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Title: TRANSFORMATION OF XYLELLA FASTIDIOSA WITH A PLASMID DNA

Author
item QIN, XIAOTING - USDA/REE/ARS/PSI/FL
item Hartung, John

Submitted to: BARC Poster Day
Publication Type: Abstract Only
Publication Acceptance Date: 4/15/2000
Publication Date: 4/20/2000
Citation: Qin, X., Hartung, J.S. 2000. Transformation of xylella fastidiosa with a plasmid dna. BARC Poster Day.

Interpretive Summary:

Technical Abstract: Xylella fastidiosa causes a serious disease of sweet orange in Brazil. The complete nucleotide sequence of a strain of this pathogen is being determined in Brazil. Strain Lar20, from sweet orange has a plasmid that we designate pXf5823. A unique Sph1 restriction enzyme site was used to clone pXf5823 in pUC19 to create Pxy1s 2 for sequence analysis. There are 5 open reading frames over 400 np within this 5823 bp plasmid. Three EcoR I fragments were separately cloned into pUC 19 to construct putative shuttle vectors designated pEcoR 10, pEcoR 11, pEcoR 12. Electroporation was used to transform Xylella fastidiosa strain Lar20 using shuttle vectors pXY1S 2, pEcoR 10, pEcoR 11, pEcoR 12. Three Xylella fastidiosa colonies were found on carbenicillin plates after tranformation with pEcoR 10. Transformation was demonstrated by reisolation of pEcoR 10 from the putative LAR20(pEcoR 10), Southern hybridization of LAR20(pEcoR 10) extracts with pUC19 as the hybridization probe, and a PCR assay using Xylella fastidiosa specific primers. Transformants grew well in the presence of 50 ug carbenicillin ml in solid and liquid media. Plasmid extracted from LAR20(pEcoR 10) was used to transform Xylella fastidiosa strain Lar20 again, and the transformation rate was increased ten-fold. Replica plating on carbenicillin and plain media showed that pEcoR 10 was unstable in LAR20. We also transformed another Xylella fastidiosa strain, Taq29, with pEcoR 10. pEcoR 10 contains a 2.5 kb fragment of pXf5823 which has homology with replication initiator protein of Pseudomonas alcaligenes. This is the first report of transformation of this species, and is a first step toward a system of genetic analysis.