viral inoculum preparation for potato plant inoculation to study cell death

Quantifying Cell Death Initiation Through Continuous Imaging

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.

Author Spotlight: A Streamlined Approach to Studying Cell Death Initiation in Hypersensitive Response 

Studying Cell Death Initiation Using a Digital Microscope

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  

To begin, use extraction bags with filter net to harvest six to eight week old potato virus infected Pentland potato plants. Add four milliliters of phosphate buffer, supplemented with DIECA for every gram of tissue. Then homogenize the plants with a hand homogenizer for one to two minutes.Next, lightly coat the first three fully developed leaves of three to four weeks old healthy potato plants with carborundum powder. Gently rub the leaves with the prepared viral inoculum. After 10 minutes, rinse the leaves clean with tap water.Finally, transfer the plants to a growth chamber to allow them to grow under controlled environmental conditions at 28 degrees Celsius for three days.

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232 Views.  National Institute of Biology. To begin, use extraction bags with filter net to harvest six to eight week old potato virus infected Pentland potato plants. Add four milliliters of phosphate buffer, supplemented with DIECA for every gram of tissue. Then homogenize the plants with a hand homogenizer for one to two minutes.Next, lightly coat the first three fully developed leaves of three to four weeks old healthy potato plants with carborundum powder. Gently rub the leaves with the prepared viral inoculum. After 10 mi...

Video: Viral Inoculum Preparation for Potato Plant Inoculation to Study Cell Death  

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.

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

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

Growing Potato Plants for Cell Death Initiation Studies

Begin by propagating healthy potato plants in test tubes containing stem node tissue culture media. After six to eight weeks, use sterile tweezers and a scalpel to cut out ten 1-centimeter-long explants containing nodes. Transfer the explants to plastic boxes containing Murashige and Skoog medium and grow them in controlled environmental conditions in a growth chamber.After two weeks, fill some pots with soil to prepare for plant transfer. Use a finger to create a 3 to 4-centimeter deep hole in the soil at the center of the pot. Now, pour water into the hole and allow it to absorb into the soil.Next, place each plant into the hole, ensuring that the leaves remain above the surface. Carefully cover the roots with soil. Cultivate these plants in a growth chamber in a controlled environment.In three to four weeks, the plants are ready to be inoculated.

Virus-Infected Potato Plant Preparation for Microscopic Lesion Recording

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.