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Transposon Mutagenesis of Xylella fastidiosa by Electroporation of Tn5 Synaptic Complexes

June 2001 , Volume 14 , Number  6
Pages  701 - 706

Magalie R. Guilhabert , 1 Les M. Hoffman , 2 David A. Mills , 3 and Bruce C. Kirkpatrick 1

1Department of Plant Pathology, University of California-Davis, 1 Shields Avenue, Davis 95616, U.S.A.; 2Epicentre Technologies, 1202 Ann Street, Madison 53713, WI, U.S.A; 3Department of Viticulture and Enology, University of California-Davis, 1 Shields Avenue, Davis 95616, U.S.A.

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Accepted 2 March 2001.

Pierce's disease, a lethal disease of grapevine, is caused by Xylella fastidiosa, a gram-negative, xylem-limited bacterium that is transmitted from plant to plant by xylem-feeding insects. Strains of X. fastidiosa also have been associated with diseases that cause tremendous losses in many other economically important plants, including citrus. Although the complete genome sequence of X. fastidiosa has recently been determined, the inability to transform or produce transposon mutants of X. fastidiosa has been a major impediment to understanding pathogen-, plant-, and insect-vector interactions. We evaluated the ability of four different suicide vectors carrying either Tn5 or Tn10 transposons as well as a preformed Tn5 transposase-transposon synaptic complex (transposome) to transpose X. fastidiosa. The four suicide vectors failed to produce any detectable transposition events. Electroporation of transposomes, however, yielded 6 × 103 and 4 × 103 Tn5 mutants per μg of DNA in two different grapevine strains of X. fastidiosa. Molecular analysis showed that the transposition insertions were single, independent, stable events. Sequence analysis of the Tn5 insertion sites indicated that the transpositions occur randomly in the X. fastidiosa genome. Transposome-mediated mutagenesis should facilitate the identification of X. fastidiosa genes that mediate plant pathogenicity and insect transmission.

© 2001 The American Phytopathological Society