Biology subjects

Schreiber, T.

Publications and source records attributed to Schreiber, T..

5 recordsLinked to original sources

bioRxiv2026

Intronised fluorophores improve reporter output and biosensor expression in plants

Genetically encoded biosensors are central to plant cell biology, but low reporter signal can limit their use. Here, we developed modular intronised fluorophores to improve reporter output in transient and stable plant expression systems. Matched nuclear-localised triple mCitrine and mScarlet3 reporters were compared with and without introduced introns in Nicotiana benthamiana and Arabidopsis thaliana. Intronisation increased fluorescence, with the magnitude of enhancement depending on the fluorophore and expression context. Immunoblot analyses supported increased reporter accumulation. In Arabidopsis, intronised reporters driven by the tissue-specific SCARECROW promoter, produced stronger fluorescence signals that remained detectable farther from the root tip than non-intronised versions. Intronised versions of the abscisic acid (ABA) biosensor ABACUS2 retained ABA responsiveness and showed a lower frequency of reduced fluorescence between generations. These modular components provide a practical toolbox for improving fluorescent reporter detection and maintaining usable biosensor expression, supporting their application in plant cell biology and quantitative imaging.

plant biology↗

bioRxiv2023

Efficient scar-free knock-ins of several kilobases by engineered CRISPR/Cas endonucleases

In plants and mammals, non-homologous end-joining is the dominant pathway to repair DNA double strand breaks, making it challenging to generate knock-in events. Using a transient assay in Nicotiana benthamiana we identified two groups of exonucleases, respectively from the Herpes Virus and from the bacteriophage T7 families, hat confer up to 38-fold increase in HDR frequencies when fused to Cas9. We achieved precise and scar-free insertion of several kilobases of DNA both in transient and stable transformation systems. In Arabidopsis thaliana, fusion of Cas9 to a Herpes Virus family exonuclease leads to 10-fold higher frequencies of knock-ins. Our results open perspectives for the routine production of knock-in and gene replacement events in plants. One-Sentence SummaryFusions of CRISPR endonucleases to specific 5'-exonucleases leads to significant increase in scar-free multikilobase knock-ins.

molecular biology↗

bioRxiv2022

DNA double strand breaks lead to de novo transcription and translation of damage-induced long RNAs in planta

DNA double strand breaks (DSBs) are lethal threats that need to be repaired. Although many of the proteins involved in the early steps of DSB repair have been characterized, recent reports indicate that damage induced long and small RNAs also play an important role in DSB repair. Here, using a Nicotiana benthamiana transgenic line originally designed as a reporter for targeted knock-ins, we show that DSBs generated by Cas9 induce the transcription of long stable RNAs (damage-induced long RNAs - dilRNAs) that are translated into proteins. Using an array of single guide RNAs we show that the initiation of transcription takes place in the vicinity of the DSB. Single strand DNA nicks are not able to induce transcription, showing that cis DNA damage-induced transcription is specific for DSBs. Our results support a model in which a default and early event in the processing of DSBs is transcription into RNA which, depending on the genomic and genic context, can undergo distinct fates, including translation into protein, degradation or production of small RNAs. Our results have general implications for understanding the role of transcription in the repair of DSBs and, reciprocally, reveal DSBs as yet another way to regulate gene expression.

molecular biology↗

bioRxiv2020

Alterations of multiple alveolar macrophage states in chronic obstructive pulmonary disease

Despite the epidemics of chronic obstructive pulmonary disease (COPD), the cellular and molecular mechanisms of this disease are far from being understood. Here, we characterize and classify the cellular composition within the alveolar space and peripheral blood of COPD patients and control donors using a clinically applicable single-cell RNA-seq technology corroborated by advanced computational approaches for: machine learning-based cell-type classification, identification of differentially expressed genes, prediction of metabolic changes, and modeling of cellular trajectories within a patient cohort. These high-resolution approaches revealed: massive transcriptional plasticity of macrophages in the alveolar space with increased levels of invading and proliferating cells, loss of MHC expression, reduced cellular motility, altered lipid metabolism, and a metabolic shift reminiscent of mitochondrial dysfunction in COPD patients. Collectively, single-cell omics of multi-tissue samples was used to build the first cellular and molecular framework for COPD pathophysiology as a prerequisite to develop molecular biomarkers and causal therapies against this deadly disease.

systems biology↗

bioRxiv2019

Oomycete small RNAs invade the plant RNA-induced silencing complex for virulence

Fungal small RNAs (sRNAs) hijack the plant RNA silencing pathway to manipulate host gene expression, named cross-kingdom RNA interference (ckRNAi). It is currently unknown how conserved and significant ckRNAi is for microbial virulence. Here, we found for the first time that sRNAs of a pathogen representing the oomycete kingdom invade the host plants Argonaute (AGO)/RNA-induced silencing complex. To demonstrate the functionality of the plant-invading oomycete Hyaloperonospora arabidopsidis sRNAs (HpasRNAs), we designed a novel CRISPR endoribonuclease Csy4/GUS repressor reporter to visualize in situ pathogen-induced target suppression in Arabidopsis thaliana host plant. By using 5 RACE-PCR we demonstrated HpasRNAs-directed cleavage of plant mRNAs. The significant role of HpasRNAs together with AtAGO1 in virulence was demonstrated by plant atago1 mutants and by transgenic Arabidopsis expressing a target mimic to block HpasRNAs, that both exhibited enhanced resistance. Individual HpasRNA plant targets contributed to host immunity, as Arabidopsis gene knockout or HpasRNA-resistant gene versions exhibited quantitative enhanced or reduced susceptibility, respectively. Together with previous reports, we found that ckRNAi is conserved among oomycete and fungal pathogens.

microbiology↗