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A Transkingdom Approach to Identify ...

Kessens, Ryan.

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A Transkingdom Approach to Identify Cell Death Regulators in Plants.

紀錄類型:	書目-電子資源 : Monograph/item
正題名/作者:	A Transkingdom Approach to Identify Cell Death Regulators in Plants./
作者:	Kessens, Ryan.
出版者:	Ann Arbor : ProQuest Dissertations & Theses, : 2018,
面頁冊數:	162 p.
附註:	Source: Dissertation Abstracts International, Volume: 80-02(E), Section: B.
Contained By:	Dissertation Abstracts International80-02B(E).
標題:	Plant pathology. -
電子資源:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=10935729
ISBN:	9780438386303

A Transkingdom Approach to Identify Cell Death Regulators in Plants.
Kessens, Ryan.

A Transkingdom Approach to Identify Cell Death Regulators in Plants. - Ann Arbor : ProQuest Dissertations & Theses, 2018 - 162 p.

Source: Dissertation Abstracts International, Volume: 80-02(E), Section: B.

Thesis (Ph.D.)--The University of Wisconsin - Madison, 2018.

Autophagy and programmed cell death (PCD) are core cellular processes essential for proper responses to stress. Despite their importance in stress tolerance, large gaps in knowledge still exist that prevent these processes from being effectively manipulated for crop improvement. The absence of conserved animal PCD regulators in plant genomes has made it challenging to draw parallels from more advanced mammalian research. In contrast, core autophagy genes are well conserved across diverse eukaryotic species but few chemical regulators of plant autophagy have been discovered and utilized for stress amelioration as they have been in humans.

ISBN: 9780438386303Subjects--Topical Terms:

3174872
Plant pathology.

A Transkingdom Approach to Identify Cell Death Regulators in Plants.
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500 $a Source: Dissertation Abstracts International, Volume: 80-02(E), Section: B.
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502 $a Thesis (Ph.D.)--The University of Wisconsin - Madison, 2018.
520 $a Autophagy and programmed cell death (PCD) are core cellular processes essential for proper responses to stress. Despite their importance in stress tolerance, large gaps in knowledge still exist that prevent these processes from being effectively manipulated for crop improvement. The absence of conserved animal PCD regulators in plant genomes has made it challenging to draw parallels from more advanced mammalian research. In contrast, core autophagy genes are well conserved across diverse eukaryotic species but few chemical regulators of plant autophagy have been discovered and utilized for stress amelioration as they have been in humans.
520 $a While obvious homologs of many animal PCD genes are absent in plant genomes, numerous studies have shown that the ectopic expression of animal anti-PCD regulators in plants can suppress cell death in response to many stresses. Specifically, an insect inhibitor of apoptosis (SfIAP) suppresses cell death induced by abiotic and biotic stress when expressed in tomato. We hypothesize that SfIAP is inhibiting the activity of endogenous pro-death regulators in tomato. However, a biochemical mechanism by which SfIAP functions in plants is still lacking. To address this deficiency, we sought to identify SfIAP-interacting partners from tomato using a yeast two-hybrid assay. Several transcription factors in the SQUAMOSA promoter binding protein (SBP) family were identified as potential binding partners. We confirmed this interaction in vivo for our top two interactors, SlySBP8b and SlySBP12a, using coimmunoprecipitation. Overexpression of SlySBP8b and -12a in N. benthamiana leaves induced tissue death characterized by the accumulation of reactive oxygen species. Additionally, growth of the necrotrophic fungal pathogen Alternaria alternata was enhanced in leaves overexpressing SlySBP8b and -12a. Fluorescence microscopy confirmed the nuclear localization of both SlySBP8b and -12a while SlySBP12a was also present at the ER membrane and deleting a putative transmembrane domain from SlySBP12a resulted in complete nuclear localization. These results support a pro-death role for SlySBP8b and -12a and suggest ER membrane tethering as a means of regulating SlySBP12a activity.
520 $a Current assays for screening large libraries to identify chemical modulators of autophagy suffer from a few key limitations. Some of these assays rely on low-throughput techniques such as immunoblotting and fluorescence microscopy while others require flow cytometers that are expensive to own and operate and require considerable expertise to operate and maintain. To address many of these problems, scientists at Promega developed a plate-based assay to assess autophagic flux in cell cultures using a novel bioluminescent reporter. The reporter consists of a small epitope tag called HiBiT fused in-frame to the N-terminus of the autophagosome marker LC3. Degradation and accumulation of LC3 in response to chemical treatment can be recorded using any plate reader equipped with a luminometer. Stable cell lines expressing the HiBiT-LC3 reporter have been successfully used to monitor autophagic flux, but a transient method of expressing the reporter would enhance the flexibility of the assay. We set out to develop a baculoviral system for transient expression of the HiBiT-LC3 reporter in multiple human cell lines. Our data show that the HiBiT-LC3 reporter can be effectively expressed in U2OS and HEK293 cells at very low viral titers while maintaining responsiveness to known autophagy regulatory compounds. In addition to baculoviral expression of the reporter, we utilized CRISPR/Cas9 and homology-directed repair with HiBiT in the donor template to tag two Arabidopsis homologs of LC3 (Atg8a and Atg8f) in Arabidopsis suspension cell protoplasts. While we were unable to establish a stable reporter line in Arabidopsis, our results with BacMam and plasmid-based expression suggest a constitutively expressed HiBiT-Atg8 reporter could be utilized in transgenic plants.
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650 4 $a Cellular biology. $3 3172791
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