The way for the genetic transformation of Xylella fastidiosa is open, in such a way that it could reduce the undesirable action of the bacterium that causes variegated chlorosis of citrus fruit (CVC), popularly called yellowing. The disease makes one third of the São Paulo orange trees unproductive. The biologist Patrícia Brant Monteiro, a researcher with the São Paulo Foundation for the Defense of Citrus Fruit Growing (Fundecitrus), with the support of FAPESP, has developed a plasmid – in this case, a laboratory built DNA sequence – from parts of the chromosome of the bacterium itself, which has managed to integrate itself into its genome. Up until this point, the Xylella had not been receptive to any kind of fragment of DNA. From now on, by transporting genes that alter the original functions of the genome, the plasmid will give origin to mutant Xylellas, whose construction – as they become routine – , will speed up the studies that aim to understand the genes of the “yellowing” disease – as much those already studied and mainly those that are unknown. This is exactly the priority goal of the Functional Genome Project, which was started last year by FAPESP.
With this plasmid, the Xylella has finally given in. And now, it has become what is called a transformant, a cell genetically modified through the introduction of externally originated DNA. A process widely used to modify the genome of other micro organisms, it was unprecedented in the case of the yellowing disease. “This is the first time that we’re making Xylella transformants”, attests the biochemist Jesus Aparecido Ferro, the coordinator of the Functional Genome Program. In his view, the project is going “to move from the remote possibility to a real possibility” of carrying out important discoveries regarding this bacterium genome. In Ferro’s opinion, there could even be an outburst of results, since the Functional Genome Project teams that are getting ready to test the genes are going to benefit directly from the new plasmid, which in its turn is of the sequencing of the Xylella genome, concluded at the beginning of the year.
Patricia’s innovation, which makes the plasmid work, essentially lies in the use of a piece of chromosome of the bacteria itself, called origin of replication, or in short OriC. It is this fragment that begins the process of chromosome replication, during the duplication of the bacterium. Another point that she put into practice and that didn’t show itself so advantageous, was to attempt to introduce into the Xylella plasmids with origins of replication coming from other bacteria such as Escherichia coli, Xanthomonas or Pseudomonas. None worked. “The plasmids made from the origins of replication of other bacteria are incompatible with the system of replication of the DNA of the Xylella”, she explained. “They might even enter into the bacterium, but they’re unstable.”
A lost year
Certainty is the son of persistence, so many were the unsuccessful attempts. Between December of 1998 and July of this year, Patrícia worked as a Fundecitrus researcher at the National Institute of La Recherche Agronomique (INRA), somewhat similar to the Brazilian Embrapa (Brazilian Agricultural Research Corporation), in the port of Bordeaux in the South of France. Her objective was the same as the other the Functional group: to study the molecular biology of the Xylella and to build a plasmid that would allow its genetic transformation. However, now she knows that she persisted for a year along the wrong path.
A bacterium very similar to the Xylella, the Xanthomonas citrii, which causes citrus canker and is the object of another sequencing program financed by FAPESP, accepts other bacteria plasmids. For this reason, it was taken as certain that the Xylella would also yield. Patrícia tested fifteen plasmids, via different methods – and nothing. In December of 1998, she imagined that she could get what she was looking for if she used part of the genome of the Xylella itself, but her very own supervisor, the Frenchman Joel Renaudin, discouraged her from going down this path. He had spent ten years before he had managed to obtain mutants of a bacterium that attacks orange trees in Europe, the Spiroplasma citri bacteria, from fragments of its own microorganism. He suggested that she try all of the plasmids available from the Xanthomonas, given the similarity that there was between the two bacteria.
Born in the state of Minas Gerais, in the town of Bocaiúva who decided at age 13 years to become a scientist when learning Mendel’s Laws while at school, Patrícia in the first moment accepted the advice. In the end, the French are internationally respected for their work on bacteria that attack the conducting ducts of plants such as the Xylella. After all, that is why that she was there. Some time later – after seeing hundreds of useless experiments and on having access to the data bank of the Xylella genome, which showed how in fact it was built up, and to that of the E. coli – she decided to bet on her own intuition.
Patricia had he assistance of Diva do Carmo Teixeira, a trained pharmaceutical chemist and the first researcher with Fundecitrus to undergo a training at the INRA between 1997 and 1998, who sent her information by e-mail about how to cultivate the Xylella,. Just before last Christmas, Patrícia began to assemble plasmids with copied strips, or to put it better, clones of the genome of the bacterium. This time there were only four mounted experiments required before she could verify, at the end of June, ten days before her scheduled return to Brazil, that one of her experiments had finally incorporated into itself the genome of the bacterium and had remained stable in the cells of the resultant offspring of the cell division process.
Precious baggage
From France, besides the dozens of test tubes containing the transformed bacteria, other test tubes with the plasmids in the form of a vanilla colored powder, and a few bottles of French wine that she had learned to appreciate, Patrícia also brought with her a plant with white flowers that promises to be very helpful in the initial stage of the tests of the mutant Xylellas, probably in two years from now. It is the Madagascar periwinkle (Catharanthus roseus), which grows easily in shaded areas. This plant is one of the natural hosts of Xylella fastidiosa, which in this case, brings on an illness denominated periwinkle wilt (PW).
The samples that Patrícia brought in her baggage – delicately set in perfume bottles with their roots wrapped by paper soaked in water – had been for more than a year and a half infected with the Xylella strain used for the sequencing of the genome, the 9a5c. They had had growing problems and the leaves were a little twisted and pointed with yellow stains. These were the symptoms not of periwinkle wilt but of CVC. Therefore, a rarity. Starting from this evidence, the Madagascar periwinkle – widely researched because of the alkaloids it contains and that are used in the treatment of some types of cancer – became one more experimental plant alternative, on which the bacteria grows more quickly than on the orange trees. In two months, the Madagascar periwinkle presents the first symptoms of yellowing, which takes between five and nine months to appear on the orange tree.
At the beginning of the year, the Functional Project researchers could not as much as count upon a plant to follow in the laboratory the development of the infection caused by the Xylella. The phytopathologist Sílvio Lopes, of the Molecular Biology Laboratory of the University of Ribeirão Preto (Unaerp), solved the problem by demonstrating that the bacterium could grow satisfactorily on a variety of tobacco (Nicotiana tabaccum), much easier than growing it on an orange tree (see Pesquisa FAPESP 53). The lack of a model plant was one of the hurdles of the Functional Project, but it was not the first. Shortly after the beginning of the project, the researchers also felt the lack of a defined culture medium for the Xylella to grow and to multiply. A few months later, the challenge was overcome with the recipe made up by the biochemists Eliana de Macedo Lemos and Lúcia Carareto Alves, of the São Paulo State University (Unesp) of Jaboticabal, who had been working for five years with the bacteria of the yellowing disease (see Notícias FAPESP 45). Patrícia dares to say that, with the possibility of creating mutants, it will be possible also to reduce the reproduction time of the Xylella, today between six and ten hours. This is almost an eternity when compared with 20 minutes of the E. coli, a bacterium widely used in laboratories to multiply plasmids.
A very successful structure
The plasmid that worked out is called p16KOC because of the three parts from which it is made: the replicated original, a strip of DNA called the promoter, both copied from the Xylella itself, and the gene that gives resistance to the antibiotic named kanamycin, originating from a plasmid of E. coli. The replicated original, OriC, which controls the process of duplication of the single chromosome of Xylella, is the greatest of the three fragments. It has 1,890 pairs of nitrogenous bases or nucleotides (adenine, cytosine, guanine or thymine), building blocks basic to the genome of any living being.
For its part formed from two pieces, the gene dnaA and dnaA Boxes, the strip of OriC allows the plasmid to multiply itself a few times in the interior of the bacterium, independent from the chromosome. This way, says the researcher, there is time for the homologous recombination to occur, as is called the exchange of parts of the genome. The homologous recombination, which gets its name from the fact that we are dealing with fragments of the organism itself, is a natural mechanism of living beings that permits the continuous re-arrangement between genes dispersed among chromosomes. “The greater the genetic variability, the greater are the chances of a species passing through natural selection and evolving”, she explains.
In this case there is a single exchange, called single crossing over, between the promoter of the plasmid and the promoter of the Xylella. Something which at first sight might appear a little strange: it is an exchange in which only one side comes out winning. Under other situations, when the operation proceeds in the so-called double crossing over, a chromosome cedes strips of DNA to another chromosome – then, indeed, there is a real exchange. However, there would also be the risk that the bacterium would not incorporate other parts of the plasmid apart from the promoter. In the single exchange, the final result is the incorporation of all of the plasmid into the chromosome of the Xylella. This is something quite uncommon. Under other circumstances, only parts of the plasmids jump to any place of the genome.
The other fragment of plasmid, also copied from the genome of the Xylella by the PCR technique (Polymerase Chain Reaction), is the P16SrRNA. It gets this name because it directs the formation of a special type of RNA, the ribosomal RNA, which makes up the ribosomes, the cells compartments where proteins are produced. Relatively small, the P16 has 831 pairs of bases. The promoters regulate the expression of the genes. In practice, they inform them when and how much of a gene is going to act in the formation of a protein – for example, to break the molecules of glucose and produce energy.
As the head of the team, the P16 directs the expression of the second strip, the gene named Kan, removed from a plasmid of E. coli. The Kan, with 1,100 pairs of bases, produces a protein that allows the bacteria to escape from the kanamycin. It is this gene that selects the bacteria whose chromosome incorporated the plasmid, when submitted to a bath of kanamycin, Kanamycin eliminates those that refused it as they are not resistant to this antibiotic.
These three fragments of DNA were added to the structure of a plasmid of E. coli, the pBS, widely used in the sequencing of the Xylella to transport and multiply fragments of the genome. However, now the path is reversed: the same carrier that transports pieces of the Xylella away from it, now becomes – with some adjusting – efficient in transporting DNA into it. The plasmid itself, independently from what was added to it, is able to duplicate itself through its own efforts, as a result of the action of the origin of the replication colE1ori which comes from the E. coli. Thus, by giving a ride to the other fragments of DNA, it makes itself able to multiply in the interior of the E. coli and of the Xylella. Consequently, it has gained versatility.
“The plasmid has to be as similar as possible so as to be accepted and to remain within the chromosome”, emphasizes the researcher. Even so, there is the risk of not even entering into the bacterium. For this reason, Patrícia didn’t give a chance to the Xylella to refuse the host. By way of a technique called electroporation, submits the yellowing bacterium to high voltage pulses of 2,500 almost twenty times the voltage of a common electrical socket. It is enough to make the external membrane of the bacteria more permeable and allows the plasmid to pass inside. Once within, the promoter P16 of the plasmid pairs itself up with the promoter of the bacterium. “This was the only point in which the operation progressed with complete success”, comments the researcher. The plasmid integrates itself into the chromosome when it is copied by the enzyme DNA polymerase, at the beginning of the process of cell division (see illustration).
Refinements
Patrícia created a perfect spy. It is able to fool Xylella, it installs its genome 378, 2 times bigger and gets by unnoticed, in such as way as to complete its mission, albeit mild. The plasmids that have been sent in so far are, to a certain extent, harmless. Their only task is check on the vulnerability of the enemy’s defenses. At the Research Center of Fundecitrus in the town of Araraquara, Patrícia is dedicating herself to the development of plasmids of a more refined version, evidently with more difficult missions. For example, by carrying genes directly involved with the pathogenicity of the Xylella instead of the promoter, the spy could in fact demonstrate how to prevent the Xylella from installing itself on the orange trees and interrupting the conduction of water and mineral salts in the xylem, in the most internal conducting ducts of the more advanced vegetables.
At the head of the project Production and Description of Non-Pathogenic Mutants of Xylella fastidiosa, which has R$ 42,800 and a further US$ 45,400 of funding by FAPESP, Patrícia has created a plasmid in which she has shortened the strip of OriC. It only has one of the parts, the region of the dnaABox, which functions in the same way, with one advantage: since it is smaller, the chances of linking with other parts of the chromosome are smaller. Through using alternatives such as this, she believes that it will be possible to annul, block, or knock out the undesirable genes, so that they no longer express themselves. Another possibility is to add to he genome of the bacterium genes that give some benefit to the plant, in such a way as to inform the plant that the Xylella is installing itself in the xylem before it is choked up. “Here is where we begin the work and I don’t know how long it’ll last” she sighed, still with remnants of that lovely accent from the north of the State of Minas Gerais. The plasmid, in her opinion, can now carry any gene.
If there is enough time, she would also like to work with the glassy-winged sharpshooter, the insects that transmit the Xylella to oranges. “Who knows if it’ll not be possible to make the bacterium produce a protein that can kill these little insects?” she wonders. Once again the researcher from Minas says that she has only a vague notion of how to go about developing the work. However, she made a similar type of comment at the beginning of her search for genes in order to construct her plasmids.
Profile:
Patrícia Brant Monteiro, 34 years of age, graduated in Biology from the Federal University of Minas Gerais (UFMG), in the city of Belo Horizonte, took her master’s and doctorate at the University of Sao Paulo (USP) and her post-doctorate at the University of North Carolina in the United States. She has been a researcher with the Fundecitrus Foundation since 1998.
Project: Production and description of Non-Pathogenic Mutants of Xylella fastidiosa
Investment: R$ 42,860.00 and a further US$ 45,410.45
