scholarly journals Cytological and Gene Profile Expression Analysis Reveals Modification in Metabolic Pathways and Catalytic Activities Induce Resistance in Botrytis cinerea Against Iprodione Isolated From Tomato

2020 ◽  
Vol 21 (14) ◽  
pp. 4865
Author(s):  
Ambreen Maqsood ◽  
Chaorong Wu ◽  
Sunny Ahmar ◽  
Haiyan Wu
Keyword(s):  
Enzymatic Activity ◽  
Rna Sequencing ◽  
Aspartic Acid ◽  
Mycelial Growth ◽  
Plant Pathogens ◽  
Fruits And Vegetables ◽  
Transcriptomic Analysis ◽  
Cell Membrane Permeability ◽  
Wild Type ◽  
Tomato Field

Grey mold is one of the most serious and catastrophic diseases, causing significant yield losses in fruits and vegetables worldwide. Iprodione is a broad spectrum agrochemical used as a foliar application as well as a seed protectant against many fungal and nematode diseases of fruits and vegetables from the last thirty years. The extensive use of agrochemicals produces resistance in plant pathogens and is the most devastating issue in food and agriculture. However, the molecular mechanism (whole transcriptomic analysis) of a resistant mutant of B. cinerea against iprodione is still unknown. In the present study, mycelial growth, sporulation, virulence, osmotic potential, cell membrane permeability, enzymatic activity, and whole transcriptomic analysis of UV (ultraviolet) mutagenic mutant and its wild type were performed to compare the fitness. The EC50 (half maximal effective concentration that inhibits the growth of mycelium) value of iprodione for 112 isolates of B. cinerea ranged from 0.07 to 0.87 µg/mL with an average (0.47 µg/mL) collected from tomato field of Guangxi Province China. Results also revealed that, among iprodione sensitive strains, only B67 strain induced two mutants, M0 and M1 after UV application. The EC50 of these induced mutants were 1025.74 μg/mL and 674.48 μg/mL, respectively, as compared to its wild type 1.12 μg/mL. Furthermore, mutant M0 showed higher mycelial growth sclerotia formation, virulence, and enzymatic activity than wild type W0 and M1 on potato dextrose agar (PDA) medium. The bctubA gene in the mutant M0 replaced TTC and GAT codon at position 593 and 599 by TTA and GAA, resulting in replacement of phenyl alanine into leucine (transversion C/A) and aspartic acid into glutamic acid (transversion T/C) respectively. In contrast, in bctubB gene, GAT codon at position 646 is replaced by AAT and aspartic acid converted into asparagine (transition G/A). RNA sequencing of the mutant and its wild type was performed without (M0, W0) and with iprodione treatment (M-ipro, W-ipro). The differential gene expression (DEG) identified 720 unigenes in mutant M-ipro than W-ipro after iprodione treatment (FDR ≤ 0.05 and log2FC ≥ 1). Seven DEGs were randomly selected for quantitative real time polymerase chain reaction to validate the RNA sequencing genes expression (log fold 2 value). The gene ontology (GO) enrichment and Kyoto encyclopedia genes and genomes (KEGG) pathway functional analyses indicated that DEG’s mainly associated with lysophopholipase, carbohydrate metabolism, amino acid metabolism, catalytic activity, multifunctional genes (MFO), glutathione-S transferase (GST), drug sensitivity, and cytochrome P450 related genes are upregulated in mutant type (M0, M-ipro) as compared to its wild type (W0, W-ipro), may be related to induce resistant in mutants of B. cinerea against iprodione.

scholarly journals Pharmacological Characteristics and Efficacy of Fluazinam Against Corynespora cassiicola, Causing Cucumber Target Spot in Greenhouses

Plant Disease ◽  
2020 ◽  
Vol 104 (9) ◽  
pp. 2449-2454
Author(s):  
Tao Li ◽  
Qian Xiu ◽  
Jie Zhang ◽  
Jian Xin Wang ◽  
Ya Bing Duan ◽  
...  
Keyword(s):  
Spore Germination ◽  
Mycelial Growth ◽  
Plant Pathogens ◽  
Resistance Management ◽  
Cell Membrane Permeability ◽  
Corynespora Cassiicola ◽  
In Planta ◽  
Action Mode ◽  
Target Spot

Cucumber target spot, caused by Corynespora cassiicola, is a devastating fungal disease in greenhouses in China. Lack of resistant cultivars and unscientific use of fungicides aggravated the difficulty to manage this disease. In recent years, resistance of C. cassiicola to benzimidazoles, quinone outside inhibitors, and succinate dehydrogenase inhibitors has occurred in China. Here, we tested the fluazinam sensitivity distribution of 79 C. cassiicola isolates from different provinces in China based on mycelial growth inhibition. The EC50 values of fluazinam ranged from 0.1002 to 0.3129 µg/ml with a mean of 0.2136 ± 0.0495 µg/ml, and the sensitivity frequency was normally distributed (P = 0.2083, Shapiro–Wilk test). Meanwhile, the EC50 values for spore germination inhibition ranged from 0.0992 to 0.2278 µg/ml with a mean of 0.1499 ± 0.0504 µg/ml. This indicated that fluazinam exhibited an excellent in vitro fungicidal activity on both mycelial growth and spore germination. In addition, fluazinam also exhibited a good in planta control efficacy on detached cucumber leaves in the protective and curative assays. Moreover, the biological and physiological characteristics of C. cassiicola as affected by fluazinam were determined. Fluazinam not only significantly inhibited respiration and adenosine triphosphate production but also caused the increase of cell membrane permeability and the dysfunctions of cellular homeostasis. Interestingly, we found that fluazinam especially damaged vacuole structures, causing the redistribution of vacuole substances. Taken together, our findings provide not only essential references for resistance management of C. cassiicola but also interesting insights for further revealing the action mode of fluazinam against plant pathogens.

scholarly journals Chemical Composition and Antimicrobial Properties of Mentha × piperita cv. ‘Kristinka’ Essential Oil

Plants ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1567
Author(s):  
Ippolito Camele ◽  
Daniela Gruľová ◽  
Hazem S. Elshafie
Keyword(s):  
Antifungal Activity ◽  
Xanthomonas Campestris ◽  
Fruits And Vegetables ◽  
Antimicrobial Properties ◽  
Pathogenic Fungi ◽  
Cell Membrane Permeability ◽  
Penicillium Expansum ◽  
Mentha Piperita ◽  
Plant Essential Oils ◽  
Main Components

Several economically important crops, fruits and vegetables are susceptible to infection by pathogenic fungi and/or bacteria postharvest or in field. Recently, plant essential oils (EOs) extracted from different medicinal and officinal plants have had promising antimicrobial effects against phytopathogens. In the present study, the potential microbicide activity of Mentha × piperita cv. ‘Kristinka’ (peppermint) EO and its main constituents have been evaluated against some common phytopathogens. In addition, the cell membrane permeability of the tested fungi and the minimum fungicidal concentrations were measured. The antifungal activity was tested against the following postharvest fungi: Botrytis cinerea, Monilinia fructicola, Penicillium expansum and Aspergillus niger, whereas antibacterial activity was evaluated against Clavibacter michiganensis, Xanthomonas campestris, Pseudomonas savastanoi and P. syringae pv. phaseolicola. The chemical analysis has been carried out using GC-MS and the main components were identified as menthol (70.08%) and menthone (14.49%) followed by limonene (4.32%), menthyl acetate (3.76%) and β-caryophyllene (2.96%). The results show that the tested EO has promising antifungal activity against all tested fungi, whereas they demonstrated only a moderate antibacterial effect against some of the tested bacteria.

scholarly journals Functional Assessment of 12 Rare Allelic CYP2C9 Variants Identified in a Population of 4773 Japanese Individuals

2021 ◽  
Vol 11 (2) ◽  
pp. 94
Author(s):  
Masaki Kumondai ◽  
Akio Ito ◽  
Evelyn Marie Gutiérrez Rico ◽  
Eiji Hishinuma ◽  
Akiko Ueda ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Three Dimensional ◽  
Warfarin Dose ◽  
Catalytic Efficiency ◽  
Therapeutic Window ◽  
Wild Type ◽  
Drug Metabolizing Enzyme ◽  
Novel Variants ◽  
Metabolizing Enzyme ◽  
Almost All

Cytochrome P450 2C9 (CYP2C9) is an important drug-metabolizing enzyme that contributes to the metabolism of approximately 15% of clinically used drugs, including warfarin, which is known for its narrow therapeutic window. Interindividual differences in CYP2C9 enzymatic activity caused by CYP2C9 genetic polymorphisms lead to inconsistent treatment responses in patients. Thus, in this study, we characterized the functional differences in CYP2C9 wild-type (CYP2C9.1), CYP2C9.2, CYP2C9.3, and 12 rare novel variants identified in 4773 Japanese individuals. These CYP2C9 variants were heterologously expressed in 293FT cells, and the kinetic parameters (Km, kcat, Vmax, catalytic efficiency, and CLint) of (S)-warfarin 7-hydroxylation and tolbutamide 4-hydroxylation were estimated. From this analysis, almost all novel CYP2C9 variants showed significantly reduced or null enzymatic activity compared with that of the CYP2C9 wild-type. A strong correlation was found in catalytic efficiencies between (S)-warfarin 7-hydroxylation and tolbutamide 4-hydroxylation among all studied CYP2C9 variants. The causes of the observed perturbation in enzyme activity were evaluated by three-dimensional structural modeling. Our findings could clarify a part of discrepancies among genotype–phenotype associations based on the novel CYP2C9 rare allelic variants and could, therefore, improve personalized medicine, including the selection of the appropriate warfarin dose.

scholarly journals Conversion of secretory proteins into membrane proteins by fusing with a glycosylphosphatidylinositol anchor signal of alkaline phosphatase

1994 ◽  
Vol 301 (2) ◽  
pp. 577-583 ◽  
Author(s):  
K Oda ◽  
J Cheng ◽  
T Saku ◽  
N Takami ◽  
M Sohda ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Endoplasmic Reticulum ◽  
Aspartic Acid ◽  
Chimeric Protein ◽  
Attachment Site ◽  
In Vitro Translation ◽  
Placental Alkaline Phosphatase ◽  
Wild Type ◽  
Aspartic Acid Residue

Placental alkaline phosphatase (PLAP) is initially synthesized as a precursor (proPLAP) with a C-terminal extension. We constructed a recombinant cDNA which encodes a chimeric protein (alpha GL-PLAP) comprising rat alpha 2u-globulin (alpha GL) and the C-terminal extension of PLAP. Two molecular species (25 kDa and 22 kDa) were expressed in the COS-1 cell transfected with the cDNA for alpha GL-PLAP. Only the 22 kDa form was labelled with both [3H]stearic acid and [3H]ethanolamine. Upon digestion with phosphatidylinositol-specific phospholipase C the 22 kDa form was released into the medium, indicating that this form is anchored on the cell surface via glycosylphosphatidylinositol (GPI). A specific IgG raised against a C-terminal nonapeptide of proPLAP precipitated the 25 kDa form but not the 22 kDa form, suggesting that the 25 kDa form is a precursor retaining the C-terminal propeptide. When a mutant alpha GL-PLAP, in which the aspartic acid residue is replaced with tryptophan at a putative cleavage/attachment site, was expressed in COS-1 cells, the 25 kDa precursor was the only form found inside the cell and retained in the endoplasmic reticulum, as judged by immunofluorescence microscopy. In vitro translation programmed with mRNAs coding for the wild-type and mutant forms of alpha GL-PLAP demonstrated that the C-terminal propeptide was cleaved from the wild-type chimeric protein, but not from the mutant one. This gave rise to the 22 kDa form attached with a GPI anchor, suggesting that GPI is covalently linked to the aspartic acid residue (Asp159) of alpha GL-PLAP. Taken together, these results indicate that the C-terminal propeptide of PLAP functions as a signal to render alpha GL a GPI-linked membrane protein in vitro and in vivo in cultured cells, and that the chimeric protein constructed in this study may be useful for elucidating the mechanism underlying the cleavage of the propeptide and attachment of GPI, which occur in the endoplasmic reticulum.

scholarly journals IMMU-35. TRANSCRIPTIONALLY DEFINED IMMUNE CONTEXTURE IN HUMAN GLIOMAS AT SINGLE-CELL RESOLUTION

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii112-ii112
Author(s):  
Pravesh Gupta ◽  
Minghao Dang ◽  
Krishna Bojja ◽  
Tuan Tran M ◽  
Huma Shehwana ◽  
...  
Keyword(s):  
Dendritic Cell ◽  
Single Cell ◽  
Rna Sequencing ◽  
Immune Cell ◽  
Wild Type ◽  
Sequencing Data ◽  
Human Gliomas ◽  
Cell Clusters ◽  
Recurrent Gliomas ◽  
Antigen Presenting

Abstract The brain tumor immune microenvironment (TIME) continuously evolves during glioma progression and a comprehensive understanding of the glioma-centric immune cell repertoire beyond a priori cell types and/or states is uncharted. Consequently, we performed single-cell RNA-sequencing on ~123,000 tumor-derived immune cells from 17-pathologically stratified, IDH (isocitrate dehydrogenase)-differential primary, recurrent human gliomas, and non-glioma brains. Our analysis delineated predominant 34-myeloid cell clusters (~75%) over 28-lymphoid cell clusters (~25%) reflecting enormous heterogeneity within and across gliomas. The glioma immune diversity spanned functionally imprinted phagocytic, antigen-presenting, hypoxia, angiogenesis and, tumoricidal myeloid to classical cytotoxic lymphoid subpopulations. Specifically, IDH-mutant gliomas were enriched for brain-resident microglial subpopulations in contrast to enhanced bone barrow-derived infiltrates in IDH-wild type, especially in a recurrent setting. Microglia attrition in IDH-wild type -primary and -recurrent gliomas were concomitant with invading monocyte-derived cells with semblance to dendritic cell and macrophage/microglia like transcriptomic features. Additionally, microglial functional diversification was noted with disease severity and mostly converged to inflammatory states in IDH-wild type recurrent gliomas. Beyond dendritic cells, multiple antigen-presenting cellular states expanded with glioma severity especially in IDH-wild type primary and recurrent- gliomas. Furthermore, we noted differential microglia and dendritic cell inherent antigen presentation axis viz, osteopontin, and classical HLAs in IDH subtypes and, glioma-wide non-PD1 checkpoints associations in T cells like Galectin9 and Tim-3. As a general utility, our immune cell deconvolution approach with single-cell-matched bulk RNA sequencing data faithfully resolved 58-cell states which provides glioma specific immune reference for digital cytometry application to genomics datasets. Resultantly, we identified prognosticator immune cell-signatures from TCGA cohorts as one of many potential immune responsiveness applications of the curated signatures for basic and translational immune-genomics efforts. Thus, we not only provide an unprecedented insight of glioma TIME but also present an immune data resource that can be exploited to guide pragmatic glioma immunotherapy designs.

540 Transcriptionally defined immune landscape in human gliomas

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A576-A576
Author(s):  
Pravesh Gupta ◽  
Minghao Dang ◽  
Krishna Bojja ◽  
Huma Shehwana ◽  
Tuan Tran ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cell ◽  
Single Cell ◽  
Rna Sequencing ◽  
Immune Cell ◽  
Myeloid Cell ◽  
Wild Type ◽  
Cell Clusters ◽  
Md Anderson ◽  
Antigen Presenting

BackgroundBrain immunity is largely myeloid cell dominated rather than lymphoid cells in healthy and diseased state including malignancies of glial origins called as gliomas. Despite this skewed myeloid centric immune contexture, immune checkpoint and T cell based therapeutic modalities are generalizably pursued in gliomas ignoring the following facts i) T cells are sparse in tumor brain ii) glioma patients are lymphopenic iii) gliomas harbor abundant and highly complex myeloid cell repertoire. We recognized these paradoxes pertaining to fundamental understanding of constituent immune cells and their functional states in the tumor immune microenvironment (TIME) of gliomas, which remains elusive beyond a priori cell types and/or states.MethodsTo dissect the TIME in gliomas, we performed single-cell RNA-sequencing on ~123,000 tumor-derived sorted CD45+ leukocytes from fifteen genomically classified patients comprising IDH-mutant primary (IMP; n=4), IDH-mutant recurrent (IMR; n=4), IDH-wild type primary (IWP; n=3), or IDH-wild type recurrent (IWR; n=4) gliomas (hereafter referred as glioma subtypes) and two non-glioma brains (NGBs) as controls.ResultsUnsupervised clustering analyses delineated predominant 34-myeloid cell clusters (~75%) over 28-lymphoid cell clusters (~25%) reflecting enormous heterogeneity within and across glioma subtypes. The glioma immune diversity spanned functionally imprinted phagocytic, antigen-presenting, hypoxia, angiogenesis and, tumoricidal myeloid to classical cytotoxic lymphoid subpopulations. Specifically, IDH-mutant gliomas were predominantly enriched for brain-resident microglial subpopulations in contrast to enriched bone barrow-derived infiltrates in IDH-wild type especially in a recurrent setting. Microglia attrition in IWP and IWR gliomas were concomitant with invading monocyte-derived cells with semblance to dendritic cell and macrophage like transcriptomic features. Additionally, microglial functional diversification was noted with disease severity and mostly converged to inflammatory states in IWR gliomas. Beyond dendritic cells, multiple antigen-presenting cellular states expanded with glioma severity especially in IWP and IWR gliomas. Furthermore, we noted differential microglia and dendritic cell inherent antigen presentation axis viz, osteopontin, and classical HLAs in IDH subtypes and, glioma-wide non-PD1 checkpoints associations in T cells like Galectin9 and Tim-3. As a general utility, our immune cell deconvolution approach with single-cell-matched bulk RNA sequencing data faithfully resolved 58-cell states which provides glioma specific immune reference for digital cytometry application to genomics datasets.ConclusionsAltogether, we identified prognosticator immune cell-signatures from TCGA cohorts as one of many potential immune responsiveness applications of the curated signatures for basic and translational immune-genomics efforts. Thus, we not only provide an unprecedented insight of glioma TIME but also present an immune data resource that can be exploited for immunotherapy applications.Ethics ApprovalThe brain tumor/tissue samples were collected as per MD Anderson internal review board (IRB)-approved protocol numbers LAB03-0687 and, LAB04-0001. One non-tumor brain tissue sample was collected from patient undergoing neurosurgery for epilepsy as per Baylor College of Medicine IRB-approved protocol number H-13798. All experiments were compliant with the review board of MD Anderson Cancer Center, USA.ConsentWritten informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal

scholarly journals RNA Sequencing Reveals Rice Genes Involved in Male Reproductive Development under Temperature Alteration

Plants ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 663
Author(s):  
Sudthana Khlaimongkhon ◽  
Sriprapai Chakhonkaen ◽  
Keasinee Tongmark ◽  
Numphet Sangarwut ◽  
Natjaree Panyawut ◽  
...  
Keyword(s):  
High Temperature ◽  
Rna Sequencing ◽  
Reproductive Development ◽  
Yield Reduction ◽  
World Population ◽  
Rice Grain ◽  
Wild Type ◽  
Rice Plants ◽  
Male Development ◽  
Temperature Conditions

Rice (Oryza sativa L.) is one of the most important food crops, providing food for nearly half of the world population. Rice grain yields are affected by temperature changes. Temperature stresses, both low and high, affect male reproductive development, resulting in yield reduction. Thermosensitive genic male sterility (TGMS) rice is sterile at high temperature and fertile at low temperature conditions, facilitating hybrid production, and is a good model to study effects of temperatures on male development. Semithin sections of the anthers of a TGMS rice line under low (fertile) and high (sterile) temperature conditions showed differences starting from the dyad stage, suggesting that genes involved in male development play a role during postmeiotic microspore development. Using RNA sequencing (RNA-Seq), transcriptional profiling of TGMS rice panicles at the dyad stage revealed 232 genes showing differential expression (DEGs) in a sterile, compared to a fertile, condition. Using qRT-PCR to study expression of 20 selected DEGs using panicles of TGMS and wild type rice plants grown under low and high temperature conditions, revealed that six out of the 20 selected genes may be unique to TGMS, while the other 14 genes showed common responses to temperatures in both TGMS and wild-type rice plants. The results presented here would be useful for further investigation into molecular mechanisms controlling TGMS and rice responses to temperature alteration.

scholarly journals GENETIC EVIDENCE FOR INTERACTION BETWEEN NONHOMOLOGOUS PROTEINS IN YEAST AND A CASE OF SUPPRESSION AT THE HIS1 LOCUS

Genetics ◽  
1980 ◽  
Vol 94 (2) ◽  
pp. 327-339 ◽  
Author(s):  
Richard Snow
Keyword(s):  
Enzyme Activity ◽  
Low Temperature ◽  
Enzymatic Activity ◽  
Temperature Effect ◽  
Conformational Change ◽  
Minimal Medium ◽  
Genetic Evidence ◽  
Structural Genes ◽  
Wild Type ◽  
Is Strain

ABSTRACT The HIS1 and THR4 loci are the structural genes for phosphoribosyl-ATP pyrophosphorylase and threonine synthetase, respectively. The allele his1-IS has no enzyme activity at 30", but does have activity at 15" provided the cell contains the wild-type THR4 allele or a suppressing allele at another locus, designated SUP(his1-1S). Under these conditions, cells with the hisl-IS mutation are capable of growth on minimal medium at 15". Three kinds of reversions of a hisl-IS thr4 sup(his1-IS) strain to histidine prototrophy have been obtained: (1) his1-IS locus reversions to HIS1 that restore growth without added histidine at 30", (2)  thr4 reversions to THR4 that simultaneously eliminate the requirement for threonine and restore the low-temperature effect on the his1-IS allele, and (3)mutations from sup to SUP. The SUP allele is not an ochre suppressor, and it is not linked to either HISI, THR4 or a centromere. It may represent a missense suppressor. I t is proposed that the effect ofTHR4 is caused by aggregation of the wild-type threonine synthetase with defective his1-IS monomers, causing a favorable conformational change in the histidine protein that restores limited enzymatic activity. This can be regarded as a case of complementation between nonhomologous proteins.

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