Recombinant expression and biochemical characterization of the SCP/TAPS domains of TcPR-1f and TcPR-1g receptor kinases from Theobroma cacao

• Grant number: 23/04646-4
• Support Opportunities: Scholarships in Brazil - Doctorate
• Start date: September 01, 2023
• End date: December 31, 2026
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Jorge Maurício Costa Mondego
• Grantee: Jaqueline Bauer Uber
• Host Institution: Instituto Agronômico (IAC). Agência Paulista de Tecnologia dos Agronegócios (APTA). Secretaria de Agricultura e Abastecimento (São Paulo - Estado). Campinas , SP, Brazil

Abstract

The cocoa culture is of great importance worldwide and for Brazilian agriculture, mainly due to the chocolate industry, with an increase in demand for its production. However, this productive increase faces challenges, one of which is the incidence of pests and pathogens. Caused by the fungus Moniliophthora perniciosa, the disease called witches' broom (BV) was one of those responsible for drastically reducing cocoa production in Brazil from the 1980s onwards. Its management is complex, which opens doors to the search for new methods to combat VB. One of the ways to generate knowledge that can be applied to face plant diseases is the molecular study of the interaction between plants and pathogens. One of the most documented proteins as a plant defense marker is called PR-1 (Pathogenicity-Related 1), belonging to the SCP/TAPS superfamily. Cocoa, among the 13 PR-1 proteins it has, has two PR-1 that stand out for having similar extension to kinase domains, being similar to kinase-type receptors (RLKs). These proteins have been named TcPR-1f and TcPR-1g, or collectively PR-1RK. Preliminary experiments with Microtom tomato overexpressing PR-1RK suggest that both would be involved in the modulation of defense against M. perniciosa. Furthermore, the kinase activity of PR-1RK was confirmed. In vivo assays in Pry1,Pry2 mutant yeasts demonstrated that PR-1RKs complemented the cholesterol-binding function, indicating that such proteins bind sterols. Seeking to deepen the knowledge about the biological activity of such proteins, this work aims to obtain the SCP/TAPS protein domains of the PR-1RK through cloning, expression in E. Coli and purification in an affinity column, so that the determination of their preferred steroidal ligand using ITC and MST techniques. This ligand will be inoculated into plants of wild Microtom and microtom overexpressing PR-1RK, to evaluate its biological effect on the modulation of plant defense response. (AU)