Transformation of the entomopathogenic fungus Paecilomyces fumosoroseus with Agrobacterium tumefaciens
2006
Lima, I.G.P. | Duarte, R.T.D. | Furlaneto, L. | Baroni, C.H. | Fungaro, M.H.P. | Furlaneto, M.C.
To test the suitability of the Agrobacterium tumefaciens-mediated transformation (AMT) method with Paecilomyces fumosoroseus, a fungal pathogen that causes diseases in a wide range of insects including whiteflies. Conidia of P. fumosoroseus were successfully transformed to hygromycin B resistance using the hph gene of Escherichia coli as the selectable marker. Transformation frequencies were 58·3 ± 18·5, 98·3 ± 24·8 and 169·7 ± 35·5 (±SEM) transformants per 10⁵, 10⁶ and 10⁷ target conidia respectively. After confirmation by PCR, transformants were subjected to Southern analysis, and the results revealed that 45% (four of nine) of the transformants contained single-copy integration of the T-DNA. In our AMT system, we efficiently transformed conidia of P. fumosoroseus. The employment of this method circumvents time-consuming protoplast preparation and allows the isolation of transformants containing single-copy integration of the T-DNA. Considering the efficiency of Ag. tumefaciens-mediated transformation, this method represents a useful tool for insertional mutagenesis to characterize genes that are important for the pathogenicity of P. fumosoroseus.
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