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Genomics

Revelation in details

Research shows genes and mechanisms that may help in the fight against the bacterium that causes yellowing disease

MIGUEL BOYAYANFive years ago, almost nothing was known about the Xylella fastidiosa bacterium, which causes one of citriculture’s worst scourges, Citrus Variegated Chlorosis (CVC), also known as the yellowing disease, which is responsible for annual losses in the order of US$ 100 million just in São Paulo, the main producing state in Brazil. The situation has changed radically. Over the last year, as the 21 research studies of the Xylella fastidiosa Functional Genome Project, financed by FAPESP, came to maturity, the mechanisms by which this microorganism infects the orange trees and grows inside them have become clear.

A series of genes and proteins take place in this process. These genes and proteins have recently been discovered and counted in abundance – there are, for example, 30 important genes that have been identified at the Agronomic Institute of Campinas, and 30 proteins that are essential for the bacterium, discovered at the São Paulo State University of Campinas (Unicamp).

Research has now started, in fact, that ought to make it possible to disable these genes and proteins in order to prevent the bacterium from spreading around. The final result will probably take the form of a vaccine against the yellowing disease. Up till now, the person who has made most headway towards this solution is João Lúcio Azevedo, a researcher who works at the Luiz de Queiroz College of Agriculture (Esalq) of the University of São Paulo and at the University of Mogi das Cruzes (UMC).

Based on the most recent findings of the Functional Genome projects, which involves almost 80 researchers from public and private universities and institutes, Azevedo implanted in the genome of two harmless bacteria that live with XylellaPantoea agglomerans and Methylobacterium sp, a gene that helps to digest fastidian gum – a gelatinous substance that is probably essential for the formation of the colonies of Xylella and for the bacteria to stick to the walls of the vessels that transport water and nutrients in the orange trees. That is how, together, the gum and the colonies, in an integrated action, clog the channels where the nutrients circulate, in a slow process that makes the trees wither little by little.

In the next three months, Azevedo will find out if the chosen bacteria really do hold back, in the orange trees, the formation of fastidian gum, the composition of which was detailed last year by the team led by Eliana Lemos, from the São Paulo State University (Unesp) in Jaboticabal. It was another important victory in the fight against the yellowing disease. It is now known that this compound, vital to the bacteria and deadly to orange trees, is a mixture of four kinds of sugar, less dense and viscous than the xanthan gum produced by another bacterium that infects citrus, beans and cabbage, Xanthomonas citri, the sequencing of which was concluded at the end of 2000.

According to Eliana, the discovery of the structure of fastidian gum facilitates the search for mechanisms that can prevent it from being formed, as it specifies the targets to be pursued and avoids a blind battle or a lack of explanations as to why a tactic worked or otherwise. As the team from Unesp has managed to make Xylella produce fastidian gum, when cultivated in the laboratory under special conditions, a path arises that is being followed more closely. As the processes for production are optimized, fastidian gum is emerging as an alternative to the similar gum produced by X. citri and employed in industry as a thickener for paper, paints and foodstuffs.

Blocked vessels
A vaccine that can hold back the gum and, as a consequence, the growth of the bacteria, is not the only prospect than the São Paulo group is working on. Also on the basis of the sequencing of this bacterium’s genome, which started in 1998 and was concluded in 2000, the researchers found a molecule that controls genes linked to the development of the disease in orange trees.

Called diffusible signal factor, it is derived from a fatty acid (a kind of fat) and is probably a result of the action of two genes, according to Marcio Lambais, one of the authors of the discovery, also from Esalq. It still remains to detail its chemical structure, but they are already thinking of producing this molecule in the laboratory and apply it to the plant, in such a way as to be able to prevent the activation of the genes that help the disease to advance. “It would work like a pesticide”, in Lambais’s plans.

When jointly analyzed the results increasingly strengthen the idea that the bacterium’s aggressiveness – or virulence – is directly connected with its capacity to form colonies, to fix itself on the walls of the xylem (the vessels that carry water and mineral salts all over the plant), and so to cause it to become clogged up – processes in which fastidian gum seems to be essential. Indeed, everything points to the blockage of these vessels being the main factor that harms so intensely the metabolism of the orange trees, in the assessment of Eduardo Caruso Machado, from the IAC.

In partnership with a team from Esalq, Machado discovered that the quantity of water that reaches the leaves is almost 60% lower in diseased orange trees. It does not matter if it is raining heavily, as is common in November and December in the north of the state of São Paulo, where one third of the world’s production of oranges comes from: the infected plants look as if they were suffering from a lack of water, even with the ground sodden – they become withered, with wilted leaves and small fruit.

But if the trees reach this state only a few years after being infected, the reduction in the flow of water – the most immediate cause of the yellowing disease manifesting itself – takes place much earlier: only three months after the trees being contaminated with Xylella . Noticed in the laboratory, this is a sign that escapes the producer, who only manages to note the infection from the outer appearance of the tree and its fruits, at a more advanced stage of the disease.Internally, there are other complications.

Studies indicate that the orange trees lose half their capacity for carrying out photosynthesis, the process by which reserves of energy (glucose) are produced. There are small pores on the surface of the leaves, called stomata, that allow the entry of carbon dioxide, which is indispensable for photosynthesis. If the xylem is clogged, even partially, and water does not reach the leaves, the stomata close, as if the plant were going through a time of drought. As a consequence, the energy production cycle loses efficiency.

But the clogging of the vessels alone does not explain the reduction in photosynthesis. The researchers believe that the bacterium must produce specific substances – toxins not known yet – that help block the production of energy. This conclusion emerged from experiments with contaminated oranges, but placed in apparently very favorable conditions, with an abundance of water and a supply of carbon dioxide 140 times superior to the normal one. It would be a way of eliminating the influences of the stomata and of the lack of water, but even so, to everyone’s surprise, the photosynthesis of the diseased orange trees was 20% lower – a loss, therefore, small but not entirely eliminated.

Genes for sticking
Marcos Antonio Machado, the coordinator of another team from the Agronomic Institute, looked into the problem from another angle; instead of analyzing the metabolism of the plants, he examined the activity of Xylella’s genes. His work went more deeply into the conclusions of other groups from the Functional Genome project, who had warned that the colonies of bacteria and their sticking to the walls of the plant’s vessels were decisive in the transformations that make the tree wither and stop producing.

Machado compared the expression of 2,205 Xylella genes in two situations of cultivation in the laboratory: the first of them, called a primary isolation condition, contained bacteria that had recently been taken from infected plants, which preserved their capacity for swiftly colonizing the plants; the other, secondary isolation, was made up of bacteria removed long before, that had propagated themselves 40 times and multiplied far more slowly on being inoculated into the orange trees, according to the tests that had already been carried out previously.

Using a biochip – a slide that makes it possible to distinguish active genes from the inactive ones – produced by the group from Mogi das Cruzes, Machado found that about 30 genes behave in a different way, with responses that depend essentially on their kinship with the bacterium taken from the plant. Generally speaking, the bacteria that are genetically more similar to the Xylella found in the plant itself, kept under the condition of primary isolation, show a greater capacity for causing the disease – their so-called pathogenicity – than those from the other group. In the first case, a larger number of genes connected with the process of adhesion to the plant were found to be active.

In another joint work, this time with Gustavo Goldman, from USP’s College of Pharmacy in Ribeirão Preto, Marcos Machado examined the degree of expression of these genes. He compared the two situations of growth and found that, in primary isolation, there were genes related to sticking some 20 times more active than in the other condition of growth. – and, accordingly, he was able to confirm the importance of the genes for adhesion.

Could it be that the same effect would manifest itself in other plants? Oddly enough, the generation of bacteria close to the one taken from the plant – with more copies of the adhesion genes – infected the Madagascar periwinkle (Catharanthus roseus), in a similar way as it did oranges. It was an important finding. “Pathogenicity seems not to be related to the plant, but rather, essentially, to the bacterium”, Machado comments.

Slow growth
The team led by José Camillo Novello, from Unicamp, worked with the product of the genes: the proteins, which, in principle, if they were blocked, would help to stem the advance of the yellowing disease. After an initial selection, the researchers from the Protein Chemistry Laboratory identified 142 proteins produced in greater quantity by Xylella , but they devoted themselves to 30 of them, associated with the capacity for sticking, with absorbing nutrients and with the toxicity of the bacterium. They are proteins that arouse interest, in particular because the bacterium exports them to the intercellular medium, as the researchers describe in an article to be published in February’s Proteomics .

“These proteins are potential targets for fighting the yellowing disease”, says Marcus Smolka, one of the members of Novello’s team, “because in principle it is easier to produce a compound that acts on a protein that is outside the bacterium than inside it”.

Analyzing the pattern of these proteins, the group from Unicamp found that Xylella does not have a mechanism, common in other bacteria, that makes it possible to produce proteins quickly. According to Smolka, this characteristic helps to explain Xylella’s slow growth, as it takes from eight to ten hours to split into two, while Escherichia coli takes no more than 20 minutes. “It all leads one to believe that Xylella cannot manage to respond in an efficient way to the substances produced by the plant’s defense system”, he comments. “Perhaps that is why it only manages to grow in aggregates that offer a form of protection”.

Surprises
But science is not just made of advances. Last year, the bacterium seemed near to being dominated, but it proved to be tougher than expected and posed new challenges for the researchers, in an indication that the next stages of the task may be more complicated than was imagined. Unexpected results arose when the attempt was made to contaminate orange trees and tobacco plants with genetically modified bacteria, in which researcher Patricia Monteiro, then with the Fund for Citrus Plant Protection (Fundecitrus), had disabled genes related with its pathogenicity.

The analyses carried out to date indicate that the strain used – isolated in Jales, in the São Paulo interior, and known by the abbreviation J1a12 – is not virulent. This is bad, because this way one cannot get to know if disabling the genes would really work, to the point of preventing the yellowing disease from developing. The other more aggressive strain, 9a5C, sequenced during the Xylella Genome project, proved to be resistant to genetic transformation.”It may be that J1a12 just causes the symptoms to show far later, for growing more slowly”, Patricia comments.

In search of more aggressive strains, whose genes can be disabled, the researchers found a promising alternative: B111, isolated in Bebedouro, and capable of contaminating orange trees with more efficiency than the Jales strain, but even so, with less virulence than 9a5C. Once this stage is overcome, everything should become easier, since Beatriz Mendes and Francisco Mourão, both from USP, have increased their mastery over the technique for producing genetically modified orange trees – besides the Hamlin variety, obtained in 2001, they have produced transgenic trees of the Pera, Valencia and Natal varieties. The laboratory where they work is ready to receive the genes that resist the yellowing disease as soon as they are found by the other research groups.

The Project
Functional Genome of Xylella Fastidiosa, with 21 Individual ResearchProjects; Modality FAPESP Genome Program; Coordinators
Jesus Aparecido Ferro – Unesp and Ana Claúdia Rasera da Silva and Luiz Eduardo Aranha Camargo – USP; Investment R$ 2,048,228.98 and US$ 2,211,758.01

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