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Boost for Brazilian science

A generation of young researchers was selected to take part in the genome sequencing project, started 20 years ago, for the Xylella fastidiosa bacterium

Fundecitrus Genomic map of the 2.7 million base pairs of the X. fastidiosa chromosomeFundecitrus

During the morning of May 1, 1997, biologist Fernando Reinach, who at the time was a professor at the Chemistry Institute of the University of São Paulo (IQ-USP), called physicist José Fernando Perez, then scientific director of FAPESP. Since the preceding year, they had been discussing new ways to invest in the scientific training of Brazilian researchers in the field of molecular genetics, considered key to the field of biotechnology. From the long conversation between the two, the first project for sequencing a genome in Brazil emerged: Xylella fastidiosa, the bacterium that causes citrus variegated chlorosis (CVC), one of the worst orange pests in the state of São Paulo. Launched on October 14 of that year, the project involved 35 laboratories and 191 researchers from several state institutions and various disciplines, which were integrated virtually by the Organization for Nucleotide Sequencing and Analysis (ONSA) Network. In addition to initiating research on molecular genetics, the sequencing of the first plant phytopathogen in the world represented a new way of doing science in Brazil, and thus contributed to the education and training of a generation of young scientists. Many used the knowledge and experience they acquired to start their own firms. Others changed the focus of their research, established new scientific collaboration networks and advanced in their careers in universities and elsewhere.

The mapping of the Xylella genome brightened the outlook for genetic research in Brazil. Other sequencing endeavors followed, such as the Genoma Cana project, completed in November 2000, with the goal of sequencing selected parts of sugarcane DNA and identifying genes with economically important characteristics. Other projects sprang up at almost the same time, such as the Cancer Genome project, completed in March 2002, and the sequencing of the genetic code of the Xanthomonas citri bacterium, which causes citrus canker, completed in August 2001. These scientific undertakings helped train researchers and improve the infrastructure of the largest laboratories in São Paulo, thus hastening the development of molecular biology in Brazil.

Cover of the journal Nature, which said that the sequencing of the phytopathogen was a success story for Brazilian bioscience

The experience acquired by taking part in the project and the promising market for biotechnology products and solutions also motivated a number of researchers to go into business. In 2002, two years after the sequencing was completed, computer scientist João Paulo Kitajima left the Bioinformatics Laboratory (LBI) at the University of Campinas Institute of Computing (IC-Unicamp) where he worked to set up Alellyx with other project participants. Alellyx is a research and development company for biotechnology products that focuses on generating and marketing patents in applied genomics (see Pesquisa FAPESP Issue nº 74).

Kitajima joined the bioinformatics team shortly after the Xylella genome project began. “I started as a bioinformatics analyst at LBI along with bioinformaticians João Meidanis and João Carlos Setubal. Then I started to assist with coordinating the bioinformatics part of the project, so I was the third leader named “João,” he says. At the time, due to the need for speedy communication among the labs that were involved, the project managers decided to set up a centrally coordinated virtual network that was physically scattered over the research centers in the state of São Paulo. The ONSA Network went live in December 1997, connecting the laboratories and establishing links among the researchers so they could develop genetic protocols, share information, solve problems and adapt and adjust techniques.

Fundecitrus Bacteria-transmitting insectFundecitrus

The network had a bioinformatics system so researchers could incorporate information from the sequencing of the bacterium’s genetic material. Setubal, Meidanis and Kitajima were in charge of maintaining the flow of information. “We were responsible for obtaining the sequencing data that the labs produce and processing them on computers to develop a complete genome,” Setubal says. Meidanis, who came into contact with bioinformatics during his doctoral work at the University of Wisconsin-Madison in the United States in 1989, recalls that “we didn’t know if the groups would have sufficient Internet bandwidth access to send us all the genomic information.”

“At Alellyx I had the opportunity to coordinate the applied research and specialize in the field of regulatory processes for transgenic organisms,” says biologist Jesus Aparecido Ferro of the School of Agricultural and Veterinary Sciences at São Paulo State University (Unesp) in Jaboticabal, and one of the founders of the company. Ferro was also one of the coordinators of the Xylella Functional Genome program, launched in June 1999. The purpose of the project was to investigate the function of the genes identified during the sequencing, and thus to understand how Xylella triggers CVC.

Eduardo Cesar Ana Claúdia Rasera, João Kitajima, Jesus Ferro, Paulo Arruda and João Setubal at the launch of biotechnology company Alellyx, in 2002Eduardo Cesar

Alellyx was sold along with CanaVialis in 2008 to multinational Monsanto for $290 million (the equivalent of R$980 million today). “In 2009, I went into the field of human health,” Kitajima says. “I worked as a bioinformatician at the Israel Institute of Education and Research at Albert Einstein Hospital until 2011.” More recently, he and other partners founded Mendelics Análise Genômica, a customized genetic testing company.

For Meidanis, the project was a major turning point in his life. “I became a hybrid of academic and company manager,” he says. Today he divides his time between his position as professor at Unicamp and entrepreneur at Scylla Bioinformática, a software company he founded that works in the fields of genomics and proteomics.

Adri Felden/Ag. Argos Award ceremony for the São Paulo Medal of Scientific and Technological Merit…Adri Felden/Ag. Argos

Ambitious project
The Xylella genome project was launched at a time when research was having a considerable impact on the field of molecular biology. But this science was brand-new in Brazil. “Perez realized that scientific production in this area was growing in other countries, and that Brazilian researchers had to be trained for us to boost our position on the international science scene,” Reinach says at his office at the Pitanga Fund, which raises money from investors interested in technology-based endeavors. That was the start of a lengthy scientific effort to develop the project. “The older researchers weren’t convinced,” he says. “It was an ambitious and risky project that would involve large sums of money,” according to Reinach, who at the time was one of the few Brazilian researchers with experience in the field of molecular biology and scientific entrepreneurship (he founded the first company in Brazil that performs paternity tests, Genomic Engenharia Molecular).

The choice of which microorganism would be sequenced was made weeks before the project was launched. “The genome had to be large enough to involve many researchers and small enough for us to be able to complete it,” says Perez in his office at Recepta Biopharma, a biotechnology firm he founded in 2005 that performs research and develops compounds that have the potential to fight cancer.

Adri Felden/Ag. Argos …given by the government of São Paulo State to the 191 researchers involved in the genome projectAdri Felden/Ag. Argos

At that time, the organism that best met these prerequisites was Thiobacillus ferrooxidans, used in biomining; however, no Brazilian groups had succeeded in growing the Xylella bacterium. “In September 1997, the Citriculture Defense Fund (Fundecitrus) expressed interest in participating in the project if Xylella was chosen,” Perez says. “I mentioned the difficulties involved in selecting the bacterium, but they replied by saying that a French researcher knew how to grow it.” They were referring to Joseph Bové of the National Institute of Agricultural Research (INRA in French), in Bordeaux, France. Bové was working in molecular genetics and was able to provide the bacterium, while Frédéric Laigret, from Bordeaux 2 University, would support the establishment of a DNA bank for the bacterium to supply the fragments to be sequenced. “They convinced us and we chose Xylella,” Perez says.

The project was launched with a $12 million investment from FAPESP and $400,000 more from Fundecitrus. This was the largest sum to date for a scientific project in Brazil. Of the 70 laboratories that applied to take part in the project, 35 were selected by an international committee consisting of Steve Oliver of the University of Manchester in England, André Goffeau of the Catholic University of Leuven in Belgium, and John Sgouros of the Imperial Cancer Research Foundation in England. This committee was placed in charge of supervising and running the project.

Adri Felden/Ag. Argos Former governor of São Paulo State Mário Covas giving biochemist Andrew Simpson (at left) the award for his leadership in coordinating the projectAdri Felden/Ag. Argos

Since the Brazilian researchers had little experience in the field of molecular biology, the decision was made to have DNA and bioinformatics coordinators for the project as well as central sequencing laboratories to train the researchers and generate the necessary sequences. Biochemist Andrew Simpson, from the Ludwig Institute for Cancer Research in São Paulo, was placed in charge of coordinating the DNA. The central sequencing laboratories were the Molecular Biology Laboratory at IQ-USP, which Reinach headed up at that time, and the Center for Molecular Biology and Genetic Engineering (CBMEG) at Unicamp, coordinated by biologist Paulo Arruda. The laboratories that were chosen were given automatic DNA sequencers, reagents and technical assistance. Xylella would be mapped using 99 cosmids, which are fragments of DNA that carry and multiply parts of the genome to be studied.

Less than a year after the project was launched, 90% of the Xylella genetic code had been sequenced. There were still a few gaps in the total sequence of the genome. To bridge the gaps and confirm the order of the cosmids, Simpson developed alternative experimental approaches at the Ludwig Institute laboratories while he attempted to connect the cosmids to those that were contiguous to them so as to order the sequences and describe the genome completely.

Gilberto Alves Group of scientists responsible for sequencing Xylella, meeting with Fernando Henrique Cardoso, former president of Brazil (in beige suit), at the Alvorada PalaceGilberto Alves

New opportunities
Geneticist Anamaria Camargo joined Simpson’s team at the Ludwig Institute in 1998 while she was a postdoctoral researcher. Her work consisted of bridging the gaps in the Xylella genome with researchers Cláudia Monteiro-Vitorello, Elizabeth Martins, Mariana Cabral de Oliveira, Marie-Anne Van Sluys, Marilis do Valle Marques and Ana Cláudia Rasera. “Today I am working in the area of cancer genomics, using much of what I learned in the Xylella project,” says Camargo, coordinator of the Molecular Oncology Research Center at the Sírio-Libanês Institute of Education and Research in São Paulo. Her colleague, biologist Marilis do Valle Marques, was invited to join the project by physicist Suely Lopes Gomes, a professor at IQ-USP. “I took part in the analysis and assembly of the sequences and the assembly and annotation of the pXF51 plasmid, which proved to be challenging because it has a duplicate region, making it difficult to assemble the sequence,” explains Marques, a professor in the Microbiology Department of the Institute of Biomedical Sciences (ICB) at USP.

The project was completed in January 2000 and consisted of sequencing 2.7 million base pairs of the Xylella chromosome. On July 13 of that year, Nature dedicated the cover of the journal to this work. In Brazil, the researchers received awards and tributes, such as the São Paulo Medal of Scientific and Technological Merit, awarded by the government of São Paulo State (see Pesquisa FAPESP Issue nº 51).

Eduardo Cesar | Léo Ramos Chaves Van Sluys and Oliveira acquired experience in Brazil and led projects in the United StatesEduardo Cesar | Léo Ramos Chaves

For Simpson, thanks to the success of the Xylella genome project, he was able to lead other projects, such as the Cancer Genome. “As a result of that work, I was promoted to the position of scientific director of the Ludwig Institute in New York,” he says. Years later he returned to Brazil as chief executive officer of Orygen, a biotechnology company that develops, produces and sells therapeutic antibodies and vaccines.

The Xylella genome project turned into an opportunity for many young scientists to become independent researchers. After participating in the project, biologist Marcelo Briones from the Microbiology Department of the Federal University of São Paulo (Unifesp) became one of the coordinators of the Cancer Genome project along with Simpson. At that time, the equipment used in his laboratory to sequence the Xylella cosmids helped him sequence genes expressed in cancers that have a high incidence in Brazil, such as cervical cancer and cancer of the head and neck. In 2000, after completing the two projects, Briones obtained funding from the Howard Hughes Medical Institute in the United States to conduct research on infectious and parasitic diseases in Brazil. “I would not have obtained the funding without the equipment and knowledge I acquired during the Xylella and Cancer projects,” he notes.

Márcia Minilo Perez and Reinach: focusing on new ways of training Brazilian researchersMárcia Minilo

“My entire career is based on the study of Xylella,” says biologist Alessandra Alves de Souza from the Sylvio Moreira Citrus Research Center at the Agronomic Institute of Campinas (IAC) in Cordeirópolis, in inland São Paulo State. De Souza received her undergraduate degree in Pernambuco State and then went to São Paulo where she began work on her master’s degree at the Luiz de Queiroz College of Agriculture (ESALQ) at USP. In her PhD work at Unicamp, she conducted comparative analyses of the expression of Xylella genes associated with pathogenicity and the formation of a biofilm that connects the community of microorganisms that attack plants. This led her to study a molecule known as N-acetilcisteine (NAC) as a possible alternative for controlling CVC. NAC is a compound used to clear respiratory tracts and reduce biofilms formed by bacteria that cause disease in humans. In preliminary testing, NAC proved effective in breaking through biofilms produced by Xylella, lessening the symptoms of CVC. Recently, de Souza and biologist Simone Picchi, also from IAC, opened CiaCamp, a research firm that develops NAC-based fertilizers (see Pesquisa FAPESP Issue nº 247). Today de Souza is working on a project funded by the European Commission to fight a variant of Xylella that is destroying olive trees in the Puglia region of Italy.

In her postdoctoral research at Michigan State University, biologist Cláudia Monteiro-Vitorello had already worked on the mitochondrial DNA sequencing of a fungus that causes diseases in chestnut trees in the United States. When she returned to Brazil in 1998, she was invited to join the team of agronomist Luiz Lehmann Coutinho of the Animal Science Department at ESALQ-USP. “During the Xylella project, I studied at the National Scientific Computing Laboratory (LNCC) in Rio de Janeiro,” she says. There she met Amos Bairoch, coordinator of Swiss-Prot, a Swiss database of proteins. “As a result of that meeting and the Xylella project, I was hired to work at Swiss-Prot on annotations for bacterial genomes,” she recalls. In 2008 Monteiro-Vitorello returned to academia as an assistant professor of microbiology at the Federal University of the ABC in Santo André, São Paulo State. In 2009 she went to ESALQ-USP, where she works in the field of genomics on the plant-pathogen interaction involving diseases of sugarcane.

Francisco Tanakae Elliot Kitajima/ESALQ-USP Microscopic image of biofilms formed by Xylella inside plantsFrancisco Tanakae Elliot Kitajima/ESALQ-USP

For biologist Marie-Anne Van Sluys, from the Botany Department of the Biosciences Institute (IB) at USP, the Xylella genome project was an opportunity to turn her studies on the interaction between bacteria and plants into the main focus of her research. After completing the sequencing of the Xylella genome, Van Sluys and biologist Mariana Cabral de Oliveira, also from IB-USP, headed a project to decode the genomes of a strain of Xylella responsible for the destruction of grapevines in the United States and the Leifsonia xyli subsp xyli bacterium, which attacks sugarcane stalks. “We joined a network headed up by the Joint Genome Institute in the United States to unlock the DNA of two more strains of Xylella that attack almond trees and that live in a plant called oleander,” she says.

“It was a time of much interaction with researchers from a variety of fields,” says Oliveira, who took part in every stage of the Xylella genome project, from sequencing to the macrostructural and comparative analysis of the genomes. Later, this paved the way for her to transfer the techniques of large-scale sequence analysis to her study of algae. For Perez, the project showed that Brazil is capable of conducting collaborative research that involves the scientific community, the government and private initiative. “Even more, with this project, Brazil changed its perception of its capabilities and its place in the world of science.”