Carbonic anhydrases contribute to mitochondrial function, conidial development, and pathogenicity of <i>Magnaporthe oryzae</i>

Carbonic anhydrases (CAs) are metalloenzymes widely present in biological systems, capable of catalyzing the reversible hydration of carbon dioxide (CO₂) to form bicarbonate (HCO₃⁻) and protons (H⁺). This biochemical reaction plays a crucial role in various physiological processes. Previous studies have shown that MoCA1, a carbonic anhydrase in <i>Magnaporthe oryzae</i>, is localized in the mitochondria and is involved in conidial development and pathogenicity. However, the functions of other carbonic anhydrases in <i>M. oryzae</i>, such as MoCA2, MoCA4, and MoCA6, remain largely unexplored. Through subcellular localization analysis using DsRED-tagged fusion proteins, it was found that MoCAs are also localized in the mitochondria, and they exhibit interactions within this organelle. When the genes encoding <i>MoCAs</i> were deleted, the resulting mutants Δ<i>MoCA2</i>, Δ<i>MoCA4</i>, and Δ<i>MoCA6</i> exhibited significant defects in conidial development and pathogenicity. Additionally, these mutants showed a marked decrease in mitochondrial membrane potential, indicating an apoptotic state, along with reduced intracellular ATP levels and downregulated expression of genes related to ATP synthesis. In nitrogen metabolism studies, the mutants displayed corresponding phenotypic changes, with significant downregulation of nitrogen metabolism-related gene expression. Taken together, the results of this study demonstrate that mitochondrial MoCAs play a critical role in conidiation and pathogenic mechanisms in <i>M. oryzae</i>. They function by coordinating the regulation of mitochondrial function, ATP synthesis, and the glutamine-glutamate metabolic pathway, thereby maintaining cellular pH homeostasis. These results provide important theoretical insights for the identification and development of novel targets for the control of <i>M. oryzae</i>.IMPORTANCERice blast disease, caused by the fungal pathogen <i>Magnaporthe oryzae</i>, represents a major threat to global rice production, resulting in significant yield losses annually. During infection, <i>M. oryzae</i> encounters multiple environmental stresses within the host, such as nitrogen limitation, elevated bicarbonate (HCO₃⁻) levels, and hypoxic conditions. These challenges necessitate robust mechanisms to maintain intracellular pH and metabolic stability, which are crucial for pathogen survival and virulence. Our study highlights the essential role of the carbonic anhydrase family of enzymes in regulating intracellular pH homeostasis in <i>M. oryzae</i>. These findings provide valuable insights into the molecular strategies employed by plant pathogens to adapt to hostile host environments, potentially informing the development of novel disease management approaches.