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Genomics

A double dose of progress

Sequencing of the grape Xylella helps to understand yellowing disease

In mid June, when they concluded the sequencing of the genetic code of the Xylella fastidiosa bacterium that causes Pierce’s disease, an devastating ailment for the vines in California, the researchers on the Agronomic and Environmental Genome project, one of the branches of FAPESP’s Genome Program, knew that they had in their hands something that was not just good news for the wine-growers of the west coast of America.

The news was also of interest to the orange planters in the state of São Paulo, who may be benefited with the conclusion of the work, in an indirect way. How so? Because the genetic map of the grape Xylella has provided important clues for a better understanding of the genome of the first plant pathogen to be sequenced in Brazil (and in the world); the lineage of the Xylella fastidiosa that causes CVC, the Citrus Variegated Chlorosis, the well-known yellowing disease that attacks orange groves.

Comparing the genetic material of the two strains, the researchers identified stretches that may be associated with the process of infection by Xylella in citrus fruit. “These areas deserve to be the object of study in future, so that we can establish whether they really are connected with the disease caused by the bacterium in the orange plantations”, says Marie-Anne Van Sluys, from the Institute of Biosciences of the University of São Paulo (IB/USP), one of the coordinators of the project that mapped the DNA of the grape Xylella.

One of these stretches is a sequence of 70,000 base pairs (the chemical units that make up the DNA of an organism), found only in bacteria specialized in attacking oranges. The sequence is present in the genome of the CVC Xylella, and a part of it is found in the genome of Xanthomonas citri, the bacterium that causes citrus canker and whose genetic code was deciphered by the FAPESP Genome Program. This sequence was not however identified in the grape Xylella, nor in another variety of Xanthomonas, the campestris, which does not affect orange plantations.

Another finding of the researchers from São Paulo: the genome of the grape Xylella has several copies of genes that are possibly associated with the process of the sticking of bacterium to the vector or host. The scientists suspect that the bacterium would not manage to stay stuck to the insect that transmits it, or to the contaminated plant, without these genes.

Similar, but not the same
The genomes of the two Xylellas are very similar, but not the same. The one of the bacterium specialized in attacking the grape is smaller and does not show some of the stretches identified in the orange pathogen. It has around 2.5 million base pairs, 200,000 less than the one that causes the yellowing disease. The genes codified by the two genomes are the same in over 90% of the cases. “We did however find many internal rearrangements”, says Mariana Cabral de Oliveira, who is another coordinator of the project, also from the IB/USP. “Each Xylella shows its own order of appearance of some genes or genetic regions.”

For the while, the grape Xylella shows 3,400 regions that are candidates for being recognized as genes (the CVC Xylella has around 2,800 genes). The researchers are avoiding calling these regions genes, because they know that the number is not yet definitive. “The quantity of genes should drop to around 2,600, as we refine the process of annotating the genome”, says João Paulo Kitajima, of the State University of Campinas (Unicamp), the bio-information technology coordinator for the grape Xylella.

Annotating a genome means identifying the regions that are genes and to say, whenever possible, which are the proteins that these genes make. The initial data of the sequencing of the bacterium of the grape are the result of an automated process of annotation, carried out with the help of a computer program. The next step will be to carry out annotation by hand, which is more detailed.

Agreements
The sequencing of the grape Xylella is part of a cooperation agreement signed in August last year between FAPESP and the United States Department of Agriculture (USDA), a body that is tantamount to an American Ministry of Agriculture. FAPESP and the USDA split fifty-fifty the total cost of the enterprise, US$ 500,000. Of the US$ 250,000 invested in the project by the USDA, one part of the amount came from the department’s own budget, and the other part in money transferred from the American Vineyard Foundation (AVF), an association of Californian wine-makers and by the California Department of Food and Agriculture (CDFA).

Worried by the advance of Pierce’s disease among the vines in California, which is the chief wine-making region of the country, the Farm Research Service of the USDA decided to invite scientists from São Paulo to carry out the sequencing of the bacterium that causes this ailment. After all, they had already done the same job on the Xylella that causes CVC.

In a development of this partnership, FAPESP has just signed a new agreement with the Americans. The researchers from São Paulo will be assembling and annotating the genomes of two varieties of Xylella fastidiosa that have been almost completely sequenced in the United States, one of which attacks the almond tree, while the other infects the oleander plant. The invitation to carry out this work was made last year, but only now has it been formalized. US$ 100,000 will be invested in the project, with FAPESP going in with 50% of this amount, the USDA with US$ 25,000, and the AVF with a similar amount. For the São Paulo researchers, putting together another two Xylella genomes will enrich even more their knowledge of this bacterium.

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