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Tools to study bacterium now complete

Researchers now have a complete genome sequence of another strain of Xylella fastidiosa — the bacterium that has turned some California vineyards into grape graveyards.

Agricultural Research Service scientists have created tools to study the genes that enable this plant pathogen to do its dirty work. Such knowledge will speed up the chances of finding new ways to control it.

Since the insect that most effectively transmits X. fastidiosa arrived in southern California in 1999, the pathogen has caused $12 million in losses to grape growers there. In Brazil, which boasts the largest citrus industry worldwide, X. fastidiosa damages 35 percent of sweet orange trees in Sao Paulo state. And it infects up to 70 percent of Brazil's coffee bushes, limiting yields.

To learn how each gene functions, researcher knock out that gene, note what changes occur, then restore the gene to validate its function. ARS plant pathologists Xiaoting Qin and John S. Hartung at the Fruit Laboratory in Beltsville, Md., created two tools from circles of DNA known as plasmids. One plasmid delivers a knockout punch to existing genes. The other can be used to haul in functional genes to replace the damaged ones.

Inside most bacteria are several small plasmids in addition to the central strand of DNA. To produce the knockout tool, Qin and Hartung coaxed one X. fastidiosa bacterium to accept plasmids from two E. coli bacteria. This results in foreign DNA being inserted directly into X. fastidiosa's central strand, disabling whatever gene lies at the insertion site.

The researchers have inserted DNA for a green fluorescent protein at several different sites in the X. fastidiosa genome. Not only did this knock out different genes, the fluorescent protein enabled researchers to “see” the pathogen's movement in the infected plant.

To restore a disabled gene's function, the researchers inserted a critical piece of DNA from X. fastidiosa into an E. coli plasmid. This hybrid plasmid shuttled a functional gene into the pathogen without disabling other of its genes.

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