**3. Pathogenesis**

As fungal pathogens have an enormous impact on plant production worldwide, the strategies they use to infect plants and to cause disease are a topic of great interest (Van De Wouw & Howlett, 2011). Knowledge of the pathogenicity/virulence factors essential for fungal infections is very important because it represent the targets that researches must attack in the fight against these pathogens (Fernandez-Acero *et al.,* 2011).

We define pathogenicity gene as those necessary for disease development but not essential for pathogen to complete its lifecycle *in vitro.* Pathogenicity genes are of interest not only to increase our overall knowledge of disease process, but also because any such gene could became a target for disease control.

The types of genes essential for pathogenesis depend on the infection process of a particular fungus. Some fungi degrade the cuticle and cell wall to enter the plant; others form specialized structures, such as appressoria, to penetrate the epidermis, while others enter the host through wounds or natural openings (Idnurm & Howlett, 2001). Once the fungus has colonized plant, it may grow obtaining nutrients from its host without killing cells (as a biotroph); some fungi produce specialized infection structures (e.g. haustoria) during biotrophic stage. Other fungi species act killing host cells with the use of toxins (as necrotrophs) or act as hemibiotroph (biotroph and necrotroph at different stages if infection). Toxins often are major components of the arsenal or virulence determinants by necrotrophic fungi. They can be host specific or non host specific, and they kill or disable functions of host cells (Van De Wouw & Howlett, 2011).

A number of fungal mechanisms and molecules have been shown to contribute to fungal pathogenicity or virulence, understood as the capacity to cause damage in a host, in absolute or relative terms. Among them, cell wall degrading proteins, inhibitory proteins, and

biology to inform chemistry (Xu *et al*., 2007). The accumulation of proteomic information of fungal plant pathogens may be an incentive to the development of new and environmentally friendly fungicides. Particularly, Proteomics is another is a highthroughput technology that allows an in depth study of the sets of proteins synthesized in a specific sample at any specific moment. By protein profile comparison between samples, the proteins involved in specific biological processes may be revealed. One of the most interesting applications of the proteomics is its use in discovering new protein targets for drug design including fungicides (Fernandez-Acero *et al.,* 2010; Ferrer-Alarcon *et al.;* 2009). It

The accumulation of information over the last decades, relating to a) fungal molecular genetic data, b) pathogenicity/virulence factors and c) proteomic approaches, has led to the appearance of several web-accessible databases which contribute to the fungal scientific community's development in this field. More than 50 genomes of pathogenic fungi are published in the Broad Institute Database for public perusal (www.broadinstitute.org/science/projects/fungal-genomeinitiative); and further data in the Phytopathogenic Fungi and Oomycete EST Database, COGEME, http://cogeme.ex.ac.uk/). In spite of the incredible amount of biological information about fungal plant pathogens, there

As fungal pathogens have an enormous impact on plant production worldwide, the strategies they use to infect plants and to cause disease are a topic of great interest (Van De Wouw & Howlett, 2011). Knowledge of the pathogenicity/virulence factors essential for fungal infections is very important because it represent the targets that researches must

We define pathogenicity gene as those necessary for disease development but not essential for pathogen to complete its lifecycle *in vitro.* Pathogenicity genes are of interest not only to increase our overall knowledge of disease process, but also because any such gene could

The types of genes essential for pathogenesis depend on the infection process of a particular fungus. Some fungi degrade the cuticle and cell wall to enter the plant; others form specialized structures, such as appressoria, to penetrate the epidermis, while others enter the host through wounds or natural openings (Idnurm & Howlett, 2001). Once the fungus has colonized plant, it may grow obtaining nutrients from its host without killing cells (as a biotroph); some fungi produce specialized infection structures (e.g. haustoria) during biotrophic stage. Other fungi species act killing host cells with the use of toxins (as necrotrophs) or act as hemibiotroph (biotroph and necrotroph at different stages if infection). Toxins often are major components of the arsenal or virulence determinants by necrotrophic fungi. They can be host specific or non host specific, and they kill or disable

A number of fungal mechanisms and molecules have been shown to contribute to fungal pathogenicity or virulence, understood as the capacity to cause damage in a host, in absolute or relative terms. Among them, cell wall degrading proteins, inhibitory proteins, and

involves the identification and early validation of disease-associated targets.

is no commercial fungicide developed from a molecular approach.

attack in the fight against these pathogens (Fernandez-Acero *et al.,* 2011).

**3. Pathogenesis** 

became a target for disease control.

functions of host cells (Van De Wouw & Howlett, 2011).

enzymes involved in the synthesis of toxins are included. These virulence factors are typically involved in evolutionary arms races between plants and pathogens (Gonzalez Fernandez *et al*., 2010).

Knowledge of the pathogenic cycle and virulence factors of the fungus is crucial for designing effective crop protection strategies, including the development of resistant plant genotypes through classical plant breeding or genetic engineering, fungicides or the use of biological control strategies (Gonzalez-Fernandez *et al.,* 2010). The determination of a specific factor as virulence or pathogenicity has been achieved by constructing defective mutants in the specific genes. The infection power of the analyzed mutants should at least decrease or disappear compared to the wild type, if the deflections of these genes in mutants produce a loss of vegetative lesion, it is logical to assume that the inhibition of this enzyme or set of enzymes by targeted strategies, should produce new fungicides. In this context, the use of natural products or related compounds as specific enzymes inhibitors is an archetype, as they would be species specific and the environmental impact would be reduced to a minimum (Fernandez-Acero *et al.,* 2011).

A diversity of fungi, oomycetes secrete proteins and other molecules to different cellular compartments of their hosts to modulate plant defense circuitry and enable parasitic colonization, these molecules have been called "effectors". The usage of the term "effector" became popular in the field of plant-microbe interactions with the discovery that plant pathogenic gram-negative bacteria utilize a specialized machinery to deliver proteins inside host cells. More recently, a broader range of plant microbiologists have adopted the term effector and its associated concepts (Abramovitch et al., 2006). This term is now also routinely used in the fungal and oomycete literature and is becoming increasingly popular in nematology to describe secreted proteins that exert some effect on plant cells (Hogenhout et al., 2009).

Some effectors are avirulence proteins and have a 'gene-for gene' relationship with resistance proteins in the host. When a fungal avirulence gene is mutated, hosts with the corresponding resistance gene no longer detect the pathogen; this leads to a compatible interaction. Host-specific proteinaceous toxins that have an 'inverse' gene-for-gene relationship with the host, whereby the interaction leads to disease such genes would be classified as pathogenicity genes (Oliver & Solomon, 2010). Small proteins encoded by fungal genes involved at various stage of infection, alter host cell structure and function facilitate infection. These proteins are often cysteine rich (hogenhout *et al*., 2009).

Fungi use signaling cascades to respond to changes in the environment by altering their gene expression. The interruption of these signaling genes results in the loss and/ or reduction in pathogenicity, as well as pleiotrophic effects on cellular processes, including mating, conidiation, growth rate and toxin production. Therefore, it is difficult to determine which aspect of fungal physiology is responsible for the loss of pathogenicity. The components of these signal transduction cascades may represent targets for the development of fungicides (Van De Wouw & Howlett, 2011). *Phytophtora infestans,* one of the most destructive pathogen of potato in the history, have a remarkable speed of adaptation to control strategies such as genetically resistant cultivars, comparison with two other *Phytophthora* genomes showed rapid turnover and extensive expansion of specific families of secreted disease effector proteins, including many genes that are induced during infection

Target-Site-Specific Screening System for Antifungal Compounds 187

compounds are structurally based on natural products and interfere with the ubiquinol

There are numerous antifungal compounds that were discovered from diverse microbial sources using traditional activity-based screening techniques. These microbial compounds showed potent control efficacy against various plant diseases, including chronic diseases which are difficult to control with conventional synthetic fungicides. Advances in screening systems directed to specific targets of fungal metabolism have increased the opportunities to discover novel antifungal agents with selectivity over non-target organisms. Microbial metabolites have also been exploited as a source for non-fungicidal disease control agents that do not inhibit vegetative hyphal growth, but rather interfere specifically with the infection process of pathogenic fungi, such as spore germination, the formation of penetration structures and sporulation (Seok & Kook, 2007). Infection structures of phytopathogenic fungi are modified hyphae specialized for the invasion of plant tissue, initial events are adhesion to the cuticule and directed growth of the germ tube on the plant surface (Mendgen *et al.,* 1996). The specificity of microbial fungicides is a highly preferred characteristic in terms of impacting the environment, where it is closely related to the occurrence of fungicide resistance. The most recently developed fungicides from microbial metabolites, the strobilurins, provide a cue for the high risk of resistance development of

These compounds have long been applied to control fungal diseases of rice, vegetables and fruits, and their effectiveness in controlling these diseases has been tested in the field and proven over many years. The importance of microbial fungicides, compared to synthetic compounds, may have been under evaluated in the past due to the limitation in their activity spectrum and in certain instances, the development of resistance (Knight *et al.,* 1997). Nevertheless, the excellent fungicidal activity of these microbial metabolites and their potential as lead candidates for further fungicide development continue to stimulate research and screening for antifungal microbial metabolites, some examples of this

**Kasugamycin** is an amino-sugar compound discovered from the metabolites of *Streptomyces kasugaensis, in vivo* studies have shown that kasugamycin efficiently suppresses the development of *M. grisea* mycelia on rice plants in both preventive and curative treatments, to overcome potential resistance problems, mixtures of kasugamycin with different

**Polyoxins** are peptidylpyrimidine nucleoside antibiotics isolated from the culture broth of *Streptomyces cacaoi* var. *asoensis*, such excellent characteristics come from the fact that polyoxins selectively inhibit the synthesis of cell-wall chitin in sensitive fungi but have no

**Validamycin A** produced by *Streptomyces hygroscopicus* var. *limoneus* has been effective in controlling rice sheath blight caused by *R. solani*. Validamycin A is converted within fungal cells to validoxylamine A, an extremely strong inhibitor of trehalose. The mode of action of validamycin A is favorable for biological selectivity, because vertebrates do not depend on

synthetic fungicides having different modes of action are currently in use.

adverse effects on organisms lacking chitinous cell walls.

the hydrolysis of trehalose (Doumbou *et al.,* 2001).

cytochrome C oxidoreductase (Foster & Thines, 2009).

site-specific fungicides.

compounds are:

stage. These fast-evolving effector genes are localized to highly dynamic and expanded regions of the *P*. *infestans* genome. This probably plays a crucial part in the rapid adaptability of the pathogen to host plants and underpins its evolutionary potential (Haas *et al.,* 2009). Other example of pathogen trainer of virulence factors is *Botrytis cinerea* to date have been identified a wide range of compounds and enzymes that uses the fungus to exerts its pathogenicity, thanks to the arsenal of degrading enzymes, *B. cinerea* is able to feed on different plant tissues, this fungus shares conserved virulence factors with other phytopathogens (Choquer *et al.,* 2007). *Botrytis cinerea* is a ascomycete necrothrofic, this fungi alone is responsible for 10% of the global fungicide market (Fernandez-Acero *et al.,* 2011), is thought to enter the host mainly by producing degrading enzymes and causing an oxidative burst, specifically because secretes nonspecific phytotoxins to kill cells from a large spectrum of plants. Among the numerous metabolites isolated from fermentation broths, the most well known is the sesquiterpene botrydial (Deighton *et al.,* 2001). This fungus is notably equipped with multiple cell wall-degrading enzymes that allow plant tissue colonization and the release of carbohydrates for consumption. Pectin, the major host cell wall component, can be degraded by a set of fungal pectinases (Ten Have *et al,* 2001; Choquer *et al,* 2007). Xilanase (Xyn11A) from *Botrytis cinerea* contributes to the infection process with the necrotizing and not with the xylan hydrolyzing activity. The main contribution of the xylanase Xyn11A to the infection process of *B. cinerea* is to induce necrosis of the infected plant tissue. A conserved 30-amino acids region on the enzyme surface, away from the xylanase active site, is responsible for this effect and mediates binding to plant cells (Noda *et al,* 2010).

New technologies like proteomics are very good tools for obtain information about proteins secreted by pathogenic fungi. Nowadays, there is lot of candidate pathogenicity genes, but there is stagnation for their functional analysis, since experiments are time-consuming and difficult to realize for some fungi. However, genomic tools are already providing a much more integrated picture of pathogenicity mechanisms, compared with the previous focus on individual genes. Many fungal genes affecting disease progression are involved in growth and development, and there are few genes for which the only effect is on disease, proteomics allowed to identify between 10 and 100 different biological functions from each gene (Van De Wouw & Howlett, 2011; Fernandez-Acero *et al.,* 2011). This is an important progress in the new fungicide discovery because pathogenic genes with function in growth and development could be target site for new fungicides.

### **4. Morphogenesis**

Since fungi are a eukaryotic organism, they have diverse metabolic profiles similar to many mammalian and plant. Hence, several antifungal agents discovered to be potentially active against plant pathogenic fungi have failed to survive during the testing process because the target site of the fungicide is found in another organism (Thines *et al.,* 2004).

The most successful fungicide in the market today acts 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 progresses 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

stage. These fast-evolving effector genes are localized to highly dynamic and expanded regions of the *P*. *infestans* genome. This probably plays a crucial part in the rapid adaptability of the pathogen to host plants and underpins its evolutionary potential (Haas *et al.,* 2009). Other example of pathogen trainer of virulence factors is *Botrytis cinerea* to date have been identified a wide range of compounds and enzymes that uses the fungus to exerts its pathogenicity, thanks to the arsenal of degrading enzymes, *B. cinerea* is able to feed on different plant tissues, this fungus shares conserved virulence factors with other phytopathogens (Choquer *et al.,* 2007). *Botrytis cinerea* is a ascomycete necrothrofic, this fungi alone is responsible for 10% of the global fungicide market (Fernandez-Acero *et al.,* 2011), is thought to enter the host mainly by producing degrading enzymes and causing an oxidative burst, specifically because secretes nonspecific phytotoxins to kill cells from a large spectrum of plants. Among the numerous metabolites isolated from fermentation broths, the most well known is the sesquiterpene botrydial (Deighton *et al.,* 2001). This fungus is notably equipped with multiple cell wall-degrading enzymes that allow plant tissue colonization and the release of carbohydrates for consumption. Pectin, the major host cell wall component, can be degraded by a set of fungal pectinases (Ten Have *et al,* 2001; Choquer *et al,* 2007). Xilanase (Xyn11A) from *Botrytis cinerea* contributes to the infection process with the necrotizing and not with the xylan hydrolyzing activity. The main contribution of the xylanase Xyn11A to the infection process of *B. cinerea* is to induce necrosis of the infected plant tissue. A conserved 30-amino acids region on the enzyme surface, away from the xylanase active site, is responsible for this effect and mediates

New technologies like proteomics are very good tools for obtain information about proteins secreted by pathogenic fungi. Nowadays, there is lot of candidate pathogenicity genes, but there is stagnation for their functional analysis, since experiments are time-consuming and difficult to realize for some fungi. However, genomic tools are already providing a much more integrated picture of pathogenicity mechanisms, compared with the previous focus on individual genes. Many fungal genes affecting disease progression are involved in growth and development, and there are few genes for which the only effect is on disease, proteomics allowed to identify between 10 and 100 different biological functions from each gene (Van De Wouw & Howlett, 2011; Fernandez-Acero *et al.,* 2011). This is an important progress in the new fungicide discovery because pathogenic genes with function in growth

Since fungi are a eukaryotic organism, they have diverse metabolic profiles similar to many mammalian and plant. Hence, several antifungal agents discovered to be potentially active against plant pathogenic fungi have failed to survive during the testing process because the

The most successful fungicide in the market today acts 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 progresses 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

target site of the fungicide is found in another organism (Thines *et al.,* 2004).

binding to plant cells (Noda *et al,* 2010).

**4. Morphogenesis** 

and development could be target site for new fungicides.

compounds are structurally based on natural products and interfere with the ubiquinol cytochrome C oxidoreductase (Foster & Thines, 2009).

There are numerous antifungal compounds that were discovered from diverse microbial sources using traditional activity-based screening techniques. These microbial compounds showed potent control efficacy against various plant diseases, including chronic diseases which are difficult to control with conventional synthetic fungicides. Advances in screening systems directed to specific targets of fungal metabolism have increased the opportunities to discover novel antifungal agents with selectivity over non-target organisms. Microbial metabolites have also been exploited as a source for non-fungicidal disease control agents that do not inhibit vegetative hyphal growth, but rather interfere specifically with the infection process of pathogenic fungi, such as spore germination, the formation of penetration structures and sporulation (Seok & Kook, 2007). Infection structures of phytopathogenic fungi are modified hyphae specialized for the invasion of plant tissue, initial events are adhesion to the cuticule and directed growth of the germ tube on the plant surface (Mendgen *et al.,* 1996). The specificity of microbial fungicides is a highly preferred characteristic in terms of impacting the environment, where it is closely related to the occurrence of fungicide resistance. The most recently developed fungicides from microbial metabolites, the strobilurins, provide a cue for the high risk of resistance development of site-specific fungicides.

These compounds have long been applied to control fungal diseases of rice, vegetables and fruits, and their effectiveness in controlling these diseases has been tested in the field and proven over many years. The importance of microbial fungicides, compared to synthetic compounds, may have been under evaluated in the past due to the limitation in their activity spectrum and in certain instances, the development of resistance (Knight *et al.,* 1997). Nevertheless, the excellent fungicidal activity of these microbial metabolites and their potential as lead candidates for further fungicide development continue to stimulate research and screening for antifungal microbial metabolites, some examples of this compounds are:

**Kasugamycin** is an amino-sugar compound discovered from the metabolites of *Streptomyces kasugaensis, in vivo* studies have shown that kasugamycin efficiently suppresses the development of *M. grisea* mycelia on rice plants in both preventive and curative treatments, to overcome potential resistance problems, mixtures of kasugamycin with different synthetic fungicides having different modes of action are currently in use.

**Polyoxins** are peptidylpyrimidine nucleoside antibiotics isolated from the culture broth of *Streptomyces cacaoi* var. *asoensis*, such excellent characteristics come from the fact that polyoxins selectively inhibit the synthesis of cell-wall chitin in sensitive fungi but have no adverse effects on organisms lacking chitinous cell walls.

**Validamycin A** produced by *Streptomyces hygroscopicus* var. *limoneus* has been effective in controlling rice sheath blight caused by *R. solani*. Validamycin A is converted within fungal cells to validoxylamine A, an extremely strong inhibitor of trehalose. The mode of action of validamycin A is favorable for biological selectivity, because vertebrates do not depend on the hydrolysis of trehalose (Doumbou *et al.,* 2001).

Target-Site-Specific Screening System for Antifungal Compounds 189

A major challenge for drug development today is how best to employ the new genome wide technologies to identify which metabolic pathways and gene products are critical for disease establishment and progression and thus to increase the probability of finding novel fungicide targets. Novel targets can be located either in biochemical or signaling 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, hostrecognition/pre-penetration processes, host colonization 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

We would like to conclude by stating that antifungal targets-site are extremely diverse. However, substances that acts on these target-sites needs to fulfill several prerequisites such as antifungal activity *in vivo* and lack of effects on the host cells. Furthermore, resistance mechanisms need to be excluded as far as possible. Therefore, investigation of the target site and the mode of action of an antifungal compound can be explored by statistical learning algorithms. Performance and applicability of the statistical learning methods in studying "fungal-target likeness" may be further improved by incorporation of new information from advances in genomic, proteomics, pathogenesis and morphogenesis studies. Efficiency and accuracy of statistical learning methods in the prediction of fungal-target like proteins can also be enhanced from new progress in

Abramovitch, R. B., Anderson, J. C. & Martin, G. B. (2006). Bacterial elicitation and evasion of plant innate immunity. *Nature Reviews Molecular Cell Biology*. 7:601-611. Albeck, S., Alzari, P., Andreini, C. , Banci, L., Berry, M., Bertini, I.,Cambillau, C., Canard, B.,

Barakat, H. Spielvogel, A., Hassan, M., El-Desouky A., El-Mansy H., Rath, F. Meyer, V. &

Stahl, U. (2010). The antifungal protein AFP from *Aspergillus giganteus* prevents secondary growth of different *Fusarium* species on barley. *Applied Microbiology and* 

Carter, L., Cohen, S. X., Diprose, J. M., Dym, O., Esnouf, R. M., Felder, C., Ferron, F., Guillemot, F., Hamer, R., Ben Jelloul, M., Laskowski, R. A., Laurent, T., Longhi, S. Lopez, R., Luchinat, C., Malet, H., Mochel, T., Morris, R. J., Moulinier, L., Oinn, T.,Pajon, A., Peleg, Y., Perrakis, A., Poch, O., Prilusky, J., Rachedi, A., Ripp, R., Rosato, A., Silman, I., Stuart, D. I., Sussman, J. L., Thierry, J.C., Thompson, J. D.,Thornton, J. M., Unger, T.,Vaughan, B., Vranken, W., Watson, J. D., Whamondg, G. & Henrickg K. (2006). SPINE bioinformatics and data management aspects of high throughput structural biology. *Acta Chrystallographic*. D62:1184-

affecting neutral or benign species (Foster & Thines, 2009).

learning algorithms and sequence descriptors.

**5. Conclusion** 

**6. References** 

1195.

*Biotechnology*. 87:617–624.

Promising microbial metabolites continue to be discovered using traditional activity-based screening procedures against various plant pathogenic fungi. In particular, many of the sitespecific antifungal metabolites have recently been discovered from microbial metabolites. These microbial metabolites include non-fungicidal compounds that interfere with the infection process of pathogenic fungi, and specific inhibitors of the fungal biosynthetic pathways for chitin, fatty acids and nucleic acids (Seok & Kook, 2007).

Other kind of antifungal agents are proteins; antifungal proteins have been isolated from various organisms ranging from bacteria, plants, insects and amphibians to human beings. Both their fungal target site and their mode of action are extremely diverse. In order for it to be applied, an antifungal protein needs to fulfill several prerequisites such as antifungal activity *in vivo* and lack of effects on the host cells. Furthermore, resistance mechanisms need to be excluded as far as possible. Therefore, investigation of the target site and the mode of action of an antifungal protein should reveal whether the protein is suitable for an application (Theis *et al.,* 2005). The antifungal protein (AFP) are abundantly secreted by the filamentous fungus *Aspergillus giganteus*, this cysteine-rich protein have ability to disturb the integrity of fungal cell walls and plasma membranes but does not interfere with the viability of other eukaryotic systems (Barakat *et al.,* 2010; Meyer, 2008).

Severe membrane alterations in *A. niger* were observed, whereas the membrane of *P. chrysogenum* was not affected after treatment with AFP. The protein localized predominantly to a cell wall attached outer layer which is probably composed of glycoproteins, as well as to the cell wall of *A. niger*. It was found to accumulate within defined areas of the cell wall, pointing towards a specific interaction of AFP with cell wall components. In contrast, very little protein was bound to the outer layer and cell wall of *P. chrysogenum*. The protein was found to act in a dose-dependent manner: it was fungistatic when applied at concentrations below the minimal inhibitory concentration, but fungicidal at higher concentrations. Using an *in vivo* model system was demonstrated that AFP indeed prevented the infection of tomato roots (*Lycopersicon esculentum*) by the plant-pathogenic fungus *Fusarium oxysporum* f. sp. *Lycopersici* (Theis *et al*., 2005).

The fungal profilins, small actin-binding proteins that share limited homology to human profilin, can operate as a potential drug target, since these proteins are essential for the growth of most eukaryotic cells, including *S. cerevisiae* (Witke, 2004). Addition, the existence of structural information can support the design of structure-based ligands for profilins. Peptides are generally used as lead compounds in drug development, to design a novel peptide ligand, an *in vitro* evolution approach has often been used. Although this approach can be used without three-dimensional (3D) structural information about the target protein, it requires laborious experimental procedures, including library constructions and the screening of bioactive peptide ligands. In this respect, if information on the structure and the active site of the target protein is known, an *in silico* approach based on the 3D structure of the target protein is a useful approach to designing the peptide ligand. The validity of the profilin as antifungal drug target was evaluated by Ueno *et al*. 2010, amino acid alignments showed the low homology between human and fungal profilins. This implies that the fungal profilin could be a target with high selectivity. Furthermore, a mouse infection study showed that the suppression of profilin expression attenuated the fungal burden in the kidney and indicated that the profilin was required for survival in the host's body (Ueno *et al.,* 2010).

A major challenge for drug development today is how best to employ the new genome wide technologies to identify which metabolic pathways and gene products are critical for disease establishment and progression and thus to increase the probability of finding novel fungicide targets. Novel targets can be located either in biochemical or signaling 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, hostrecognition/pre-penetration processes, host colonization 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 (Foster & Thines, 2009).
