Mutational analysis of the mitotic exit GTPase MoTem1 reveals its role in development, stress adaptation,...

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.