scholarly journals Comparative Proteomics Reveals the Potential Targets of BcNoxR, a Putative Regulatory Subunit of NADPH Oxidase of Botrytis cinerea

2016 ◽  
Vol 29 (12) ◽  
pp. 990-1003 ◽  
Author(s):  
Hua Li ◽  
Zhanquan Zhang ◽  
Chang He ◽  
Guozheng Qin ◽  
Shiping Tian
Keyword(s):  
Nadph Oxidase ◽  
Botrytis Cinerea ◽  
Proteomic Analysis ◽  
Fungal Pathogens ◽  
Molecular Mechanisms ◽  
Quantitative Polymerase Chain Reaction ◽  
Tomato Fruit ◽  
Polymerase Chain Reaction Analysis ◽  
Regulatory Subunit ◽  
Wild Type

The NADPH oxidase (NOX) complex has been shown to play a crucial role in stress response and in the virulence of various fungal pathogens. The underlying molecular mechanisms of NOX, however, remain largely unknown. In the present study, a comparative proteomic analysis compared changes in protein abundance in wild-type Botrytis cinerea and ΔbcnoxR mutants in which the regulatory subunit of NOX was deleted. The ΔbcnoxR mutants exhibited reduced growth, sporulation, and impaired virulence. A total of 60 proteins, representing 49 individual genes, were identified in ΔbcnoxR mutants that exhibited significant differences in abundance relative to wild-type. Reverse transcription-quantitative polymerase chain reaction analysis demonstrated that the differences in transcript levels for 36 of the genes encoding the identified proteins were in agreement with the proteomic analysis, while the remainder exhibited reverse levels. Functional analysis of four proteins that decreased abundance in the ΔbcnoxR mutants indicated that 6-phosphogluconate dehydrogenase (BcPGD) played a role in the growth and sporulation of B. cinerea. The Δbcpgd mutants also displayed impaired virulence on various hosts, such as apple, strawberry, and tomato fruit. These results suggest that NOX can influence the expression of BcPGD, which has an impact on growth, sporulation, and virulence of B. cinerea.

scholarly journals NADPH Oxidase Is Crucial for the Cellular Redox Homeostasis in Fungal Pathogen Botrytis cinerea

2019 ◽  
Vol 32 (11) ◽  
pp. 1508-1516
Author(s):  
Hua Li ◽  
Shiping Tian ◽  
Guozheng Qin
Keyword(s):  
Nadph Oxidase ◽  
Botrytis Cinerea ◽  
Fungal Pathogen ◽  
Molecular Mechanisms ◽  
Redox Homeostasis ◽  
Signal Transduction Pathway ◽  
Regulatory Subunit ◽  
Wild Type ◽  
Mutant Proteins ◽  
In The Wild

During interactions, both plants and pathogens produce reactive oxygen species (ROS). Plants generate ROS for defense induction, while pathogens synthesize ROS for growth, sporulation, and virulence. NADPH oxidase (NOX) complex in the plasma membrane represents a main protein complex for ROS production in pathogens. Although NOX plays a crucial role in pathogenicity of pathogens, the underlying molecular mechanisms of NOX, especially the proteins regulated by NOX, remain largely unknown. Here, we applied an iodoacetyl tandem mass tag-based redox proteomic assay to investigate the protein redox dynamics in deletion mutant of bcnoxR, which encodes a regulatory subunit of NOX in the fungal pathogen Botrytis cinerea. In total, 214 unique peptidyl cysteine (Cys) thiols from 168 proteins were identified and quantified in both the wild type and ∆bcnoxR mutant. The Cys thiols in the ∆bcnoxR mutant were generally more oxidized than those in the wild type, suggesting that BcNoxR is essential for maintaining the equilibrium of the redox state in B. cinerea. Site-specific thiol oxidation analysis indicated that 142 peptides containing the oxidized thiols changed abundance significantly in the ∆bcnoxR mutant. Proteins containing these differential peptides are classified into various functional categories. Functional analysis revealed that one of these proteins, 6-phosphate dehydrogenase, played roles in oxidative stress response and pathogenesis of B. cinerea. These results provide insight into the potential target proteins and the ROS signal transduction pathway regulated by NOX.

scholarly journals Olfactomedin 4 contributes to hydrogen peroxide-induced NADPH oxidase activation and apoptosis in mouse neutrophils

2018 ◽  
Vol 315 (4) ◽  
pp. C494-C501 ◽  
Author(s):  
Wenli Liu ◽  
Yueqin Liu ◽  
Hongzhen Li ◽  
Griffin P. Rodgers
Keyword(s):  
Hydrogen Peroxide ◽  
Nadph Oxidase ◽  
Molecular Mechanisms ◽  
Oxidase Inhibitor ◽  
Superoxide Production ◽  
Wild Type ◽  
Escherichia Coli Bacteria ◽  
Induced Apoptosis ◽  
Nadph Oxidase Activation ◽  
Deficient Mice

Neutrophils increase production of reactive oxygen species, including superoxide, hydrogen peroxide (H2O2), and hydroxyl radical, to destroy invading microorganisms under pathological conditions. Conversely, oxidative stress conditions, such as the presence of H2O2, induce neutrophil apoptosis, which helps to remove neutrophils after inflammation. However, the detailed molecular mechanisms that are involved in the latter process have not been elucidated. In this study, we investigated the potential role of olfactomedin 4 (Olfm4) in H2O2-induced superoxide production and apoptosis in mouse neutrophils. We have demonstrated that Olfm4 is not required for maximal-dosage PMA- and Escherichia coli bacteria-induced superoxide production, but Olfm4 contributes to suboptimal-dosage PMA- and H2O2-induced superoxide production. Using an NADPH oxidase inhibitor and gp91phox-deficient mouse neutrophils, we found that NAPDH oxidase was required for PMA-stimulated superoxide production and that Olfm4 mediated H2O2-induced superoxide production through NADPH oxidase, in mouse neutrophils. We have shown that neutrophils from Olfm4-deficient mice exhibited reduced H2O2-induced apoptosis compared with neutrophils from wild-type mice. We also demonstrated that neutrophils from Olfm4-deficient mice exhibited reduced H2O2-stimulated mitochondrial damage and membrane permeability, and as well as reduced caspase-3 and caspase-9 activity, compared with neutrophils from wild-type mice. Moreover, the cytoplasmic translocation of the proapoptotic mitochondrial proteins Omi/HtrA2 and Smac/DIABLO in response to H2O2was reduced in neutrophils from Olfm4-deficient mice compared with neutrophils from wild-type mice. Our study demonstrates that Olfm4 contributes to H2O2-induced NADPH oxidase activation and apoptosis in mouse neutrophils. Olfactomedin 4 might prove to be a potential target for future studies on inflammatory neutrophil biology and for inflammatory disease treatment.

scholarly journals NADPH Oxidases Are Involved in Differentiation and Pathogenicity in Botrytis cinerea

2008 ◽  
Vol 21 (6) ◽  
pp. 808-819 ◽  
Author(s):  
Nadja Segmüller ◽  
Leonie Kokkelink ◽  
Sabine Giesbert ◽  
Daniela Odinius ◽  
Jan van Kan ◽  
...  
Keyword(s):  
Botrytis Cinerea ◽  
Nicotinamide Adenine Dinucleotide ◽  
Amino Acid Sequences ◽  
Host Tissue ◽  
Regulatory Subunit ◽  
Phytopathogenic Fungus ◽  
Wild Type ◽  
Nadph Oxidases ◽  
Knock Out

Nicotinamide adenine dinucleotide (NADPH) oxidases have been shown to be involved in various differentiation processes in fungi. We investigated the role of two NADPH oxidases in the necrotrophic phytopathogenic fungus, Botrytis cinerea. The genes bcnoxA and bcnoxB were cloned and characterized; their deduced amino acid sequences show high homology to fungal NADPH oxidases. Analyses of single and double knock-out mutants of both NADPH oxidase genes showed that both bcnoxA and bcnoxB are involved in formation of sclerotia. Both genes have a great impact on pathogenicity: whereas bcnoxB mutants showed a retarded formation of primary lesions, probably due to an impaired formation of penetration structures, bcnoxA mutants were able to penetrate host tissue in the same way as the wild type but were much slower in colonizing the host tissue. Double mutants showed an additive effect: they were aberrant in penetration and colonization of plant tissue and, therefore, almost nonpathogenic. To study the structure of the fungal Nox complex in more detail, bcnoxR (encoding a homolog of the mammalian p67phox, a regulatory subunit of the Nox complex) was functionally characterized. The phenotype of ΔbcnoxR mutants is identical to that of ΔbcnoxAB double mutants, providing evidence that BcnoxR is involved in activation of both Bcnox enzymes.

Promoter Methylation and Decreased Expression of Mir-124 In Myelodysplastic Syndromes

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4014-4014
Author(s):  
Yuesheng Meng ◽  
Qiao Xia ◽  
Jun Hu
Keyword(s):  
Myelodysplastic Syndromes ◽  
Promoter Methylation ◽  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Quantitative Polymerase Chain Reaction ◽  
Methylation Status ◽  
Polymerase Chain Reaction Analysis ◽  
Promoter Regions ◽  
Hematopoietic Stem

Abstract Abstract 4014 The myelodysplastic syndromes (MDS) are a heterogeneous group of clonal disorders of hematopoietic stem/progenitor cells. Although demethylating agents such azacytidine and decitabine have been widely used to treat MDS, the underlying molecular mechanisms remain obscure. Abnormalities of microRNAs (miRNA) have been recently associated with hematological malignancies including MDS. The miR-124 was initially demonstrated to modulate neurogenesis. It was recently shown that EVI1-induced methylation and silencing of miR-124 were present in murine MDS cells. In the retrospective study we evaluated methylation status and expression levels of miR-124 in fifteen MDS patients (subtypes included RCUD, RCMD, RAEB-1, RAEB-2 and CMML). Genomic DNA samples were modified with bisulfite and methylation at three promoter regions of miR-124 was examined with methylation-specific real time quantitative polymerase chain reaction analysis (MQPCR). In general, we observed an increased methylation levels of miR-124 in MDS patients than that in normal bone marrow (NBM, P<0.01). In accordance with this, marked depression of miR-124 was seen in six patients when compared with NBM (more than 2 times lower), as determined with quantitative reverse-transcriptive PCR assay. Moreover, there were higher degrees of promoter methylation in cases with depressed miR-124 than that in remaining cases. A negative correlation between the expression and methylation levels was statistically significant (R= -0.498, P<0.01). The change of miR-124 was not directly related to short-term clinical response or prognosis, possibly due to limited size of the sample. However, the miR-124 amount returned to basal levels in two cases (RCMD and CMML subtypes respectively) after low-dose decitabine therapy and DNA methylation of all three loci disappeared. Continued work is underway to accumulate more cases and make long-term clinical follow-up. In conclusion, this primary work suggested a possible role of the methylation-mediated silencing of miR-124 in the pathogenesis or disease progression of MDS. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Renal molecular mechanisms underlying altered Na+ handling and genesis of hypertension during adulthood in prenatally undernourished rats

2014 ◽  
Vol 111 (11) ◽  
pp. 1932-1944 ◽  
Author(s):  
Leucio D. Vieira-Filho ◽  
Edjair V. Cabral ◽  
Juliane S. Farias ◽  
Paulo A. Silva ◽  
Humberto Muzi-Filho ◽  
...  
Keyword(s):  
Lipid Peroxidation ◽  
Nadph Oxidase ◽  
Atpase Activity ◽  
Proximal Tubule ◽  
Molecular Mechanisms ◽  
Renal Tissue ◽  
Regulatory Subunit ◽  
Ang Ii ◽  
Down Regulation ◽  
Cox 2

In the present study, we investigated the development of hypertension in prenatally undernourished adult rats, including the mechanisms that culminate in dysfunctions of molecular signalling in the kidney. Dams were fed a low-protein multideficient diet throughout gestation with or without α-tocopherol during lactation. The time course of hypertension development followed in male offspring was correlated with alterations in proximal tubule Na+-ATPase activity, expression of angiotensin II (Ang II) receptors, and activity of protein kinases C and A. After the establishment of hypertension, Ang II levels, cyclo-oxygenase 2 (COX-2) and NADPH oxidase subunit expression, lipid peroxidation and macrophage infiltration were examined in renal tissue. Lipid peroxidation in undernourished rats, which was very intense at 60 d, decreased at 90 d and returned to control values by 150 d. During the prehypertensive phase, prenatally undernourished rats exhibited elevated renal Na+-ATPase activity, type 2 Ang II receptor down-regulation and altered protein kinase A:protein kinase C ratio. Stable late hypertension coexisted with highly elevated levels of Ang II-positive cells in the cortical tubulointerstitium, enhanced increase in the expression of p47phox (NADPH oxidase regulatory subunit), marked down-regulation of COX-2 expression, expanded plasma volume and decreased creatinine clearance. These alterations were reduced when the dams were given α-tocopherol during lactation. The offspring of well-nourished dams treated with α-tocopherol exhibited most of the alterations encountered in the offspring of undernourished dams not treated with α-tocopherol. Thus, alterations in proximal tubule Na+ transport, subcellular signalling pathways and reactive oxygen species handling in renal tissue underpin the development of hypertension.

scholarly journals Amylopectin Induces Fumonisin B1 Production by Fusarium verticillioides During Colonization of Maize Kernels

2005 ◽  
Vol 18 (12) ◽  
pp. 1333-1339 ◽  
Author(s):  
B. H. Bluhm ◽  
C. P. Woloshuk
Keyword(s):  
Quantitative Polymerase Chain Reaction ◽  
Fusarium Verticillioides ◽  
Animal Health ◽  
Fumonisin B1 ◽  
Polymerase Chain Reaction Analysis ◽  
Defined Medium ◽  
Wild Type ◽  
Fungal Genes ◽  
High Amylose ◽  
Maize Kernels

Fusarium verticillioides, a fungal pathogen of maize, produces fumonisin mycotoxins that adversely affect human and animal health. Basic questions remain unanswered regarding the interactions between the host plant and the fungus that lead to the accumulation of fumonisins in maize kernels. In this study, we evaluated the role of kernel endosperm composition in regulating fumonisin B1 (FB1) biosynthesis. We found that kernels lacking starch due to physiological immaturity did not accumulate FB1. Quantitative polymerase chain reaction analysis indicated that kernel development also affected the expression of fungal genes involved in FB1 biosynthesis, starch metabolism, and nitrogen regulation. A mutant strain of F. verticillioides with a disrupted α-amylase gene was impaired in its ability to produce FB1 on starchy kernels, and both the wild-type and mutant strains produced significantly less FB1 on a high-amylose kernel mutant of maize. When grown on a defined medium with amylose as the sole carbon source, the wild-type strain produced only trace amounts of FB1, but it produced large amounts of FB1 when grown on amylopectin or dextrin, a product of amylopectin hydrolysis. We conclude that amylopectin induces FB1 production in F. verticillioides. This study provides new insight regarding the interaction between the fungus and maize kernel during pathogenesis and highlights important areas that need further study.

scholarly journals The Glutamate Receptor Plays a Role in Defense against Botrytis cinerea through Electrical Signaling in Tomato

2021 ◽  
Vol 11 (23) ◽  
pp. 11217
Author(s):  
Shuxian Feng ◽  
Caizhe Pan ◽  
Shuting Ding ◽  
Qiaomei Ma ◽  
Chaoyi Hu ◽  
...  
Keyword(s):  
Botrytis Cinerea ◽  
Fungal Pathogen ◽  
Fungal Pathogens ◽  
Plant Immunity ◽  
Main Role ◽  
Wild Type ◽  
Tomato Plants ◽  
Electrical Signals ◽  
Electrical Signaling ◽  
The Relationship

Plant glutamate-like receptor genes (GLRs) are homologous to mammalian ionotropic glutamate receptors genes (iGluRs). Although GLRs have been implicated in plant defenses to biotic stress, the relationship between GLR-mediated plant immunity against fungal pathogens and electrical signals remains poorly understood. Here, we found that pretreatment with a GLR inhibitor, 6,7-dinitriquinoxaline-2,3-dione (DNQX), increased the susceptibility of tomato plants to the necrotrophic fungal pathogen Botrytis cinerea. Assessment of the glr3.3, glr3.5 and glr3.3/glr3.5 double-mutants upon B. cinerea infection showed that tomato GLR3.3 and GLR3.5 are essential for plant immunity against B. cinerea, wherein GLR3.3 plays the main role. Analysis of the membrane potential changes induced by glutamate (Glu) or glycine (Gly) revealed that amplitude was significantly reduced by knocking out GLR3.3 in tomato. While treatment with Glu or Gly significantly increased immunity against B. cinerea in wild-type plants, this effect was significantly attenuated in glr3.3 mutants. Thus, our data demonstrate that GLR3.3- and GLR3.5-mediated plant immunity against B. cinerea is associated with electrical signals in tomato plants.

scholarly journals Deletion of the Regulatory Subunit of Protein Kinase A in Aspergillus fumigatus Alters Morphology, Sensitivity to Oxidative Damage, andVirulence

2006 ◽  
Vol 74 (8) ◽  
pp. 4865-4874 ◽  
Author(s):  
Wei Zhao ◽  
John C. Panepinto ◽  
Jarrod R. Fortwendel ◽  
Lauren Fox ◽  
Brian G. Oliver ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Protein Kinase ◽  
Oxidative Damage ◽  
Aspergillus Fumigatus ◽  
Fungal Pathogens ◽  
Regulatory Subunit ◽  
Wild Type ◽  
Dependent Protein Kinase ◽  
Dependent Protein ◽  
Opportunistic Fungal Pathogen

ABSTRACT Aspergillus fumigatus is an important opportunistic fungal pathogen. The cAMP-dependent protein kinase (PKA) signaling pathway plays an important role in regulating morphology, growth, and virulence in a number of fungal pathogens of plants and animals. We have constructed a mutant of A. fumigatus that lacks the regulatory subunit of PKA, pkaR, and analyzed the growth and development, sensitivity to oxidative damage, and virulence of the mutant, along with those of the wild type and a complemented mutant. Both growth and germination rates of the mutant are reduced, and there are morphological abnormalities in conidiophores, leading to reduced conidiation. Conidia from the ΔpkaR mutant are more sensitive to killing by hydrogen peroxide, menadione, paraquat, and diamide. However, the hyphae of the mutant are killed to a greater extent only by paraquat and diamide, whereas they are less susceptible to the effects of hydrogen peroxide. In an immunosuppressed mouse model, intranasally administered conidia of the mutant are significantly less virulent than those of the wild type or a complemented mutant. Unregulated PKA signaling is detrimental to the virulence of A. fumigatus, perhaps through the reduced susceptibility of the mutant to damage by oxidizing agents and reduced growth kinetics.

scholarly journals Cyclin D1 Expression Mediated by Phosphatidylinositol 3-Kinase through mTOR-p70S6K-Independent Signaling in Growth Factor-Stimulated NIH 3T3 Fibroblasts

1999 ◽  
Vol 19 (2) ◽  
pp. 1346-1358 ◽  
Author(s):  
Noriko Takuwa ◽  
Yasuhisa Fukui ◽  
Yoh Takuwa
Keyword(s):  
Cell Cycle ◽  
Growth Factor ◽  
Cyclin D1 ◽  
Molecular Mechanisms ◽  
D1 Protein ◽  
Regulatory Subunit ◽  
Phase Cell ◽  
Wild Type ◽  
Cyclin D1 Protein ◽  
Nih 3T3

ABSTRACT Phosphatidylinositol (PI) 3-kinase is required for G1to S phase cell cycle progression stimulated by a variety of growth factors and is implicated in the activation of several downstream effectors, including p70S6K. However, the molecular mechanisms by which PI 3-kinase is engaged in activation of the cell cycle machinery are not well understood. Here we report that the expression of a dominant negative (DN) form of either the p110α catalytic or the p85 regulatory subunit of heterodimeric PI 3-kinase strongly inhibited epidermal growth factor (EGF)-induced upregulation of cyclin D1 protein in NIH 3T3(M17) fibroblasts. The PI 3-kinase inhibitors LY294002 and wortmannin completely abrogated increases in both mRNA and protein levels of cyclin D1 and phosphorylation of pRb, inducing G1 arrest in EGF-stimulated cells. By contrast, rapamycin, which potently suppressed p70S6K activity throughout the G1 phase, had little inhibitory effect, if any, on either of these events. PI 3-kinase, but not rapamycin-sensitive pathways, was also indispensable for upregulation of cyclin D1 mRNA and protein by other mitogens in NIH 3T3 (M17) cells and in wild-type NIH 3T3 cells as well. We also found that an enforced expression of wild-type p110 was sufficient to induce cyclin D1 protein expression in growth factor-deprived NIH 3T3(M17) cells. The p110 induction of cyclin D1 in quiescent cells was strongly inhibited by coexpression of either of the PI 3-kinase DN forms, and by LY294002, but was independent of the Ras-MEK-ERK pathway. Unlike mitogen stimulation, the p110 induction of cyclin D1 was sensitive to rapamycin. These results indicate that the catalytic activity of PI 3-kinase is necessary, and could also be sufficient, for upregulation of cyclin D1, with mTOR signaling being differentially required depending upon cellular conditions.

scholarly journals Actin Is Required for Cellular Development and Virulence of Botrytis cinerea via the Mediation of Secretory Proteins

mSystems ◽  
2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Hua Li ◽  
Zhanquan Zhang ◽  
Guozheng Qin ◽  
Chang He ◽  
Boqiang Li ◽  
...  
Keyword(s):  
Botrytis Cinerea ◽  
Virulence Factors ◽  
Proteomic Analysis ◽  
Crucial Role ◽  
Pathogenic Fungi ◽  
Extracellular Proteins ◽  
Secretory Proteins ◽  
Wild Type ◽  
Cell Wall Degrading Enzymes ◽  
Vital Component

ABSTRACT Actin is a vital component of the cytoskeleton of living cells and is involved in several complex processes. However, its functions in plant-pathogenic fungi are largely unknown. In this paper, we found that deletion of the Botrytis cinerea actin gene bcactA reduced growth and sporulation of B. cinerea and lowered virulence. Based on iTRAQ (isobaric tags for relative and absolute quantification)-based proteomic analysis, we compared changes of the secretome in ΔbcactA and wild-type strains. A total of 40 proteins exhibited significant differences in abundance in ΔbcactA mutants compared with the wild type. These proteins included 11 down-accumulated cell wall-degrading enzymes (CWDEs). Among them, two CWDEs, cellobiohydrolase (BcCBH) and β-endoglucanase (BcEG), were found to contribute to the virulence of B. cinerea, indicating that bcactA plays a crucial role in regulating the secretion of extracellular virulence factors. These findings unveil previously unknown functions of BcactA to mediate the virulence of B. cinerea and provide new mechanistic insights into the role of BcactA in the complex pathogenesis of B. cinerea. IMPORTANCE The cytoskeleton is an important network that exists in cells of all domains of life. In eukaryotic cells, actin is a vital component of the cytoskeleton. Here, we report that BcactA, an actin protein in B. cinerea, can affect the growth, sporulation, and virulence of B. cinerea. Furthermore, iTRAQ-based proteomic analysis showed that BcactA affects the abundance of 40 extracellular proteins, including 11 down-accumulated CWDEs. Among them, two CWDEs, cellobiohydrolase (BcCBH) and β-endoglucanase (BcEG), contributed to the virulence of B. cinerea, indicating that bcactA plays a crucial role in regulating extracellular virulence factors. These findings unveil previously unknown functions of BcactA in mediating growth, sporulation, and virulence of B. cinerea.

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