Questo lavoro è stato sostenuto dal Fondo austriaco della scienza (FWF, P20619 e P22988 a BZ).

Microonde preparazione del campione assistita <ol><li> Prima dell&#39;inizio di preparazione del campione bisogna programmare il processore automatico per tessuti microonde microscopia elettronica (EM Leica AMW; Microsystems Leica, Vienna, Austria) con i seguenti protocolli (Tabella 1) per la preparazione del campione per microonde assistita TEM: <img src="/files/ftp_upload/2950/2950table1.jpg" alt="Tabella 1" /> Tabella 1. Protocollo per la preparazione del campione mediante irradiazione a microonde. Le colonne mostrano differenti (da sinistra a destra): <ol><li> Numero flaconcino (nr Vial.) Rappresenta l&#39;ordine in cui è caricato il flacone nel carosello del processore. </li><li> Fase del processo vero e proprio. </li><li> Reagenti nelle fiale. </li><li> Durata della fase attuale. </li><li> Temperatura massima che si raggiunge nella fiala prima irradiazione con microonde è spento. </li><li> Forno a microonde impostazione irradiazione: continua = incrementi di temperatura rapidaase, tenendo la temperatura impostata; Slope = aumento di temperatura moderata, temperatura finale raggiunta al termine; Pulsed = rapido aumento di temperatura, alimentazione spento finché la temperatura è scesa a 5 ° C, potenza ripreso a temperatura. </li><li> Potenza massima della radiazione a microonde. </li></ol></li><li> Fresco preparare le soluzioni per le diverse fasi descritte nel protocollo di preparazione del campione (per Agar 100 resina epossidica vedi 1.7), riempirle nei flaconi designati secondo il protocollo programmato (Tabella 1), caricare le fiale sulla giostra, quindi inserire il giostra nel processore tessuto microonde, e infine caricare il primo flaconcino in modo mono-camera. </li><li> Tagliare piccole sezioni di foglie (1mm 2) di Nicotiana tabacum infettati con virus del mosaico del tabacco (TMV), con una lama di rasoio in un piatto ceroplastica in un calo del 3% glutaraldeide (Agar Scientific Ltd., Stansted, Inghilterra) in 60mm Sørensen tampone fosfato (pH 7,2) a temperatura ambienteteratura. </li><li> Trasferire le sezioni con le pinzette sottili immediatamente nei cesti designati con una larghezza delle maglie di circa 200μm. Stack i cestelli uno sopra l&#39;altro e inserirle nella modalità mono-camera. Bisogna fare attenzione che i campioni sono costantemente ricoperti di soluzione fissativa durante il carico e accatastamento dei cestelli in modo che non si secchino. </li><li> Avviare il programmato in precedenza microonde assistita protocollo di preparazione del campione per la fissazione, la disidratazione e l&#39;infiltrazione. </li><li> Durante la preparazione del campione viene eseguita automaticamente dal processore tessuto microonde continuare con colorazione negativa con il materiale vegetale residuo come descritto nella sezione 3 (colorazione negativa). </li><li> Preparata fresca Agar 100 resina epossidica miscelando i seguenti componenti come descritto: riempimento 24g Agar 100, 16g dodecenyl anidride succinica, e 10 g di anidride metil nadic (per tutti i componenti vedere Agar Scientific Ltd., Stansted, Inghilterra) in una tazza di plastica, di calore a40 ° C e mescolare bene. Aggiungere 1,2 g di benzile dimetilammina e mescolare accuratamente. Riempire Agar 100 resina epossidica nella polimerizzazione designato forma appena prima del protocollo di preparazione del campione giunge al termine (ad esempio durante la fase 22 in tabella 1). </li><li> Dopo che il protocollo è terminata (dopo passo 22 nella tabella 1) rilasciare i cestelli sovrapposti contenenti i campioni infiltrate dalla modalità mono-camera nel flaconcino ultimo della giostra. Rimuovere la giostra dal dispositivo a microonde, Affianca i cestelli e caricarle utilizzando pinzette sottili nelle forme di polimerizzazione designati. Bisogna fare attenzione che i campioni sono sempre ricoperti di Agar 100 resina epossidica durante disimpilamento e il caricamento in modo che non si secchino. </li><li> Impilare le forme di polimerizzazione sopra l&#39;altro. Bisogna fare attenzione che i campioni sono sempre ricoperti di Agar 100 resina epossidica durante accatastamento e carico in modo che non si secchino. </li><li> Rimuovere le fiale precedentemente utilizzati dalla giostra del Tiss microondeue processore, caricarlo con i cesti impilati e inserire la giostra nel processore tessuto forno a microonde. </li><li> Avviare il protocollo di polimerizzazione programmato in precedenza (tabella 1). </li><li> Mentre polimerizzazione viene effettuata automaticamente dal processore tessuto microonde, esaminare griglie negativamente colorati con un microscopio elettronico a trasmissione [per esempio Philips CM10 TEM, FEI (ex Philips), Eindhoven, Paesi Bassi] ed eseguire analisi di immagine come descritto nel capitolo 3 e 4 ( colorazione negativa e analisi d&#39;immagine). </li><li> Dopo che il protocollo è terminata rimuovere le forme di polimerizzazione dal mono-modalità camera, Unstack le forme di polimerizzazione e polimerizzate rimuovere i blocchi contenenti i campioni. Essi sono ora pronti per essere sezionato con un microtomo. </li></ol> 2. Taglio e sezionamento <ol><li> Inserisci uno o più blocchi in porta-campioni separati per ultrasottile sezionamento con il campione di incollaggio superiore a circa 1 cm dal supporto. </li><li>Tagliare il blocco con un trimmer campione per TEM (ad esempio Leica Reichert Ultratrim, Leica Microsystems) in modo che una faccia blocco di max. 1 mm in lunghezza e in larghezza 200μm che contiene materiale fogliare quanto possibile si ottiene (Block Size faccia potrebbe dover essere adattato alle dimensioni del coltello diamante). </li><li> Sezione del blocco con un ultramicrotomo (es. Reichert Leica Ultracut S; Microsystems Leica) utilizzando un coltello di diamante con un angolo di 45 ° coltello (es. DIATOME Ultra 45, Gröpl, Tulln, Austria). Spessore della sezione deve essere regolato a circa 70 a 90 nm e la velocità di taglio dovrebbe essere di circa 1 mm / s. Raccogliere più sezioni con un (Agar Scientific Ltd.) di rame ricoperto di nichel o formvar 200 griglia a maglie quadrate. </li><li> Post-macchia le sezioni sulla griglia di partenza con citrato di piombo (Agar Scientific Ltd., citrato di piombo 1,1 g sciolti in 42 ml di acqua bidistillata e 8ml 1N NaOH) per 5 minuti in una capsula di Petri parzialmente pieni di NaOH per creare un senza CO 2 ambiente e per 15 minuti con acetato di uranile 1% (Agar Scientific Ltd.) disciolto in acqua distillata a temperatura ambiente. Lavare le griglie con acqua distillata per 1 minuto tra ogni fase post-colorazione. Far asciugare le griglie in una casella della griglia. </li><li> Esaminare le sezioni con un microscopio elettronico a trasmissione (es. Philips CM10 TEM, FEI, Eindhoven, Paesi Bassi). </li></ol> 3. Colorazione negativa <ol><li> Raccolto circa 100mg di TMV materiale infetto foglia e preparare linfa grezzo mediante omogeneizzazione del materiale per 2 minuti con una lama di rasoio in un vetrino in 100 microlitri di 60mm Søfrensen tampone fosfato (pH 7,2). </li><li> Trasferire 20μl di omogenato risultante sul primo pozzetto di un vetrino rivestito teflon con 4 o più pozzetti (in alternativa è anche possibile trasferire l&#39;omogeneizzato su un pezzo di parafilm). </li><li> Inserire una griglia formvar rivestito sopra l&#39;omogenato con l&#39;(Agar Scientific Ltd.) formvar lato patinato rivolto verso la caduta e incubate per 5 minuti. </li><li> Lavare la griglia 2 volte per 2 minuti ciascuno disponendo la griglia sopra due gocce di 200μl 60mm Sørensen tampone fosfato (pH 7,2). </li><li> Incubare la griglia per 1 minuto con una appena preparata di acido fosfotungstico al 2% (Agar Scientific Ltd.) in soluzione 60mm Sørensen tampone fosfato (pH 6,5). </li><li> Rimuovere la griglia e lasciare asciugare in una scatola griglia. </li><li> Esaminare la griglia con un microscopio elettronico a trasmissione (es. Philips CM10 TEM, FEI, Eindhoven, Paesi Bassi). Prendere almeno 10 immagini scelte a caso virioni colorazione negativa (almeno 10 o più virioni dovrebbero essere visibili su ogni immagine) con il microscopio elettronico a trasmissione ad un ingrandimento di primaria 21000X o superiore. Bisogna fare attenzione che tutte le immagini hanno lo stesso ingrandimento. </li></ol> 4. Analisi delle immagini <ol><li> Misurare la lunghezza e la larghezza di almeno 100 particelle scelte a caso singolo virus sulle micrografie prese dai campioni di colorazione negativa utilizzando qualsiasi software di analisi immagine del computer [per esempio cellule D (Olympus, Vita e Scienza dei Materiali Europe GmbH, Hamburg, Germania) con lo strumento di analisi di particelle o Optimas 6.5.1-(Media Cybernetics Inc., Bethesda, Maryland, USA)]. </li><li> Calcolare medie e deviazioni standard per ottenere una lunghezza e la larghezza media delle particelle virali individuate tramite metodi di colorazione negativa e TEM. </li><li> Confronta lunghezza e caratteristiche ultrastrutturali (ottenuta in 2.5) con dimensioni di virus e alterazioni ultrastrutturali indotte da malattie virali noti in letteratura per identificare chiaramente le malattie virali. </li></ol> 5. Rappresentante dei risultati: Dopo assistite campione microonde preparazione tipica alterazioni ultrastrutturali TMV indotte quali grandi aree contenenti virioni allineati in forma parallela potrebbe essere osservato con il microscopio elettronico a trasmissione nel citosol di infetto Nicotiana cellule tabacum (Fig. 1A). Inoltre, nella linfa grezza di TMV-infetta lascia particelle di TMV potrebbe essere osservata come flessuosi, astiformi strutture dopo colorazione negativa (Fig. 1B). L&#39;analisi delle immagini di 100 particelle virali ha rivelato una dimensione media di TMV di 280 nm di lunghezza e 17Nm di larghezza (Fig. 2). L&#39;ultrastruttura in TMV cellule infettate e le dimensioni dei virioni in questo studio sono risultati in accordo con proprietà ultrastrutturali TMV-indotte nel tabacco e l&#39;intervallo di dimensioni delle particelle di TMV precedentemente riportato in letteratura 9-15. <img src="/files/ftp_upload/2950/2950fig1.jpg" alt="Figura 1" /> Figura 1. Micrografie elettroniche di trasmissione di TMV-cellule infettate da foglia e virioni. A) L&#39;immagine mostra l&#39;ultrastruttura delle cellule del mesofillo TMV-infettati foglie di Nicotiana tabacum dopo la preparazione del campione centrale assistita a microonde. Si noti la grande area di virioni allineate parallele che hanno accumulato nel citosol.C = cloroplasto con amido (St), M = mitocondrio, N = nucleo, V = vacuolo, Bar = 2μm. B) L&#39;immagine mostra virioni che sono stati individuati mediante colorazione negativa la linfa delle foglie infette. <img src="/files/ftp_upload/2950/2950fig2.jpg" alt="Figura 2" /> Figura 2. Dimensione relativa di virioni. Distribuzione relativa della lunghezza e della larghezza del TMV-particelle dopo colorazione negativa la linfa delle foglie infette come apparivano al microscopio elettronico. I valori medi (media lunghezza / larghezza ± deviazione standard) sono stati calcolati da 100 virioni.

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Nessun conflitto di interessi dichiarati.

Forno a microonde assistita preparazione del campione impianto per TEM ha dimostrato di fornire i dati ultrastrutturali veloci e affidabili nel giro di poche ore 3,4,16. La conservazione multa strutturale di organelli e membrane ottenuti con il metodo utilizzato in questo studio era simile a campioni convenzionali e cryofixed 5,15 con il vantaggio di una drastica riduzione fissazione campione e l&#39;incorporamento di tempo da 3 giorni o più per circa 2 ore. Questo rappresenta il protocollo più veloce preparazione del campione per TEM attualmente disponibili in letteratura. Il metodo descritto in questo studio combinato microonde assistita impianto per la preparazione del campione TEM con metodi di colorazione negativa che ha consentito una identificazione chiara e rapida delle alterazioni ultrastrutturali TMV indotte e l&#39;agente virale stessa. TMV alterazioni indotte ultrastrutturali poteva essere analizzata dopo il taglio, sezionamento e post-colorazione entro circa 4 ore dopo l&#39;inizio del fissaggio nella trasmissione elettron microscopio. Utilizzando la modalità completamente automatica preparazione del provino liberato il ricercatore di condurre colorazione negativa nel frattempo, al fine di determinare le dimensioni e la larghezza del agente virale. Quindi, possiamo concludere che questo metodo permette una diagnosi chiara e rapida delle malattie delle piante virus in circa mezza giornata, che è di grande importanza per un uso futuro in esperimenti di agricoltura e scientifico in impianti fitopatologia. Poiché questo metodo potrebbe anche essere utilizzato per la diagnosi rapida di malattie umane e animali ha un grande potenziale per la futura applicazione in medicina veterinaria e patologia.

<table border="1"><tbody><tr><td> Nome del reagente / attrezzature </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti </td></tr><tr><td> Glutaraldeide </td><td> Agar Scientific Ltd. </td><td> R1312 </td><td></td></tr><tr><td> Osmio tetrossido </td><td> Agar Scientific Ltd. </td><td> R1022 </td><td></td></tr><tr><td> Agar resina 100 </td><td> Agar Scientific Ltd. </td><td> R1043 </td><td></td></tr><tr><td> Dodecenyl anidride succinica </td><td> Agar Scientific Ltd. </td><td> R1051 </td><td></td></tr><tr><td> Metile anidride nadic </td><td> Agar Scientific Ltd. </td><td> R1081 </td><td></td></tr><tr><td> Benzyl dimetilammina </td><td> Agar Scientific Ltd. </td><td> R1060 </td><td></td></tr><tr><td> Citrato di piombo </td><td> Agar Scientific Ltd. </td><td> R1210 </td><td></td></tr><tr><td> Acetato di uranile </td><td> Agar Scientific Ltd. </td><td> R1260A </td><td></td></tr><tr><td> Acido fosfotungstico </td><td> Agar Scientific Ltd. </td><td> R1213 </td><td></td></tr><tr><td> Formvar </td><td> Agar Scientific Ltd. </td><td> R1202 </td><td></td></tr><tr><td colspan="4"></td></tr><tr><td> Leica EM AMW </td><td> Leica Microsystems </td><td></td><td></td></tr><tr><td> Leica (Reichert) Ultratrim </td><td> Leica Microsystems </td><td></td><td> Post più recente modello è disponibile </td></tr><tr><td> Leica (Reichert) Ultracut S </td><td> Leica Microsystems </td><td></td><td> Post più recente modello è disponibile </td></tr><tr><td> DIATOME Ultra 45 </td><td> Gröpl </td><td></td><td></td></tr><tr><td> Philips CM10 TEM </td><td> FEI (ex Philips) </td><td> &lt;/ Td&gt; <td> Post più recente modello è disponibile </td></tr><tr><td> Cella D </td><td> Olympus Inc. </td><td></td><td></td></tr><tr><td> Optimas 6.5.1 </td><td> Media Cybernetics Inc. </td><td></td><td> Post più recente versione è disponibile </td></tr></tbody></table>

microwave assisted rapid diagnosis of plant virus diseases by transmission electron microscopy

Le indagini sulle alterazioni ultrastrutturali indotte dal virus sono spesso necessario individuare chiaramente malattie virali nelle piante. Con la preparazione del campione convenzionale per la microscopia elettronica a trasmissione (TEM) che tali indagini può richiedere diversi giorni 1,2 e non sono quindi adatti per una rapida diagnosi di malattie virali delle piante. Fissazione microonde può essere utilizzata per ridurre drasticamente il tempo di preparazione del campione per TEM con risultati simili ultrastrutturali come osservato dopo convenzionalmente preparazione del campione 3-5. Molti diversi dispositivi a microonde su misura disponibile, che può essere impiegata per il fissaggio successo e incorporamento di campioni biologici per TEM 5-8. In questo studio abbiamo dimostrato il virus del mosaico del tabacco (TMV) piante infettate Nicotiana tabacum che è possibile diagnosticare alterazioni ultrastrutturali in foglie in circa mezza giornata utilizzando preparazione del campione assistita microonde per TEM. Abbiamo scelto di compiere questo studio con un dispositivo a microonde disponibile in commercio come effettua la preparazione del campione quasi completamente automatico 5 in contrasto con gli altri dispositivi disponibili dove molti passi devono essere ancora eseguite manualmente 6-8 e sono quindi più tempo e consumare manodopera. Come preparazione del campione è eseguita in modo completamente automatico la colorazione negativa di particelle virali nel linfa dei restanti TMV-foglie infette e l&#39;esame del seguente ultrastruttura e le dimensioni possono essere eseguite durante la fissazione e l&#39;inclusione.

Watch this Scientific Journal Video about Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by Transmission Electron Microscopy at JoVE.com

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

Questo studio descrive un metodo che permette la diagnosi rapida e chiara delle malattie delle piante virus in circa mezza giornata, utilizzando una combinazione di preparazione dell&#39;impianto assistita campione microonde per microscopia elettronica a trasmissione e metodi di colorazione negativa.

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 ...

microwave-assisted-rapid-diagnosis-plant-virus-diseases-transmission

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JoVE Journal

Immunology and Infection

13.4K Views.  University of Graz. Questo studio descrive un metodo che permette la diagnosi rapida e chiara delle malattie delle piante virus in circa mezza giornata, utilizzando una combinazione di preparazione dell'impianto assistita campione microonde per microscopia elettronica a trasmissione e metodi di colorazione negativa.

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

On-Site Molecular Detection of Soil-Borne Phytopathogens Using a Portable Real-Time PCR System

On-site diagnosis of plant diseases can be a useful tool for growers for timely decisions enabling the earlier implementation of disease management strategies that reduce the impact of the disease. Presently in many diagnostic laboratories, the polymerase chain reaction (PCR), particularly real-time PCR, is considered the most sensitive and accurate method for plant pathogen detection. However, laboratory-based PCRs typically require expensive laboratory equipment and skilled personnel. In this study, soil-borne pathogens of potato are used to demonstrate the potential for on-site molecular detection. This was achieved using a rapid and simple protocol comprising of magnetic bead-based nucleic acid extraction, portable real-time PCR (fluorogenic probe-based assay). The portable real-time PCR approach compared favorably with a laboratory-based system, detecting as few as 100 copies of DNA from Spongospora subterranea. The portable real-time PCR method developed here can serve as an alternative to laboratory-based approaches and a useful on-site tool for pathogen diagnosis.

Rapid and accurate detection of plant pathogens on-site, especially soil-borne pathogens, is crucial to prevent further inoculum production and proliferation of plant diseases in the field. The method developed here using a portable real-time PCR detection system enables on-site diagnosis under field conditions.

Rilevamento in loco molecolare di fitopatogeni terricoli, utilizzando un sistema PCR in tempo reale portatile

On-site diagnosis of plant diseases can be a useful tool for growers for timely decisions enabling the earlier implementation of disease management ...

Ricerca

Ambiente

Potato Virus X-Based microRNA Silencing (VbMS) In Potato.

Virus-based microRNA silencing (VbMS) is a rapid and efficient tool for functional characterization of microRNAs (miRNAs) in plants. The VbMS system has been developed and applied for various plant species including Nicotiana benthamiana, tomato, Arabidopsis, cotton, and monocot plants such as wheat and maize. Here, we describe a detailed protocol using PVX-based VbMS vectors to silence endogenous miRNAs in potato. To knock down the expression of a specific miRNA, target mimic (TM) molecules of miRNA of interest are designed, integrated into plant virus vectors, and expressed in potato by Agrobacterium infiltration to bind directly to the endogenous miRNA of interest and block its function.

Potato Virus X-Based microRNA Silencing VbMS In Potato.

We present a detailed protocol for potato virus X (PVX)-based microRNA silencing (VbMS) system to functionally characterize endogenous microRNAs (miRNAs) in potato. Target mimic (TM) molecules of miRNA of interest are integrated into the PVX vector and transiently expressed in potato to silence the target miRNA or miRNA family.

Virus della patata Silenziamento del microRNA basato sull'X (VbMS) nella patata.

Virus-based microRNA silencing (VbMS) is a rapid and efficient tool for functional characterization of microRNAs (miRNAs) in plants. The VbMS system has ...

Fluorescence in situ Hybridizations (FISH) for the Localization of Viruses and Endosymbiotic Bacteria in Plant and Insect Tissues

Fluorescence in situ hybridization (FISH) is a name given to a variety of techniques commonly used for visualizing gene transcripts in eukaryotic cells and can be further modified to visualize other components in the cell such as infection with viruses and bacteria. Spatial localization and visualization of viruses and bacteria during the infection process is an essential step that complements expression profiling experiments such as microarrays and RNAseq in response to different stimuli. Understanding the spatiotemporal infections with these agents complements biological experiments aimed at understanding their interaction with cellular components. Several techniques for visualizing viruses and bacteria such as reporter gene systems or immunohistochemical methods are time-consuming, and some are limited to work with model organisms&#160;and involve complex methodologies. FISH that targets RNA or DNA species in the cell is a relatively easy and fast method for studying spatiotemporal localization of genes and for diagnostic purposes. This method can be robust and relatively easy to implement when the protocols employ short hybridizing, commercially-purchased probes, which are not expensive. This is particularly robust when sample preparation, fixation, hybridization, and microscopic visualization do not involve complex steps. Here we describe a protocol for localization of bacteria and viruses in insect and plant tissues. The method is based on simple preparation, fixation, and hybridization of insect whole mounts and dissected organs or hand-made plant sections, with 20 base pairs short DNA probes conjugated to fluorescent dyes on their 5&#39; or 3&#39; ends. This protocol has been successfully applied to a number of insect and plant tissues, and can be used to analyze expression of mRNAs or other RNA or DNA species in the cell.

Fluorescence in situ Hybridizations FISH for the Localization of Viruses and Endosymbiotic Bacteria in Plant and Insect Tissues

We describe here a simple fluorescence in situ hybridization (FISH) method for the localization of viruses and bacteria in insect and plant tissues. This protocol can be extended for the visualization of mRNA in whole mount and microscopic sections.

Fluorescenza<em&gt; In situ</em&gt; Ibridazioni (FISH) per la localizzazione di virus e batteri endosimbiontica a Plant e Insetti tessuti

Fluorescence in situ hybridization (FISH) is a name given to a variety of techniques commonly used for visualizing gene transcripts in eukaryotic cells ...

Immunologia e infezioni

A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

The isolation of giant viruses is of great interest in this new era of virology, especially since these giant viruses are related to protists. Giant viruses may be potentially pathogenic for many species of protists. They belong to the recently described order of Megavirales. The new lineage Faustovirus that has been isolated from sewage samples is distantly related to the mammalian pathogen African swine fever virus. This virus is also specific to its amoebal host, Vermamoeba vermiformis, a protist common in health care water systems. It is crucial to continue isolating new Faustovirus genotypes in order to enlarge its genotype collection and study its pan-genome. We developed new strategies for the isolation of additional strains by improving the use of antibiotic and antifungal combinations in order to avoid bacterial and fungal contaminations of the amoeba co-culture and favoring the virus multiplication. We also implemented a new starvation medium to maintain V. vermiformis in optimal conditions for viruses co-culture. Finally, we used flow cytometry rather than microscopic observation, which is time-consuming, to detect the cytopathogenic effect. We obtained two isolates from sewage samples, proving the efficiency of this method and thus widening the collection of Faustoviruses, to better understand their environment, host specificity and genetic content.

A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

We describe here the latest advances in viral isolation for the characterization of new genotypes of Faustovirus, a new asfarvirus-related lineage of giant viruses. This protocol can be applied to the high throughput isolation of viruses, especially giant viruses infecting amoeba.

Una strategia rapido per l&#39;isolamento di nuovi Faustoviruses da campioni ambientali Utilizzando<em&gt; vermiformis Vermamoeba</em

The isolation of giant viruses is of great interest in this new era of virology, especially since these giant viruses are related to protists. Giant ...

A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

Here, we present a simple and rapid protocol to detect and assess the extent of cell-to-cell macromolecular transport in planta. In this protocol, a fluorescently tagged-protein of interest is transiently expressed in plant tissue following biolistic delivery of its encoding DNA construct. The intra- and intercellular distribution of the tagged protein is then analyzed by confocal microscopy. We describe this technology in detail, providing step-by-step protocols to assay and evaluate the extent of symplastic protein transport in three plant species, Arabidopsis thaliana, Nicotiana benthamiana and N. tabacum (tobacco).

A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

Macromolecular trafficking between plant cells can be assessed by transiently expressing a fluorescently-tagged protein of interest and analyzing its intra- and intercellular distribution by confocal microscopy.

Una cellula-cellula Macromolecole saggio di trasporto In Planta Utilizzando Bombardamento Biolistic

Here, we present a simple and rapid protocol to detect and assess the extent of cell-to-cell macromolecular transport in planta. In this protocol, a ...

Biologia

Transmitting Plant Viruses Using Whiteflies

Whiteflies, Hemiptera: Aleyrodidae, Bemisia tabaci, a complex of morphologically indistinquishable species5, are vectors of many plant viruses. Several genera of these whitefly-transmitted plant viruses (Begomovirus, Carlavirus, Crinivirus, Ipomovirus, Torradovirus) include several hundred species of emerging and economically significant pathogens of important food and fiber crops (reviewed by9,10,16). These viruses do not replicate in their vector but nevertheless are moved readily from plant to plant by the adult whitefly by various means (reviewed by2,6,7,9,10,11,17). For most of these viruses whitefly feeding is required for acquisition and inoculation, while for others only probing is required. Many of these viruses are unable or cannot be easily transmitted by other means. Therefore maintenance of virus cultures, biological and molecular characterization (identification of host range and symptoms)3,13, ecology2,12, require that the viruses be transmitted to experimental hosts using the whitefly vector. In addition the development of new approaches to management, such as evaluation of new chemicals14 or compounds15, new cultural approaches1,4,19, or the selection and development of resistant cultivars7,8,18, requires the use of whiteflies for virus transmission. The use of whitefly transmission of plant viruses for the selection and development of resistant cultivars in breeding programs is particularly challenging7. Effective selection and screening for resistance employs large numbers of plants and there is a need for 100% of the plants to be inoculated in order to find the few genotypes which possess resistance genes. These studies use very large numbers of viruliferous whiteflies, often several times per year.
Whitefly maintenance described here can generate hundreds or thousands of adult whiteflies on plants each week, year round, without the contamination of other plant viruses. Plants free of both whiteflies and virus must be produced to introduce into the whitefly colony each week. Whitefly cultures must be kept free of whitefly pathogens, parasites, and parasitoids that can reduce whitefly populations and/or reduce the transmission efficiency of the virus. Colonies produced in the manner described can be quickly scaled to increase or decrease population numbers as needed, and can be adjusted to accommodate the feeding preferences of the whitefly based on the plant host of the virus.
There are two basic types of whitefly colonies that can be maintained: a nonviruliferous and a viruliferous whitefly colony. The nonviruliferous colony is composed of whiteflies reared on virus-free plants and allows the weekly availability of whiteflies which can be used to transmit viruses from different cultures. The viruliferous whitefly colony, composed of whiteflies reared on virus-infected plants, allows weekly availability of whiteflies which have acquired the virus thus omitting one step in the virus transmission process.

Improving our understanding of and our ability to manage many of the insect-transmitted plant viruses requires the use of the vector. Insect transmission of plant viruses is a tritrophic interaction, and as such requires the manipulation of insects, virus, and plant. Vectors must be reared in large numbers and manipulated in such a way as to insure high rates of transmission to test plants. The basics of rearing and manipulating the whitefly, Bemisa tabaci (Hemiptera: Aleyrodidae), a vector of plant viruses belonging to several emerging plant virus genera, representing a large number of economically significant viruses is presented.

Trasmissione di virus vegetali Utilizzo Aleurodidi

Whiteflies, Hemiptera: Aleyrodidae, Bemisia tabaci, a complex of morphologically indistinquishable species5, are vectors of many plant viruses. Several ...

Tandem High-pressure Freezing and Quick Freeze Substitution of Plant Tissues for Transmission Electron Microscopy

Since the 1940s transmission electron microscopy (TEM) has been providing biologists with ultra-high resolution images of biological materials. Yet, because of laborious and time-consuming protocols that also demand experience in preparation of artifact-free samples, TEM is not considered a user-friendly technique. Traditional sample preparation for TEM used chemical fixatives to preserve cellular structures. High-pressure freezing is the cryofixation of biological samples under high pressures to produce very fast cooling rates, thereby restricting ice formation, which is detrimental to the integrity of cellular ultrastructure. High-pressure freezing and freeze substitution are currently the methods of choice for producing the highest quality morphology in resin sections for TEM. These methods minimize the artifacts normally associated with conventional processing for TEM of thin sections. After cryofixation the frozen water in the sample is replaced with liquid organic solvent at low temperatures, a process called freeze substitution. Freeze substitution is typically carried out over several days in dedicated, costly equipment. A recent innovation allows the process to be completed in three hours, instead of the usual two days. This is typically followed by several more days of sample preparation that includes infiltration and embedding in epoxy resins before sectioning. Here we present a protocol combining high-pressure freezing and quick freeze substitution that enables plant sample fixation to be accomplished within hours. The protocol can readily be adapted for working with other tissues or organisms. Plant tissues are of special concern because of the presence of aerated spaces and water-filled vacuoles that impede ice-free freezing of water. In addition, the process of chemical fixation is especially long in plants due to cell walls impeding the penetration of the chemicals to deep within the tissues. Plant tissues are therefore particularly challenging, but this protocol is reliable and produces samples of the highest quality.

Obtaining high-quality transmission electron microscopy images is challenging, especially in the case of plant cells, which have abundant large water-filled vacuoles and aerated spaces. Tandem high-pressure freezing and quick freeze substitution greatly reduce preparation time of plant samples for TEM while producing samples with excellent ultrastructural preservation.

Tandem congelamento ad alta pressione e congelamento rapido Sostituzione di tessuti vegetali per la microscopia elettronica a trasmissione

Since the 1940s transmission electron microscopy (TEM) has been providing biologists with ultra-high resolution images of biological materials. Yet, ...

Immunofluorescent Labeling of Plant Virus and Insect Vector Proteins in Hemipteran Guts

Most plant viruses in nature are transmitted from one plant to another by hemipteran insects. A high population density of the vector insects that are highly efficient at virus transmission plays a key role in virus epidemics in fields. Studying virus-insect vector interactions can advance our understanding of virus transmission and epidemics with the aim of designing novel strategies to control plant viruses and their vector insects. Immunofluorescence labeling has been widely used to analyze interactions between pathogens and hosts and is used here in the white-backed planthopper (WBPH, Sogatella furcifera), which efficiently transmits the southern rice black streaked dwarf virus (SRBSDV, genus Fijivirus, family Reoviridae), to locate the virions and insect proteins in the midgut epithelial cells. Using laser scanning confocal microscopy, we studied the morphological characteristics of midgut epithelial cells, cellular localization of insect proteins, and the colocalization of virions and an insect protein. This protocol can be used to study virus activities in insects, functions of insect proteins, and interactions between virus and vector insect.

This protocol for immunofluorescent labeling of both plant virus proteins and vector insect proteins in excised insect guts can be used to study interactions among virus and vector insects, insect protein functions and molecular mechanisms underlying virus transmission.

Marcatura immunofluorescente di virus vegetali e proteine di insetti vettori nell'intestino degli emitteri

Most plant viruses in nature are transmitted from one plant to another by hemipteran insects. A high population density of the vector insects that are ...

Detecting Virus and Salivary Proteins of a Leafhopper Vector in the Plant Host

Insect vectors horizontally transmit many plant viruses of agricultural importance. More than one-half of plant viruses are transmitted by hemipteran insects that have piercing-sucking mouthparts. During viral transmission, the insect saliva bridges the virus-vector-host because the saliva vectors viruses, and the insect proteins, trigger or suppress the immune response of plants from insects into plant hosts. The identification and functional analyses of salivary proteins are becoming a new area of focus in the research field of arbovirus-host interactions. This protocol provides a system to detect proteins in the saliva of leafhoppers using the plant host. The leafhopper vector Nephotettix cincticeps infected with rice dwarf virus (RDV) serves as an example. The vitellogenin and major outer capsid protein P8 of RDV vectored by the saliva of N. cincticeps can be detected simultaneously in the rice plant that N. cincticeps feeds on. This method is applicable for testing the salivary proteins that are transiently retained in the plant host after insect feeding. It is believed that this system of detection will benefit the study of hemipteran-virus-plant or hemipteran-plant interactions.

This protocol demonstrates how to use the plant host to detect salivary proteins of leafhopper and plant viral proteins released by leafhopper vectors.

Rilevamento del virus e delle proteine salivari di un vettore cicalina nell'ospite della pianta

Insect vectors horizontally transmit many plant viruses of agricultural importance. More than one-half of plant viruses are transmitted by hemipteran ...

Quantificazione dell'inizio della morte cellulare attraverso l'imaging continuo

Studying Cell Death Initiation Using a Digital Microscope

Hypersensitive response (HR)-conferred resistance is an effective defense response that can be determined by the N resistance genes. HR is manifested as the formation of cell death zones on inoculated leaves. Here, a protocol for studying the rate of cell death initiation by imaging inoculated leaves in the time between the cell death initiation and the cell death appearance using a digital microscope is presented. The digital microscope enables a continuous imaging process in desired intervals, which allows an accurate determination of cell death initiation rate up to minutes exactly, as opposed to hours in traditional methods. Imaging with the digital microscope is also independent of light and can therefore be used during day and night without disturbing the circadian rhythm of the plant. Different pathosystems resulting in programmed cell death development could be studied using this protocol with minor modifications. Overall, the protocol thus allows simple, accurate, and inexpensive identification of cell death initiation rate.

Autore in primo piano: Un approccio semplificato allo studio dell'inizio della morte cellulare nella risposta ipersensibile 

Here we present a protocol for studying the rate of programmed cell death initiation by continuously imaging inoculated leaves following cell death induction.

Studiare l'inizio della morte cellulare utilizzando un microscopio digitale

Hypersensitive response (HR)-conferred resistance is an effective defense response that can be determined by the N resistance genes. HR is manifested as ...

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

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