Author
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QIN, XIAOTING - VISITING SCIENTIST, ARS |
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Hartung, John |
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Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal Publication Acceptance Date: 4/14/2004 Publication Date: 7/31/2004 Citation: Qin, X., Hartung, J.S. 2004. Use of triparental mating system to introduce green fluorescent protein marked transposon insertions in the plant pathogen xylella fastidiosa.. Applied and Environmental Microbiology. 49 (3):211-216. Interpretive Summary: Xylella fastidiosa causes Pierce's Disease of grapevines in the United States and Citrus Variegated Chlorosis and Coffee Leaf Scorch diseases in Brazil. The citrus strain of Xylella fastidiosa became the first plant pathogen to have its genome sequence completely determined by a research consortium in Brazil. In order to exploit the genome sequence information and control the disease, genetic analysis is required to assign roles for genes in the disease process. We have for the first time introduced foreign DNA into Xylella fastidiosa by mating with strains of another bacterium. The DNA we have introduced inserts itself into host genes, creating strains that have specific mutations. In our work, we inserted a gene that encodes green fluorescent protein, and so our mutants fluoresce with a bright green color when illuminated with blue light. This 'green label' makes the bacteria easy to observe in plants and will improve studies of the movement of the pathogen in plants. We have inoculated our marked strains into both sweet orange and tobacco, and the strains colonized the plant vascular system in a typical manner. When we recovered the bacteria from the plants eight weeks later, we observed that the intensity of the green fluorescent signal produced by the bacteria had been significantly increased compared to the signal observed from strains maintained continuously in laboratory culture. The increased fluorescence was transient, and fluorescence returned to the base level after further culture in the laboratory. We have established the basis for genetic analysis of Xylella fastidiosa, which will be very useful for scientists seeking to control these diseases in the United States and Brazil. Technical Abstract: Xylella fastidiosa, a gram-negative bacterial plant pathogen, causes many serious diseases of fruit trees in North America. In Brazil the pathogen causes the recently described Citrus Variegated Chlorosis and Coffee Leaf Scorch diseases. A consortium of researchers in Brazil has determined the complete nucleotide sequence of the genome of a citrus strain of the pathogen. However, in order to exploit the genomic sequence data to enable effective disease control, systems for genetic manipulation of the pathogen are necessary, but have thus far been completely lacking. We report the introduction of foreign DNA into a citrus strain of Xylella fastidiosa by use of a triparental mating system. With this system, we have introduced a mini-Tn5 transposon that encodes a Green Fluorescent Protein (GFP) gene optimized for expression in bacteria. The mini-Tn5 derivative was inserted into different sites of the genome in independent transconjugants as determined by Southern blotting. The GFP gene was also expressed well in Xylella fastidiosa, and to different levels in different transconjugants. Four independent transconjugants were separately used to inoculate sweet orange and tobacco seedlings. When the relative fluorescence of the transconjugants that had been passed through either tobacco or sweet orange was compared to that of the same transconjugant maintained continuously in vitro, we observed that passage through either plant host significantly, but transiently, increased the level of expression of the GFP. We have developed a system for the introduction of marked mutations which will be useful for both in vitro and in planta analysis of gene expression of Xylella fastidiosa. |
