Questa ricerca è stata sostenuta da una sovvenzione da parte del National Institute of Health e l&#39;Università del Texas ad Arlington istituito fondi a MM.

1. Le piante che crescono e batteri Preparazione <ol><li> Per iniziare questa procedura seminare Arabidopsis su un 01:01:01 v: v: v miscela di substrato (Redi-terra spina e mescolare piantina, Sun Gro), vermiculite fine e perlite. </li><li> Far crescere le piantine in una camera di crescita (22 ° C, 12 ore di luce di 100 micromol / mm 2 / sec giorno e 65 ± 5% di umidità) e di acqua se necessario. Le piante sono pronte per l&#39;uso in 4-6 settimane, quando sono giovani foglie completamente espanse prima della bullonatura. </li><li> Due giorni prima di iniziare l&#39;esperimento preparare la cultura batterio. Streak Pseudomonas syringae da stock glicerolo su medio LB modificato (10 g / L triptone, 5 g / L estratto di lievito, 5 g / L NaCl pH 7,0, e 1,5% agar) e incubare per 24 ore a 28 ° C. Utilizzare la cultura freschi per preparare l&#39;inoculo e iniziano sempre piastre di coltura dalle scorte glicerolo come batteri diventa meno virulenti dopo sub-coltura. </li><li> Utilizzare i batteri che crescono sulla piastra per iniziare una coltura liquida 10 ml di P. syringae in una beuta da ml 50. Incubare la cultura notte a 28 ° C con forte agitazione fino a raggiungere densità ottica o OD a 600 nm tra 0,8 e 1. </li><li> Misurare la densità ottica a 600 e raccogliere le cellule per centrifugazione a 1360 xg per 10 minuti. Risospendere le cellule in acqua distillata in modo che il diametro finale è di 0,2. Questo corrisponde a OD 10 8 unità formanti colonia (CFU) / ml. Con i batteri pronti, procedere per preparare le foglie ed eseguire il test. </li></ol> 2. Foglia di colorazione e di incubazione con i batteri <ol><li> Al fine di macchiare le prime foglie preparare 20 ioduro di propidio mM o la soluzione PI in acqua. 10 ml di soluzione è sufficiente a macchiare 3 foglie piccole alla volta. </li><li> Recuperare le piante dalla camera di crescita e con una pinza staccare 3 giovani, completamente ampliata foglie. Immergere le foglie intere nella soluzione PI per 5 minuti. Quindi rimuovere le foglie e sciacquarli brevemente con acqua distillata. </li><li> Posto accanto una foglia con la superficie inferiore rivolta verso il basso su un vetrino da microscopio. Tagliare la foglia con una lama di rasoio affilato in quattro pezzi esclusa la vena a metà in modo che la foglia adagiata sulla diapositiva. </li><li> Ad incubare i batteri con le foglie, aggiungere 300 ml di sospensione batterica con i pezzi di foglia nella diapositiva. Assicurarsi che la superficie inferiore della foglia è in contatto con l&#39;inoculo. </li><li> Incubare i campioni nelle stesse condizioni ambientali che sono state adibite alla coltura delle piante. Al momento per osservare al microscopio le foglie, il trasferimento dei pezzi foglia di una nuova diapositiva con minore superficie rivolta verso l&#39;alto. Si noti che scivolano copertura non viene utilizzata durante il montaggio le diapositive. Il campione stessa foglia può essere ripreso più volte durante l&#39;incubazione e un corso di tempo può essere impostato in funzione degli obiettivi della ricerca. </li></ol> 3. Microscopia, di misura e analisi dei dati <ol><li> Ecco un microscopio confocale a scansione laser (LSM 510 Meta, Carl Zeiss Inc.) è utilizzato per esaminare le foglie. Osservare la superficie inferiore delle foglie per rilevare la fluorescenza di PI (eccitazione 453 nm, emissione di 543 e 620 nm). </li><li> Utilizzando i campioni stessa foglia di catturare immagini di stomi nel tempo. Salvare le immagini per misurare la larghezza della apertura stomatica. </li><li> Misura larghezza stomatica apertura di almeno 60 stomi per ogni trattamento ad ogni punto ora con l&#39;immagine del browser LSM. </li><li> Calcolare l&#39;errore medio e standard per la larghezza di apertura stomatica. Significatività statistica dei risultati può essere calcolato usando 2-code, Studente appaiati saggio t-test. </li></ol> 4. Immagini rappresentante della risposta stomatica di incubazione con i batteri <ol><li> Ecco una tipica immagine microscopica (usando un obiettivo 20x) di una superficie fogliare di Arabidopsis con microscopio confocale a scansione laser in vista in campo chiaro (Figura 1). </li><li> Macchie di ioduro di propidio la parete cellulare delle cellule vitali aumentare la visibilità delle cellule di guardia, oltre a consentire l&#39;identificazione di cellule non danneggiate per la raccolta dei dati (Figura 2). Una serie di aperture dei pori stomatica può essere visto in questi micrografie, da stomi completamente chiuso al largo stomi aperti. </li><li> Stomi completamente chiuso sono identificati con la forma delle cellule di guardia (Figura 3). La larghezza di apertura è considerato 0 micron. </li><li> Per stomi aperti (Figura 4) la larghezza apertura mostrata è misurato utilizzando una linea retta tracciata attraverso la più ampia superficie del poro stomatica. </li><li> Questi risultati sperimentali sono tipici di questo test stomi. Arabidopis le foglie sono state incubate al buio con tre diversi ceppi di Pseudomonas syringae. Solo il batterio Pst DC3000 è in grado di aprire scuro chiuso stomi, misurata dalla larghezza di apertura selezionati in modo casuale 60 stomi per ceppo batterico (Figura 5). </li></ol&gt; <img src="/files/ftp_upload/2185/2185fig1.jpg" alt="Figura 1" /> Figura 1. Micrografia della superficie di una foglia di Arabidopsis. Un campo di vista della superficie fogliare con microscopio confocale a scansione laser (LSCM) utilizzando il 20x obiettivo. Si noti che l&#39;apertura degli stomi non è così evidente rispetto alla vista aiutato dalla colorazione fluorescente ioduro di propidio (Figura 2). <img src="/files/ftp_upload/2185/2185fig2.jpg" alt="Figura 2" /> Figura 2. Micrografia della superficie di un propidio ioduro macchiato di foglia di Arabidopsis. Un campo di vista della superficie fogliare con microscopio confocale a scansione laser (LSCM) utilizzando il 20x obiettivo. Nota una gamma di apertura dei pori stomatica. Frecce gialle sono puntati a chiudere completamente stomi e frecce verdi sono puntati a largo stomi aperti. <img src="/files/ftp_upload/2185/2185fig3.jpg" alt="Figura 3" /> Figura 3. Completamente stomi chiudere identificato dalla forma e l&#39;apertura tra le cellule di guardia. La larghezza di apertura è considerato 0 micron. <img src="/files/ftp_upload/2185/2185fig4.jpg" alt="Figura 4" /> Figura 4. Stomi aperti che mostrano l&#39;ampiezza apertura in micron. Misure sono state prese utilizzando il browser LSCM sulla base di una linea retta tracciata attraverso la più ampia superficie del poro stomatica. <img src="/files/ftp_upload/2185/2185fig5.jpg" alt="Figura 5" /> Figura 5. Apertura degli stomi delle foglie di Arabidopsis incubate con tre ceppi di Pseudomonas syringae pv pomodoro:.. DC3118, DB29, DC3000 e piante sono stati tenuti al buio durante il periodo di sperimentazione. I risultati sono mostrati come media (n = 50-70) ± errore standard. Significatività statistica è stata rilevata con due code di Student t-test. L&#39;esperimento è stato ripetuto tre volte con risultati simili.

<ol>
	<li>Brooks, D. M., Hernandez-Guzman, G., Kloek, A. P., Alarcon-Chaidez, F., Sreedharan, A., Rangaswamy, V., Penaloza-Vazquez, A., Bender, C. L., Kunkel, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+a+well-defined+series+of+coronatine+biosynthetic+mutants+of+Pseudomonas+syringae+pv.+tomato+DC3000.">Identification and characterization of a well-defined series of coronatine biosynthetic mutants of Pseudomonas syringae pv. tomato DC3000.</a> Mol Plant-Microbe Interact. 17, 162-174 (2004).</li><li>Katagiri, F., Thilmony, R., He, S. Y., Somerville, C. R., Meyerowitz, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Arabidopsis+thaliana-Pseudomonas+syringae+interaction.">The Arabidopsis thaliana-Pseudomonas syringae interaction.</a> The Arabidopsis Book. , (2002).</li><li>Ma, S. W., Morris, V. L., Cuppels, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+DNA+region+required+for+production+of+the+phytotoxin+coronatine+by+Pseudomonas+syringae+pv.+tomato.">Characterization of a DNA region required for production of the phytotoxin coronatine by Pseudomonas syringae pv. tomato.</a> Mol Plant-Microbe Interact. 4, 69-74 (1991).</li><li>Melotto, M., Underwood, W., Koczan, J., Nomura, K., He, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+stomata+function+in+innate+immunity+against+bacterial+invasion.">Plant stomata function in innate immunity against bacterial invasion.</a> Cell. 126, 969-980 (2006).</li></ol>

Nessun conflitto di interessi dichiarati.

Abbiamo presentato una procedura semplice da misurare apertura degli stomi nei tessuti foglia intatta che permette una valutazione della risposta stomatica facile per diversi trattamenti. Anche se abbiamo presentato i risultati utilizzando il sistema modello Arabidopsis / Pseudomonas syringae, il saggio foglia intatta stomi possono essere potenzialmente eseguita con tutta la pianta-batterio combinazione. Il protocollo può essere facilmente modificato per soddisfare esigenze di crescita di altre piante e batteri patogeni. Il principio generale e la procedura rimane la stessa. Inoltre, questo metodo può essere utile ai ricercatori che desiderano studiare in uscita funzionale delle cellule di guardia non solo di vivere microbi, ma anche ad altri stimoli e agli agenti chimici in condizioni che consentono di mantenere l&#39;ambiente naturale della foglia. Foglia intera può essere difficile per l&#39;immagine grazie al suo spessore e la topografia irregolare. Questo problema può essere alleviato rimuovendo la vena metà della foglia in modo che adagiata sul vetrino e usando la microscopia confocale. Tuttavia, altri tipi di microscopio a fluorescenza può essere utilizzato per ottenere immagini ad alta risoluzione della superficie fogliare.

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<td>Propidium iodide</td>
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assessing stomatal response to live bacterial cells using whole leaf imaging

Stomi sono aperture naturali nell&#39;epidermide impianto responsabile per lo scambio di gas tra l&#39;interno delle piante e dell&#39;ambiente. Esse sono formate da una coppia di cellule di guardia, che sono in grado di chiudere il poro stomi in risposta ad una serie di fattori esterni, incluso l&#39;intensità della luce, concentrazione di anidride carbonica, e umidità relativa (RH). Il poro stomi è anche la via principale per l&#39;ingresso degli agenti patogeni in foglie, un passo cruciale per lo sviluppo della malattia. Recenti studi hanno rivelato che la chiusura del poro è efficace nel ridurre lo sviluppo batterico malattia in piante di Arabidopsis, parte integrante della pianta immunità innata. In precedenza, abbiamo utilizzato le bucce epidermica per valutare la risposta degli stomi per vivere batteri (Melotto et al 2006.), Ma mantenendo condizioni ambientali favorevoli per entrambe le bucce impianto epidermiche e le cellule batteriche è stato impegnativo. Epidermide foglia può essere mantenuto vivo e sano con MES di buffer (10 mM KCl, 25 mM MES-KOH, pH 6.15) per gli esperimenti elettrofisiologici di cellule di guardia. Tuttavia, questo buffer non è appropriato per ottenere la sospensione batterica. D&#39;altra parte, le cellule batteriche possono essere tenuti in vita in acqua, che non è proprio per mantenere le bucce epidermico per un lungo periodo di tempo. Quando un galleggia sull&#39;acqua buccia epidermica, le cellule della buccia che sono esposti ad asciugare all&#39;aria entro 4 ore limitando i tempi per condurre l&#39;esperimento. Un metodo ideale per valutare l&#39;effetto di uno stimolo particolare sulle cellule di guardia dovrebbe presentare la minima interferenza alla fisiologia degli stomi e per l&#39;ambiente naturale della pianta il più possibile. Noi, quindi, sviluppato un nuovo metodo per valutare la risposta degli stomi di vivere i batteri in cui ferendo foglia e la manipolazione è notevolmente ridotto al fine di fornire un saggio facilmente riproducibili e affidabili stomatica. Il protocollo si basa sulla colorazione delle foglie intatte con ioduro di propidio (PI), di incubazione di colorazione foglia con sospensione batterica, e l&#39;osservazione delle foglie sotto microscopio confocale a scansione laser. Infine, questo metodo consente l&#39;osservazione dello stesso campione di foglia di vivere per lunghi periodi di tempo a stretto contatto con le condizioni che simulano le condizioni naturali in cui sono attaccate le piante da agenti patogeni.

Watch this Scientific Journal Video about Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging at JoVE.com

Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

Abbiamo sviluppato un protocollo semplice e riproducibile per l&#39;accesso risposta stomatica di batteri vivi. Questo metodo riduce ferimento e la manipolazione della foglia rispetto all&#39;utilizzo di bucce epidermica riferito in precedenza.

Valutazione della risposta stomatica a Live cellule batteriche utilizzando Imaging Whole Leaf

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of ...

assessing-stomatal-response-to-live-bacterial-cells-using-whole-leaf

Research

JoVE Journal

Immunology and Infection

18.2K Views.  University of Texas at Arlington. Abbiamo sviluppato un protocollo semplice e riproducibile per l'accesso risposta stomatica di batteri vivi. Questo metodo riduce ferimento e la manipolazione della foglia rispetto all'utilizzo di bucce epidermica riferito in precedenza.

Video: Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

Utilizzando luciferasi di infezioni batteriche immagine in topi

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

Ricerca

Immunologia e infezioni

Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external environment. The cell can either recover from this insult, which requires active membrane repair processes, or else die depending on the amount of toxin exposure and cell type1. In addition, these toxins induce strong inflammatory responses in infected hosts through activation of immune cells, including macrophages, which produce an array of pro-inflammatory cytokines2. Many Gram positive bacteria produce cholesterol binding toxins which have been shown to contribute to their virulence through largely uncharacterized mechanisms.
Morphologic changes in the plasma membrane of cells exposed to these toxins include their sequestration into cholesterol-enriched surface protrusions, which can be shed into the extracellular space, suggesting an intrinsic cellular defense mechanism3,4. This process occurs on all cells in the absence of metabolic activity, and can be visualized using EM after chemical fixation4. In immune cells such as macrophages that mediate inflammation in response to toxin exposure, induced membrane vesicles are suggested to contain cytokines of the IL-1 family and may be responsible both for shedding toxin and disseminating these pro-inflammatory cytokines5,6,7. A link between IL-1&beta; release and a specific type of cell death, termed pyroptosis has been suggested, as both are caspase-1 dependent processes8. To sort out the complexities of this macrophage response, which includes toxin binding, shedding of membrane vesicles, cytokine release, and potentially cell death, we have developed labeling techniques and fluorescence microscopy methods that allow for real time visualization of toxin-cell interactions, including measurements of dysfunction and death (Figure 1). Use of live cell imaging is necessary due to limitations in other techniques. Biochemical approaches cannot resolve effects occurring in individual cells, while flow cytometry does not offer high resolution, real-time visualization of individual cells. The methods described here can be applied to kinetic analysis of responses induced by other stimuli involving complex phenotypic changes in cells.

Methods for purifying the cholesterol binding toxin streptolysin O from recombinant E. coli and visualization of toxin binding to live eukaryotic cells are described. Localized delivery of toxin induces rapid and complex changes in targeted cells revealing novel aspects of toxin biology.

Visualizzazione delle risposte tossina batterica provocata con diretta microscopia a fluorescenza cellulare

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external ...

Bioluminescent Bacterial Imaging In Vivo

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of bacteria in gene and cell therapy for cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and concurrently tumor sites. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor bearing mice. Visualization of commensal bacteria in the Gastrointestinal tract (GIT) by BLI is also described. This powerful, and cheap, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research, in particular gene therapy, and infectious disease. This video outlines the procedure for studying lux-tagged E. coli in live mice, demonstrating the spatial and temporal readout achievable utilizing BLI with the IVIS system.

Bioluminescent Bacterial Imaging In Vivo

This article describes the administration of lux-tagged bacteria to mice and subsequent in vivo analysis using IVIS bioluminescence imaging.

Bioluminescent Imaging batterica<em&gt; In Vivo</em

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of ...

Visualizing Protein-DNA Interactions in Live Bacterial Cells Using Photoactivated Single-molecule Tracking

Protein-DNA interactions are at the heart of many fundamental cellular processes. For example, DNA replication, transcription, repair, and chromosome organization are governed by DNA-binding proteins that recognize specific DNA structures or sequences. In vitro experiments have helped to generate detailed models for the function of many types of DNA-binding proteins, yet, the exact mechanisms of these processes and their organization in the complex environment of the living cell remain far less understood. We recently introduced a method for quantifying DNA-repair activities in live Escherichia coli cells using Photoactivated Localization Microscopy (PALM) combined with single-molecule tracking. Our general approach identifies individual DNA-binding events by the change in the mobility of a single protein upon association with the chromosome. The fraction of bound molecules provides a direct quantitative measure for the protein activity and abundance of substrates or binding sites at the single-cell level. Here, we describe the concept of the method and demonstrate sample preparation, data acquisition, and data analysis procedures.

Photoactivated localization microscopy (PALM) combined with single-molecule tracking allows direct observation and quantification of protein-DNA interactions in live Escherichia coli cells.

Visualizzare le interazioni proteina-DNA in cellule vive batteriche Uso fotoattivati ​​singola molecola di monitoraggio

Protein-DNA interactions are at the heart of many fundamental cellular processes. For example, DNA replication, transcription, repair, and chromosome ...

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised patients, the elderly, and pregnant women. The most common manifestations of listeriosis in humans include meningitis, encephalitis, and fetal abortion. A significant but much less documented sequelae of invasive L. monocytogenes infection involves the heart. The death rate from cardiac illness can be up to 35% despite treatment, however very little is known regarding L. monocytogenes colonization of cardiac tissue and its resultant pathologies. In addition, it has recently become apparent that subpopulations of L. monocytogenes have an enhanced capacity to invade and grow within cardiac tissue. This protocol describes in detail in vitro and in vivo methods that can be used for assessing cardiotropism of L. monocytogenes isolates. Methods are presented for the infection of H9c2 rat cardiac myoblasts in tissue culture as well as for the determination of bacterial colonization of the hearts of infected mice. These methods are useful not only for identifying strains with the potential to colonize cardiac tissue in infected animals, but may also facilitate the identification of bacterial gene products that serve to enhance cardiac cell invasion and/or drive changes in heart pathology. These methods also provide for the direct comparison of cardiotropism between multiple L. monocytogenes strains.

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes causes fetal infections in pregnant women and meningitis in susceptible populations. Subpopulations of bacteria can colonize cardiac tissue, causing myocarditis in patients and laboratory animals. Here we present a protocol that describes how to assess L. monocytogenes cardiac cell invasion in vitro and cardiac colonization in infected animals.

Valutazione dell'invasione batterica delle cellule cardiache in coltura e colonizzazione cardiaca nei topi infetti utilizzando listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised ...

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

In the absence of specialized mobile immune cells, plants utilize their localized programmed cell death and Systemic Acquired Resistance to defend themselves against pathogen attack. The contribution of a specific Arabidopsis gene to the overall plant immune response can be specifically and quantitatively assessed by assaying the pathogen growth within the infected tissue. For over three decades, the hemibiotrophic bacterium Pseudomonas syringae pv. maculicola ES4326 (Psm ES4326) has been widely applied as the model pathogen to investigate the molecular mechanisms underlying the Arabidopsis immune response. To deliver pathogens into the leaf tissue, multiple inoculation methods have been established, e.g., syringe infiltration, dip inoculation, spray, vacuum infiltration, and flood inoculation. The following protocol describes an optimized syringe infiltration method to deliver virulent Psm ES4326 into leaves of adult soil-grown Arabidopsis plants and accurately screen for enhanced disease susceptibility (EDS) towards this pathogen. In addition, this protocol can be supplemented with multiple pre-treatments to further dissect specific immune defects within different layers of plant defense, including Salicylic Acid (SA)-Triggered Immunity (STI) and MAMP-Triggered Immunity (MTI).

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

Quantification of pathogen growth is a powerful tool to characterize various Arabidopsis thaliana (hereafter: Arabidopsis) immune responses. The method described here presents an optimized syringe infiltration assay to quantify the Pseudomonas syringae pv. maculicola ES4326 growth in adult Arabidopsis leaves.

Batterica Leaf Infiltrazione Assay per Fine Caratterizzazione di risposte di difesa delle piante che utilizzano il<em&gt; Arabidopsis thaliana-Pseudomonas syringae</em&gt; Patosistema

In the absence of specialized mobile immune cells, plants utilize their localized programmed cell death and Systemic Acquired Resistance to defend ...

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila express gustatory receptors1,2, ion channels3-6, and ionotropic receptors7 that are involved in the detection of volatile and non-volatile sensory cues. These neurons directly contact tastants such as food, noxious substances and pheromones and therefore influence many complex behaviors such as feeding, egg-laying and mating. Electrode recordings and calcium imaging have been widely used in insects to quantify the neuronal responses evoked by these tastants. However, electrophysiology requires specialized equipment and obtaining measurements from a single taste sensillum can be technically challenging depending on the cell-type, size, and position. In addition, single neuron resolution in Drosophila can be difficult to achieve since taste sensilla house more than one type of chemosensory neuron. The live calcium imaging method described here allows responses of single gustatory neurons in live flies to be measured. This method is especially suitable for imaging neuronal responses to lipid pheromones and other ligand types that have low solubility in water-based solvents.

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

The forelegs and proboscis of Drosophila contain a rich repertoire of gustatory sensory neurons. Here, we present a method using calcium imaging to measure physiological responses from sensory neurons in the foreleg and proboscis of live flies upon exogenous application of a gustatory pheromone.

Misurare risposte fisiologiche di<em&gt; Drosophila</em&gt; Neuroni sensoriali a Lipid feromoni utilizza Live Calcio Imaging

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila ...

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research investigating immunological responses against A. fumigatus has been limited by the lack of consistent and reliable assays for measuring the antifungal activity of specific immune cells in vitro. A new method is described to assess the antifungal activity of primary monocytes and neutrophils from human donors against A. fumigatus using FLuorescent Aspergillus REporter (FLARE) conidia. These conidia contain a genetically encoded dsRed reporter, which is constitutively expressed by live FLARE conidia, and are externally labeled with Alexa Fluor 633, which is resistant to degradation within the phagolysosome, thus allowing a distinction between live and dead A. fumigatus conidia. Video microscopy and flow cytometry are subsequently used to visualize the interaction between conidia and innate immune cells, assessing fungicidal activity whilst also providing a wealth of information on phagocyte migration, phagocytosis and the inhibition of fungal growth. This novel technique has already provided exciting new insights into the host-pathogen interaction of primary immune cells against A. fumigatus. It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.
It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Here, we describe a protocol to assess antifungal activity of primary human immune cells in real-time using fluorescent Aspergillus reporter conidia in conjunction with live-cell video microscopy and flow cytometry. Generated data provide insight into host cell-Aspergillus interactions such as fungicidal activity, phagocytosis, cell migration and inhibition of fungal growth.

Immagini dal vivo di antifungini attività da Human neutrofili e monociti primaria in risposta a<em&gt; A. fumigatus</em

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research ...

Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay

In all animals, innate immunity provides an immediate and robust defense against a broad spectrum of pathogens. Humoral and cellular immune responses are the main branches of innate immunity, and many of the factors regulating these responses are evolutionarily conserved between invertebrates and mammals. Phagocytosis, the central component of cellular innate immunity, is carried out by specialized blood cells of the immune system. The fruit fly, Drosophila melanogaster, has emerged as a powerful genetic model to investigate the molecular mechanisms and physiological impacts of phagocytosis in whole animals. Here we demonstrate an injection-based in vivo phagocytosis assay to quantify the particle uptake and destruction by Drosophila blood cells, hemocytes. The procedure allows researchers to precisely control the particle concentration and dose, making it possible to obtain highly reproducible results in a short amount of time. The experiment is quantitative, easy to perform, and can be applied to screen for host factors that influence pathogen recognition, uptake, and clearance.

Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay

This protocol describes an in vivo phagocytosis assay in adult Drosophila melanogaster to quantify phagocyte recognition and clearance of microbial infections.

Valutare la risposta immunitaria cellulare del moscerino della frutta, Drosophila melanogaster, utilizzando un test di fagocitosi In Vivo

In all animals, innate immunity provides an immediate and robust defense against a broad spectrum of pathogens. Humoral and cellular immune responses are ...

Three-dimensional Imaging of Bacterial Cells for Accurate Cellular Representations and Precise Protein Localization

The shape of a bacterium is important for its physiology. Many aspects of cell physiology such as cell motility, predation, and biofilm production can be affected by cell shape. Bacterial cells are three-dimensional (3D) objects, although they are rarely treated as such. Most microscopy techniques result in two-dimensional (2D) images leading to the loss of data pertaining to the actual 3D cell shape and localization of proteins. Certain shape parameters, such as Gaussian curvature (the product of the two principal curvatures), can only be measured in 3D because 2D images do not measure both principal curvatures. Additionally, not all cells lie flat when mounting and 2D imaging of curved cells may not accurately represent the shapes of these cells. Accurately measuring protein localization in 3D can help determine the spatial regulation and function of proteins. A forward convolution technique has been developed that uses the blurring function of the microscope to reconstruct 3D cell shapes and to accurately localize proteins. Here, a protocol for preparing and mounting samples for live cell imaging of bacteria in 3D both to reconstruct an accurate cell shape and to localize proteins is described. The method is based on simple sample preparation, fluorescent image acquisition, and MATLAB-based image processing. Many high-quality fluorescent microscopes can be simply modified to take these measurements. These cell reconstructions are computationally intensive and access to high-throughput computational resources is recommended, although not necessary. This method has been successfully applied to multiple bacterial species and mutants, fluorescent imaging modalities, and microscope manufacturers.

This protocol explains how to prepare and mount bacterial samples for live three-dimensional imaging and how to reconstruct the three-dimensional shape of E. coli from those images.

Immagini tridimensionali di cellule batteriche per rappresentazioni cellulari accurate e localizzazione precisa delle proteine

The shape of a bacterium is important for its physiology. Many aspects of cell physiology such as cell motility, predation, and biofilm production can be ...