Mutational analysis of the mitotic exit GTPase MoTem1 reveals its role in development, stress adaptation, pathogenicity and global gene regulation in Magnaporthe oryzae

The mitotic exit network (MEN), regulated by the small GTPase Tem1, plays a crucial role in coordinating cytokinesis and cell cycle progression in eukaryotes. In this study, we identified MoTem1, a functional homolog of Saccharomyces cerevisiae Tem1, in the rice blast fungus Magnaporthe oryzae, and investigated its role in mitotic regulation and pathogenesis. Using targeted mutagenesis, we generated a series of mutant strains: ΔMotem1 (knockout), MoTem1-OE (overexpression), as well as MoTem1-CA (constitutively active) and MoTem1-DN (dominant-negative) variants created via single-nucleotide substitutions. Phenotypic characterization revealed that MoTem1's activity states are critical for fungal growth, development, stress tolerance, and pathogenicity. While ΔMotem1 and MoTem1-CA strains showed reduced virulence, the MoTem1-DN mutant exhibited hypervirulence. Transcriptomic profiling and weighted gene co-expression network analysis (WGCNA) identified chitin synthase MoCHS1 as a downstream gene whose expression is directly or indirectly influenced by MoTem1 activity states. Pharmacological inhibition of chitin synthesis using Polyoxin B in MoTem1-CA showed increased sensitivity, confirming a decreased expression of chitin synthase in the MoTem1-CA strain. Subcellular localization studies revealed GTP-dependent spindle pole body (SPB) targeting, with inactive MoTem1 failing to localize to SPBs, while constitutive MEN activation in MoTem1-CA disrupted spindle position checkpoint (SPOC) controls, resulting in multinucleate hyphae and a range of developmental defects. In conclusion, our work establishes MoTem1 not merely as a cell cycle regulator, but as a global upstream factor that influences nuclear division, cell wall integrity, and broadly reshapes the genomic regulatory network to govern development and pathogenesis in M. oryzae.