Name: Video: Viral Inoculum Preparation for Potato Plant Inoculation to Study Cell Death
Uploaded: 2023-11-10T12:20:00
Duration: 58 s
Description: 237 Views. Viral Inoculum Preparation for Potato Plant Inoculation to Study Cell Death

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

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  

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JoVE Journal

Biology

237 Views. Viral Inoculum Preparation for Potato Plant Inoculation to Study Cell Death

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 ...