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Publications - Cell and Metabolic Biology
Publications
Salinity is a serious challenge to global agriculture and threatens human food security. Plant cells can respond to salt stress either by activation of adaptive responses, or by programmed cell death. The mechanisms deciding the respective response are far from understood, but seem to depend on the degree, to which mitochondria can maintain oxidative homeostasis. Using plant PeptoQ, a Trojan Peptoid, as vehicle, it is possible to transport a coenzyme Q10 (CoQ10) derivative into plant mitochondria. We show that salinity stress in tobacco BY-2 cells (Nicotiana tabacum L. cv Bright Yellow-2) can be mitigated by pretreatment with plant PeptoQ with respect to numerous aspects including proliferation, expansion, redox homeostasis, and programmed cell death. We tested the salinity response for transcripts from nine salt-stress related-genes representing different adaptive responses. While most did not show any significant response, the salt response of the transcription factor NtNAC, probably involved in mitochondrial retrograde signaling, was significantly modulated by the plant PeptoQ. Most strikingly, transcripts for the mitochondrial, Mn-dependent Superoxide Dismutase were rapidly and drastically upregulated in presence of the peptoid, and this response was disappearing in presence of salt. The same pattern, albeit at lower amplitude, was seen for the sodium exporter SOS1. The findings are discussed by a model, where plant PeptoQ modulates retrograde signalling to the nucleus leading to a strong expression of mitochondrial SOD, what renders mitochondria more resilient to perturbations of oxidative balance, such that cells escape salt induced cell death and remain viable.
Publications
Site-directed methods for the generation of genetic diversity are essential tools in the field of directed enzyme evolution. The Golden Gate cloning technique has been proven to be an efficient tool for a variety of cloning setups. The utilization of restriction enzymes which cut outside of their recognition domain allows the assembly of multiple gene fragments obtained by PCR amplification without altering the open reading frame of the reconstituted gene. We have developed a protocol, termed Golden Mutagenesis that allows the rapid, straightforward, reliable and inexpensive construction of mutagenesis libraries. One to five amino acid positions within a coding sequence could be altered simultaneously using a protocol which can be performed within one day. To facilitate the implementation of this technique, a software library and web application for automated primer design and for the graphical evaluation of the randomization success based on the sequencing results was developed. This allows facile primer design and application of Golden Mutagenesis also for laboratories, which are not specialized in molecular biology.
Publications
Pathogenicity of the Gram-negative plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria depends on a type III secretion (T3S) system which translocates effector proteins into plant cells. Effector protein delivery is controlled by the T3S chaperone HpaB, which presumably escorts effector proteins to the secretion apparatus. One intensively studied effector is the transcription activator-like (TAL) effector AvrBs3, which binds to promoter sequences of plant target genes and activates plant gene expression. It was previously reported that type III-dependent delivery of AvrBs3 depends on the N-terminal protein region. The signals that control T3S and translocation of AvrBs3, however, have not yet been characterized. In the present study, we show that T3S and translocation of AvrBs3 depend on the N-terminal 10 and 50 amino acids, respectively. Furthermore, we provide experimental evidence that additional signals in the N-terminal 30 amino acids and the region between amino acids 64 and 152 promote translocation of AvrBs3 in the absence of HpaB. Unexpectedly, in vivo translocation assays revealed that AvrBs3 is delivered into plant cells even in the absence of HrpF, which is the predicted channel-forming component of the T3S translocon in the plant plasma membrane. The presence of HpaB- and HrpF-independent transport routes suggests that the delivery of AvrBs3 is initiated during early stages of the infection process, presumably before the activation of HpaB or the insertion of the translocon into the plant plasma membrane.
This page was last modified on 27 Jan 2025 .
