viral inoculum preparation for potato plant inoculation to study cell death

通过连续成像量化细胞死亡起始

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.

作者聚焦：研究超敏反应中细胞死亡起始的简化方法 

使用数码显微镜研究细胞死亡起始

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  

马铃薯植株接种的病毒接种物制备以研究细胞死亡

首先，使用带有过滤网的提取袋收获六到八周龄的马铃薯病毒感染的彭特兰马铃薯植株。每克组织加入 4 毫升磷酸盐缓冲液，补充 DIECA。然后用手动均质器将植物均质化一到两分钟。接下来，在三到四个星期大的健康马铃薯植株的前三片完全发育的叶子上轻轻涂上碳化硅粉。用准备好的病毒接种物轻轻擦拭叶子。10分钟后，用自来水将叶子冲洗干净。最后，将植物转移到生长室中，让它们在28摄氏度的受控环境条件下生长三天。

viral-inoculum-preparation-for-potato-plant-inoculation-to-study-cell

Research

JoVE Journal

Biology

Viral Inoculum Preparation for Potato Plant Inoculation to Study Cell Death

241 次观看. 首先，使用带有过滤网的提取袋收获六到八周龄的马铃薯病毒感染的彭特兰马铃薯植株。每克组织加入 4 毫升磷酸盐缓冲液，补充 DIECA。然后用手动均质器将植物均质化一到两分钟。接下来，在三到四个星期大的健康马铃薯植株的前三片完全发育的叶子上轻轻涂上碳化硅粉。用准备好的病毒接种物轻轻擦拭叶子。10分钟后，用自来水将叶子冲洗干净。最后，将植物?...

视频: 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 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 ...