrio et al. 2006). Novel gene products which are critical for appressorium formation have been uncovered by microarray-based analyses, including a subtilisin-like protease and a NAD-specific glutamate dehydrogenase (Oh et al. 2008). These studies clearly illustrate the potential of genome-wide technologies to potentiate the identification of potential new fungicide targets by narrowing the search for possible pathogenicity factors.

In Fusarium graminearum, a whole genome microarray has been used to investigate which pathways are induced during the germination of conidia, an important first step in the establishment of disease (Seong et al. 2008). Clearly some of these differentially regulated genes might prove attractive as targets for drug intervention, although obviously with such an analysis significant follow-up experiments would be required in order to identify which genes are essential for the process studied, as these are likely only a small subset of the total number identified.

Further commercially produced whole-genome microarrays are in development for other plant pathogenic fungi, including Botrytis cinerea and Mycosphaerella graminicola. It can be anticipated that the range of microarrays available and their application within plant pathology will expand greatly in the future. Whether the application of these microarrays will eventually reveal fungicide targets which are conserved across a broad range of species is open to question; however there is no doubt about the potential of this technology to generate new experimental hypothesis which could greatly facilitate the identification of novel targets for drug control within individual species.

Extensive microarray-based analysis can also proceed in the absence of a whole-genome sequence. An example is the study of Both and co-workers (2005), who looked at more than 2000 Blumeria graminis genes from an infection time-course and revealed how several genes previously implicated in disease progression as well as many new unknown genes were expressed during infection-related development. Some of the genes identified during this study might be potential candidates for fungicide targets, although proving this is not straightforward in an obligate pathogen such as Blumeria graminis.

The application of microarrays to the study of fungal pathogens of humans is also becoming increasingly commonplace. For example, Histoplasma capsulatum is an ascomycete dimorphic fungus responsible for the human disease histoplasmosis. Microarray analysis using genomic DNA-derived targets has identified genes specifically expressed in the pathogenic yeast form (Hwang et al. 2003). Again significant follow-up experiments are required to test whether any of the products of these genes are essential for virulence but, in terms of identifying potential novel targets, such studies offer valuable new hypotheses which will no doubt lead to the identification of novel drug targets which could prove valuable in future years.

6.3 Cross-Species Comparisons: Comparative Genomic

Where significant similarity exists between closely related fungal species there is a possibility to conduct cross-species comparisons using microarrays. A good example of such an application is provided by the study of Moran and co-workers (2004) who exploited Candida albicans-based genomic microarrays to compare the genome of this species to the very closely related but less virulent species C. dubliniensis. Although the vast majority of genes were found to be highly conserved, several hundred genes were identified which were either absent in C. dubliniensis or whose sequence was poorly conserved between these two species. This study not only generated several testable hypotheses which might shed light on the difference in virulence between these two species but also provided a great deal of information about the C. dubliniensis gene repertoire in the absence of a genome sequence. Interestingly several of the genes poorly conserved between the two species studied are predicted to encode transcription factors and it would therefore be of significant interest to extend these analyses by comparing the transcriptome of the two species.

6.4 Identifying Possible Drug Targets by Comparison of the Transcriptome of Mutant and Wild-Type Cells

Using whole-genome microarrays to study a major developmental switch such as germination or appressorium formation, several hundred to thousands of genes have been found to be differentially expressed (e.g. Oh et al. 2008; Seong et al. 2008). An alternative route to identify the genes involved in a particular process is to examine a mutant blocked at that developmental stage. With a view to drug development, this may be a more attractive route, as the number of target genes identified by this approach is more likely in the tens rather than in the hundreds. Such an application of microarrays is nicely illustrated by a recent study of the human pathogen Cryptococcus neoformans (Cramer et al. 2006). These workers identified genes which are transcriptionally down-regulated in a mutant which lacks a component of the cAMP signalling pathway which is required for capsule formation, a critical determinant of virulence. One of these genes was found to encode a transcription factor with similarity to yeast transcription factor Nrg1p. A C. neoformans strain lacking this transcription factor was generated and was also found to be deficient in capsule formation. Microarrays were then used to identify genes transcriptionally dependent on the product of NRG1 and among the 71 genes identified was UGD1, a gene whose product was previously implicated in capsule formation in this fungus (Moyrand and Janbon 2004). Although this gene had previously been identified, this study illustrates the potential of the combination of microarray analysis and specific mutations to uncover potentially drugable targets. In a similar manner, Odenbach and co-workers (2007) used microarrays to identify approximately 100 Magnaporthe grisea genes whose transcription depends on the Con7p transcription factor during germination of spores of this fungus. These included a chitin synthase encoding gene CHS7 which, like the con7 − mutant, is affected in the formation of the appressoria (Odenbach et al. 2007, 2009). Again chitin synthase is already considered a potential target for drug intervention in human disease, so although this is not a novel fungicide target, this study nicely illustrates the potential of using microarrays in combination with specific mutants to identify potential drug targets. In a similar manner using a human pathogenic fungus, Ngugen and Sil (2008) identified a Histoplasma capsulatum regulator Ryp1p which is required for yeast phase gene expression and identified several genes controlled by this transcription factor by comparing transcript profiles of mutant and wild-type cells.

Identification of novel fungicide targets is ultimately a process of elimination. Eliminating which factors are not good targets from the likely comparatively small number which are useful (drugable) targets is an important step in target identification. Mutant generation will continue to be a decisive experiment in this process for years to come. Any means by which it is possible to narrow the search for new targets is obviously attractive. Much research in the past few decades in the academic world has focused on signal transduction and, although it is questionable whether any of the proteins identified by such studies are themselves good targets, the mutants generated by such studies could be exploited to identify downstream components of which a small subset are likely to be essential for the process and which may include useful targets for drug intervention.

6.5 Exploring Drug Resistance Using Transcriptome Analysis: Candida albicans

A further application for microarrays within the field of drug development is in the study of resistance to fungicides. Such an approach has already been adopted by a number of groups attempting to understand the transcriptional response to fungicide treatment. In this respect by far the best studied species within fungi is the opportunistic fungal pathogen of humans Candida albicans. Two of the most commonly deployed drugs in the treatment of diseases caused by C. albicans are the polyene fungicide amphotericin B (AMB) and fungistatic azole compounds such as fluconazole. Although the mode of action of these drugs differs, resistance to both drugs within clinical isolates is often associated with induction of drug efflux transporter activity and/or alterations to the ergosterol biosynthetic enzymes (reviewed by Akins 2005). This is not surprising, as both drugs are thought to ultimately impact on membrane integrity. A broader understanding of the resistance mechanisms to these and other drugs has come from several microarray-based studies which have examined transcript profiles within amphotericin B-resistant isolates or drug-treated wild-type strains. These studies both confirmed previous reports which suggested induction of drug efflux transporter activity and/or alterations to the ergosterol biosynthetic enzymes and also led to the identification of further genes which are induced specifically on treatment with drugs of a specific class or which seem to be part of a more general response to drug challenge. Several genes whose transcription is altered by drug treatment are otherwise uncharacterised and their participation in drug response may therefore be the first clue to their function. Furthermore the specific transcript profile induced by treatment with a particular drug might also be used as a ‘fingerprint’ for drugs of that type and might assist in assigning a tentative mode of action to a drug whose targets is unknown or unclear. C. albicans is also known to commonly form biofilms which show increased resistance to commonly deployed antifugals (Chandra et al. 2001; Ramage et al. 2001). Farnesol, a quorum-sensing molecule, was shown to prevent biofilm at high concentrations and is considered a possible novel Candida control agent (Ramage et al. 2002). Microarray analysis subsequently shed some light on the mode of action which was previously not known, suggesting that the compound might in part prevent filamentous growth by inducing the transcription of the TUP gene which encodes a repressor of hyphal development (Cao et al. 2005). Again this study highlights the utility of microarray experiments in understanding drug response and mechanisms of resistance.

The response to azole fungicides has also been studied in Aspergillus fumigatus where, as is the case for C. albicans, resistance to clinically deployed drugs of this class is increasingly problematic. Several thousand genes were found to be differentially regulated in response to voriconazole (da Silva Ferreira et al. 2006). Up-regulated genes included several transporters of ABC and MFS type which might play a role in drug efflux.

The use of microarrays for the study of resistance to drug treatment can be expected to lead to valuable insights into what are the critical determinants of resistance and increasing application in this field as well as in determination of mode of action is anticipated in the future.

6.6 MPSS and SAGE

Serial analysis of gene expression (SAGE; Velculescu et al. 1995) and massively parallel signature sequencing (MPSS; Brenner et al. 2000) are related technologies which yield short sequence signatures derived from mRNAs. By comparison of the relative abundance of these signatures within libraries derived from different mRNA populations, a semi-quantitative measure of transcript abundance in a sample is obtained.

Examples of the application of these technologies are studies conducted using the plant pathogens Magnaporthe grisea (Irie at al. 2003) and Blumeria graminis (Thomas et al. 2002). Several genes dependent on cAMP were revealed using SAGE in Magnaporthe, including a number which have already been shown to be essential for pathogenicity (Irie et al. 2003). In B. graminis, an obligate pathogen, several thousand SAGE tags were sequenced and more than 100 genes differential transcribed during infection-related development were highlighted (Thomas et al. 2002). This study highlights one great advantage of these technologies: they can be applied to any organism irrespective of whether a genome sequence is available or not. A further application for such technologies is in determining which of the predicted genes within an annotated genome is really transcribed. Thus in the case of M. grisea, a large number of the genes predicted to exist in the genome by automated annotation were subsequently shown to be really transcribed using SAGE/MPSS (Gowda et al. 2006). In fact the same study indicted the existence of several thousand additional transcripts which do not correspond to any annotated gene and has therefore expanded the M. grisea gene repertoire.

SAGE has also been used to study protein kinase A (PKA)-dependent gene expression in the corn smut pathogen Ustilago maydis (Larraya et al. 2005). By analysis of transcript profiles based on the sequences of at least 40 000 tags for the wild type and two mutants in the PKA pathway, the authors were able demonstrate novel functions for PKA signalling in this organism, including a link to phosphate metabolism. Because PKA signalling is critical for virulence in this species, such analyses pinpoint downstream targets of PKA signalling which may well include potential new targets for fungicide control of corn smut.

Within fungi which are pathogenic to humans, SAGE has been employed for transcriptome analysis most extensively in Cryptococcus neoformans. One comparison used 49 224 SAGE tags from each of two protein kinase A (PKA) pathway mutants and a wild-type control under conditions known to induce capsule formation in this species (Hu et al. 2007). Among the 599 tags exhibiting altered abundance between these SAGE libraries were several corresponding to genes whose products might play a role in secretion. Furthermore, using inhibitors of secretion, the authors were able to demonstrate that capsule formation is inhibited at drug concentrations which do not affect growth in culture. This indicates that, as one might anticipate, secretion plays an important role in capsule formation. These results not only expand the understanding of the targets of PKA signalling but also highlight the potential of targeting components of the secretion machinery in order to control this species.

In conclusion SAGE and MPSS are powerful means to obtain a semi-quantitative assessment of relative transcript abundance under different conditions. There is no doubting the value of these technologies in providing information which could suggest which pathways or enzymes are likely to be activated under specific conditions. Additionally these technologies have been successfully employed in fungal species which have not yet been completely sequenced. As with microarrays, these technologies could readily be employed to suggest possible new targets for drug intervention; however, in comparison to microarrays, MPSS and SAGE have been much less broadly applied experimentally and it is therefore probable that their application in future drug development will also be peripheral.

6.7 Transcriptomics: Outlook

Microarrays and related technologies have unquestionably provided the researcher aiming to study plant–pathogen interactions with powerful tools which is are now very broadly accepted and increasingly widely used. It is likely that with ever improving technologies and decreasing prices such forms of analysis will become available to most researchers in the coming years. Nevertheless it is important to be aware that transcript abundance does not always correlate well with protein abundance. It has been common to extrapolate from the results of such analyses and suggest that certain pathways are activated in response to certain developmental or environmental conditions or as a result of certain mutations. Although this is likely to be true in the majority of cases, these technologies should at best be viewed as a very elegant means to generate hypotheses in the absence of any preconceptions of the experimental outcome. Although it is easy to be blinded by the power of this technology, any study which reports only microarray data is likely to provide very many new questions but no real answers. The real power of genome wide analysis of transcript abundance will only be realised in combination with other approaches which address the response of the organism at other levels and by genetic manipulations to address the question as to whether the observed alteration in transcript abundance has any meaningful consequences for the test organism. Genetic manipulation of most fungal pathogens remains the major bottleneck in moving from the results of genome-wide studies of transcript abundance to establishing that the product of a particular gene is really important or essential in establishing disease (and therefore a potential drug target).

6.8 Other ‘omics’

The ultimate targets of most known fungicides are proteins and there is some logic to working directly at the level of the protein. Additionally there are many examples where the major level of control which determines protein abundance is post-transcriptional. For these reasons proteome profiling represents an attractive approach to identify potential new fungicide targets. Proteome profiling studies in Aspergillus species have been used to identify proteins expressed during various physiological states of the cell (for a review, see Kim et al. 2008). Especially in the opportunistic human pathogenic fungus Aspergillus fumigatus, comparative proteome studies have been conducted in order to identify proteins involved in virulence or essential for mycelial growth that may serve as targets for antimycotic therapies. Bruneau and co-workers focussed on surface proteins thereby identifying glycosylphosphatidylinositol-anchored membrane proteins which had previously been shown to be involved in cell wall biosynthesis (Mouyna et al. 2000). It was shown that, out of five proteins with unknown function, the Ecm33 protein influences the conidial cell wall biosynthesis/cell wall morphogenesis (Chabane et al. 2006).

A proteome study conducted by Asif and co-workers (2006) led to the identification of 26 conidial cell surface proteins as potential vaccine candidates. Several of the proteins identified had no known function and may therefore be novel targets for therapeutic approaches.

In proteome studies using mutants it was recently shown that enhanced PKA activity results in the activation of stress-associated proteins, enzymes involved in protein biosynthesis, and glucose catabolism. Enzymes involved in nucleotide and amino acid biosynthesis and enzymes involved in catabolism other than glucose were down-regulated by PKA signalling (Große et al. 2008). It was furthermore found that septin and β-tubulin were down-regulated by enhanced PKA signalling, indicating that cAMP/PKA signalling is involved in the regulation of fungal morphogenesis and may therefore be of relevance for virulence. These findings are backed by mutant studies showing the relevance of PKA/cAMP signalling in Aspergillus fumigatus (Liebmann et al. 2004).

Proteomic analysis has also been carried out in the fungal human pathogen Candida glabrata, where a recent study using mutants lacking the Ace2 transcription factor which have an increased ability to cause disease. The types of proteins identified suggested that this factor controls the transcription of genes involved in cell wall biogenesis in this species (Stead et al. 2005).

In conclusion proteome analysis must been regarded as powerful tool to identify proteins required for structural integrity of the cells or for pathogenicity and it is likely that this approach might find application in the search for suitable targets for therapeutic approaches.

7 Conclusions/Outlook

Genome-wide studies typically yield large numbers of candidate genes of which it is likely that only a small subset will be essential for the process studied. So for example, of several genes identified as differentially regulated during infection-related morphogenesis in Magnaporthe grisea only three out of 16 selected for mutation were found to be essential for pathogenicity. In order to fully exploit the results of genome-wide analysis there is a clear need for streamlined strategies for high-throughput analysis of gene function. Given that a subset of genes may be essential for viability, an attractive approach to investigate whether a particular gene represent a useful fungicide target would be gene silencing because inducible gene silencing might then allow the analysis of essential genes. Again taking Magnaporthe as an example, gene silencing has been shown to be effective in this fungus (Kadotani et al. 2003) and tools for high-throughput analysis using this technology show great promise for the future (Nguyen et al. 2008). A collection of mutants deleted for all of the predicted genes, as has been developed for Saccharomyces cerevisiae, is an attractive resource; however the generation of such a resource is a major undertaking and it is likely that the intelligent use of transcription profiling combined with mutation of a subset of likely candidates is a more economical way to identify novel fungicide targets. Although the targets of most of the currently deployed fungicides were discovered by more traditional approaches, it is important to remember that genome-wide technologies are relatively new and that it may be several years before they yield their first fruits in terms of identification of new drug targets.