We thank Dr. Shahid Mukhtar for critiquing the manuscript and Dr. Xinnian Dong for the sample data analysis file. This work is supported by a NSF-CAREER award (IOS-1350244) to KPM and the UAB Biology Department.

Gli autori non hanno nulla da rivelare.

Con la diminuzione a disposizione terreni agricoli e l&#39;aumento della popolazione, i ricercatori di tutto il mondo sono sfidati con le esigenze urgenti per il miglioramento delle colture. Il rendimento può essere notevolmente influenzato da vari stress biotici e abiotici. Tra questi, l&#39;infezione patogeno è una delle principali cause di riduzione della resa delle colture, responsabile di circa il 12% delle perdite solo 45 negli Stati Uniti. Per risolvere questo problema, la ricerca massiccia è stata ...

A causa della loro natura, sessili, piante sono costantemente minacciati da una pletora di agenti patogeni che espongono diversi stili di vita e le strategie nutrizionali 1. In prima approssimazione, gli agenti patogeni biotrofici mantengono il loro ospite vivo per recuperare le sostanze nutritive, mentre agenti patogeni necrotrophic tossine attivamente segreti ed enzimi per uccidere tessuto ospite e nutrono le cellule morte 1. Un altro gruppo di agenti patogeni, hemibiotrophs chiamati, inizia il loro corso l&#39;infezione con il palco biotrofico e si sposta alla fase necrotrophic al raggiungimento di una certa soglia di accumulo patogeno 2. Al fine di difendersi efficacemente contro questi microrganismi, piante hanno sviluppato un complesso sistema immunitario innato dotato di molteplici meccanismi di sorveglianza per rilevare l&#39;attacco patogeno e innescare localizzato morte cellulare programmata 3 e sistemica la resistenza acquisita (SAR) 4. La ricerca attuale è focalizzata sulla caratterizzazione del sig essenzialenaling componenti e incrociati colloqui all&#39;interno del sistema immunitario pianta 5. Come proposto nel modello &quot;zigzag&quot; 5, il primo strato della risposta immunitaria innata pianta richiede la presenza di plasma-membrana localizzata Motivo recettori di riconoscimento (PRR) per rilevare l&#39;invasione di un microbo. PRR sono in grado di riconoscere i modelli Microbe-Associated Molecular (mAmps) e stabilire Immunità MAMP-Triggered (MTI) 6. Oltre ad indurre una upregulation trascrizionale di geni che codificano le proteine ​​PR antimicrobici 7, MTI porta ad una serie di eventi che arrestare la crescita dei patogeni, inclusa la produzione di specie reattive dell&#39;ossigeno (ROS) e reattive di azoto specie (RNS), deposizione di callosio a parete cellulare nonché l&#39;attivazione della chinasi di segnalazione molteplici vie 8. Fino ad oggi, diversi piani di gestione pluriennali sono stati identificati per innescare MTI in Arabidopsis, compresi flg22 batterica 9 </sup&gt; (Un frammento di acido 22 aminoacidi derivato da flagellina), elf18 10 (18 aminoacidi dalla batterica fattore di allungamento Tu) e un componente strutturale della parete cellulare peptidoglicani 11. Per stabilire una infezione di successo, alcuni patogeni specializzate hanno sviluppato la capacità di proteine ​​effettrici virulenza segrete negli spazi intracellulari o intercellulari, e quindi reprimere MTI e innescare suscettibilità Effector-Triggered (ETS) 12,13. Ad esempio, effettori di virulenza possono inattivare proteine ​​Mitogen-chinasi attivata (MAPK) fosforilazione cascate di MTI per indurre lo sviluppo della malattia entro il tessuto infetto 14-16. Durante la co-evoluzione dinamica tra host e patogeni, piante anche sviluppato la strategia di contrattacco di riconoscere le proteine ​​effettrici e attenuare la virulenza patogeno molecole 17. Questo riconoscimento diretto o indiretto effettrici è mediata dalla resistenza alle malattie (R) 18 proteine. La maggior parte di torlo sono membri di NB-LRR (Nucleotide Binding e leucina-ricchi ripetizioni) della famiglia 19. La percezione di un effettore avirulent da una proteina R provoca una risposta più forte e più ampia immunitario caratterizzato come Immunità Effector-Triggered (ETI) 20. Oltre inducendo l&#39;espressione di geni di difesa 21 e la produzione di metaboliti difesa 22, ETI spesso porta ad una rapida morte cellulare programmata localizzata noto come Hypersensitive Response (HR) per limitare il patogeno di diffondersi nel tessuto adiacente 3. Oltre alla morte cellulare programmata localizzato 23, le piante sono in grado di avviare un lungo periodo e risposta immunitaria a livello di sistema denominato Systemic Acquired Resistance (SAR) 4. Su sfida con un agente patogeno biotrofico, cellule vegetali innescano la biosintesi e l&#39;accumulo di un phytohormone endogena acido salicilico (SA) e delle proteine ​​PR in entrambi i tessuti locali e sistemiche 24. Attraverso til suo processo, un elevato stato di preparazione si ottiene nelle foglie non infetti che consente il montaggio risposte di difesa veloce durante una successiva infezione da un ampio spettro di agenti patogeni 24. SA ed i suoi analoghi sintetici come benzo (1,2,3) -thiadiazole-7-carbothioic estere dell&#39;acido S-metil (BTH) e acido 2,6-dichloroisonicotinic (INA) sono in grado di indurre chimicamente acido salicilico (SA) Immunità -Triggered (STI), su richiesta esterna 24. Nonexpressor di-Patogenesi correlati geni 1 (NPR1) si propone di essere uno dei recettori SA e funziona come un regolatore trascrizionale importante durante risposta di difesa SA-mediata sia nei tessuti locali e sistemiche 21,25,26. E &#39;stato definitivamente dimostrato che NPR1 è necessario per istituzione SAR e la perdita di NPR1 conduce alla drammatica suscettibilità verso Pseudomonas syringae 25. Per caratterizzare ampiamente il contributo molecolare della piantacomponenti nelle interazioni pianta-patogeno, molteplici test biologici sono stati sviluppati per misurare gli eventi di difesa specifici, tra cui i ROS scoppio 27, callosio di deposizione 28, espressione geni di difesa e l&#39;accumulo dei loro prodotti proteici 21. Mentre questi dosaggi individuali possono fornire intuizioni in una forma specifica della risposta immunitaria pianta, nessuno di essi, tuttavia, sono in grado di rappresentare la risposta di difesa completa sull&#39;intera livello di impianto. Al contrario, la quantificazione della crescita dei patogeni dopo l&#39;infezione fornisce una stima complessiva della risposta immunitaria a livello di organismi. Pertanto, lo sviluppo e l&#39;ottimizzazione di un preciso e altamente standardizzata saggio patogeno inoculazione è fondamentale per alimentare la ricerca e le scoperte sulle risposte immunitarie Arabidopsis. Pseudomonas syringae, un batterio Gram-negativi, è stato identificato come un phytopathogen in grado di causare la malattia in una vasta gamma di piante ospiti tra cui Arabidopsè 29. Poiché il sistema pianta-patogeno modello Arabidopsis - P. interazione syringae è stato ampiamente applicato per comprendere i meccanismi molecolari alla base della pianta risposte di difesa 29. Finora, oltre 50 P. patovar syringae sono stati identificati in base alla loro capacità di infettare diverse specie vegetali 30 . P. syringae pv. DC3000 pomodoro (Pst DC3000) 31 e P. syringae pv. maculicola ES4326 (PSM ES4326) 32 sono i due ceppi virulenti più utilizzati e ampiamente caratterizzate. Oltre ad essere riconosciuto dalla pianta e innescando la risposta MTI, Pst DC3000 e Psm ES4326 sono in grado di secernere proteine ​​effettrici virulenti per sopprimere MTI e attivare ETS per favorire la crescita del 31,33 patogeno. Per sezionare funzionalmente l&#39;interazione tra Arabidopsis e P. syringae, multiplo0; metodi di infezione patogeni sono stati sviluppati sulla base del metodo di consegna patogeno. Per gli impianti di terreno coltivati, agente patogeno può essere trasportata da siringa infiltrazione, infiltrazione di vuoto, tuffo inoculazione e spruzzo inoculazione 29,34. Recentemente, piantina saggio alluvione-inoculazione è stato sviluppato per eseguire grandi schermi su colture di tessuti piastre-grown giovani piante di Arabidopsis 35. Siringa infiltrazione, come una delle soluzione più utilizzata, garantisce manualmente patogeno nel nell&#39;apoplasto attraverso le aperture dell&#39;anta naturale chiamati stomi 29. Attraverso questo approccio, gli importi uguali di P. syringae può essere infiltrato nella foglia infetta e la forza della risposta immunitaria pianta è inversamente correlato ai livelli di crescita dei patogeni. Pertanto, la quantificazione della crescita dei patogeni serve come un approccio ottimale per valutare la funzione immunitaria a livello di tutta la pianta. Inoltre, siringa infiltrazione può distinguere il tessuto locale e sistemica, che può be applicabile nel caratterizzare i meccanismi molecolari alla base SAR 36. Nel seguente protocollo, si descrive una siringa infiltrazione dosaggio ottimizzato con PSM ES4326 per lo screening mutanti di Arabidopsis per una maggiore suscettibilità alle malattie (EDS). Questo protocollo si avvarrà di due genotipi di Arabidopsis: una wild-type ecotipo Columbia-0 (Col-0) piante (controllo) e mutanti npr1-1 perdita di funzione (ipersensibili) che sarà infettato con ceppo batterico virulento Psm ES4326 37. Il mutante npr1-1 porta una mutazione puntiforme all&#39;interno della sequenza consenso ankyrin-repeat della molecola NPR1, che altera la istidina altamente conservata in tirosina e rende la proteina non funzionale 25. Inoltre, una serie di modifiche del dosaggio siringa infiltrazione sono descritte che consentono la quantificazione dei difetti negli strati specifici della risposta immunitaria, compresi MTI e STI.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>MetroMix 360&#160;</td>
			<td>Grosouth</td>
			<td>SNGMM360</td>
			<td></td>
		</tr>
		<tr>
			<td>Large pots</td>
			<td>Grosouth</td>
			<td>TEKUVCC10TC</td>
			<td></td>
		</tr>
		<tr>
			<td>12x6 Inserts</td>
			<td>Grosouth</td>
			<td>LM1206</td>
			<td></td>
		</tr>
		<tr>
			<td>11x21 Flats with no holes</td>
			<td>Grosouth</td>
			<td>LM1020</td>
			<td></td>
		</tr>
		<tr>
			<td>11x21 Flats with holes</td>
			<td>Grosouth</td>
			<td>LM1020H</td>
			<td></td>
		</tr>
		<tr>
			<td>Vinyl propagation domes</td>
			<td>Grosouth</td>
			<td>CW-221</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteose Peptone</td>
			<td>Fisher Scientific</td>
			<td>DF0122-17-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Phosphate Dibasic Trihydrate&#160;</td>
			<td>MP Biomedicals</td>
			<td>151946</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar&#160;</td>
			<td>Fisher Scientific</td>
			<td>A360-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptomycin sulfate</td>
			<td>Bio Basic Inc</td>
			<td>SB0494</td>
			<td></td>
		</tr>
		<tr>
			<td>100x15mm Petri dishes</td>
			<td>Fisher Scientific</td>
			<td>FB0875713</td>
			<td></td>
		</tr>
		<tr>
			<td>150x15mm Petri dishes</td>
			<td>Fisher Scientific</td>
			<td>R80150</td>
			<td></td>
		</tr>
		<tr>
			<td>Rectangular plate</td>
			<td>Fisher Scientific</td>
			<td>12-565-450&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2 Hexahydrate</td>
			<td>Bio Basic Inc</td>
			<td>MB0328</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Bio Basic Inc</td>
			<td>GB0232</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4&#160;</td>
			<td>Bio Basic Inc</td>
			<td>MN1988</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL syringe</td>
			<td>Fisher Scientific</td>
			<td>NC9992493&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipe</td>
			<td>Fisher Scientific</td>
			<td>06-666-A</td>
			<td></td>
		</tr>
		<tr>
			<td>Grinding tubes&#160;</td>
			<td>Denville Scientific</td>
			<td>B1257</td>
			<td></td>
		</tr>
		<tr>
			<td>Caps for grinding tubes</td>
			<td>Denville Scientific</td>
			<td>B1254</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless steel grinding ball</td>
			<td>Fisher Scientific</td>
			<td>2150</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate&#160;</td>
			<td>Fisher Scientific</td>
			<td>12-556-008</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Salicylate</td>
			<td>Sigma Aldrich</td>
			<td>s3007-1kg</td>
			<td></td>
		</tr>
		<tr>
			<td>flg22 (QRLSTGSRINSAKDDAAGLQIA)</td>
			<td>Genescript</td>
			<td>Made to order</td>
			<td></td>
		</tr>
		<tr>
			<td>elf18 (Ac-SKEKFERTKPHVNVGTIG)</td>
			<td>Genescript</td>
			<td>Made to order</td>
			<td></td>
		</tr>
		<tr>
			<td>Hole puncher</td>
			<td>Staples</td>
			<td>146308</td>
			<td></td>
		</tr>
		<tr>
			<td>Biophotometer plus</td>
			<td>Eppendorf</td>
			<td>952000006</td>
			<td></td>
		</tr>
		<tr>
			<td>PowerGen High-Throughput Homogenizer</td>
			<td>Fisher Scientific</td>
			<td>02-215-503</td>
			<td></td>
		</tr>
		<tr>
			<td>Accu spin micro centrifuge</td>
			<td>Fisher Scientific</td>
			<td>13-100-675</td>
			<td></td>
		</tr>
		<tr>
			<td>Multichannel pipette (10-100 &#181;l)</td>
			<td>Eppendorf</td>
			<td>3122 000.043</td>
			<td></td>
		</tr>
		<tr>
			<td>Multichannel pipette (30-300&#181;l)</td>
			<td>Eppendorf</td>
			<td>3122 000.060</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette (20&#181;l)</td>
			<td>Eppendorf</td>
			<td>3120 000.038</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tips</td>
			<td>Fisher Scientific</td>
			<td>3552-HR</td>
			<td></td>
		</tr>
		<tr>
			<td>Sharpie permanent marker</td>
			<td>Staples</td>
			<td>507130</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL tube</td>
			<td>Eppendorf</td>
			<td>22363204</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fisher Scientific</td>
			<td>08-890</td>
			<td></td>
		</tr>
	</tbody>
</table>

bacterial leaf infiltration assay for fine characterization of plant defense responses using the arabidopsis thaliana-pseudomonas syringae pathosystem

In assenza di cellule immunitarie specializzate mobili, piante utilizzano il loro localizzata morte cellulare programmata e Sistemica resistenza acquisita per difendersi contro l&#39;attacco patogeno. Il contributo di uno specifico gene Arabidopsis per la risposta immunitaria impianto complessivo può essere specifico e quantitativamente valutata analizzando la crescita dei patogeni all&#39;interno del tessuto infetto. Per oltre tre decenni, il batterio Pseudomonas syringae pv hemibiotrophic. Maculicola ES4326 (PSM ES4326) è stato ampiamente applicato come modello patogeno per studiare i meccanismi molecolari alla base della risposta immunitaria Arabidopsis. Per offrire patogeni nel tessuto foglia, sono stabilite diversi metodi di inoculazione, ad esempio, la siringa infiltrazione, tuffo inoculazione, spray, infiltrazione vuoto e inondazioni inoculazione. Il protocollo che segue descrive un metodo siringa infiltrazione ottimizzato per fornire virulenta Psm ES4326 in foglie di adultisuolo coltivato piante di Arabidopsis e schermo per una migliore precisione predisposizione alle malattie (EDS) nei confronti di questo patogeno. Inoltre, questo protocollo può essere integrato con più pre-trattamenti per sezionare ulteriormente specifici difetti immunitari all&#39;interno di diversi livelli di difesa vegetale, compresi l&#39;acido salicilico (SA) Immunità -Triggered (STI) e Immunità MAMP-Triggered (MTI).

Il protocollo che descriviamo qui rappresenta un P. ottimizzata syringae assay siringa infiltrazione per valutare quantitativamente la risposta immunitaria in piante di Arabidopsis. Come illustrato nella figura 1, l&#39;infiltrazione siringa Psm ES4326 è seguita da estrazione patogeno e quantificazione mediante diluizioni seriali e colonie enumerazione. Come descritto al punto 3 nel testo protocollo Enhanced Malattia susc...

Watch this Scientific Journal Video about Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem at JoVE

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem | Immunology and Infection | Video

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 ...

bacterial-leaf-infiltration-assay-for-fine-characterization-plant

Research

Education

JoVE Journal

JoVE Core

Biology

Immunology and Infection

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

Plant Biology

Plant Responses to the Environment

SCIENTISTS IN ACTION

21.5K Views.  University of Alabama at Birmingham. 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.

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

Assay for Pathogen-Associated Molecular Pattern (PAMP)-Triggered Immunity (PTI) in Plants

To perceive potential pathogens in their environment, plants use pattern recognition receptors (PRRs) present on their plasma membranes. PRRs recognize conserved microbial features called pathogen-associated molecular patterns (PAMPs) and this detection leads to PAMP-triggered immunity (PTI), which effectively prevents colonization of plant tissues by non-pathogens1,2. The most well studied system in PTI is the FLS2-dependent pathway3. FLS2 recognizes the PAMP flg22 that is a component of bacterial flagellin. 
Successful pathogens possess virulence factors or effectors that can suppress PTI and allow the pathogen to cause disease1. Some plants in turn possess resistance genes that detect effectors or their activity, which leads to effector-triggered immunity (ETI)2.
We describe a cell death-based assay for PTI modified from Oh and Collmer4. The assay was standardized in N. benthamiana, which is being used increasingly as a model system for the study of plant-pathogen interactions5. PTI is induced by infiltration of a non-pathogenic bacterial strain into leaves. Seven hours later, a bacterial strain that either causes disease or which activates ETI is infiltrated into an area overlapping the original infiltration zone. PTI induced by the first infiltration is able to delay or prevent the appearance of cell death due to the second challenge infiltration. Conversely, the appearance of cell death in the overlapping area of inoculation indicates a breakdown of PTI. 
Four different combinations of inducers of PTI and challenge inoculations were standardized (Table 1). The assay was tested on non-silenced N. benthamiana plants that served as the control and plants silenced for FLS2 that were predicted to be compromised in their ability to develop PTI.

Assay for Pathogen-Associated Molecular Pattern PAMP-Triggered Immunity PTI in Plants

A cell death-based assay for PTI in Nicotiana benthamiana plants is described.

Test per patogeni associati modello molecolare (PAMP)-trigger Immunità (PTI) nelle piante

To perceive potential pathogens in their environment, plants use pattern recognition receptors (PRRs) present on their plasma membranes. PRRs recognize ...

Ricerca

Immunologia e infezioni

Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of guard cells, which are able to close the stomatal pore in response to a number of external factors including light intensity, carbon dioxide concentration, and relative humidity (RH). The stomatal pore is also the main route for pathogen entry into leaves, a crucial step for disease development. Recent studies have unveiled that closure of the pore is effective in minimizing bacterial disease development in Arabidopsis plants; an integral part of plant innate immunity. Previously, we have used epidermal peels to assess stomatal response to live bacteria (Melotto et al. 2006); however maintaining favorable environmental conditions for both plant epidermal peels and bacterial cells has been challenging. Leaf epidermis can be kept alive and healthy with MES buffer (10 mM KCl, 25 mM MES-KOH, pH 6.15) for electrophysiological experiments of guard cells. However, this buffer is not appropriate for obtaining bacterial suspension. On the other hand, bacterial cells can be kept alive in water which is not proper to maintain epidermal peels for long period of times. When an epidermal peel floats on water, the cells in the peel that are exposed to air dry within 4 hours limiting the timing to conduct the experiment. An ideal method for assessing the effect of a particular stimulus on guard cells should present minimal interference to stomatal physiology and to the natural environment of the plant as much as possible. We, therefore, developed a new method to assess stomatal response to live bacteria in which leaf wounding and manipulation is greatly minimized aiming to provide an easily reproducible and reliable stomatal assay. The protocol is based on staining of intact leaf with propidium iodide (PI), incubation of staining leaf with bacterial suspension, and observation of leaves under laser scanning confocal microscope. Finally, this method allows for the observation of the same live leaf sample over extended periods of time using conditions that closely mimic the natural conditions under which plants are attacked by pathogens.

We have developed a simple and reproducible protocol to access stomatal response to live bacteria. This method minimizes wounding and manipulation of the leaf as compared to the use of epidermal peels reported previously.

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 ...

Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by Transmission Electron Microscopy

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample preparation for transmission electron microscopy (TEM) such investigations can take several days1,2 and are therefore not suited for a rapid diagnosis of plant virus diseases. Microwave fixation can be used to drastically reduce sample preparation time for TEM investigations with similar ultrastructural results as observed after conventionally sample preparation3-5. Many different custom made microwave devices are currently available which can be used for the successful fixation and embedding of biological samples for TEM investigations5-8. In this study we demonstrate on Tobacco Mosaic Virus (TMV) infected Nicotiana tabacum plants that it is possible to diagnose ultrastructural alterations in leaves in about half a day by using microwave assisted sample preparation for TEM. We have chosen to perform this study with a commercially available microwave device as it performs sample preparation almost fully automatically5 in contrast to the other available devices where many steps still have to be performed manually6-8 and are therefore more time and labor consuming. As sample preparation is performed fully automatically negative staining of viral particles in the sap of the remaining TMV-infected leaves and the following examination of ultrastructure and size can be performed during fixation and embedding.

This study describes a method that allows the rapid and clear diagnosis of plant virus diseases in about half a day by using a combination of microwave assisted plant sample preparation for transmission electron microscopy and negative staining methods.

Forno a microonde Assist diagnosi rapida di malattie virali piante da microscopia elettronica a trasmissione

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample ...

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8&alpha;+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6.
Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6	. There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design.
We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to &beta;-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection.

Misurazione della carica batterica e Risposte immunitarie in topi infettati con Listeria monocytogenes</em

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

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 ...

Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster

The fruit fly Drosophila melanogaster is one of the premier model organisms for studying the function and evolution of immune defense. Many aspects of innate immunity are conserved between insects and mammals, and since Drosophila can readily be genetically and experimentally manipulated, they are powerful for studying immune system function and the physiological consequences of disease. The procedure demonstrated here allows infection of flies by introduction of bacteria directly into the body cavity, bypassing epithelial barriers and more passive forms of defense and allowing focus on systemic infection. The procedure includes protocols for the measuring rates of host mortality, systemic pathogen load, and degree of induction of the host immune system. This infection procedure is inexpensive, robust and quantitatively repeatable, and can be used in studies of functional genetics, evolutionary life history, and physiology.

Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster

Drosophila melanogaster is an outstanding model organism for studying innate immune systems and the physiological consequences of infection and disease. This protocol describes how to deliver robust and quantitatively repeatable bacterial infections to D. melanogaster, and how to subsequently measure infection severity and quantify the host immune response.

Sistemica infezione batterica e immunitario Difesa fenotipi in<em&gt; Drosophila melanogaster</em

The fruit fly Drosophila melanogaster is one of the premier model organisms for studying the function and evolution of immune defense. Many aspects of ...

Application of Long-term cultured Interferon-&#947; Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is controlled up to 95 % of the T cells generated during the expansion phase are eliminated (i.e., contraction phase) and memory T cells remain, sometimes for a lifetime. In humans, two functionally distinct subsets of memory T cells have been described based on the expression of lymph node homing receptors. Central memory T cells express C-C chemokine receptor 7 and CD45RO and are mainly located in T-cell areas of secondary lymphoid organs. Effector memory T cells express CD45RO, lack CCR7 and display receptors associated with lymphocyte homing to peripheral or inflamed tissues. Effector T cells do not express either CCR7 or CD45RO but upon encounter with antigen produce effector cytokines, such as interferon-&#947;. Interferon-&#947; release assays are used for the diagnosis of bovine and human tuberculosis and detect primarily effector and effector memory T cell responses. Central memory T cell responses by CD4+ T cells to vaccination, on the other hand, may be used to predict vaccine efficacy, as demonstrated with simian immunodeficiency virus infection of non-human primates, tuberculosis in mice, and malaria in humans. Several studies with mice and humans as well as unpublished data on cattle, have demonstrated that interferon-&#947; ELISPOT assays measure central memory T cell responses. With this assay, peripheral blood mononuclear cells are cultured in decreasing concentration of antigen for 10 to 14 days (long-term culture), allowing effector responses to peak and wane; facilitating central memory T cells to differentiate and expand within the culture.

Application of Long-term cultured Interferon-&amp;#947; Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle

Long-term cultured interferon-&#947; enzyme-linked immunospot assay is used as a measure of central memory responses and correlates with protective anti-mycobacterial vaccine responses. With this assay, peripheral blood mononuclear cells are stimulated with mycobacterial antigens and interleukin-2 for 14 days, enabling differentiation and expansion of central memory T cells.

Applicazione di lungo termine colta interferone-γ Enzyme Linked immunospot Assay per la valutazione effettori e memoria delle cellule T risposte a Bovino

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is ...

Characterization of Thymus-dependent and Thymus-independent Immunoglobulin Isotype Responses in Mice Using Enzyme-linked Immunosorbent Assay

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a formidable defense against invading pathogens via diverse mechanisms. One major goal of vaccination is to induce protective antigen-specific antibodies to prevent life-threatening infections. Both thymus-dependent (TD) and thymus-independent (TI) antigens can elicit robust antigen-specific IgM responses and can also induce the production of isotype-switched antibodies (IgG, IgA and IgE) as well as the generation of memory B cells with the help provided by antigen presenting cells (APCs). Here, we describe a protocol to characterize TD and TI Ig isotype responses in mice using enzyme-linked immunosorbent assay (ELISA). In this protocol, TD and TI Ig responses are elicited in mice by intraperitoneal (i.p.) immunization with hapten-conjugated model antigens TNP-KLH (in alum) and TNP-polysaccharide (in PBS), respectively. To induce TD memory response, a booster immunization of TNP-KLH in alum is given at 3 weeks after the first immunization with the same antigen/adjuvant. Mouse sera are harvested at different time points before and after immunization. Total serum Ig levels and TNP-specific antibodies are subsequently quantified using Ig isotype-specific Sandwich and indirect ELISA, respectively. In order to correctly quantify the serum concentration of each Ig isotype, the samples need to be appropriately diluted to fit within the linear range of the standard curves. Using this protocol, we have consistently obtained reliable results with high specificity and sensitivity. When used in combination with other complementary methods such as flow cytometry, in vitro culture of splenic B cells and immunohistochemical staining (IHC), this protocol will allow researchers to gain a comprehensive understanding of antibody responses in a given experimental setting.

In this paper, we describe a protocol to characterize T-dependent and T-independent immunoglobulin (Ig) isotype responses in mice using ELISA. This method used alone or in combination with flow cytometry will allow researchers to identify differences in B cell-mediated Ig isotype responses in mice following T-dependent and T-independent antigen immunization.

Caratterizzazione delle risposte di isotipo dell'immunoglobulina timo-dipendenti e indipendenti del timo in topi usando l'analisi enzima-collegata dell'immunosorbente

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a ...

Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' pathogens (Listeria monocytogenes, Shigella flexneri, Burkholderia pseudomallei, Francisella tularensis, and Rickettsia spp.) gain access to the infected cell cytosol by physically and enzymatically degrading the primary vacuolar membrane. Once in the cytosol, these pathogens both proliferate as well as generate sufficient mechanical forces to penetrate the plasma membrane of the host cell in order to infect new cells. Here, we show how this terminal step of the cellular infection cycle of L. monocytogenes (Lm) can be quantified by both colony-forming unit assays and flow cytometry and give examples of how both pathogen- and host-encoded factors impact this process. We also show a close correspondence of Lm infection dynamics of cultured cells infected in vitro and those of hepatic cells derived from mice infected in vivo. These function-based assays are relatively simple and can be readily scaled up for discovery-based high-throughput screens for modulators of eukaryotic cell function.

We describe both in vitro and in vivo infection assays that can be used to analyze the activities of host-encoding factors.

Utilizzando un agente patogeno batterico alla sonda per le risposte cellulari e organismica-livello Host

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' ...

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental procedure in which phagocytic cells are co-cultured with bacterial units. The immune cells will phagocytose and kill the bacterial cultures in a complement-dependent manner. The efficiency of the immune-mediated cell killing is dependent on a number of factors and can be used to determine how different bacterial cultures compare with regard to resistance to cell death. In this way, the efficacy of potential immune-based therapeutics can be assessed against specific bacterial strains and/or serotypes. In this protocol, we describe a simplified OPKA that utilizes basic culture conditions and cell counting to determine bacterial cell viability after co-culture with treatment conditions and HL-60 immune cells. This method has been successfully utilized with a number of different pneumococcal serotypes, capsular and acapsular strains, and other bacterial species. The advantages of this OPKA protocol are its simplicity, versatility (as this assay is not limited to antibody treatments as opsonins), and minimization of time and reagents to assess basic experimental groups.

This opsonophagocytic killing assay is used to compare the ability of phagocytic immune cells to respond to and kill bacteria based on different treatments and/or conditions. Classically, this assay serves as the gold&#160;standard for assessing effector functions of antibodies raised against a bacterium as opsonin.

Anticorpli uccisione test per valutare le risposte immunologiche contro batteri patogeni

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental ...