Biology subjects

Willmund, F.

Publications and source records attributed to Willmund, F..

9 recordsLinked to original sources

bioRxiv2024

Advancing chloroplast synthetic biology through high-throughput plastome engineering of Chlamydomonas reinhardtii

Chloroplast synthetic biology holds promise for developing improved crops through improving the function of plastids. However, chloroplast engineering efforts face limitations due to the scarcity of genetic tools and the low throughput of plant-based systems. To address these challenges, we here established Chlamydomonas reinhardtii as a prototyping chassis for chloroplast synthetic biology. We developed an automation workflow that enables the generation, handling, and analysis of thousands of transplastomic strains in parallel, expanded the repertoire of selection markers for chloroplast transformation, established new reporter genes, and characterized over 140 regulatory parts, including native and synthetic promoters, UTRs, and intercistronic expression elements. We integrated the system within the Phytobrick cloning standard and demonstrate several applications, including a library-based approach to develop synthetic promoter designs in plastids. Finally, we provide a proof-of-concept for prototyping novel traits in plastids by introducing a chloroplast-based synthetic photorespiration pathway and demonstrating a twofold increase in biomass production. Overall, our study advances chloroplast engineering, and provides a promising platform to rapidly prototype chloroplast manipulations before their transfer into higher plants and crops.

synthetic biology↗

bioRxiv2023

Guidelines for performing ribosome profiling in plants including structural analysis of rRNA fragments

Ribosome profiling (or Ribo-seq) is a technique that provides genome-wide information on the translational landscape (translatome). Across different plant studies, variable methodological setups have been described which raises questions about the general comparability of data that were generated from diverging methodologies. Furthermore, a common problem when performing Ribo-seq are abundant rRNA fragments that are wastefully incorporated into the libraries and dramatically reduce sequencing depth. To remove these rRNA contaminants, it is common to perform preliminary trials to identify these fragments because they are thought to vary depending on nuclease treatment, tissue source, and plant species. Here, we compile valuable insights gathered over years of generating Ribo-seq datasets from different species and experimental setups. We highlight which technical steps are important for maintaining cross experiment comparability and describe a highly efficient approach for rRNA removal. Furthermore, we provide evidence that many rRNA fragments are structurally preserved over diverse nuclease regimes, as well as across plant species. Using a recently published cryo-electron microscopy (cryo-EM) structure of the tobacco 80S ribosome, we show that the most abundant rRNA fragments are spatially derived from the solvent-exposed surface of the ribosome. The guidelines presented here shall aid newcomers in establishing ribosome profiling in new plant species and provide insights that will help in customizing the methodology for individual research goals.

plant biology↗

bioRxiv2023

Networked proteins redundantly interact with VAP27 and RABG3 to regulate membrane tethering at the vacuole and beyond

Biological processes in eukaryotes depend on the spatio-temporal compartmentalization of their cells. Integrity and positioning of organelles on the other hand rely on the organization of the actin cytoskeleton. Previously, it has been shown that changes of the plants largest organelle, the vacuole, depend on a functional actin organization. The connection between actin filaments and the vacuole is established by the family of Networked (NET) 4 proteins and, consequently, altering NET4 abundance impacts vacuolar morphology. However, the precise regulatory mechanism is unknown and gene deletions of NET4 did not result in a global growth phenotype. Here, we show that NET4 functions redundantly with NET3, interacting with RABG3-GTPases at the vacuole to allow for homotypic fusion or, alternatively, the generation of endoplasmic reticulum (ER) - vacuole contact sites. We found that ER-resident NET3 is able to interact with RABG3 residing at the tonoplast and that NET4 interacts with the contact site protein VAP27-1 at the ER. Generation of net3 net4 triple mutants by CRSIPR-guided mutagenesis helped us to overcome functional redundancy, resulting in impaired plant growth and development. Our results demonstrate how diversification of NET genes led to functional redundancy between different family members to create cellular plasticity of vascular plants. We hypothesize that establishment of a direct ER-vacuole connection enables direct lipid and protein transfer which is especially important in young and fast-growing cells. Availability of lipids would facilitate rapidly expanding vacuoles, which are the basis for high cell elongation rates and eventually fast plant growth.

cell biology↗

bioRxiv2023

Utilizing high resolution ribosome profiling for the global investigation of gene expression in Chlamydomonas reinhardtii

Ribosome profiling (Ribo-seq) is a powerful method for the deep analysis of translation mechanisms and regulatory circuits during gene expression. Here, we established an optimized and high resolution Ribo-seq protocol for the unicellular model alga Chlamydomonas reinhardtii (Chlamydomonas). Comparing different nuclease treatments for the extraction and sequencing of ribosome-protected fragments (RPFs) and parallel RNA-seq, provided deep insight into translational dynamics and post-transcriptional control of gene expression, thoroughly covering more than 10,000 different transcripts. Our high quality Ribo-seq protocol captures the 3-nucleotide movement of elongating ribosomes along nuclear and chloroplast transcripts. Detailed analysis of the ribosomal offsets on transcripts uncovers presumable transition states during translocation of elongating ribosomes within the 5- and 3-sections of transcripts and features of eukaryotic translation termination. These offsets reveal drastic differences between the nature of cytosolic and chloroplast translation mechanisms. Chloroplast translation is further characterized by heterogenous RPF size distribution. We found that local accumulation of small RPFs correlates with local slowdown of psbA translation, possibly revealing an uncharacterized regulator step during PsbA/D1 synthesis. Further analyses of RPF distribution along specific cytosolic transcripts revealed characteristic patterns of translation elongation exemplified for the major light harvesting complex proteins, LHCs. Moreover, our Ribo-seq data can be utilized to survey coding sequence annotations and the expression preference of alternatively spliced transcripts in Chlamydomonas. We made these features easily accessible for the research community by attaching our Ribo-seq data to the most recent Chlamydomonas reference genome.

systems biology↗

bioRxiv2023

Competition co-immunoprecipitation reveals interactors of the chloroplast CPN60 chaperonin machinery

The functionality of essential metabolic processes in chloroplasts depends on a balanced integration of nuclear-and chloroplast-encoded polypeptides into the plastids proteome. The chloroplast chaperonin machinery is an essential player in chloroplast protein folding with a more intricate structure and subunit composition compared to the orthologous GroEL/ES chaperonin of Escherichia coli. However, its exact role in chloroplasts remains obscure, mainly because of a very limited knowledge about the folded substrates. We employed the competition immunoprecipitation method for the identification of the chaperonins substrates in Chlamydomonas reinhardtii. Co-immunoprecipitation of the target complex in the presence of increasing amounts of isotope-labelled competitor epitope and subsequent mass spectrometry analysis specifically allowed to distinguish true interactors from unspecifically co-precipitated proteins. Besides known substrates such as RbcL, we revealed numerous new substrates with high confidence. Identified substrate proteins differ from bulk chloroplast proteins by a higher content of beta-sheets, lower alpha-helical content and increased aggregation propensity. Immunoprecipitations performed with a subunit of the co-chaperonin lid revealed the ClpP protease as a specific partner complex, with altered interactions during heat stress, pointing to a close collaboration of these machineries to maintain protein homeostasis in the chloroplast.

molecular biology↗

bioRxiv2022

OHP2 is not required for psbA translation in Chlamydomonas

In land plants and cyanobacteria, co-translational association of chlorophyll (Chl) to the nascent D1 polypeptide, a reaction center protein of photosystem II (PSII), requires a Chl binding complex consisting of a short-chain dehydrogenase (HCF244/Ycf39) and One-Helix Proteins of the LHC superfamily (OHP1 and OHP2 in chloroplasts). Here, we show that an ohp2 mutant of the green alga Chlamydomonas reinhardtii fails to accumulate core PSII subunits, in particular D1. Extragenic suppressors arise at high frequency, suggesting the existence of another route for Chl association to PSII. The ohp2 mutant can be complemented by the Arabidopsis ortholog. In contrast to land plants, where psbA translation is prevented in the absence of OHP2, ribosome profiling experiments show that the Chlamydomonas mutant translates the psbA transcript over its full length. Pulse labelling suggests that D1 is degraded during or immediately after translation. The translation of other PSII subunits is affected by assembly-controlled translational regulation (the CES process). Proteomics show that HCF244, a translation factor which associates with and is stabilized by OHP2 in land plants, still partly accumulates in the Chlamydomonas ohp2 mutant, explaining the persistence of psbA translation. Several Chl biosynthesis enzymes overaccumulate in the mutant membranes. Partial inactivation of the D1-degrading FtsH protease restores a low level of PSII activity in an ohp2 background, but not photoautotrophy. Taken together, our data suggest that OHP2 is not required for psbAD1 translation in Chlamydomonas, but necessary for its stabilization.

plant biology↗

bioRxiv2021

Systems approaches identify the consequences ofmonosomy in somatic human cells

Chromosome loss that results in monosomy is detrimental to viability, yet, it is frequently observed in cancers. How cancers survive with monosomy is unknown. Using p53 deficient monosomic cell lines, we found that chromosome loss impairs proliferation and genomic stability. Transcriptome and proteome analysis revealed a partial compensation of the gene dosage changes that mitigates the effects of chromosome loss. Monosomy triggers global gene expression changes that differ from the effects of trisomy. We show that ribosome biogenesis and translation were commonly downregulated in monosomic cells, likely due to haploinsufficiency of ribosomal genes. The ensuing ribosome biogenesis stress triggers the p53 pathway and G1 arrest when TP53 is reintroduced into monosomic cells. Accordingly, impaired ribosome biogenesis and p53 inactivation are associated with monosomy in cancer. Our first systematic study of monosomy in human cells explains why monosomy is so detrimental and how loss of p53 enables its incidence in cancer.

systems biology↗

bioRxiv2020

CRISPR/Cas with ribonucleoprotein complexes and transiently selected telomere vectors allows highly efficient marker-free and multiple genome editing in Botrytis cinerea

CRISPR/Cas has become the state-of-the-art technology for genetic manipulation in diverse organisms, enabling targeted genetic changes to be performed with unprecedented efficiency. Here we report on the first establishment of robust CRISPR/Cas editing in the important necrotrophic plant pathogen Botrytis cinerea based on the introduction of optimized Cas9-sgRNA ribonucleoprotein complexes (RNPs) into protoplasts. Editing yields were further improved by development of a novel strategy that combines RNP delivery with transiently stable telomeres containing vectors, which allowed temporary selection and convenient screening of marker-free editing. We demonstrate that this approach provides vastly superior editing rates compared to existing CRISPR/Cas-based methods in filamentous fungi, including the model plant pathogen Magnaporthe oryzae. The high performance of telomere vector-mediated coediting was demonstrated by random mutagenesis of codon 272 of the sdhB gene, a major determinant of resistance to succinate dehydrogenase inhibitor (SDHI) fungicides by in bulk replacement of the codon 272 with codons encoding all 20 amino acids. All exchanges were found at similar frequencies in the absence of selection but SDHI selection allowed the identification of novel amino acid substitutions which conferred differential resistance levels towards different SDHI fungicides. The increased efficiency and easy handling of RNP-based cotransformation is expected to greatly facilitate molecular research in B. cinerea and other fungi.

genetics↗

bioRxiv2019

The proteomic inventory reveals the chloroplast ribosome as nexus within a diverse protein network

Chloroplast gene expression is tightly regulated and majorly controlled on the level of protein synthesis. Fine-tuning of translation is vital for plant development, acclimation to environmental challenges and for the assembly of major protein complexes such as the photosynthesis machinery. However, many regulatory mediators and the interaction network of chloroplast ribosomes are not known to date. We report here on a deep proteomic analysis of the plastidic ribosome interaction network in Chlamydomonas reinhardtii cells. Affinity-purification of ribosomes was achieved via endogenous affinity tagging of the chloroplast-encoded protein Rpl5, yielding a specific enrichment of >650 chloroplast-localized proteins. The ribosome interaction network was validated for several proteins and provides a new source of mainly conserved factors directly linking translation with central processes such as protein folding, photosystem biogenesis, redox control, RNA maturation, energy and metabolite homeostasis. Our approach provided the first evidence for the existence of a plastidic co-translational acting N-acetyltransferase (cpNAT1). Expression of tagged cpNAT1 confirmed its ribosome-association, and we demonstrated the ability of cpNAT1 to acetylate substrate proteins at their N-terminus. Our dataset establishes that the chloroplast protein synthesis machinery acts as nexus in a highly choreographed, spatially interconnected protein network and underscores its wide-ranging regulatory potential during gene expression. ONE-SENTENCE SUMMARYAffinity purification of Chlamydomonas reinhardtii chloroplast ribosomes and subsequent proteomic analysis revealed a broad spectrum of interactors ranging from global translation control to specific pathways.

biochemistry↗