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The discoveries are multiplying

Recently concluded, the mapping of Xanthomonas citri will begin comparative genomics within the country, the quickest way to study genes

Just before Christmas eve, the sequencing of the genome of Xanthomonas citri, the bacteria which causes citric cancer, an old and serious problem in the citricultural world, was completed. For the period over which it was done, only fourteen months, this work was proof of the maturing of the work methods and of the team itself – in a large part, the same that had participated this year in the mounting of the genomes of other bacteria Xylella fastidiosa, which provokes Citrus Variegated Clorose (CVC) or yellowing, another pest of the orange tree and that of the sugar cane.

The project of Xanthomonas will also inaugurate within the country comparative genomics: it begins by putting side by side the genetic material of the micro organisms, to discover how the genes work and to find means of reducing the agricultural loses. Also well advanced, is the classification – or, as they say, the annotation – of the estimated 4,500 genes which make up the single chromsome of Xanthomonas the second phytopathogen mapped in Brazil and one of the first in the world.

Of the total, 593 genes are associated with metabolic processes of the production of energy, 365 in the synthesis of amino acids and other molecules which help in the working of enzymes, 486 in the formation of macro molecules (proteins, carbohydrates and lipids), 310 in cellular processes (the transport of substances, the division of cells and mobility) and 292 are related to the pathogenics, virulence and adaptation of the bacteria. At the end of November, there were still 1,530 in the category of hypothetical i.e. of uncertain function.

This final stage could finish at the same time as the conclusion of the mounting of the basic skeleton, with large pieces of the genome of the other bacteria which begun to be mapped in September: the Xanthomonas campestris, very similar to that which has just finished being recognized. The researchers believe that, perhaps in March, they will already have the detailed genome. The group has gained experience. “This time nobody got worried, wondering if it would or would not be all right.” commented João Meidanis, coordinator of the Central Laboratory of Bioinformatics, of the State University of Campinas (Unicamp), which processes the information of the sequencing laboratories – and since 1997 has been accompanying the pioneers of genomics in Brazil.

As well as confidence, they also gained time at some stages. The Xylella was entirely mapped by way of DNA plasmids – sequences of DNA which carry and multiply parts of the genome which is being studied. One of the researchers, Anete Pereira de Souza, of Unicamp, went to Heidelberg in Germany to learn how to make the so called library of DNA plasmids, the primary material for the sequencing of the genome which represents the entire chromosome.

With Xanthomonas it was different. In the 11 sequencing laboratories – spread over the University of São Paulo (USP), the State Paulista University (Unesp) and Unicamp, under the coordination of two centers, one in the Faculty Agrarian and Veterinary Sciences (FCAV) in Unesp at Jaboticabal and the other in the Chemistry Institute of USP – firstly a scaffold was made – a virtual map or skeleton of the genome. The framework was mounted with close to 2,000 DNA plasmids, each one with 35,000 to 50,000 pairs of bases of nucleotides (adenine, cytosine, guanine and thymine).

The two points of each DNA plasmid were mapped, in such a way that afterwards they could be fitted in and gave a general vision of the genome. A more detailed vision came about as soon as the method of the shotguns was applied – smaller clones of between 1,500 and 3,00 pairs of bases, literally fired over the genome, which superimposed themselves entirely over it, reproducing its structure. Since August of last year, when the work began, the researchers have produced 205,000 shotguns, whose processes of mounting were known since the Xylella bacteria.

The novelty is that this time the DNA plasmids as well were made entirely in Brazil. Luiz Roberto Furlan, a researcher at the central laboratory of the genome of Unesp in Jaboticabal, will not for a long time forget the day on which the coordinators of the project gave him the task of constructing the library of DNA plasmids. Dr. Furlan, better known since the time of his student days as Cedral, because of the town in the Paulista interior where he was born, knew the importance of the mission, but didn’t have the least idea as to how to carry it out. Journeys to Germany? Don’t even think about it! Everything would be done right here.

In resume: two months later, Cedral had recreated the methodology and was an expert in handling the vector Lawrist, a piece of circular DNA which incorporates fragments of the genome. “I didn’t believe it when the first DNA plasmid came out right.” he said “I thought I had done something wrong.” Another difference is that the Bioinformatic Laboratory of Unicamp centralized the analysis of the genome, but was able to count on the reinforcement of two centers of bioinformatics created in Jaboticabal and at USP.

It was, in this way, much easier though Xanthomonas had a genome with 5.2 million pares of bases, the double of that of Xylella . To it were dedicated 51 researchers, a discrete group when compared with the 207 of Xylella and the 140 of the sugar cane genome. Up until now, including the sequencing of the campestris, there has been an investment of US$ 2.2 million, less than half of the allocation of US$ 5.4 million destined by FAPESP.

The tranquility with which the researchers have been working can be seen in the nickname with which they have been treating Xanthomonas on a day to day basis – simply it was Xanthô. In the scientific articles it was called Xac, an abbreviation of Xanthomonas axonopodis pv citri (pv signifies patovar, a classification based on the type of plant attacked by the bacteria).The Xylella , until today, is simply Xylella , without any nickname. The team lived through difficult moments at the end of May of this year, when the skeleton of the genome was ready. It had 95 parts. The problem was that they didn’t fit together directly. Nothing to despair about for one of the coordinators of the project, Ana Claudia Rasera da Silva, of the Chemical Institute of USP.

Since the age of ten she has spent entire days on the carpet of the front room of her house mounting jigsaw puzzles of 2 or 3 thousand pieces, such as one of a medieval castle surrounded by flowers which decorates her laboratory. For her the genome is “only a little larger jigsaw puzzle with millions of pieces.” Ana Claudia guarantees that, in spite of the impasses, she doesn’t lose her patience nor her sense of humor at any time.

On the screen of the computer, the known fragments extend in straight lines and are often parallel because of the common parts. Between them there are close to 30 blank areas, without any information. Formally, they shouldn’t exist: the thousands of shotguns fired over the genome would appear to be sufficient as to not leave any fragment undiscovered.

Precious repetitions
A number of weeks passed before Ana Claudia and Meidanis managed to outline the problem: the blank areas were, in truth, repeated fragments – and the computer didn’t know what to do with them. In order to understand what were these repetitions, Ana Claudia, with the help of Dr. Marcelo Trindade and of professor Shaker Chuk Farah, both also from the Chemical Institute of USP, took up some artesian work: they eliminated the repeated parts and the pieces fell into place. With time, they came to see that in them were the most gratifying characteristics – and, if we have been able to tell the story of the researchers correctly, the most dazzling – of the genome of Xanthomonas.

Half of the repetitions were transposons – the jumping genes which jump from one point to another of the chromosome or even from one chromosome to another. They appear to be capable of inducing their neighbors to express themselves or to make other genes inactive. The team of Marie-Anne Van Sluys, of the Institute of Biosciences of USP, have already identified 51 of them. One of them is Xatn1 (informally called xatinho 1 [nuisance 1]), which repeats itself 18 times in the genome; the xatinho 2, repeats only five times. According to Marie-Anne, there are strong indications that another xatinho could have come from another bacteria, Pseudomonas, by way of a mechanism called horizontal transference of genes, which occurs between different species.

It was difficult to discover that the other half of the repetitions – also elucidated by way of DNA plasmids – were the secondary structures of DNA. They form loops which work like clips, and more seriously, impede an enzyme, polymerase, slipping along the molecule and characterizes it: that is when the areas without any information come about. In six months of work, the researchers managed to unwind the majority of the loops. At the end of November there were only two missing loops which put back for some weeks the celebration of the conclusion of the genome. “We knew right from the start that it would be difficult.” recognized Jesus Aparecido Ferro, a researcher of Unesp at Jaboticabal and one of the coordinators of the project.

With almost allof the problems resolved, the researchers now want to understand the specific mechanisms which might explain, for example, why the Xanthomonas citri causes citric cancer and the X. campestris, very similar, attacks cabbages. Another doubt: how many bacteria of the same species can there be? The first comparisons indicate that there are high similarities in the genes, though the organization of them is very different.

Evolution dilemma
Another close relative, the X. albilineans, that the teams from Jaboticabal and São Paulo pretend to sequence next year, is slow to reproduce, something like Xylella, and only lives in the xylem of the sugar cane. Biologically speaking, it has more in common with thecitri. It is thought that it might be the link between the two species. The comparisons of Xylella with Xanthomonas have already begun. The amino acid sequences – the molecules which form the proteins – of both are very similar. The first is, let us say, more good looking. It almost doesn’t have transposons and loops, for example. “The impression is that Xylella is an Xanthomonas which has shrunk or that the Xanthomonas is an Xylella which is enlarged.” joked Ana Claudia.

“No matter what, both the organisms had a common ancestor,” said Sergio Russo Matioli, a specialist in evolution of the Institute of Biosciences of USP. “If we were to have evidence that the genome of their ancestor had been the size of Xanthomonas, then there would have been a shrinking of Xylella, should it have been the contrary, then there was an amplification for Xanthomonas.” However, if the original species had had a genome of intermediary size, it could both have increased in the genome of the lineage which originated Xanthomonas and diminished in the lineage which produced Xylella .

This field of evolution runs into the difficulty of finding registered fossils of bacteria. Even then, the sequencing of the complete genomes has helped enormously in the understanding of evolutionary curiosities. Some time ago, for example, when the sequencing of Escherichia coli was completed, the bacteria used as a model for genetic and biochemical studies, something extraordinary was found: half of the genome had an unknown function.

The idea that this part of the genome could be involved in activities outside of the Petri dish, on which the researchers put the organisms which they wish to study, quickly gained adherence. For this reason Matioli remembers that “If this hypothesis had been correct, then the only studies which take into consideration the natural environment could clear up the function of all the genes of the micro organisms.

Ana Claudia confesses: “The genome of Xanthomonas is more sophisticated.” One of the arguments is that the Xanthomonas has a density of bases of guanine and cytosine considerably higher (65%). In Xylella it is of 53%. Consequently, the DNA of the former is more stable, but as well it is more difficult to beinterpreted by the enzyme polymerase – for this reason it is difficult to find, understand and take apart the loops. Even at that it is a polemic point. “Every genome has its charm.” remembered Meidanis. From his point of view, the familiarity which one has with each organism weighs heavily. Of Xylella, he remembered “we understood almost nothing”, whereas the Xanthomonas was reasonably well understood, including with some genes already described.

Getting to the facts, the Xylella reproduces slowly between 20 and 30 days. It depends on the insects, the cigarrinhas, to spread them and to survive in the insect itself or in the sap juice of the conducting ducts of plants like the orange tree or the grape vine. It is tempting, though risky, to say that the Xanthomonas lives under better conditions: it reproduces more quickly, every two days and it is a free living bacteria: it spreads through the air, through the water and through the soil.

Over the next few years, it is probable that the information which spring from the genome will assist, principally, to find the weak points of Xanthomonas citri. As a matter of fact, we are already beginning to see how the bacteria causes citric cancer. Important in this process are the genes avr (of virulence, responsible for the pathogenesicity). There are four copies of them in the two cytoplasts – circular DNAs, a lot smaller than the chromosome. The vegetable cell only actions the defense mechanism if it recognizes the proteins generated by the avr. However, what appears to happen is that the Xanthomonas manages to trick the alert mechanisms, infiltrating itself and carries out the conquest of the host cell.

Close to 20 proteins participate in the process of infection, the so called secretion system type 3, nonexistent in Xylella. “It is possible that some proteins reach the nucleus and alter the genetic expression of the plant.” said Ronaldo Bento Quaggio, a researcher with an unusual formation: he is a physicist and worked on films (he is also graduated in the Cinema) before taking his post graduation and becoming a professor at the Chemical Institute of USP. There is also working in the team the Canadian researcher Shaker Farah, who estimates that there are between 50 and 80 proteins involved in the pathogenesicity – the development of citric cancer.

The doubts add to the conquests. After the initial fright, appraised Cedral “There was a notable scientific growth within the team.” The laboratory of Unesp at Jaboticabal, for example, prepared itself to produce stocks of DNA, now of all types – shotguns, DNA plasmids or even larger clones, used in the study of the human genome – for other institutions.

They could now only think on providing a service as the methods of work have changed enormously. At Jaboticabal, the selection of the colonies with the clones – before done by hand using toothpicks – are today done by a robot endowed with a selection of needles which it itself cleans and sterilizes. The robot can make between 80 and 100 plates each with 96 holes in four hours. Manually the same task would have taken an entire day for eight or ten people.

In the laboratories dedicated to the genome project in the State of São Paulo, gaining favor is the idea of maximum possible automation in the sequencing for the team to subject themselves only to the analysis of the results. A goal which is apparently viable: in the Xylella the sequencing was still very laborious, with many manual stages, but with the new machines it is possible to put the DNA in place for sequencing in the morning and to obtain the results in the late afternoon. It could be here – in the work to attribute functions to the genes – that is the strength of the Brazilian team. “We know how to take note.” assured Cedral. Something similar occurred some years ago in Formula 1 racing: intuitive and audacious Brazilian drivers won races in cars made in other countries.

Devastating bacteria

The curriculum of the genre Xanthomonas is not something modest. Not so much for the number of species, only 20, but for its range: it attacks 392 plants, with specific varieties of beans, rice, manioc, cotton, corn, sugar cane, wheat and Soya bean, as examples. “There is a Xanthomonas for almost all of the cultivated plants, above all in hot climates.” stated Rui Pereira Leite Jr., a researcher with the Agronomy Institute of Parana (Iapar), which accompanied the work of the Paulista group. It was he who provided the samples of X. citri to be sequenced. “In cold climates the Xanthomonas almost don’t appear.”

Whilst the Xylella is a problem restricted principally to Brazil and the Argentine, the bacteria of the other genres destroy plants in Asia, in Africa and in North America. In Brazil, it is found from the States of Rio Grande do Sul to Rio Grande do Norte. Citric cancer is only one of the diseases caused by the Xanthomonas. Described for the first time in 1889 in Japan, it is actually endemic in all of South East Asia.

It arrived in Brazil in 1957 and since then has only advanced. “If citric cancer has installed itself definitely in the country, some varieties of citrus fruits will no longer be able to be cultivated, such as the Galician and Sicilian lemon, oranges of the type bay and hamlin and the grapefruit, which are more susceptible to the bacteria.” said Dr. Leite Jr. According to him, because of this pest, the Argentine no longer cultivates these varieties and at the moment it is the growers in Florida, in the United States, who are losing their sleep, preoccupied by the same possibility.

The citric cancer is associated with the burrowing larva of the citrus (Phyllocnistis citrella), detected in Brazil in 1996. The larva makes passageways in the leaves through which the Xanthomonas enter. Proliferating, they make salient lesions on the leaves and fruit which ends up falling off. To avoid the spread of the pest, there is no other way: it is necessary to remove the contaminated trees. In 1999, the State of São Paulo eliminated close to 1 million trees. The bill to be paid is also high: during the last two years, the control and the eradication cost close to R$ 50 million. “The combat of citric cancer improved considerably.” he stated “If it continues in this manner, we might be able to keep the situation under control.”

Genome Xanthomonas
Project of FAPESP Genome Program
Dr. Jesus Aparecido Ferro – Faculty of Agrarian and Veterinary Sciences of Unesp at Jaboticabal; and Dr. Fernando de Castro Reinach and Dr. Ana Claudia Rasera da Silva – Chemical Institute of USP
US$ 2,210,328.17