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Three new attack routes against the pest

Article published in Nature compares the genomes of the Xanthomonas citri and campestri and shows how to avoid citric canker, which makes orange trees unproductive

FUNDECITRUSAn infected orange tree: the Xanthomonas citri shows itself on the external parts of the fruit and the leavesFUNDECITRUS

Almost a year and a half after the conclusion of the sequencing, which unveiled the genetic identity of the bacterium Xanthomonas citri, the orange tree pestilence, researchers at the ONSA laboratory network – formed for the sequencing of another enemy of orange trees, the Xylella fastidiosa – have attained two further conquests. First, they have pointed out directions for combating the X. citri beginning with the analysis of only one hundred of their thousands of genes, which will have an impact on all of agricultural pathogenic research, since the study of the citri was carried out through a comparison with its cousin Xanthomonas campestri.

And this has a very favorable characteristic: it can infect the Arabidopsis thaliana, a model plant for genetic studies, whose genome was recently sequenced. The other conquest, after the sequencing of the citri and together with the suggestions for combating it, is the sequencing of the campestri. The knowledge generated will not only benefit citrus farming. It can be extended to the main plants that feed human beings, since the genre of the Xanthomonas is made up from twenty species that attack some 392 vegetables, among them beans, rice, cassava, cotton, corn, sugarcane wheat and soya.

The comparison of the sequences of the two bacteria, carried out by sixty nine researchers from eleven ONSA network laboratories, made the pages of the 23rd of May issue of the magazine Nature. This scientific recognition reaffirms national leadership in the research of agricultural pathogens: of the four pathogens already sequenced throughout the world, three of them have been done here in Brazil. “With the genomes of the two Xanthomonas concluded, we have a perfect model to study the relationship bacterium-plant, identifying exactly the genes involved in this interaction. This will serve as a base for the understanding of other agricultural pests”, explains Jesus Aparecido Ferro, of the Agrarian and Veterinary Sciences College of the São Paulo State University (FCAV-Unesp), in the town of Jaboticabal. Ferro split with Ana Cláudia Rasera da Silva, from the Chemical Institute of the University of São Paulo (IQ-USP), the coordination of the Xanthomonas Genome Project, within FAPESP’s Genome Program.

The work involved an in-depth study of three possible attack routes, considered to be the most promising. One starts with the gene PTHA, which is known to be related to the abnormal cell proliferation that the X. citri brings about. In the scientific paper it was proven that the X. campestri does not have this gene, but in this bacterium a large number of enzymes which degrade the cellular walls of the plants (the Polygalactoranase) were identified, and this explains the massive degradation of the tissue that the pathogen causes.

Various genes are probably codifiers of these enzymes and the researchers are preparing a project about the genome to prove this relationship. They hope that this will open up a route towards the development of substances that will block the enzymes, essential for the bacterium to digest the plant. The second route came from the search for what determines that each species of the Xanthomonas only attacks certain plants. The key to this specialization is in the virulent genes of the bacteria – they codify proteins capable of bringing into action the defense system of some vegetables, but not of others. There is only an infection when the virulent genes of a bacterium hoodwink the resistance genes of the plant. It is a precise interaction that clearly restricts the number of possible hosts for each Xanthomonas.

The virulent genes are not necessarily the same ones that cause the pathogenicity, but, in the case of X. citri, there is a coincidence. The PTHA, which causes abnormal cellular proliferation, is also the virulence gene. The transfer of this gene to another species of Xanthomonas turns the bacterium incapable of infecting its habitual host, the rice plant, because the PTHA triggered the plant’s defenses.

Besides the PTHA, the study identified, as a result of the action of the virulence genes, a series of proteins on the surface of the bacterium related to the synthesis of antigen -O. It is believed that this antigen hoodwinks the defense system. “The resistance gene remains on the surface of the plant and the antigen-O, on the surface of the bacterium, in a similar interaction to that which occurs in animals between antigens and anti-bodies”, explains Ana Claudia. The infection occurs if the plant does not recognize an antigen -O as a stranger. In the Xanthomonas citri close to ten enzymes have been identified that result in a specific antigen-O.

“We want to study the enzymes more and the genes that are synthesized because we believe that, if we block their synthesis with some kind of substance, the plant will probably react against the bacterium, making itself resistant”, comments Ana Claudia. This substance will be applied to the fruit tree and could function as a type of vaccine. Although it would act against the bacterium and not on the plant directly, the treatment would turn the orange trees resistant to the canker by unmasking the aggressor towards the defense system.

The third means of attack is focused on the mechanisms of the proliferation of the citri . The campestri is systemic – it spreads itself out from within and causes the degradation of the tissues -, while the citri conquers areas on the external part, through which it is disseminated by the action of wind. The comparison of the genomes has allowed for the identification of specific metabolisms of the bacteria related to the parts of the plant where each of them lives.

The physicist Ronaldo Bento Quaggio discovered that the campestri assimilates nitrogen from the nitrates and nitrites within the soil, and in a lesser quantity, from the sap of the xylem (the plant’s circulation system), in which the bacterium lives. On the other hand, the citri , which lives in the intercellular spaces, does not have this capacity: it manages nitrogen by breaking down proteins into peptides, which are absorbed with the help of a transporter, the PPA, which assists in placing the peptides within the bacterium.

One manner of combating the citrus canker would be an inhibitor that would impede the bacterium from synthesizing this PPA: the X. citri would then be incapable of obtaining its food and would simply die off from inanition. Both the Xanthomonas and the Arabidopsis , the model plant for the studies in this area, are of easy genetic manipulation. In the opinion of the researchers, it is simple to inhibit or to insert genes into the DNA (deoxyribonucleic acid, the carrier of the genetic code present in each cell) of these organisms so as to ascertain the function of each gene.

The research centered itself on the comparison of the genome of the X. citri with that of X. campestri through the combination of two contradictory factors: the genetic closeness of the two bacteria and the fact that they have manners of reacting that are very distinct. While the citri spreads itself over the external part, causing a type of canker (cancer) on the leaves and fruit, the campestri provokes the so called black death of the cruciferous, somewhat like leprosy, which installs itself and spreads throughout the circulatory system as happens with kales and cabbages. Arabidopsis and similar plants.

The rationale that inspired the comparison was simple: if two bacteria so genetically close to each other have mechanisms of proliferation and effects so diverse, it should be enough to go in search of the differences in their DNAs in order to find the genes responsible for the proliferation and pathogenicity of each of them. The comparison thus promised to be enlightening, and it was.In each of the Xanthomonas the vital genes that make possible the taking over of the respective host, were identified. The study suggests, as an efficient manner to combat the pestilences, the development of a substance that inhibits these genes, killing off the bacteria through inanition. The team further believes that they have discovered the bacteria’s genes responsible for hoodwinking the plants’ defensive systems. Inhibiting them could turn the plants resistant to the disease.

Another route, as yet only slightly explored, is linked to the unusual quantity of genes of chemotaxis found in the Xanthomonas. They are the genes that determine the attraction for certain chemical substances – in this case serine, an amino acid abundantin the leaves during photosynthesis. “These genes could be linked to the capacity for the bacterium to penetrate into the plant”, says Ana Claudia. “If this hypothesis were to be proven, we could investigate the best form of inhibiting these genes.” The study has opened up perspectives for various other genome projects, according to Ferro, which promise to evolve much more rapidly than that of the Xylella, the first to have been sequenced in Brazil, which causes the yellowing disease.

“With the Xylella“, adds Ferro, “we had to overcome three hurdles that don’t exist with the Xanthomonas: the slowness in the growth of the bacteria, the long time that the infection takes to install itself in the host and the fact that the methods of conventional genetic manipulation didn’t work with the Xylella“. However, with the Xanthomonas, as well as growing quickly, there is an alternative host (Arabidopsis) that also develops the disease rapidly as well as a DNA that can be easily manipulated. The genomes of the two Xanthomonas were matched up with that of the Xylella fastidiosa, their closest relative – it is estimated that the genres of the Xanthomonas and Xylella had, in terms of evolution, separated some 160 million years ago.

The comparison showed that the close to 200 genes of the X. campestris that are not found in the X. citri are the portion of its genome most similar to that of the Xylella: they could be related to the specificity of the host, since both of them attack citrus fruit.The study pointed out the importance of comparative genomic analysis. The initial plan was not to sequence all of the campestri, of considerably less economic importance than the X. citri. “But quickly we perceived that, in spite of being very close in appearance, the two genomes had large parts of circular DNA locked into different locations, which made the comparison impossible without a complete sequencing”, explains Ana Claudia. The amount of work expanded and made the Xanthomonas the first Brazilian project to exceed the expected time limit.

A detailed look
4,322 genes of the X. citri were sequenced, containing some 5.1 million nucleotides, as well as the 4,079 genes of the X. campestri. In the citri, the researchers believe that they understand the function of some 2,700 genes, of which some three hundred are possible targets for the development of ways in which to combat the bacterium and one hundred – among them the genes related to adhesion and to the cytotoxicity of the two bacteria – have already been explored in Nature. The next step is to match up the genes of the citri with those of the Xanthomonas that also bring on canker, but in other vegetal species.

The project
Xanthomonas Genome; Modality Project of FAPESP’s Genome Program; Coordinators Jesus Aparecido Ferro – Unesp of Jaboticabal; and Fernando de Castro Reinach and Ana Claudia Rasera da Silva – USP; Investment US$ 2,210,328.17