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Forgot password? Old Password. New Password. Password Changed Successfully Your password has been changed. Proteomics is very useful for mapping proteomes, isolation of subproteomes and studying protein-protein interaction [ 8 ]. Moreover, proteomic studies are crucial in order to understand protein post-translational modifications PTMs.
There are more than PTMs described, allowing us to identify between 10 and different biological functions from each gene. This compares with around 32, different genes estimated in the human genome, due to alternative splicing, sequence deletions, and PTMs occurring during protein biosynthesis. It is estimated that the total human proteome consists of over a million different protein species [ 9 ]. Proteomic studies have various applications from discovering proteins expressed during infection, to vaccine candidates.
One of the most interesting applications of the proteomics is its use in discovering new protein targets for drug design [ 5 , 10 ]. It involves the identification and early validation of disease-associated targets. Despite the fact that most of the current drug targets are proteins, less than human proteins are actually being used as drug targets. In recent years, pharmaceutical research and development spending has increased, but the number of drugs has not [ 9 ].
USD million and 10 years is required for a pharmaceutical company to bring a new compound to the market. Most of these advances have been applied to solve medical problems. The use of proteomics to search new targets and develop fungicides is still mostly unknown. Fungal phytopathogens are one of the most serious problems in agriculture. They produce large losses to the farmers during plant development, storage and distribution. Moreover, public awareness of the use of chemicals in the food chain, plus the fungicide associated environmental problems, are modifying the rules for fungicide selection and design.
In this review, we will summarize future prospects with regard to fungicide design based on target selection and identification using proteomic approaches. Fungal phytopathogens show a tremendous level of versatility.
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No part of the plant structure is exempt from fungal infections, for example, sweet fruits, leaves, and roots. Moreover, all plant developmental stages may suffer a fungal attack as in planta during fruit distribution. This capacity is supported by a complex fungal life and infection cycle. Fungal plant pathogens have two different reproductive roles Figure 1. During the asexual reproduction cycle anamorphs the fungus produces spores conidia, zygospores, etc. The sexual reproduction teleomorphs is mediated by the creation of sexual gametes, such as B. In most cases, fungal spores asexual and meiotic act as one of the main survival structures.
These organisms are able to maintain their activity in soil or plant debris during winter, waiting for spring to germinate and infect a new harvest. To infect the plant, fungal species need to break through the plant cell walls. This step is mediated by the appressorium that produces a complex set of enzymes and toxins to disarm the plant defenses.
When this is done, a thin invasion hypha penetrates into the plant tissues. To transform the plant biomass into fungal biomass, the thin hypha is transformed into a specific structure named haustorium, which is responsible for plant digestive degradation. Phytopathogenic fungi invade and colonize plant tissues, producing the first disease symptoms. The fungal mycelium then continues its invasion, regularly producing more asexual reproduction structures i. Fungal plant pathogens are a group of microorganisms that show a very high versatility during their infection cycles [ 1 ].
This versatility allows them to infect a wide variety of crops, and also the capacity to survive during unfavorable seasons, such as winter, staying ready to infect when the weather conditions become optimal [ 2 ]. They employ diverse strategies to infect and colonize the plants, and they also establish a complex interaction between fungus species and their hosts [ 13 , 14 ]. Common strategies phytopathogenic fungi use include forming specialized infection structures i.
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Therefore, a considerable number of genes, which encode these proteins, metabolites and toxins, are involved in the infection cycles of phytopathogenic fungi. To avoid misunderstandings of these terms, a pathogenicity factor is defined as a gene, protein or toxin that is necessary for the development of the disease, but is not essential for completing the pathogen life cycle in vitro.
Pathogenicity refers to the capacity of a pathogen to cause disease. Virulence factors are those that are able to regulate the intensity of the infection, and virulence can be considered as the degree of pathogenicity of the corresponding gene, protein or toxin [ 1 ]. Over the years, advances in fungal molecular biology have allowed a better understanding of the strategies used by these phytopathogens. More than 50 genomes of pathogenic fungi are published in the Broad Institute Database for public perusal www. Between these genera, there are human and plant pathogens, including important phytopathogenic species such as: Aspergillus nidulans , A.
This database contains more than one thousand entries, and therefore is a catalogue of experimentally verified pathogenicity, virulence and effector genes from fungal, Oomycete and bacterial pathogens, which infect animal, plant, fungal and insect hosts. Most of the proteomic results achieved during the last years by several groups are also collected in several public databases.
Proteomic techniques have contributed a high amount of data related to the proteins that fungi synthesize and secrete to the environment to complete their infection cycles [ 6 ]. Proteomic approaches, in which complex samples containing hundreds of proteins are identified in an experimental setup, offer a whole new perspective [ 10 ]. Therefore, Proteomics is a powerful potential tool for dissecting the molecular mechanisms underlying fungus-plant interactions [ 30 ]. Valuable knowledge is being obtained from these studies in the functional analysis of gene products and cellular pathways, and proteomics is being used to discover the proteins involved in particular disease stages [ 2 ].
Many other advances have been achieved over the years, demonstrating the usefulness of proteomic techniques in identifying proteins of biological relevance in their infection cycles. Up to four hundred protein spots were resolved in 2-DE after optimizing a protocol for protein extraction using phosphate buffer, followed by TCA-acetone precipitation. Due to the absence of genomic data on B. From this initial proteome map, most of the identified spots may play a crucial role as pathogenicity or virulence factors, including some housekeeping enzymes, such as malate and glyceraldehyde dehydrogenases [ 31 ].
In the last update of the proteome map of B.
As has been mentioned above, the determination of a specific factor as virulence or pathogenicity has been achieved by constructing defective mutants in the specific genes. In both cases, the infection power of the analyzed mutants should at least decrease or disappear compared to the wild types. If the deflections of these genes in mutants produce a loss of vegetal 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 enzyme inhibitors is an archetype, as they would be species specific and the environmental impact would be reduced to a minimum. Antifungal biocides are represented by a wide range of chemical agents.
In fact, it is believed that there is a common series of events starting with interaction at the cell surface followed by passage of a biocide through the fungal cell wall to reach its target site s , but little information is available on the ways by which uptake into fungal cells is achieved, despite longstanding studies of biocide adsorption to yeasts and moulds [ 33 ].
Disease Control in Crops: Biological and Environmentally-Friendly Approaches
There is thus a growing need to establish mechanisms of action for biocides to assist in the design of new compounds or combinations of compounds, in order to understand resistance mechanisms and to provide a focus for toxicological attention. Biocides comprise a heterogeneous group of chemical agents often well characterized in their behavior in diverse applications. Several biocides possess significant antifungal activities that depend on several factors, including concentration, pH, temperature, organic load, interfering substances, and the types of cells.
The activity of biocides against fungal microorganisms is not as well documented as their activity against bacteria. In general, biocides are less active against filamentous fungi than against non-sporulating bacteria [ 34 — 37 ]. But, it is often assumed that fungal inactivation arises by mechanisms similar to, or identical with, those responsible for the destruction of non-sporulating bacteria [ 35 , 38 ]. Studies about the mode of action of antifungal biocides suggest that, unlike antibiotics for which selective action against specific cell targets is fundamental to their clinical value, biocides may act at one or several sites [ 39 , 40 ].
Antifungal substances target a range of cellular loci, from cell wall, plasma membrane to respiratory functions, enzymes and genetic material Table 1. For example Aldehydes, such as gluteraldehyde, have broad spectrum of activity against fungi [ 40 ], which acts by virtue of its intermolecular cross-linking effects on amino groups in cell wall protein [ 41 ].
Exploitation of Diversity within Crops—the Key to Disease Tolerance?
The presence of polymers, such as chitin on cell surfaces, indicates that this former is a potentially reactive site for cross-linking agents gluteraldehyde and formaldehyde [ 35 ]. Other reports indicated that gluteraldehyde agglutinates cells of Candida lipolytica and Saccharomyces carlsbergensis increase their settling rate on the outer cell layers as a result of using an antifungal agent [ 42 ]. For some antifungal agents, such as chlorhexidine, quaternary ammonium compounds QACs , organic acids, esters and alcohols, cell membrane is probably their major target site.
These biocides act by interfering with the structure or permeability of the cell membrane, subsequently altering its barrier function [ 35 , 38 , 43 ]. Thus, chlorhexidine induces leakage of intercellular materials and causes protoplast lysis. It has been observed that bisbiguandine inhibited filamentation probably as a result of some enzyme inhibition at the cytoplasmic membrane level [ 45 ]. Similarly, the toxic effect of some QACs agents against yeast cells resulted from disorganization of the plasma membrane, followed by inactivation of cell enzymes [ 46 ].
Ethanol disrupts the fungal plasma membrane of S. Esters are known as fungistatic agents and, at high concentrations, affect the plasma membrane causing leakage of intracellular constituents. Strobilurin fungicide QoIs , such as azoxystrobin and kresoxim-methyl, are the most important group of fungicide because they are highly effective against a wide variety of fungal pathogen.
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