Bicarbonate (HCO₃⁻) transporter family including the anion exchanger (AE) group is involved in multiple physiological processes through regulating acid-base homeostasis. HCO₃⁻ transporters have been extensively studied in mammals, but fungal homologues of AE are poorly understood. Here, we characterized the AE group member (MoAE4) in Magnaporthe oryzae. MoAE4 exhibits more sequence and structure homologies with the reported AE4 and BOR1 proteins. In addition to the common sublocalization on cytomembrane, MoAE4 also localizes on tonoplast. Yeast complementation verified that MoAE4 rescues boron sensitivity and endows NaHCO₃ tolerance in the BOR1 deleted yeast. MoAE4 gene is bicarbonate induced in M. oryzae; and loss of MoAE4 (ΔMoAE4) resulted in mycelial growth inhibited by NaHCO₃. Lucigenin fluorescence quenching assay confirmed that ΔMoAE4 accumulated less HCO₃⁻ in vacuole and more HCO₃⁻ in cytosol, revealing a real role of MoAE4 in bicarbonate transport. ΔMoAE4 was defective in conidiation, appressorium formation, and pathogenicity. More H₂O₂ was detected to be accumulated in ΔMoAE4 mycelia and infected rice cells. Summarily, our data delineate a cytomembrane and tonoplast located HCO₃⁻ transporter, which is required for development and pathogenicity in M. oryzae, and revealing a potential drug target for blast disease control.

Summary: ATP‐dependent Lon proteases function in bacterial pathogenesis by regulating the expression of the Type III secretion system; however, little is known about how Lon proteases regulate fungal pathogenesis. We previously investigated Lon‐binding proteins involved in fungal pathogenesis that interact with PrePL, the smallest Magnaporthe oryzae Lon‐binding protein. Here, we show that Lon cleaves PrePL and produces Pc, an extracellular 11‐kDa isoform with catalase and peroxidase activity. The Δ PrePL loss‐of‐function strain showed stronger sporulation and accelerated disease development, suggesting a temporally specific negative regulatory mechanism controlled by PrePL in disease progression. Neither the truncated Pc, nor the full‐length PrePL missing the Lon cleavage site complemented the Δ PrePL phenotype, suggesting that full‐length PrePL and Pc both function in fungal development. PrePL targeted to the mitochondria undergoes hydrolysis by Lon to produce Pc, which accumulates in the fungal apoplast. Importantly, recombinant Pc induced plant defence responses and cell death after being infiltrated into selected plant leaves, indicating that it functions as an avirulence factor. This work thus reveals a novel pathogenic factor in the fungal Lon‐mediated pathway. Additionally, our results provide new insight into the functions of a full‐length protein and its cleaved isoform in fungal pathogenesis.

Carbonic anhydrases (CAs) are metalloenzymes widely present in biological systems, capable of catalyzing the reversible hydration of carbon dioxide (CO₂) to form bicarbonate (HCO ₃ ⁻) and protons (H ⁺ ). This biochemical reaction plays a crucial role in various physiological processes. Previous studies have shown that MoCA1, a carbonic anhydrase in Magnaporthe oryzae , is localized in the mitochondria and is involved in conidial development and pathogenicity. However, the functions of other carbonic anhydrases in M. oryzae , 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 MoCAs were deleted, the resulting mutants Δ MoCA2 , Δ MoCA4 , and Δ MoCA6 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 M. oryzae . 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 M. oryzae . IMPORTANCE: Rice blast disease, caused by the fungal pathogen Magnaporthe oryzae , represents a major threat to global rice production, resulting in significant yield losses annually. During infection, M. oryzae encounters multiple environmental stresses within the host, such as nitrogen limitation, elevated bicarbonate (HCO ₃ ⁻) 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 M. oryzae . 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.

Cordycepin, a nucleoside analog naturally produced by Cordyceps fungi, exhibits broad-spectrum biological activity and has been reported to inhibit both prokaryotic and eukaryotic microorganisms, although its antifungal mechanisms remain poorly understood. In this study, we systematically evaluated the inhibitory effects of cordycepin on the growth, development, and pathogenicity of Magnaporthe oryzae, the causal agent of rice blast, and further elucidated its mode of action through transcriptomic analysis and gene function validation. Cordycepin significantly inhibited hyphal growth and reduced both conidial production and germination in a dose-dependent manner. Moreover, it severely impaired appressorium formation and increased the frequency of malformed structures, ultimately leading to attenuated virulence. Transcriptome profiling revealed that cordycepin reprogrammed multiple metabolic and signaling pathways, including nitrogen metabolism, fatty acid metabolism, melanin and chitin biosynthesis, and protein kinase networks. Notably, two carbonic anhydrase genes, MoCA1 and MoCA5, were significantly downregulated and identified as candidate genes responsive to cordycepin. Functional analysis showed that MoCA5 localizes to mitochondria and interacts with MoCA1; deletion of either gene resulted in disrupted mitochondrial membrane potential, reduced ATP synthesis, and decreased pathogenicity. Collectively, our results indicate that cordycepin suppresses the growth and virulence of M. oryzae through coordinated modulation of mitochondrial function and nitrogen metabolism - associated pathways. These findings provide new insights into the antifungal action of cordycepin and identify potential molecular components involved in fungal metabolic adaptation to cordycepin stress.

Carbonic anhydrases (CAs) are a class of zinc-containing metalloenzymes widely present in the biological world, catalyzing the reversible hydration of CO₂ to form HCO₃⁻ and H⁺. These enzymes play essential roles in pH homeostasis, gas exchange, metabolic regulation, and virulence expression in pathogens. In fungi, CAs mainly belong to the α- and β-classes and have undergone extensive diversification during evolution. In plant pathogenic fungi, the functions of CAs have extended beyond traditional metabolic roles, evolving into key “environmental adaptation and virulence regulatory factors.” This review takes Magnaporthe oryzae as a model organism and integrates recent advances in CA research across various microorganisms. It systematically summarizes the classification diversity, structural features, subcellular localization, and biological functions of fungal CAs. Particular emphasis is placed on the molecular profile, mitochondrial localization, physical interaction network, and multiple functional roles of the MoCA family members in conidial development, appressorium formation, oxidative stress response, HCO₃⁻ homeostasis, nitrogen metabolism, and mitochondrial energy metabolism. Based on these findings, this study proposes a multi-layered analytical framework integrating CA molecular characteristics, mitochondrial functional regulation, and fungal pathogenicity. It explores the potential of targeting fungal CAs for the development of novel selective fungicides and highlights key research directions, aiming to provide theoretical insights into plant-fungal interactions and innovative strategies for disease control.

With growing global recognition of the nutritional value of blueberries, their cultivation has expanded significantly across various regions. However, gray mold disease, caused by Botrytis cinerea, remains a major challenge in postharvest storage and transportation. In this study, two strains—Trichoderma asperellum BBR–A and B. cinerea BC7–1—were isolated and identified from the blueberry rhizosphere and diseased leaves. Co-culture assays demonstrated that BBR–A exhibits potent antagonistic activity, rapidly suppressing the growth of the pathogen BC7–1 through mycoparasitic behaviors, including hyphal coiling, penetration, and degradation. Transcriptomic analysis revealed that upon interaction with BC7–1, BBR–A undergoes extensive reprogramming of carbon metabolism, with coordinated upregulation of genes encoding degradative enzymes, transporters, and stress-related proteins. Congo red staining and enzymatic activity assays further confirmed the enhanced secretion of extracellular enzymes and the increased cell wall-degrading capacity of BBR–A. On detached blueberry leaves, seedlings, and fruits, BBR–A treatment significantly delayed lesion expansion and reduced disease severity. Metabolomic profiling further demonstrated that BBR–A induces the marked accumulation of phenolic compounds, polyamines, and other antioxidant- and immunity-related metabolites in blueberry leaves, thereby maintaining host redox homeostasis and enhancing stress resilience. Collectively, these findings indicate that T. asperellum BBR–A effectively suppresses B. cinerea through multiple mechanisms, including antagonism, mycoparasitism, metabolic regulation, and the induction of plant defense responses, highlighting its strong potential for development as a biocontrol agent against blueberry gray mold.