MAGNAPORTHE ORYZAE SEPTINS: A STRUCTURAL APPROACH

Septins are ubiquitous in fungi and animals and are potentially involved in many critical processes beyond cytokinesis[1]. These proteins bind to GTP and can assemble into hetero-oligomers, linear filaments, and higher-order structures such as bundles and networks[2]. In the rice blast fungus, Magnaporthe oryzae, a filamentous network forms at the base of the appressorium through four main septins (MoSEP3-6), which assemble into a ring that colocalizes with actin filaments. This ring provides cortical rigidity for the protrusion of a rigid penetration peg that breaches the leaf surface[3]. The aim of this research is to characterize the septins of Magnaporthe oryzae. MoSEP3-HIS/MoSEP4-STREP was expressed and purified, showing degradation and instability, which eluted as a dimer in 300 mM NaCl on SEC. More stable constructs such as MoSEP3HLa0G-HIS/MoSEP4-STREP were confirmed as tetramers by SEC-MALS and cryo-electron microscopy. This construct is currently undergoing crystallization, along with MoSep3HLa0G-HIS/MoSep4HLa0G. The MoSEP5-HIS/MoSEP6 construct, analyzed by SEC-MALS, showed it was a tetramer in a buffer with 150 mM NaCl and a dimer in 300 mM NaCl. The nucleotide content showed only GDP, suggesting active catalytic activity in both proteins, unlike in Saccharomyces cerevisiae. Negative stain electron microscopy and cryo-electron microscopy of the MoSEP5-HIS/MoSEP6 tetramer in 150 mM NaCl revealed the tetramer structure at a resolution of 3.4 Å and the dimer at 2.8 Å. The MoSep5HLa0G-HIS/MoSep6HLa0G construct was crystallized, resulting in the validation of the 3D structure at 1.57 Å resolution, presenting only GDP. The octamer composed of MoSEP3/MoSEP4-STREP and MoSEP5-HIS/MoSEP6, when purified in 500 mM NaCl, resulted in non-equimolar protein fractions, possibly due to the disruption of NC interfaces. At 300 mM NaCl, all four septins eluted nearly equimolarly, and SEC-MALS confirmed a molecular mass close to the theoretical octamer value. The octamer only presented GDP, unlike in S. cerevisiae where septins bind GTP as well. Additionally, a cryo-EM 3D model was obtained for an octamer including a construct without the C-terminal of MoSep5 (5HLα0G), with a global resolution of 3.5 Å. Oligomers were also evaluated at low concentration (10 nM) using mass photometry, revealing octamers, hexamers, tetramers, dimers, and monomers.