@phdthesis{Spinti2021,
  author    = {Spinti, Daniela},
  title     = {Proteasomal protein turnover during defense priming in Arabidopsis},
  doi       = {10.25932/publishup-50590},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-505909},
  school      = {Universit{\"a}t Potsdam},
  pages     = {x, 164},
  year      = {2021},
  abstract  = {The ubiquitin-proteasome-system (UPS) is a cellular cascade involving three enzymatic steps for protein ubiquitination to target them to the 26S proteasome for proteolytic degradation. Several components of the UPS have been shown to be central for regulation of defense responses during infections with phytopathogenic bacteria. Upon recognition of the pathogen, local defense is induced which also primes the plant to acquire systemic resistance (SAR) for enhanced immune responses upon challenging infections. Here, ubiquitinated proteins were shown to accumulate locally and systemically during infections with Psm and after treatment with the SAR-inducing metabolites salicylic acid (SA) and pipecolic acid (Pip). The role of the 26S proteasome in local defense has been described in several studies, but the potential role during SAR remains elusive and was therefore investigated in this project by characterizing the Arabidopsis proteasome mutants rpt2a-2 and rpn12a-1 during priming and infections with Pseudomonas. Bacterial replication assays reveal decreased basal and systemic immunity in both mutants which was verified on molecular level showing impaired activation of defense- and SAR-genes. rpt2a-2 and rpn12a-1 accumulate wild type like levels of camalexin but less SA. Endogenous SA treatment restores local PR gene expression but does not rescue the SAR-phenotype. An RNAseq experiment of Col-0 and rpt2a-2 reveal weak or absent induction of defense genes in the proteasome mutant during priming. Thus, a functional 26S proteasome was found to be required for induction of SAR while compensatory mechanisms can still be initiated. E3-ubiquitin ligases conduct the last step of substrate ubiquitination and thereby convey specificity to proteasomal protein turnover. Using RNAseq, 11 E3-ligases were found to be differentially expressed during priming in Col-0 of which plant U-box 54 (PUB54) and ariadne 12 (ARI12) were further investigated to gain deeper understanding of their potential role during priming. PUB54 was shown to be expressed during priming and /or triggering with virulent Pseudomonas. pub54 I and pub54-II mutants display local and systemic defense comparable to Col-0. The heavy-metal associated protein 35 (HMP35) was identified as potential substrate of PUB54 in yeast which was verified in vitro and in vivo. PUB54 was shown to be an active E3-ligase exhibiting auto-ubiquitination activity and performing ubiquitination of HMP35. Proteasomal turnover of HMP35 was observed indicating that PUB54 targets HMP35 for ubiquitination and subsequent proteasomal degradation. Furthermore, hmp35-I benefits from increased resistance in bacterial replication assays. Thus, HMP35 is potentially a negative regulator of defense which is targeted and ubiquitinated by PUB54 to regulate downstream defense signaling. ARI12 is transcriptionally activated during priming or triggering and hyperinduced during priming and triggering. Gene expression is not inducible by the defense related hormone salicylic acid (SA) and is dampened in npr1 and fmo1 mutants consequently depending on functional SA- and Pip-pathways, respectively. ARI12 accumulates systemically after priming with SA, Pip or Pseudomonas. ari12 mutants are not altered in resistance but stable overexpression leads to increased resistance in local and systemic tissue. During priming and triggering, unbalanced ARI12 levels (i.e. knock out or overexpression) leads to enhanced FMO1 activation indicating a role of ARI12 in Pip-mediated SAR. ARI12 was shown to be an active E3-ligase with auto-ubiquitination activity likely required for activation with an identified ubiquitination site at K474. Mass spectrometrically identified potential substrates were not verified by additional experiments yet but suggest involvement of ARI12 in regulation of ROS in turn regulating Pip-dependent SAR pathways. Thus, data from this project provide strong indications about the involvement of the 26S proteasome in SAR and identified a central role of the two so far barely described E3-ubiquitin ligases PUB54 and ARI12 as novel components of plant defense.},
  language  = {en}
}
@phdthesis{Oberkofler2022,
  author    = {Oberkofler, Vicky},
  title     = {Molecular basis of HS memory in Arabidopsis thaliana},
  doi       = {10.25932/publishup-56954},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-569544},
  school      = {Universit{\"a}t Potsdam},
  pages     = {181},
  year      = {2022},
  abstract  = {Plants can be primed to survive the exposure to a severe heat stress (HS) by prior exposure to a mild HS. The information about the priming stimulus is maintained by the plant for several days. This maintenance of acquired thermotolerance, or HS memory, is genetically separable from the acquisition of thermotolerance itself and several specific regulatory factors have been identified in recent years. On the molecular level, HS memory correlates with two types of transcriptional memory, type I and type II, that characterize a partially overlapping subset of HS-inducible genes. Type I transcriptional memory or sustained induction refers to the sustained transcriptional induction above non-stressed expression levels of a gene for a prolonged time period after the end of the stress exposure. Type II transcriptional memory refers to an altered transcriptional response of a gene after repeated exposure to a stress of similar duration and intensity. In particular, enhanced re-induction refers to a transcriptional pattern in which a gene is induced to a significantly higher degree after the second stress exposure than after the first. This thesis describes the functional characterization of a novel positive transcriptional regulator of type I transcriptional memory, the heat shock transcription factor HSFA3, and compares it to HSFA2, a known positive regulator of type I and type II transcriptional memory. It investigates type I transcriptional memory and its dependence on HSFA2 and HSFA3 for the first time on a genome-wide level, and gives insight on the formation of heteromeric HSF complexes in response to HS. This thesis confirms the tight correlation between transcriptional memory and H3K4 hyper-methylation, reported here in a case study that aimed to reduce H3K4 hyper-methylation of the type II transcriptional memory gene APX2 by CRISPR/dCas9-mediated epigenome editing. Finally, this thesis gives insight into the requirements for a heat shock transcription factor to function as a positive regulator of transcriptional memory, both in terms of its expression profile and protein abundance after HS and the contribution of individual functional domains. In summary, this thesis contributes to a more detailed understanding of the molecular processes underlying transcriptional memory and therefore HS memory, in Arabidopsis thaliana.},
  language  = {en}
}