Septins from Magnaporthe oryzae: a strctural approach

• Grant number: 22/00152-4
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• Start date: April 01, 2022
• End date: March 31, 2026
• Field of knowledge: Biological Sciences - Biophysics - Molecular Biophysics
• Principal Investigator: Richard Charles Garratt
• Grantee: Luis Alberto Valverde Fernández
• Host Institution: Instituto de Física de São Carlos (IFSC). Universidade de São Paulo (USP). São Carlos , SP, Brazil
• Associated research grant: 20/02897-1 - Septin filaments: structure, polymerization and role in pathologies, AP.TEM

Abstract

More than 50 years ago, septins were first described in Saccharomyces cerevisiae. Initially, studies showed that they formed a collar around the budding neck, which was important for the recruitment of proteins for cytokinesis. Today, it is known that septins are ubiquitous in fungi and animals, and may be involved in many important processes in the cell besides cytokinesis, such as phagocytosis, ciliogenesis, cell polarization and morphogenesis, in addition to others that require membrane restructuring and/or support proteins. A dramatic example is seen in Magnaporthe oryzae which is the causative agent of rice blast, the most devastating disease that affects cultivated rice. To invade the host, the fungus develops a specialized cellular structure called an appressorium, which allows the fungus to enter the plant tissue. The appressoria generate enormous internal turgor pressure that creates the force necessary to physically break the plant's cuticle. A network of toroidal actin filaments forms at the base of the appressorium and this network is structured by four septins (sep3, sep4, sep5 and sep6), which form a dynamic ring structure, which co-locates with the actin filaments. Septins, therefore, provide the cortical rigidity and membrane curvature necessary for the protrusion of a rigid penetration peg to breach the leaf surface. Due to the extraordinary role that septins play in appressorium formation, structural information about them would be of great value, including for the development of compounds to delay blast disease. The objectives of this proposal are the structural characterization of M. oryzae septins, individually and in their complexes. Different constructs of the main septins will be expressed using heterologous expression systems in bacteria for preliminary characterization (oligomerization status, nucleotide content, hydrolytic activity, etc.). The purified proteins will be used for crystallization experiments either individually or in combinations guided by current knowledge about the arrangement found in S. Cerevisiae. Structures will be determined using conventional methodologies. In parallel, negative stain electron microscopy assays will be performed to confirm the organization of the complexes and then cryo-EM, if feasible, to determine their structures. (AU)