Heterologous transposition in Colletotrichum gloeosporioides

HETEROLOGOUS TRANSPOSITION IN COLLETOTRICHUM GLOEOSPORIOIDES Li Destri Nicosia M.G.1, Cacciola S.O.2, Agosteo G.E.,1 Magnano di San Lio G.1 1Dipartimento di Agrochimica ed Agrobiologia, Mediterranean University of Reggio Calabria, P.zza S. Francesco di Sales, 4 – 89061 Gallina (RC), phone: +390965689057; fax: +390965689049; e-mail: giulia.lidestri@unirc.it; 2Dipartimento di Scienze Entomologiche, Fitopatologiche, Microbiologiche agrarie e Zootecniche, Plant Pathology Section, University of Palermo, Italy Colletotrichum gloeosporioides (Penz.) Penz. et Sacc. is a genetically species complex, infecting a wide range of host plants, including olive, causing anthracnose. An insertional mutagenesis strategy, using the heterologous transposon impala of Fusarium oxysporum, is being developed with the aim of isolating genes involved in the pathogenicity process of olive anthracnose. A phenotypic excision test was used to detect transposon excisions. An impala construction, carrying the transposon inserted into the promoter region of the Aspergillus nidulans nitrate reductase (niaD) gene, was introduced by transformation into a stable C. gloeosporioides nia- mutant. Revertants able to grow on nitrate were then selected among the niaD::impala transformants. Transposition of impala in C. gloeosporioides was demonstrated by analysing the genomic DNA of the nia+ revertants in Southern blotting experiments. It was demonstrated that the transposon excised from the niaD gene and it reinserted in the genome (Fig.1). In the upper blot hybridization with a niaD probe is shown. Upon transposon excision nia+ revertants presented a new band (2.7 Kb), as indicated by a circle, corresponding to the niaD wt signal. In the lower blot, hybridization was performed by an impala probe and it demonstrated the reinsertion of the transposon excised. Revertants showed the appearance of an impala band in a new position with respect to the original transformant, as indicated by squares. The high percentage of reinsertion observed is an important feature for the development of a transposon tagging strategy using impala. Further demonstration of impala excision was the typical impala footprints found into the niaD site of excision (Fig. 2). Pathogenicity assays on different host species, including olive and apple fruits, are currently being carried out. Preliminary results of a screening test on apples suggest that some revertants have a lower ability to colonise plant tissue than the original transformant.