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The progressive encircling of yellowing disease

Tobacco, proteins and gum are sources in the study of Xylella

The researchers of the Project Functional Genome are announcing three new scientific contributions to the elucidation of the mechanisms of pathogeniticy of the bacterium Xylella fastidiosa, the agent which causes Citrus Variegated Chlorosis (CVC), the popularly named “amarelinho” (yellowing). After the conclusion of the sequencing of the genes of this microorganism, 21 groups of scientists are working hard  to obtain results on genome Xylella in order to understand how the bacteria works and, afterwards, to develop strategies to free the Brazilian orange trees from the pest of CVC.

The first new development solves one of the problems of studying Xylella which is the lack of an experimental model plant to study the disease. Professor Sílvio Lopes, of the Molecular Biology Laboratory of the University of  Ribeirão Preto (Unaerp), concluded a study, together with his team, which indicates a variety of tobacco (Nicotiana tabaccum) which could be used as an experimental model. When artificially inoculated with the bacteria Xylella, the tobacco allows a rapid colonization by the pathogen of its vascular system, and expresses the symptoms in its leaves within a short period of time.

Another study is a bank of data on the proteins synthesized by the Xylella, available through the Protein Chemistry Laboratory of the State University of  Campinas (Unicamp). According to the researcher Marcus Bustamante Smolka, a map with 816 bacterium proteins are receiving final preparations before shortly being available via the Internet, serving other institutions which are working on the Functional Genome Program.

A third study identified a gum produced by the bacteria which allows it to be fixed in the xylem (the vascular system of the plant) and on the sucking apparatus of the insects which transmit the sickness. “The Xylella live in locations which are very turbulent the gum assured its survival”, explains Felipe Rodrigues da Silva, Researcher from the Center of Molecular Biology and Genetic Engineering of Unicamp. “We detected that the bacteria makes this type of gum and we managed to induce it to produce the substance in the laboratory. Now we are analyzing the biochemistry of the gum – called fastiana – which is a polysaccharide, a type of sugar.” In the future, the study of this gum and of the proteins of Xylella could contribute to the elaboration of a bactericide or of another strategy for eliminating the CVC.

Orange trees  won’t do
All the new discoveries are available for the work of other laboratories, in an integration which has been a characteristic of the Genome Program of FAPESP. In the tobacco, for example, it will be possible to test the function of the different genes which, apparently, are involved in the ability of Xylella to colonize and to cause sickness in the orange trees. The study, which identified this plant as an experimental model, was recently accepted for publication by the magazine Plant Disease, of the American Phytopathological Society.

The orange trees take a long time to show the symptoms and not all are infected when they are artificially inoculated with the pathogen. “Sometimes,  we inoculate ten trees and only three or five present symptoms after three months to a year, invalidating its use in the research”, explains Lopes. “With the tobacco plants which were inoculated the opposite happened. All of them expressed symptoms in their leaves in the maximum 60 days after inoculation.  It is worth recalling that the tobacco plant was the only vegetable specie which presented a high susceptibility to Xylella within the more than 20 species researched.”

Another new fact in the Functional Genome Project is the re-structuring of the researchers into four sub-areas. “The division aims to speed up the development of the project and to increase the interchange between the groups”, explains professor Jesus Aparecido Ferro, of  School of Agricultural and Veterinary Science of the São Paulo State University (Unesp) of Jaboticabal, one of the two coordinators of the project, along with Ana Cláudia Rasera, of the Chemical Institute of the University of São Paulo (USP).

The decision to re-structure Functional Genome came from a suggestion by the steering committee of the foreign advisors who analyzed the progress of the program and indicated a new organization for the studies. The advice was accepted by the Scientific Director of FAPESP who summoned the complete group for a meeting on the 27th of April last, when the new strategies were announced and discussed. Thus, it was defined that each subgroup will have a sub-coordinator and two external advisors. These

sub-coordinators as well as the coordinators of the project are going to meet monthly and analyze the goals agreed on the previous month. All who participate will have a new tool for communicating with each other. Via the site: the researchers will write updated reports each month on the course of the research. “Each group will tell what went well and what didn’t go well. The negative attempts are important so that there is no repetition of the proceeds and no time is wasted uselessly”, explains  professor Ferro.

The dance of the groups
The sub-areas are divided into four groups: growth, transformations, DNA-array proteome and physiology. The first is going to establish methods of growing the bacterium Xylella in cultures to be used for all the researchers of the project. This group is coordinated by Márcio Rodrigues Lambais, of the Department of Science and Soil of the College of Agriculture, Luiz de Queiróz (Esalq) of USP. The group of transformation is developing techniques of genetic manipulation to obtain a system which does the transferring and the breaking off of the genes from the bacterium.  The coordination is with Suely Lopes Gomes, of the Department of Biochemistry of the Chemical Institute of USP, and with Marilis do Valle, of the Department of Microbiology of the Institute of Biomedical sciences (ICB) of USP.

The group of DNA-array proteome is identifying which of the genes are responsible for the infection of the plant and which of them generate the disease. In this sub-area as well are housed the groups which identify and analyze all the proteins synthesized by the Xylella. The coordination is by José Camillo Novello, of the Department of Biochemistry of Unicamp, and of Regina Costa de Oliveira, of the Integrated Center of Biotechnology of the University of Mogi das Cruzes. In the final sub-area, that of physiology, groups of study on ecology, physiology, biochemistry, and epidemiology of bacterium are united, under the co-ordination of Luis Eduardo Aranha Camargo, of Esalq-USP.

The reorganization of the studies of the Functional Genome Program represents more than a strategy in the battle to beat the CVC. It is a contribution which generates knowledge for this new operational and collective way of study, as yet unique as a research program in Brazilian science.