Name: Viral Inoculum Preparation for Potato Plant Inoculation to Study Cell Death
Uploaded: 2023-11-10T12:20:00
Duration: 58 s
Description: Watch this Scientific Journal Video about Viral Inoculum Preparation for Potato Plant Inoculation to Study Cell Death at JoVE.com

viral inoculum preparation for potato plant inoculation to study cell death

Kvantifisering av celledødsinitiering gjennom kontinuerlig avbildning

Potato is one of the world&#39;s most widely grown food crops, fourth place behind rice, wheat, and corn. However, potato production can be severely affected by potato virus Y (PVY), which is currently considered its most important virus pathogen1,2. In potato plants cv. Rywal, several strains of PVY (including PVY strain N-Wilga) trigger hypersensitive response (HR)-conferred resistance, where the pathogen&#39;s restriction to the infection site manifests as necrotic lesions on inoculated leaves3. In this pathosystem, HR is mediated by Ny-1 resistance gene, which is temperature-dependent, as plants grown at lower temperatures efficiently develop necrotic lesions, whereas in plants grown constitutively at elevated (28 &#176;C) temperature, abortion of resistance is demonstrated as lack of lesion formation and systemic virus spread3,4. When the plants are transferred to a lower temperature (22 &#176;C), cell death is initiated, which can be exploited to follow the rate of cell death initiation by imaging inoculated leaves in the time between the cell death initiation and the cell death appearance.
This protocol demonstrates a simple method for the determination of cell death initiation rate using a digital microscope. By imaging the inoculated leaves after transferring the plant from 28 &#176;C to 22 &#176;C, a digital microscope enables continuous observation of the leaf in desired intervals. Unlike the use of other methods (for example, confocal microscopy or observation of lesion formation with the naked eye), this allows determination of the exact time of lesion formation and therefore, the cell death initiation rate up to minutes exactly, as opposed to hours in aforementioned methods5,6. Digital microscope use is also independent of light and can therefore be used during day and night. This protocol can also be used to identify components involved in cell death initiation or to determine the effects of different components on cell death initiation rate if used plants are transgenic and have altered levels of components of interest.

Forfatter Spotlight: En strømlinjeformet tilnærming til å studere celledødsinitiering i overfølsom respons 

Studerer celledødsinitiering ved hjelp av et digitalt mikroskop

To begin, transfer the virus infected potato plants to a growth chamber maintained at 22 degrees Celsius. Pick a plant for observation and use adhesive tape to fully immobilize the second inoculated leaf. Next, connect the digital microscope to the computer and launch the image capture software.Place the leaf under the microscope. Then use the dial to focus on the surface of the leaf. To adjust the camera settings, click the settings button.Now set the brightness to 64, the contrast to 14 and the hue to zero. Next, adjust the white balance, then set the saturation to 47, sharpness to zero and gamma to five. Click on the image capture icon and select time lapsed video option.Next, set image capture for every 15 minutes for a total of 24 hours. Press the start button to begin the image capture. To save the captured images, select all images and click the save icon.Now choose the export options, then set dots per inch to maximum. Once saved, delete all images from the program. To edit the images, launch an editing software like ImageJ.Click on file, then select import, and choose image sequence to import the time sequences of images from a single field of vision. Paste the path of the directory of saved pictures, and press okay. Once the conversion is complete, ImageJ will automatically launch an internal video player displaying the finished video.Click on file, then save as and select the AVI format to export the video file. When a small window opens, set the frame rate to 0.3 FPS, then press okay to save the video as an AVI file. Three to four week old inoculated potato plants with at least three to four fully developed leaves were used for digital microscopic analysis.The same area on the inoculated leaf was observed at 15 minute intervals to determine the lesion occurrence and expansion over time. At 14 hours of observation, no lesion was visible. About 90 minutes later, a visible lesion was recorded.The lesion started to expand two hours later, and continued expansion over the next eight and a half hours.

Watch this Scientific Journal Video about Viral Inoculum Preparation for Potato Plant Inoculation to Study Cell Death at JoVE.com

Viral Inoculum Preparation for Potato Plant Inoculation to Study Cell Death  

Viral inokulumforberedelse for potetplanteinokulering for å studere celledød

For å begynne, bruk ekstraksjonsposer med filternett for å høste seks til åtte uker gamle potetvirusinfiserte Pentland-potetplanter. Tilsett fire milliliter fosfatbuffer, supplert med DIECA for hvert gram vev. Deretter homogeniserer plantene med en håndhomogenisator i ett til to minutter.Deretter belegger du de tre første fullt utviklede bladene av tre til fire uker gamle sunne potetplanter med karborundumpulver. Gni forsiktig bladene med det forberedte virale inokulumet. Etter 10 minutter, skyll bladene rene med vann fra springen.Til slutt, overfør plantene til et vekstkammer for å tillate dem å vokse under kontrollerte miljøforhold ved 28 grader Celsius i tre dager.

Research

JoVE Journal

Biology

Viral Inoculum Preparation for Potato Plant Inoculation to Study Cell Death

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 ...