Roy Choudhury, Sayan
(2021).
Nuclear protein dynamics during pattern-triggered immunity in Arabidopsis thaliana.
PhD thesis, Universität zu Köln.
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Abstract
Perception of microbe-associated molecular patterns (MAMPs) by cell surface localized pattern recognition receptors (PRRs) triggers pattern-triggered immunity (PTI). PTI is associated with massive, rapid transcriptional reprogramming that occurs within 30 min after MAMP perception. However, it remains unclear how signal for MAMP recognition at the plasma membrane is relayed into the nucleus to trigger transcriptional reprograming. I hypothesized that investigating nuclear and total protein abundance changes during PTI would provide insights into this. However, existing protocols for nuclear isolation required for nuclear proteomics have shortcomings. In this study, I modified a nuclear isolation protocol to facilitate detection of nuclear proteins in Arabidopsis thaliana. Mass spectrometry-based comparison with an existing nuclear isolation protocol showed that my method not only increased detection of nuclear proteins but also reduced contaminants, especially plastidial proteins compared with the previous protocol. I used this method to investigate nuclear protein dynamics during the MAMP flg22-induced PTI. I found that flg22-induced PTI resulted in significant changes in nuclear protein abundances with distinct responses at different time points. For instance, the transcription factor WRKY47 showed increased abundance in the nucleus at 3 and 9 h after flg22 treatment but was not detected in the total fraction. Consistently, mutant plants deficient in WRKY47 showed compromised resistance against a bacterial pathogen. These results point to the significance of nuclear proteomics. In addition, by comparing nuclear and total proteome changes upon flg22-treatment, I found proteins showing signatures of movements between the cytosol and nucleus including the kinases- NADK1 and PCRK1 showing signatures of nuclear import and a phosphatase- MKP1 and a transcription factor TOE1 showing signatures of nuclear export. I also profiled nuclear and total proteomes at earlier time points to investigate protein abundance changes which likely precede transcriptional reprogramming. A hypothesis for rapid transcriptional activation is de-repression: flg22 perception triggers inhibition or disappearance of high-turnover negative regulators, which leads to transcriptional activation of flg22-responsive genes. This is supported by the data that flg22 and the translational inhibitor cycloheximide triggers highly overlapped gene expression changes. However, I found that flg22 and cycloheximide triggered very different protein abundance changes, suggesting that flg22 and cycloheximide do not share the same mechanism but do activate the common set of genes. I found that nuclear proteome at 15 min after flg22-treatment showed downregulation of a number of proteins including RIN4-a negative regulator of PTI, SSP5-a phosphatase and transcriptional repressor, and MVQ1- a MPK3/6 substrate. Since, this downregulation was observed exclusively at 15 min in the nuclear fraction, downregulation of some of those proteins may be important for transcriptional reprogramming in response to flg22. Taken together, this thesis advances the field of nuclear proteomes in plants and provides insights into nuclear protein dynamics during flg22-trigerred PTI.
| Item Type: | Thesis (PhD thesis) | ||||||||
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| URN: | urn:nbn:de:hbz:38-355591 | ||||||||
| Date: | January 2021 | ||||||||
| Language: | English | ||||||||
| Faculty: | Faculty of Mathematics and Natural Sciences | ||||||||
| Divisions: | Außeruniversitäre Forschungseinrichtungen > MPI for Plant Breeding Research | ||||||||
| Subjects: | Natural sciences and mathematics | ||||||||
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| Date of oral exam: | 30 November 2020 | ||||||||
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| Funders: | DAAD, MPIPZ | ||||||||
| Refereed: | Yes | ||||||||
| URI: | http://kups.ub.uni-koeln.de/id/eprint/35559 |
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