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Genetic engineering against Xylella

Research defines new strategies for fighting the pest of the orange

EDUARDO ALVES / ESALQ-USPColonies of Xylella growing inside the vessels that carry the sap: action is starting to be containedEDUARDO ALVES / ESALQ-USP

The first genetically modified variety of sweet oranges, which opens the way for the production of disease resistant orange trees, and a series of mutant bacteria, in which genes considered harmful to the plants were disabled. These are part of the novelties at the 1st Xylella fastidiosa Functional Genome Symposium, which took place in Serra Negra from December 10th to 13th. It was there that the coordinators of the 21 research groups of the Functional Genome project financed by FAPESP announced the strategies for the fight against Xylella fastidiosa, the bacterium that causes the yellowing disease, citrus variegated chlorosis (CVC).

Transmitted by an insect – the cicada -, this is a scourge that, with symptoms that can be more serious or milder, affects 65 million orange trees in the state of São Paulo (36% of the total), and makes about six million of them unproductive each year. The researchers’ targets are clear: they are the genes that allow Xylella to unleash the disease (giving it its pathogenic Nature) or which determine the aggressiveness (virulence) with which the plant will be infected. The work, which started two years ago, now converges towards the search for mechanisms – like genetically altered plants or bacteria – that can block the action of these harmful genes, and allow more efficient insecticides to be developed, to prevent the cicada from transmitting the bacterium to healthy plants, or to lead to the development of resistant plants, producing proteins capable of impeding Xylella survival.

Thus a new stage starts in the fight against the bacterium that causes the yellowing disease, launched in 2000 with the victorious sequencing of its genome, in a program financed by FAPESP. Besides the techniques already in use – the control of the insects that transmit it and the use of saplings free from contamination, which at least prevent the problem spreading to plants that are still intact -, it will be possible to make use of the pest, that bring losses to São Paulo citrus growers estimated at US$ 100 million a year.

Before an audience of about 100 researchers at the Serra Negra symposium, Beatriz Mendes, from the Center for Nuclear Energy in Agriculture (Cena), and Francisco Alves Mourão Filho, of the Luiz de Queiroz College of Agriculture (Esalq), both of the University of São Paulo (USP), presented the first variety of genetically modified sweet orange, obtained with the use of adult tissue.

Decisive conquest
In 2000, Beatriz and Mourão had already obtained a transgenic plant from young citrus tissue, that is, taken straight after the germination of the seed, but they were not satisfied with the result: the plant can take from five to eight years to bear fruit. They worked for a year and a half assessing the factors that influence the plant’s conditions for growth – such as the medium for cultivation, the time of incubation and the temperature. Finally, they were successful with an orange tree of the Hamlin variety, using adult tissue, which bears fruit earlier, in about two years. It is a conquest that promises to be decisive for the next stage of the project, which is forecast to end in the middle of this year.

“When we have a gene that gives the plant resistance against the bacterium, we will be able to produce plants for testing in the field in a couple of years or so”, Beatriz reckons. More research should mature this year. In the front line of the results is João Lúcio de Azevedo, the coordinator of the Integrated Nucleus of Biotechnology (NIB) of the University of Mogi das Cruzes (UMC) and retired professor from Esalq. He is developing a Xylella mutant in which the gene of the endoglycanase A enzyme, one of those associated with the production of fastidian gum, can be blocked. This is the gum that the bacterium uses to stick to the xylem, the system of vessels that carries water and mineral salts all through the plant.

Fastidian gum is also connected with the clogging of the xylem, and, as a consequence, with the manifestations of the symptoms of the disease – the yellow blotches – on the leaves of the orange tree. Before 2002 is out, Azevedo hopes to obtain the first results from inoculating the modified bacterium into the periwinkle (Catharantus roseus), used as a model for this kind of experiment, as well as in citrus fruit. “If it works”, says he, “we may extend the technique to bacteria of the Xantomonas genus, which attacks citrus fruit and green vegetables.”

To go forward, the researches don’t just rely on information on how the bacterium causes the disease and the aggressiveness with which the plant is infected. This data has now been taken further by the knowledge built up about the Xylella genome, whose sequencing could count on almost all the researchers who are now taking part in the Functional Genome project. There has also been a lot of progress in the knowledge of the proteins produced by the causing agent of the yellowing disease – 130 of them have now been identified by the Protein Chemistry Laboratory of the Institute of Biology of the State University of Campinas (Unicamp) – and, in a wider manner, in the epidemiology of the disease.

Experiments carried out, particularly at Esalq, show that both the yellowing disease and the cicada propagate themselves more intensely in the hotter regions of the state, where the shortage of water is common. Accordingly, in the area of the municipalities of Barretos and Bebedouro, in the north of the state, 48% of the orange trees are infected, while in the vicinity of Limeira and Itapetininga, to the south, they are no more than 17%, according to a survey carried out in 2001 by the Fund for the Defence of Citrus Growing (Fundecitrus), maintained by the farmers.

“The most important stages have now been overcome”, comments Jesus Aparecido Ferro, from the São Paulo State University (Unesp) in Jaboticabal, one of the coordinators of Functional Genome: “From now onwards, progress will certainly be quicker.” It will be quicker still if Fundecitrus biologist Patrícia Brant Monteiro’ wish is fulfilled: she has produced stable mutant bacteria for 12 genes, which govern, among other things, the pathogenic Nature and the production of toxins for the plant, or of polysaccharides (sugars) that willdestroy the xylem.

Both Patrícia and Azevedo are working on the technique of interrupting genes. It is the “reading” of a gene that determines the production of specific proteins. When a sequence of DNA is put in the middle of a gene, this reading is disturbed, and so it is “switched off”. The bacterium that results from this modification is a mutant: it now carries the altered gene in its genetic material. Patrícia was a pioneer in the construction of a mutant Xylella that resists the incorporation in its genome of exotic (coming from other organisms) genetic material. She overcame the problem by using as a vector a plasmid (a segment of circular DNA) developed in a laboratory and which contained a small sequence of genetic material from the bacterium itself.

This work has opened the way for other groups. Researchers from USP Marilis Marques, of the Institute of Biomedical Sciences, and Suely Gomes, of the Institute of Chemistry, have also achieved success in the production of mutant bacteria. Using a different strategy, they developed a plasmid that made it possible to incorporate exotic DNA in the Xylella genome. Marilis and Sueli developed colonies of Xylella that have the gspD gene altered. This is the gene that is responsible for the production of a protein that forms channels in the wall of the bacterium, through which the enzymes that destroy the vessels of the xylem are secreted.

Tests recently done with the use of radioactive material for marking genes confirmed that one in every eight colonies of mutant bacteria keep incorporated the altered DNA sequence, even after ten reproductive cycles. “We have begun to master the technique for transforming Xylella, which is important to determine the function of each gene in the disease”, Marilis reveals. She is now trying to produce mutant bacteria using transposons (bits of DNA that change place in the chromosome) as vectors, instead of plasmids. The advantage of this would be to obtain, using a single kind of vector, various colonies of transformed bacteria, each one with a different gene switched off.

Integrated research
Mastering the technique for producing mutants was equally essential for the group in Piracicaba coordinated by Sérgio Pascholati, from Esalq. He worked on the identification of genes that codify exoenzymes – proteins that the bacterium produces and that function to obtain nutrients and to colonize the plant. Availing himself of information on the Xylella genome, he identified eight possible exoenzymes, and, in laboratory tests, characterized three of them: they are three cellulases, enzymes that digest and transform it into glucose, an essential molecule for any organism to obtain energy. The next stage: developing bacteria with altered genes that prevent the production of these proteins.

Another gene that the Piracicaba researchers want to switch off is the Xf1940, which produces the methionine sulfoxide reductase enzyme. This enzyme is part of the mechanism for the bacterium to adhere to the wall of the xylem and to other bacteria, to form colonies, according to the model developed by Breno Leite, from Pascholati’s team. Methionine could also be related to fixing Xylella in the cicada’s oral apparatus. They believe they can arrive at a mechanism for controlling the disease, if they block the action of this gene.

Pascholati works in conjunction with specialists from Fundecitrus, who are also connected with Azevedo’s team and the NIB in Mogi das Cruzes, which also interacts with Esalq and the Agronomic Institute of Campinas (IAC). A single strategy is not being sought to hold back the damage caused by the yellowing disease in citrus fruit growing in São Paulo. Azevedo’s own team, besides blocking genes, is working on another way of controlling the scourge: through endophytic microorganisms, bacteria that live together with Xylella in the orange tree, but which do not cause disease in the plant.

His group has identified nine genera of citrus endophytic bacteria. Among them is Pantoea agglomerans, into which the researchers have now managed to introduce the gene that produces xanthanase. This is the enzyme that prevents the formation of xanthan gum, produced by bacteria of the Xantomonas genus and similar to fastidian gum.

Selected targets
“We have to test all the possibilities”, says coordinator Ferro, from Unesp: “We do not know which one will work.” Patrícia intends to test the mutants in plants before the end of this year, but knows that there are uncertainties: “We will be lucky to get a non-pathogenic Xylella, since half the genes from organisms in general codify proteins whose functions are still unknown.” For the fight against a pest to be efficient, the scientists had to create tools that help to determine the best targets. Amongst them, there is the microarray, also called biochip, a microscope slide where the genes of the bacteria are deposited. In just one go, the biochip indicates which genes, among all in the genome – are more active in a given situation.

It was the group led by Regina de Oliveira, from the NIB of Mogi das Cruzes, which concluded the third version of Xylella‘s biochip, with around 2,500 genes – 93% of the roughly 2,700 that make up the bacterium’s genetic material. “It’s as if we were to take a photograph of the cell’s genetic expression at a given moment”, explains Luiz Nunes, from the NIB. He has already identified a set of genes that the bacterium puts into action, in response to oxidative stress, for example – which occurs when it is attacked by reactive forms of oxygen, like oxygen peroxide, released by the host plant’s defensive system.

Regina and Nunes work in cooperation with Sílvio Lopes, of the Biotechnology Unit of the University of Ribeirão Preto (Unaerp), which is studying the activity of the genes of the strains of Xylella that infect different plants. “We have now identified various genes that we believe to be connected with the pathogenic Nature of the bacterium and of its specificity regarding the host”, says Nunes. Marcos Antônio Machado, from the IAC’s Center for Citrus Growing, made use of the biochip to compare the genic expression of the bacterium in two situations of growth: in primary isolation, when it is just taken out of the plant, and after successive cultivations, after 25 reproductive cycles in laboratory. In the first state, Xylella develops slowly in an artificial culture, but, when inoculated, colonizes the plant rapidly. In the second case, the opposite happens.

Protein bank
The comparison between the two situations showed that some genes, linked to the capacity for adhesion, become less active when Xylella is cultivated outside the plant. “These results prove that the capacity for colonization is associated with the capacity for aggregation”, says Machado. “Perhaps we may be able to develop some way of reducing the action of these genes.” To get to know the genes that are most active in the bacterium in a given situation, the specialists from the Protein Chemistry Laboratory of Unicamp’s Institute of Biology are adopting a different approach.

Instead of analyzing the genes directly, they are watching the final result: the proteins. In four months, this group from Campinas under the coordination of José Camillo Novello, the only one to study Xylella‘s Proteome (set of proteins), has identified 130 proteins produced by the bacterium. The main ones are associated with the processes of adhesion and aggregation, to the capturing and storing of iron, and to the elimination of toxins. Also found were membrane proteins, which act in the capture of nutrients. Until the end of the current stage of the Functional Genome project, expected to finish in the middle of the year, the Unicamp team hopes to form a database with 250 to 300 proteins characterized.

Culture medium
The Serra Negra meeting also marked the overcoming of one of the most complicated stages of Functional Genome: the development of a defined culture medium, in which all the nutrients are known – vitamins, minerals, hydrocarbons and amino-acids – that are necessary for the growth of the bacterium. At the end of two years of work, researchers Eliana de Macedo Lemos and Lúcia Carareto Alves, from the School of Agrarian and Veterinary Sciences of Unesp at Jaboticabal, have produced a minimum culture medium that has as its sole source of nitrogen the aspartic acid (C4H7NO4).

“The study of the metabolic aspects shows that the bacterium can grow in a relatively simple medium, in conditions that are very close to those of xylem”, says Eliana. Deciding the culture medium is an important tool for those working on Xylella‘s physiology or genetics, as it makes it possible to get to know the genes expressed in a given condition, and helps with the formation of mutant forms of the bacterium. Watch out, Xylella.