Transformation of the entomopathogenic fungus Paecilomyces fumosoroseus using T-DNA from Agrobacterium tumefaciens .

Marcia Cristina Furlaneto, Ivan Gláucio Paulino Lima, Rubens Tadeu Delgado Duarte, Luciana Furlaneto, and Maria Helena Pelegrinelli Fungaro.

Author address: 

Universidade de Londrina, Microbiologia, Londrina, Parana Brazil

Abstract: 

Paecilomyces fumosoroseus causes diseases in the whitefly Bemisia tabaci which is responsable for damage to a variety of high value crops worldwide. Agrobacterium tumefaciens-mediated transformation was successfully applied to P. fumosoroseus. Conidia of P. fumosoroseus were transformed to hygromycin B resistance using the hph gene of Escherichia coli as the selective trait, under the control of a heterologous gpd promoter and trpC terminator from Aspergillus nidulans. The efficiency of transformation was up to 74, 127 and 247 transformants per 105, 106 and 107 target conidia, respectively. Following 5 serial passages of transformants on non-selective medium, 100% of the transformants were found to be mitotically stable by a conidial germination test. A.tumefaciens-mediated transformation yielded stable transformants capable of growing on increased concentrations of hygromycin B (up to 900 ug/ml).The presence of hph gene was confirmed by PCR. Southern analysis revealed that the P. fumosoroseus transformants contained a randomly integrated single ou multiple T-DNA copies in the genome. Inverse PCR will be used to recover the P. fumosoroseus DNA flanking the T-DNA. Considering the efficiency and flexibility of A. tumefaciens-mediated transformation, this method appears to be an efficient alternative to other insertional mutagenesis techniques in characterizing genes that are important for the pathogenicity of P. fumosoroseus. Acknowledgements: CNPq/CAPES/CPG-UEL
2005

abstract No: 

572

Full conference title: 

23rd Fungal Genetics Conference
    • Fungal Genetics Conference 23rd (2002)