Journal: Applied and Environmental Microbiology

Article Title: Carbonic anhydrases contribute to mitochondrial function, conidial development, and pathogenicity of Magnaporthe oryzae

doi: 10.1128/aem.02488-25

Figure Lengend Snippet: MoCA2, MoCA4, and MoCA6 subcellular localization, interaction with MoCA1, and function in Magnaporthe oryzae . ( A ) Subcellular localization in hyphae and conidia. Red fluorescence signals of MoCA2-RFP, MoCA4-RFP, and MoCA6-RFP protein of hyphae and conidia were examined by confocal microscopy and colocalized with Mito-Tracker Green on mitochondria. Scale bar = 10 μm. ( B ) Y2H between MoCA2, MoCA4, MoCA6, and MoCA1. MoCA2-AD, MoCA4-AD, MoCA6-AD, and MoCA1-BD were co-transformed into yeast strain Y2H Gold and cultured on SD−Leu−Trp and SD−Leu−Trp−His media. pGADT7-MoCA2/pGBKT7-MoCA1, pGADT7-MoCA4/pGBKT7-MoCA1, and pGADT7-MoCA6/pGBKT7-MoCA1, experimental group; pGADT7-T/pGBKT7-53 , positive control group; pGADT7/pGBKT7-MoCA1, negative control group; pGADT7-T/pGBKT7-lam , blank group. ( C ) BiFC assay demonstrating the interaction between MoCA2, MoCA4, MoCA6, and MoCA1. YFP fluorescence signals were observed in N. benthamiana leaves following co-expression of MoCA2-YC, MoCA4-YC, and MoCA6-YC (C-terminal fragment of YFP) with MoCA1-YN (N-terminal fragment of YFP). JC-1 staining confirmed mitochondrial membrane localization. Negative controls included MoCA2-YC, MoCA4-YC, MoCA6-YC, and YN alone. Scale bar = 5 μm.

Article Snippet: Wild-type and MoCA1 , MoCA2 , MoCA4 , MoCA6 knockout mutant strains were cultured in the PDA medium containing CA inhibitor-acetazolamide (Ace) (MCE, China) ( ) at a concentration of 50 nM in the experimental group and 0 nM in the control group at a temperature of 28°C, and the lesion area was counted on the 7th day.

Techniques: Fluorescence, Confocal Microscopy, Transformation Assay, Cell Culture, Positive Control, Negative Control, Bimolecular Fluorescence Complementation Assay, Expressing, Staining, Membrane

Journal: Applied and Environmental Microbiology

Article Title: Carbonic anhydrases contribute to mitochondrial function, conidial development, and pathogenicity of Magnaporthe oryzae

doi: 10.1128/aem.02488-25

Figure Lengend Snippet: Analysis of synergistic effects of MoCA genes. ( A ) Colony morphology of WT, ∆ MoCA1 , and ∆ MoCA2 , ∆ MoCA4 , and ∆ MoCA6 on media with 0 nM and 50 nM carbonic anhydrase inhibitor. ( B ) Colony area measurement and statistical analysis. ( C ) Statistical analysis of MoCA gene expression in ACE of the WT strain. ( D ) Statistical analysis of MoCA1 gene expression in WT, Δ MoCA2 , Δ MoCA4 , and Δ MoCA6 . ( E ) Statistical analysis of MoCA2 gene expression in WT, Δ MoCA1 , Δ MoCA4 , and Δ MoCA6 . ( F ) Statistical analysis of MoCA4 gene expression in WT, Δ MoCA1 , Δ MoCA2 , and Δ MoCA6 . ( G ) Statistical analysis of MoCA6 gene expression in WT, Δ MoCA1 , Δ MoCA2 , and Δ MoCA4 . Error bars represent mean ± SD from three replicates. ns, P > 0.05; * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: Wild-type and MoCA1 , MoCA2 , MoCA4 , MoCA6 knockout mutant strains were cultured in the PDA medium containing CA inhibitor-acetazolamide (Ace) (MCE, China) ( ) at a concentration of 50 nM in the experimental group and 0 nM in the control group at a temperature of 28°C, and the lesion area was counted on the 7th day.

Techniques: Gene Expression

Journal: Applied and Environmental Microbiology

Article Title: Carbonic anhydrases contribute to mitochondrial function, conidial development, and pathogenicity of Magnaporthe oryzae

doi: 10.1128/aem.02488-25

Figure Lengend Snippet: Pathogenicity and invasive hyphal growth mechanisms of MoCA2 , MoCA4 , and MoCA6 . ( A ) Spray inoculation assay on rice leaves. Conidia suspensions of the WT, Δ MoCAs , and Δ MoCAs / CAs were used to infect rice leaves. ( B ) Lesion area analysis. ( C ) Statistical analysis of relative fungal growth. ( D ) Rice leaf sheath infection assay. The conidial suspension of indicated strains was injected into a rice sheath. Representative photographs of infectious hyphae were taken after 24, 48, and 72 h of incubation at 25°C. Scale bar = 10 μm. IH, infectious hyphae. ( E ) The infection rate was calculated according to the number of type 1 to type 4 events. The infection status of more than 100 germinated conidia per leaf sheath was scored at 24, 48, and 72 hpi. Type 1 has no penetration; type 2 only has a penetration peg or a single infectious hypha (IH); type 3 has more than two IH in one rice cell; type 4 has extensive IH into adjacent rice cells. Error bars represent at least three independently repeated standard deviations. Error bars represent at least three independently repeated standard deviations. ns, P > 0.05, * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: Wild-type and MoCA1 , MoCA2 , MoCA4 , MoCA6 knockout mutant strains were cultured in the PDA medium containing CA inhibitor-acetazolamide (Ace) (MCE, China) ( ) at a concentration of 50 nM in the experimental group and 0 nM in the control group at a temperature of 28°C, and the lesion area was counted on the 7th day.

Techniques: Infection, Suspension, Injection, Incubation

Journal: Applied and Environmental Microbiology

Article Title: Carbonic anhydrases contribute to mitochondrial function, conidial development, and pathogenicity of Magnaporthe oryzae

doi: 10.1128/aem.02488-25

Figure Lengend Snippet: The role of MoCA2 , MoCA4, and MoCA6 in ATP synthesis. ( A ) Mitochondrial membrane potential test using the JC-1 dye. Confocal microscopy was used for observation, with CCCP as a positive control. Scale bar = 10 µm. (B）Ratio of red to green fluorescence intensity indicating mitochondrial membrane potential. ( C ) Statistical analysis of the ATP content of mutant strains. ( D ) Statistical analysis of ATP synthase-related gene expression in WT and knockout strains of M. oryzae . Error bars represent mean ± SD from three replicates. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: Wild-type and MoCA1 , MoCA2 , MoCA4 , MoCA6 knockout mutant strains were cultured in the PDA medium containing CA inhibitor-acetazolamide (Ace) (MCE, China) ( ) at a concentration of 50 nM in the experimental group and 0 nM in the control group at a temperature of 28°C, and the lesion area was counted on the 7th day.

Techniques: Membrane, Confocal Microscopy, Positive Control, Fluorescence, Mutagenesis, Gene Expression, Knock-Out

Journal: Applied and Environmental Microbiology

Article Title: Carbonic anhydrases contribute to mitochondrial function, conidial development, and pathogenicity of Magnaporthe oryzae

doi: 10.1128/aem.02488-25

Figure Lengend Snippet: The role of MoCA2, MoCA4, and MoCA6 in nitrogen metabolism. ( A ) Strains inoculated in MM (abundant-nitrogen medium), MM-N (nitrogen-free medium), MM-N (+Gln) (nitrogen-free medium with glutamic acid), and MM-N (+Glu) (nitrogen-free medium with glutamine). The hyphae were stained with Congo red dye and photographed for images. ( B ) Statistical analysis of colony size after 7 days of cultivation in abundant-nitrogen medium, nitrogen-free medium, nitrogen-free medium with glutamic acid, and nitrogen-free medium with glutamine. ( C ) Statistical analysis of gene expression in nitrogen metabolism of WT, Δ MoCA2 , Δ MoCA4 , and Δ MoCA6 . Error bars represent at least three independently repeated standard deviations. ns, P > 0.05; * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: Wild-type and MoCA1 , MoCA2 , MoCA4 , MoCA6 knockout mutant strains were cultured in the PDA medium containing CA inhibitor-acetazolamide (Ace) (MCE, China) ( ) at a concentration of 50 nM in the experimental group and 0 nM in the control group at a temperature of 28°C, and the lesion area was counted on the 7th day.

Techniques: Staining, Gene Expression

Journal: Cell Communication and Signaling : CCS

Article Title: Disruption of the biorhythm in gastric epithelial cell triggers inflammation in Helicobacter pylori -associated gastritis by aberrantly regulating NFIL3 via CagA activated ERK-SP1 pathway

doi: 10.1186/s12964-025-02302-z

Figure Lengend Snippet: H. pylori transcriptionally increased gastric epithelial NFIL3 expression via ERK-SP1 pathway. ( A ) AGS cells were subjected to infection with H. pylori 11,637 or 26,695 (MOI = 100, 24 h) either in the same (lower) chamber or in a separate (upper) chamber of a Transwell apparatus. The expression levels of NFIL3 were assessed using qRT-PCR ( n = 3) and WB. ( B ) The expression of NFIL3 in AGS cells infected with H. pylori TN2GF4 or its virulence mutant strains was evaluated through the GEO database ( GSE60661 ). ( C ) NFIL3 expression was further examined in AGS cells infected with H. pylori 11,637 or the Δ cagA mutant, utilizing qRT-PCR ( n = 3) and WB. ( D ) The expression of NFIL3 was also analyzed in AGS cells infected with H. pylori 26,695 or the Δ vacA mutant through qRT-PCR ( n = 3) and WB. ( E ) The expression levels of NR1D1 were investigated in AGS cells infected with H. pylori TN2GF4 or virulence mutant strains utilizing the GEO database ( GSE60661 ). ( F ) Potential transcription factor binding sites were predicted via the PROMO website, focusing on a 2000 bp segment of the NFIL3 promoter. ( G ) A conserved sequence of putative SP1 binding sites (red line) was identified in both human and mouse models. ( H ) WB and qRT-PCR ( n = 3) analyses of NFIL3 were conducted in AGS cells post-treatment with H. pylori 11,637, U0126, and Mithramycin A (MMA). ( I ) Following a 24-hour transfection with SP1 siRNA, AGS cells were subsequently infected with H. pylori 11,637 for an additional 24 h. The expression levels of NFIL3 and SP1 were analyzed using qRT-PCR ( n = 3) and WB. ( J ) NFIL3 promoter luciferase reporter assays were conducted in AGS cell lines treated with H. pylori 11,637, U0126, Mithramycin A (MMA), and the Δ cagA mutant. ( K ) Luciferase reporter assay for NFIL3-Luc and mutant-Luc. n.s. P > 0.05, ** P < 0.01, and *** P < 0.001

Article Snippet: We utilized several strains of H. pylori , specifically the cagA and vacA -positive strain 26,695 (ATCC 700392), the vacA knockout mutant strain 26,695 (referred to as Δ vacA herein), the cagA and vacA -positive strain NCTC 11,637 (ATCC 43504, designated as H. pylori 11637 in this manuscript), the cagA knockout mutant strain NCTC 11,637 (termed Δ cagA in this article), and the cagA and vacA -positive strain PMSS1 (pre-mouse Sydney strain 1) [ , ].

Techniques: Expressing, Infection, Quantitative RT-PCR, Mutagenesis, Binding Assay, Sequencing, Transfection, Luciferase, Reporter Assay