10th INTERNATIONAL VERTICILLIUM SYMPOSIUM 16-20 ...

IDENTIFICATION AND CHARACTERIZATION OF A MFS-TRANSPORTER GENE FROM VERTICILLIUM DAHLIAE INTHE INTERACTION WITH OLIVE (OLEA EUROPAEA L.)CARMEN NAVARRO-RAYA 1 , ENRIQUETA MOYANO 2 , JOSELIN BENÍTEZ-ALFONSO 2 , KATHERINE F. DOBINSON 3, 4 , JUAN MUÑOZ-BLANCO 2 , ANDJESÚS MERCADO-BLANCO 11 Dep. Protección de Cultivos, Instituto de Agricultura Sostenible (CSIC), Apartado 4084, 14080Córdoba (jesus.mercado@ias.csic.es), and2 Dep. Bioquímica y Biología Molecular, Edificio Severo Ochoa, Universidad de Córdoba, Campus deRabanales, 14071- Córdoba, Spain;3 Southern Crop Protection and Food Research Centre, AAFC and 4 Department of Biology, Universityof Western Ontario, London, Canadá.Verticillium wilt of olive (VO) is one of the most serious diseases affectingthis woody crop worldwide, and may cause severe losses and plant death. Severity ofattacks by Verticillium dahliae depends upon virulence of the pathogen isolates,which can be classified into defoliating (D) and nondefoliating (ND) pathotypes basedon their ability to cause or not defoliation of green leaves from shoots and twigs,respectively. Investigations of VO have traditionally focused on studies ofpathogenicity and resistance mechanisms, primarily from biochemical,epidemiological and physiological viewpoints. However, knowledge is scarce of themolecular basis, either for resistance in olive cultivars or pathogenicity/virulence ofpathotypes of V. dahliae. In order to identify genes involved in the V. dahliae-oliveinteraction, two suppression subtractive hybridization libraries were obtained,potentially enriched in genes whose expression is induced (up regulated) or repressed(down regulated) during infection of olive by V. dahliae. Sequencing of some 1000ESTs has enabled us to select several ESTs whose sequences showed high sequencesimilarity to genes from other fungal species. Here, we present the identification,characterization and disruption by transposon mutagenesis of a putative majorfacilitator superfamily (MFS) transporter gene of V. dahliae. DNA and proteinsequence analyses have revealed high similarity with members of this gene familyfound in different fungi. Blast analysis revealed that the deduced protein sequencewas 60% identical to that of Cryptococcus neoformans. Gene expression studies(quantitative real-time RT-PCR) indicated that the putative V. dahliae MFStransportergene is expressed under different growth conditions, and even induced inculture medium simulating the xylem fluid. Finally, targeted gene disruption hasbeen accomplished by inserting the hygromycin-resistance gene cassette into severalpositions within the MFS coding region. Several disruption plasmids are available,and mutants are expected to be produced in a representative isolate of the V. dahliaeD pathotype by A. tumefaciens-mediated transformation. Mutants will be valuable tostudy the role of this gene in the V. dahliae-olive interaction.53