Project description:The experiment was conducted to examine the influence of non-chloroplast genomes rearangements on chloroplast transcription in cucumber
Project description:Saccharopolyspora spinosa is an industrial rare actinomycete capable of producing important environmental-friendly biopesticides, spinosyns. However, exploitation of S. spinosa has been limited due to its genetic inaccessibility and lack of effective genome engineering tools. Here, we characterized the activity of an endogenous type I-B CRISPR-Cas system as well as its recognized protospacer adjacent motifs (PAMs) based on bioinformatics analysis combined with plasmid interference assay in S. spinosa. By reprogramming this endogenous CRISPR-Cas system, we achieved 100% editing efficiency for gene deletion. Using this tool, the genetic barrier composed of the Restriction-Modification (RM) systems was systematically disarmed. We showed that by disarming one type I RM system and two type II RM systems simultaneously, the transformation efficiency of foreign plasmids was significantly increased. Based on the engineered strain with simultaneous deletion of these three RM genes, we achieved the deletion of 75-kb spinosyns biosynthetic gene cluster as well as gene insertion at high efficiency. Collectively, we developed a reliable and high-efficiency genome editing tool based on the endogenous type I CRISPR-Cas system combined with the disarmament of the RM systems in S. spinosa. This is the first time to establish an endogenous CRISPR-Cas-based genome editing tool in the non-model industrial actinomycetes.
Project description:Chloroplast biogenesis represents a crucial step in seedling development, and is essential for the transition to autotrophic growth in plants. This light-controlled process relies on the transcription of nuclear and plastid genomes that drives the effective assembly and regulation of the photosynthetic machinery. Here we reveal a novel regulation level for this process by showing the involvement of chromatin remodelling in the coordination of nuclear and plastid gene expression for proper chloroplast biogenesis and function. The two Arabidopsis homologs of the yeast EPL1 proteins, core components of the NuA4 histone acetyl-transferase complex, are essential for the correct assembly and performance of chloroplasts. EPL1 proteins are necessary for the coordinated expression of nuclear genes encoding most of the components of chloroplast transcriptional machinery, specifically promoting H4K5Ac deposition in these loci. These data unveil a key participation of epigenetic regulatory mechanisms in the coordinated expression of the nuclear and plastid genomes.
Project description:Chloroplasts are organelles responsible for photosynthesis. They originated form a procaryotic ancestor in the process of endosymbiosis and contain their own genomes. The chloroplast genome is packaged into a chromatin-like structure known as the nucleoid. The internal arrangement of the nucleoid, molecular mechanisms of DNA packaging and connection of the nucleoid structure to gene expression remain poorly understood. We show that Arabidopsis thaliana chloroplast nucleoids have a unique organization driven by DNA binding to the thylakoid membranes. Membrane association of specific DNA regions is correlated with high levels of transcription, high protein occupancy and reduced DNA accessibility. Genes with low levels of transcription are further away from the membranes, have lower protein occupancy and higher DNA accessibility. Gene-specific disruption of transcription in sigma factor mutants causes a corresponding reduction in membrane association, indicating that RNA polymerase activity causes DNA tethering to the membranes. We propose that transcription organizes the chloroplast nucleoid into a transcriptionally active membrane-associated core and a less active Periphery.
Project description:Chloroplasts are organelles responsible for photosynthesis. They originated form a procaryotic ancestor in the process of endosymbiosis and contain their own genomes. The chloroplast genome is packaged into a chromatin-like structure known as the nucleoid. The internal arrangement of the nucleoid, molecular mechanisms of DNA packaging and connection of the nucleoid structure to gene expression remain poorly understood. We show that Arabidopsis thaliana chloroplast nucleoids have a unique organization driven by DNA binding to the thylakoid membranes. Membrane association of specific DNA regions is correlated with high levels of transcription, high protein occupancy and reduced DNA accessibility. Genes with low levels of transcription are further away from the membranes, have lower protein occupancy and higher DNA accessibility. Gene-specific disruption of transcription in sigma factor mutants causes a corresponding reduction in membrane association, indicating that RNA polymerase activity causes DNA tethering to the membranes. We propose that transcription organizes the chloroplast nucleoid into a transcriptionally active membrane-associated core and a less active Periphery.