Cantoral, Jesús Manuel

Carbú, María

Fernández-Acero, Francisco Javier

Garrido, Carlos

Victoria E., González-Rodríguez

Repositorio Institucional de la Universidad de Córdoba

126 3527(Ï0,&$1Ò0(52)(%5(52AbstractTwo dimensional gel electrophoresis (2-DE) plus mass spectrometry (MS) have been widely used to study the proteome of a good number of tissues, plants, animals or microorganisms. However, few of them have been carried out to study the proteome of phytopathogenic fungi. These organisms are res-ponsible of several plant diseases in different crops around the world, causing very important economic losses to the farmers. There are several reasons to explain the lack of proteomic studies describing fun-gal plant pathogen proteomes. The main aim of this work is to tackle the strategies to overcome these SUREOHPVLHE\WKHPRGL¿FDWLRQRISURWHLQH[WUDF-tion protocols, or using microbiology techniques to decrease the analytical variability.Main textFungal plant pathogens are one of the main res-ponsible of farmer losses. By way of example, the annual consumption of fungicides against phytopa-thogenic fungus Botrytis cinerea (Botrycides) moves about 10% of global fungicide market, which would mean 540 million per year [1]. Moreover, the losses increase with the value of agricultural losses, quan-WL¿HGEHWZHHQDQGPLOOLRQ(XURVSHU\HDUThe losses caused by B. cinerea in French vineyards are between 15% and 40% per year, depending on weather conditions. In Holland, B. cinerea produces DORVVLQÀRZHUFURSVDQGLQ6SDLQin the strawberry crops. Annual average expenditure on fungicides against Botrytis, is around 3 million Euros in tomato crops, 2 million in strawberry and 3 million in grapewine crops (BASF, personal com-munication). The genus Botrytis is an ascomycetes ¿ODPHQWRXVIXQJXVZKLFKLQFOXGHVDODUJHQXPEHUof pathogenic species capable of infecting a variety of crops. Some species are able to infect a single Applying proteomic technologies to dissect molecular aspect of phytopathogenic fungi, a Botrytis cinerea approachFrancisco Javier Fernández-Acero, Carlos Garrido, María Carbú, Victoria E. González-Rodríguez, Jesús Manuel CantoralLaboratory of Microbiology, Marine and Environmental Sciences Faculty, University of Cádiz, Pol. Río San Pedro s/n, Puerto Real, 11510, Cádiz, Spaincrop, such as B. tulipae, B. squamosa or B. fabae,pathogen to tulips, bean and onions respectively. However, B. cinerea is able to attack more than 200 species, including tomatoes, strawberries and gra-pes, three of the most important crops in Andalusia. 7KLVIXQJXVKDVJUHDWÀH[LELOLW\SUHIHUDEO\LVDEOHto attack fruit (grapes, strawberries, tomatoes, etc.), ÀRZHUVEXWDOVRLVDEOHWRDWWDFNVWHPVOHDYHVDQGseeds. Moreover, it is also capable to attack many different plants (grapevine, tomato, Arabidopsis,roses, etc.), in different organs of the same plant (fruits, leaves, petals and other structures of the same plant). Furthermore, the disease may appear directly “in planta” or during storage and distribu-tion of fruits. These data show the relevance of this pathogen that is being currently considered as a mo-del organism in all those experimental approaches to the study of plant-pathogen interaction.The fact that there are only few reports on the proteome of phytopathogenic fungi is mainly due WRSUREOHPVVXFKDVWKHGLI¿FXOW\LQREWDLQLQJIXQ-gal protein extracts or the lack of available fungal protein databases [2]. An effective protein extrac-tion is a key step for obtaining a good resolution avoiding streaking in the 2-DE gels. It will allow us to remove contaminants (i. e. nucleid acid, salts, lipids, pigments) which may disturb the proteins focusing. However, it is especially relevant when WKH ELRORJLFDO VRXUFH LV D¿ODPHQWRXV IXQJXVGXHto its richness in cell wall polysaccharides, and the mechanical resistance of the fungal cell wall. This problem has been overcome by using phenol ba-sed protocol [3]. On the other hand, the amount of genomic resources is increasing continuously, and DERXWIXQJDOJHQRPHSURMHFWVDUHEHLQJ¿QLVKHG(Broad Institute, MIT, Harvard). This information is FUXFLDOWRJHWVLJQL¿FDQWQXPEHURISRVLWLYHSURWHLQLGHQWL¿FDWLRQKLWV,QVSLWHRIWKHVHSUREOHPVVHHPWRbe overcame, there are still some points that become 3527(Ï0,&$1Ò0(52)(%5(52 127FULWLFDODSURWHRPLFDSSURDFKWR¿ODPHQWRXVIXQJDOproteome. In spite of other aspects, we are especia-OO\LQWHUHVWHGLQWZRPDMRU¿HOGVLWRGHFUHDVHWKHlevels of variability founded in these studies, and (ii) WRLQFUHDVHWKHEHQH¿WREWDLQHGIURPWKHODUJHOLVWRILGHQWL¿HGSURWHLQVCalculations of analytical and biological varia-bility are directed to quantify the variations associa-ted with the 2-DE experiments and differences in the level of protein expression between independent re-plicates [4]. The averages of analytical and biologi-FDOFRHI¿FLHQWVRIYDULDQFHDUHXVHGWREHFDOFXODWHG[5]. Only those changes consistently present in all the extract replicates, whose quantitative differences are higher than the corresponding biological and analytical variance, should be considered [2]. In spite of the levels obtained in fungal approaches are similar to the obtained variability in plant or micro-RUJDQLVPVWXGLHV>@:HKDGXVHGPRQRFRQLGLDOisolates in attempt to decrease these levels, but the UHVXOWV UHPDLQKLJK2Q WKH RWKHU KDQG WKH¿QDOresults obtained in a proteomic approach use to be DODUJHOLVWRILGHQWL¿HGSURWHLQV7KHUROHRIHDFKprotein is also discussed for its biological relevance, or used “in further studies”. This information is very important and interesting, but if we apply our own and particular “optics”, we assume the risk of infor-mation losses in other parallel biological process. As an interesting tool is the website PANTHER (Pro-tein ANalysis THrough Evolutionary Relationships; ZZZSDQWKHUGERUJ WKDW DOORZV WKH FODVVL¿FDWLRQRILGHQWL¿HGSURWHLQVLQGLIIHUHQWFDWHJRULHVDFFRU-ding to (i) its biological process or (ii) its molecular function, based on Gene Ontology. This information PD\KLJKOLJKWVRPHLQIRUPDWLRQZKLFKLVGLI¿FXOWWRextract directly from a protein list. In this sense, an algorithm to elucidate the drug target-likeness of a protein has been published [7]. This work could im-prove the development of new therapeutic targets to ¿JKWDJDLQVWVHYHUDOGLVHDVHV8QIRUWXQDWHO\DWWKLVmoment is not feasible to develop similar approach to analyse new candidates for fungicide design (per-sonal communication).References[1] UIPP, Union des Industries de la Proteccion des Plantes, Boulogne 2002.[2] Fernández-Acero FJ, Carbú M, Garrido C, Va-llejo I and Cantoral JM. Proteomic Advances in Phytopathogenic Fungi. Curr Proteomics 2007;4:79-88.[3] Fernández-Acero FJ, Colby T, Harzen A, Can-toral JM and Schmidt J. Proteomic analysis of the phytopathogenic fungus Botrytis cin-erea during cellulose degradation. Proteomics 2009;9:2892-902.[4] Fernández-Acero FJ, Carbú M, Garrido C, Collado IG, et al. Screening Study of Potential Lead Compounds for Natural Product-based Fungicides Against Phytophthora Species. J. Phytopathol 2006;154:616-21.[5] Jorge I, Navarro RM, Lenz C, Ariza D, et al. The Holm Oak leaf proteome: Analytical and biological variability in the protein expression OHYHODVVHVVHGE\'(DQG3URWHLQLGHQWL¿FDWLRQtandem mass spectrometry de novo sequencing and sequence similarity searching. Proteomics 2005;5:222–34.[6] Fernández-Acero FJ, Jorge I., Calvo E, Vallejo I, et al. Proteomic analysis of phytopathogenic fungus Botrytis cinerea as a potential tool for identifying pathogenicity factors, therapeutic targets and for basic research. Arch of Microbiol 2007;187:207–15.>@ +XDQ;+DQJ<DQJ;0LQJ=KL/:HL:HWDOLearning the drug target-likeness of a protein. Proteomics, 2007;7:4255-63.

Applying proteomic technology to dissect molecular aspect of phytopathogenic fungi, a Botrytis cinerea approach

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