Isolation of Fungal Infection Structures from Plant Tissue by Flow Cytometry for Cell-Specific Transcriptome Analysis

2011 ◽  
pp. 3-13 ◽  
Author(s):  
Hiroyuki Takahara ◽  
Elmar Endl ◽  
Richard O’Connell
Keyword(s):  
Flow Cytometry ◽  
Fungal Infection ◽  
Transcriptome Analysis ◽  
Plant Tissue ◽  
Infection Structures

scholarly journals Glyoxal fixation facilitates transcriptome analysis after antigen staining and cell sorting by flow cytometry

PLoS ONE ◽  
2021 ◽  
Vol 16 (1) ◽  
pp. e0240769
Author(s):  
Prasanna Channathodiyil ◽  
Jonathan Houseley
Keyword(s):  
Flow Cytometry ◽  
Transcriptome Analysis ◽  
Mammalian Cells ◽  
Single Cells ◽  
Rna Extraction ◽  
Cyclin B1 ◽  
Immunofluorescent Staining ◽  
Rna Seq ◽  
Simple Method ◽  
Staining Methods

A simple method for extraction of high quality RNA from cells that have been fixed, stained and sorted by flow cytometry would allow routine transcriptome analysis of highly purified cell populations and single cells. However, formaldehyde fixation impairs RNA extraction and inhibits RNA amplification. Here we show that good quality RNA can be readily extracted from stained and sorted mammalian cells if formaldehyde is replaced by glyoxal—a well-characterised fixative that is widely compatible with immunofluorescent staining methods. Although both formaldehyde and glyoxal efficiently form protein-protein crosslinks, glyoxal does not crosslink RNA to proteins nor form stable RNA adducts, ensuring that RNA remains accessible and amenable to enzymatic manipulation after glyoxal fixation. We find that RNA integrity is maintained through glyoxal fixation, permeabilisation with methanol or saponin, indirect immunofluorescent staining and flow sorting. RNA can then be extracted by standard methods and processed into RNA-seq libraries using commercial kits; mRNA abundances measured by poly(A)+ RNA-seq correlate well between freshly harvested cells and fixed, stained and sorted cells. We validate the applicability of this approach to flow cytometry by staining MCF-7 cells for the intracellular G2/M-specific antigen cyclin B1 (CCNB1), and show strong enrichment for G2/M-phase cells based on transcriptomic data. Switching to glyoxal fixation with RNA-compatible staining methods requires only minor adjustments of most existing staining and sorting protocols, and should facilitate routine transcriptomic analysis of sorted cells.

Using Flow Cytometry Analysis in Plant Tissue Culture Derived Plants

2018 ◽  
pp. 317-332
Author(s):  
Rosa María Escobedo-Gracia-Medrano ◽  
Martha Josefa Burgos-Tan ◽  
José Roberto Ku-Cauich ◽  
Adriana Quiroz-Moreno
Keyword(s):  
Flow Cytometry ◽  
Tissue Culture ◽  
Plant Tissue Culture ◽  
Plant Tissue ◽  
Flow Cytometry Analysis

scholarly journals Extensive Differences in Antifungal Immune Response in TwoDrosophilaSpecies Revealed by Comparative Transcriptome Analysis

2013 ◽  
Vol 2013 ◽  
pp. 1-15 ◽  
Author(s):  
Yosuke Seto ◽  
Koichiro Tamura
Keyword(s):  
Immune System ◽  
Fungal Infection ◽  
Transcriptome Analysis ◽  
Innate Immune System ◽  
Expression Patterns ◽  
Innate Immune ◽  
Antifungal Peptides ◽  
Genes Encoding ◽  
Yeasts And Molds ◽  
Immune Related Genes

The innate immune system ofDrosophilais activated by ingestion of microorganisms.D. melanogasterbreeds on fruits fermented bySaccharomyces cerevisiae, whereasD. virilisbreeds on slime flux and decaying bark of tree housing a variety of bacteria, yeasts, and molds. In this study, it is shown thatD. virilishas a higher resistance to oral infection of a species of filamentous fungi belonging to the genusPenicilliumcompared toD. melanogaster. In response to the fungal infection, a transcriptome profile of immune-related genes was considerably different betweenD. melanogasterandD. virilis: the genes encoding antifungal peptides, Drosomycin and Metchnikowin, were highly expressed inD. melanogasterwhereas, the genes encoding Diptericin and Defensin were highly expressed inD. virilis. On the other hand, the immune-induced molecule (IM) genes showed contrary expression patterns between the two species: they were induced by the fungal infection inD. melanogasterbut tended to be suppressed inD. virilis. Our transcriptome analysis also showed newly predicted immune-related genes inD. virilis. These results suggest that the innate immune system has been extensively differentiated during the evolution of theseDrosophilaspecies.

Penetration and infection of western larch seedlings by Botrytis cinerea

1989 ◽  
Vol 67 (9) ◽  
pp. 2596-2599 ◽  
Author(s):  
Frank Dugan ◽  
George M. Blake
Keyword(s):  
Fungal Infection ◽  
Botrytis Cinerea ◽  
Light Microscope ◽  
Serial Sections ◽  
Larix Occidentalis ◽  
Western Larch ◽  
Conifer Seedlings ◽  
Infection Structures ◽  
Application Rates ◽  
Whole Mounts

The fungus Botrytis cinerea Persoon (ex Fries) is an important pathogen of container-grown conifer seedlings. The examination of whole mounts and serial sections of diseased needles of western larch (Larix occidentalis Nuttall) seedlings with a light microscope confirmed that the development of fungal infection structures conforms to that observed in nonconiferous hosts. Both stomatal and cuticular penetrations were evident. Infection hyphae displayed occasional taxis toward stomata on senescent needles but not on healthy needles. The treatment of seedlings with fungicides at levels corresponding to 1 and 10% normal application rates induced no consistent changes in fungal structures.

scholarly journals Flow Cytometric Analysis of Ploidy in Rhododendron (subgenus Tsutsusi)

HortScience ◽  
2001 ◽  
Vol 36 (1) ◽  
pp. 125-127 ◽  
Author(s):  
S. De Schepper ◽  
L. Leus ◽  
M. Mertens ◽  
E. Van Bockstaele ◽  
M. De Loose ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
High Resolution ◽  
Ploidy Level ◽  
Plant Tissue ◽  
Nuclear Dna ◽  
Large Population ◽  
Flow Cytometric Analysis ◽  
Plant Tissues ◽  
Flow Cytometric ◽  
Through Flow

Ploidy level was determined for six species and 88 cultivars of the Rhododendron subgenus Tsutsusi. High-resolution flow cytometry of nuclear DNA was performed on macerated plant tissue. All plants analyzed were diploid (2n = 26) with the exception of `Euratom', `Euratom Orange', and `Red Wing', which were triploid (3n = 39), and `Casablanca Tetra', which was found to be a cytochimera: mixoploid (2n + 4n) in the LI and LII, but tetraploid in the LIII. The described method has proven to be useful in screening a large population of rhododendrons. Analysis of different organs and plant tissues was easily accomplished through flow cytometry, and has proven useful in determining the ploidy of different histogenic layers.

scholarly journals Infection of Galleria mellonella (Lepidoptera) Larvae With the Entomopathogenic Fungus Conidiobolus coronatus (Entomophthorales) Induces Apoptosis of Hemocytes and Affects the Concentration of Eicosanoids in the Hemolymph

2022 ◽  
Vol 12 ◽  
Author(s):  
Anna Katarzyna Wrońska ◽  
Agata Kaczmarek ◽  
Michalina Kazek ◽  
Mieczysława Irena Boguś
Keyword(s):  
Flow Cytometry ◽  
Fungal Infection ◽  
Galleria Mellonella ◽  
Soil Fungus ◽  
Caspase Activity ◽  
Pla2 Activity ◽  
Caspase Activation ◽  
Conidiobolus Coronatus ◽  
Factors Influencing ◽  
Apoptosis And Necrosis

Apoptosis and autophagy, the mechanisms of programmed cell death, play critical roles in physiological and pathological processes in both vertebrates and invertebrates. Apoptosis is also known to play an important role in the immune response, particularly in the context of entomopathogenic infection. Of the factors influencing the apoptotic process during infection, two of the lesser known groups are caspases and eicosanoids. The aim of this study was to determine whether infection by the entomopathogenic soil fungus Conidiobolus coronatus is associated with apoptosis and changes in caspase activity in the hemocytes of Galleria mellonella larvae, and to confirm whether fungal infection may affect eicosanoid levels in the host. Larvae were exposed for 24 h to fully grown and sporulating fungus. Hemolymph was collected either immediately after termination of exposure (F24 group) or 24 h later (F48 group). Apoptosis/necrosis tests were performed in hemocytes using fluorescence microscopy and flow cytometry, while ELISA tests were used to measure eicosanoid levels. Apoptosis and necrosis occurred to the same degree in F24, but necrosis predominated in F48. Fungal infection resulted in caspase activation, increased PGE1, PGE2, PGA1, PGF2α, and 8-iso-PGF2α levels and decreased TXB2 levels, but had no effect on TXA2 or 11-dehydro-TXB2 concentrations. In addition, infected larvae demonstrated significantly increased PLA2 activity, known to be involved in eicosanoid biosynthesis. Our findings indicate that fungal infection simultaneously induces apoptosis in insects and stimulates general caspase activity, and this may be correlated with changes in the concentrations of eicosanoids.

scholarly journals Induction, identification and genetics analysis of tetraploid Actinidia chinensis

2019 ◽  
Vol 6 (11) ◽  
pp. 191052 ◽  
Author(s):  
Shengxing Li ◽  
Xiaozhen Liu ◽  
Huiming Liu ◽  
Xianang Zhang ◽  
Qinxia Ye ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Transcriptome Analysis ◽  
Stress Resistance ◽  
Quantitative Polymerase Chain Reaction ◽  
Colchicine Treatment ◽  
Actinidia Chinensis ◽  
Flow Cytometry Analysis ◽  
Commercially Important ◽  
Polymerase Chain ◽  
Gene Expression Levels

Actinidia chinensis is a commercially important fruit, and tetraploid breeding of A. chinensis is of great significance for economic benefit. In order to obtain elite tetraploid cultivars, tetraploid plants were induced by colchicine treatment with leaves of diploid A. chinensis ‘SWFU03’. The results showed that the best treatment was dipping leaves 30 h in 60 mg l −1 colchicine solutions, with induction rate reaching 26%. Four methods, including external morphology comparison, stomatal guard cell observation, chromosome number observation and flow cytometry analysis were used to identify the tetraploid of A. chinensis . Using the induction system and flow cytometry analysis methods, 187 tetraploid plants were identified. Three randomly selected tetraploid plants and their starting diploid plants were further subjected to transcriptome analysis, real-time quantitative polymerase chain reaction (RT-qPCR) and methylation-sensitive amplification polymorphism (MSAP) analysis. The transcriptome analysis results showed that there were a total of 2230 differentially expressed genes (DEG) between the diploid and tetraploid plants, of which 660 were downregulated and 1570 upregulated. The DEGs were mainly the genes involved in growth and development, stress resistance and antibacterial ability in plants. RT-qPCR results showed that the gene expression levels of the growth and stress resistance of tetraploid plants were higher than those of diploid ones at the transcriptome level. MSAP analysis of DNA methylation results showed that tetraploid plants had lower methylation ratio than diploid ones. The present results were valuable to further explore the epigenetics of diploid and tetraploid kiwifruit plants.

scholarly journals Glyoxal fixation facilitates transcriptome analysis after antigen staining and cell sorting by flow cytometry

2020 ◽  
Author(s):  
Prasanna Channathodiyil ◽  
Jonathan Houseley
Keyword(s):  
Flow Cytometry ◽  
Transcriptome Analysis ◽  
Mammalian Cells ◽  
Single Cells ◽  
Rna Extraction ◽  
Cyclin B1 ◽  
Immunofluorescent Staining ◽  
Rna Seq ◽  
Simple Method ◽  
Staining Methods

AbstractA simple method for extraction of high quality RNA from cells that have been fixed, stained and sorted by flow cytometry would allow routine transcriptome analysis of highly purified cell populations and single cells. However, formaldehyde fixation impairs RNA extraction and inhibits RNA amplification. Here we show that good quality RNA can be readily extracted from stained and sorted mammalian cells if formaldehyde is replaced by glyoxal - a well-characterised fixative that is widely compatible with immunofluorescent staining methods. Although both formaldehyde and glyoxal efficiently form protein-protein crosslinks, glyoxal does not crosslink RNA to proteins nor form stable RNA adducts, ensuring that RNA remains accessible and amenable to enzymatic manipulation after glyoxal fixation. We find that RNA integrity is maintained through glyoxal fixation, permeabilisation with methanol or saponin, indirect immunofluorescent staining and flow sorting. RNA can then be extracted by standard methods and processed into RNA-seq libraries using commercial kits; mRNA abundances measured by poly(A)+ RNA-seq correlate well between freshly harvested cells and fixed, stained and sorted cells. We validate the applicability of this approach to flow cytometry by staining MCF-7 cells for the intracellular G2/M-specific antigen cyclin B1 (CCNB1), and show strong enrichment for G2/M-phase cells based on transcriptomic data. Switching to glyoxal fixation with RNA-compatible staining methods requires only minor adjustments of most existing staining and sorting protocols, and should facilitate routine transcriptomic analysis of sorted cells.

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