Stable Transformation of the Xylella fastidiosa Citrus Variegated Chlorosis Strain with oriC Plasmids

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Abstract
Xylella fastidiosa is a gram-negative, xylem-limited bacterium affecting economically important crops (e,g,, grapevine, citrus, and coffee). The citrus variegated chlorosis (CVC) strain of X. fastidiosa is the causal agent of this severe disease of citrus in Brazil and represents the first plant-pathogenic bacterium for which the genome sequence was determined. Plasmids for the CVC strain of X. fastidiosa were constructed by combining the chromosomal replication origin (oriC) of X. fastidiosa with a gene which confers resistance to kanamycin (Kan(r)). In plasmid p16KdAori, the oriC fragment comprised the dnaA gene as well as the two flanking intergenic regions, whereas in plasmid p16Kori the oriC fragment was restricted to the dnaA-dnaN intergenic region, which contains dnaA-box like sequences and AT-rich clusters. In plasmid p16K, no oriC sequence was present. In the three constructs, the promoter region of one of the two X, fastidiosa rRNA operons was used to drive the transcription of the Kan(r) gene to optimize the expression of kanamycin resistance in X. fastidiosa, Five CVC X. fastidiosa strains, including strain 9a5c, the genome sequence of which was determined, and two strains isolated from coffee, were electroporated with plasmid p16KdAori or p16Kori, Two CVC isolates, strains J1a12 and B111, yielded kanamycin-resistant transformants when electroporated with plasmid p16KdAori or p16Kori but not when electroporated with p16K, Southern blot analyses of total DNA extracted from the transformants revealed that, in all clones tested, the plasmid had integrated into the host chromosome at the promoter region of the rRNA operon by homologous recombination, To our knowledge, this is the first report of stable transformation in X. fastidiosa, Integration of oriC plasmids into the X. fastidiosa chromosome by homologous recombination holds considerable promise for functional genomics by specific gene inactivation.