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Title: CONSTRUCTION OF A SHUTTLE VECTOR AND TRANSFORMATION OF XYLELLA FASTIDIOSA WITH PLASMID DNA

Author
item QIN, XIAOTING - VISITING SCIENTIST
item Hartung, John

Submitted to: Current Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/26/2001
Publication Date: N/A
Citation: N/A

Interpretive Summary: Symptoms of Citrus Variegated Chlorosis disease caused by Xylella fastidiosa currently occur on 68 million sweet orange trees in Sao Paulo State Brazil. The disease is a threat to citrus in the United States. No resistant varieties are available and the pathogen is spread by at least 13 species of insects. The threat to the industry prompted an ambitious project to sequence the genome of this pathogen in Sao Paulo. This project was successful, and the genome sequence is now available. Until now however, there has not been a way to use the genome sequence data to develop control measures because there has been no system for genetic manipulation of the pathogen. Genetic manipulation is required to confirm any hypotheses about the basis of symptom expression, vector transmission etc., which will underlie any prospective control measures. We report here the first step towards the development of a genetic system with the creation of a system for the transformation of Xylella fastidiosa with DNA manipulated in the laboratory. Our system is based on a naturally occurring DNA element from Xylella fastidiosa combined with a similar element from a common laboratory bacterium. We have also for the first time introduced purified DNA into the pathogen. Our methods will be invaluable for researchers in Brazil who wish to study mechanisms of pathogenicity and control of CVC disease, and also for researchers in the United States who wish to obtain similar information about Pierce's Disease of grapevine, which is caused by similar bacteria and which is currently ravaging vineyards in California and the SE United States.

Technical Abstract: We have isolated, cloned and sequenced a 5823-bp cryptic plasmid from a strain of Xylella fastidiosa. This plasmid encodes five open reading frames (ORF) greater than 400 nucleotides each. ORF 2 encodes a protein with 37 percent amino acid identity to the replication initiator protein of plasmid pECB2 from Pseudomonas alcaligenes. This RepA protein from X. fastidiosa contains both a leucine zipper and helix turn helix motif characteristic of proteins involved in DNA replication. The sequence 5' of ORF 2 has all of the features characteristic of plasmid origins of replication as well as regulatory elements required for transcription of ORF 2. Open reading frame 2 along with the upstream origin of replication were cloned as an EcoRI fragment into pUC19 to create a shuttle vector. This construct was introduced into Xylella fastidiosa by electroporation, with selection for carbenicillin resistance. Transformation was verified by both PCR and Southern hybridization experiments. Frequency of transformation was low, but increased 10-fold when the plasmid was grown in X. fastidiosa rather than Escherichia coli prior to transformation. This work represents the first step towards the development of a system for genetic analysis of this important plant pathogen.