Potential targets

Research financed by FAPESP hopes to shed light on the mechanisms of a disease that has drastically affected Brazilian cocoa production for two decades

By Mônica Pileggi

Agência FAPESP – The key to understanding the infection process of witch’s broom disease, a plague affecting cocoa plants that is caused by the fungus Moniliopthora perniciosa, could be the alliance of two branches of biology: molecular and structural.

SMOLBnet 2.0: Structural Studies of Key Proteins for Fungal Diseases in Cocoa – witch’s broom and moniliasis – developing strategies to control and understand the pathogenicity, a project coordinated by researcher Andre Luis Berteli Ambrosio of the National Bioscience Laboratory (LNBio) hopes to shed light on the mechanisms of action of a disease that has drastically affected Brazilian cocoa production for two decades.

The project was selected in the Request for Proposals launched by the Network for Structural Biology on Advanced Scientific Topics –SMOLBnet 2.0., which is a FAPESP program aimed at promoting partnerships between research groups that have experiences in structural resolution of macromolecules through X-ray crystallography or magnetic resonance and research groups in the molecular area that develop high impact competitive projects on complex biological systems.

In the first phase of the project, the molecular biology group was charged with the task of identifying the molecules of the fungus responsible for its activity on the plant.

This task was conducted under the command of Gonçalo Amarante Guimarães Pereira, chief of the Genetics, Evolution and Bioagent Department at Universidade Estadual de Campinas’ (Unicamp) Biology Institute and LNBio research associate, along with Jorge Maurício Costa Mondego, researcher at the Agronomics Institute (IAC).

Witch’s broom disease first appeared in the Amazon, also a major cocoa producing region in Brazil. According to official documents, the fungus appeared in Bahia – Brazil’s main cocoa producing region – in 1989.

“It was devastation. The volume of cocoa plants fell to 25% of its production, dropping from 400,000 tons to 100,000 per year in 2000,” Pereira told Agência FAPESP.

After infecting the cocoa tree, the Moniliopthora perniciosa invades a place between the cells of the cocoa called the apoplastic space. There it secretes proteins that interact with others in the plant.

As a consequence, the branch of the cocoa tree hypertrophies and dries out, giving it the appearance of a broom – thus the name. The physiological process exhausts the plant, causing a reduction in fruit production.

According to Pereira, all countries in South and Central America, with the exception of Costa Rica, are contaminated by witch’s broom disease and moniliasis, a more aggressive disease caused by another fungus, the Moniliopthora roreri.

Moniliasis still has not arrived in Brazil, but the disease has been detected in border regions and scientists believe that contamination of the country’s producing regions is inevitable. All previous attempts to contain witch’s broom disease in the country have been unsuccessful.

Key and lock

One of the first steps to deter the disease is understanding the interaction among the fungus and the plant, i.e. its behavior. In 2000, a consortium led by Unicamp, with financing from the Bahia State Government and the National Council for Scientific and Technological Development (CNPq), mapped the fungus’ genome.

Since then, Pereira and colleagues have studied a series of proteins produced by the fungus and considered important for the disease. “These proteins, called targets, are the ones that if blocked have the potential to reduce or paralyze the effect of disease on the plant,” he explains. Each target is genetically coded and it is fundamental to understand its structure in order to inhibit its action on cocoa.

In total, 27 of the Moniliopthora perniciosa’s proteins were selected for the second stage of the project. “Molecular biology locates the potential targets, which are the locks. The work of structural biology consists of finding the keys to these locks,” explains Pereira.

During the “key” side of the research, structural biology will combine X-ray chrystallography and nuclear magnetic resonance. According to Ambrosio, these are currently the most advanced techniques for obtaining the atomic details of a protein, which is important for comprehension of its organization and functions.

These activities will be shared by researchers Andre Ambrosio and Sandra Martha Gomes Dias, both of LNBio, and Ana Carolina de Mattos Zeri, coordinator of the LNBio’s Multiuser Nuclear Magnetic Resonance Laboratory.

“They and their teams will be responsible for the large scale heterologous production, and afterwards, for the structural studies of the diverse proteins in the project,” says Pereira.

The scientists believe that the targets identified can help in the discovery of blockers for the two fungi. Although they are of the same genus, the moniliasis and the witch’s broom disease belong to different species.

According to Pereira, the proteins identified in witch’s broom and moniliasis are very similar. Due to the similarity of the infection processes of these diseases, the idea is to exchange information between molecular and structural biology,” comments Ambrosio.

In order to apply the knowledge in rational planning and the synthesis of the small regulatory molecules in the select proteins, the team is collaborating with professor Rafael Guido of the USP São Carlos’s Physics Institute, and professor Ronaldo Aloise Pilli of Unicamp’s Chemistry Institute.