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An imbalance in photosynthetic electron transfer is thought to be redressed by photosynthetic control of the rate of expression of genes encoding apoproteins of photosystem (PS)-I and PS-II in response to the redox state of plastoquinone (PQ), which is a connecting electron carrier. PS stoichiometry is then adjusted to enhance photosynthetic efficiency. In prokaryotes, sigma factors are well known for their participation in the control of RNA polymerase activity in transcription, whereas there have been no reports concerning their association with redox regulation. We have found that the phosphorylation of SIG1, the major sigma factor (SIG), is regulated by redox signals and selectively inhibits the transcription of the psaA gene, which encodes a PS-I protein.We produced transgenic Arabidopsis plants with or without the putative phosphorylation sites for SIG1 and demonstrated through in vivo labeling that Thr-170 was involved in the phosphorylation. We analyzed the in vivo and in vitro transcriptional responses of the transgenic Arabidopsis plants to the redox status in regard to involvement of the phosphorylation site. We revealed an enhanced phosphorylation of SIG1 under oxidative conditions of PQ in a form associated with the molecular mass of the holoenzyme. Phosphorylation of SIG1 proved crucial through a change in the promoter specificity for sustaining balanced expression of components in PS-I and PS-II and was responsible for harmonious electron flow to maintain photosynthetic efficiency.chloroplast | redox regulation | plastoquinone | RNA polymerase | Arabidopsis P lants are required for the survival of humans as food supplies and as alternative sources of energy in the form of biofuel, and are important in conservation of the environment. These contributions of plants are based on the function of chloroplasts. Chloroplasts are thought to have originated from ancestral cyanobacteria, which became symbionts in eukaryotic host cells. Plants have evolved as sessile organisms that use their chloroplasts to exploit solar energy. They are unable to escape from undesirable environmental conditions and must adapt to survive. When plants are exposed to sunlight, their chloroplast photosystems (PSs) excite electrons donated by water, which are transferred to NADP + and other recipients. Altered light conditions influence the efficiency of electron excitation in PS-I and PS-II, leading to an imbalance in photosynthetic electron transfer (1-3) and injury caused by uncoupled excess energy. This imbalance is thought to be redressed by the photosynthetic control of PS stoichiometry (2), where the rate of expression of genes for apoproteins of PS-I and PS-II is regulated in response to the redox state of plastoquinone (PQ) (4, 5). However, it is not known how the regulation is achieved in a signal transduction pathway through redox states of PQ. We have focused on the activity of RNA polymerase as one of the critical steps. The transcription of most photosynthesis genes in the chloroplast is accomplished with bacterial...

BackgroundClustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome editing permits the rapid production of genetically engineered mice. To make the most of this innovative technology, a streamlined procedure is needed for the robust construction of CRISPR/Cas9 vectors, the efficient preparation of mouse oocytes, and refined genotyping strategies. Although we previously demonstrated the applicability of oocyte cryopreservation technologies and various genotyping methods in the production of transcription activator-like effector nuclease-mediated genome editing in mice, it has not yet been clarified whether these techniques can be applied to the CRISPR/Cas9-mediated generation of knockout mice. In this study, we investigated easy, efficient, and robust methods of creating knockout mice using several CRISPR/Cas9 systems.ResultsWe constructed three types of CRISPR/Cas9 vectors expressing: 1) single guide RNA (gRNA) and Cas9 nuclease, 2) two gRNAs and Cas9 nickase, and 3) two gRNAs and FokI-dCas9, targeting the same genomic locus. These vectors were directly microinjected into the pronucleus of freeze-thawed fertilized oocytes, and surviving oocytes were transferred to pseudopregnant ICR mice. Cas9 nuclease resulted in the highest mutation rates with the lowest birth rates, while Cas9 nickase resulted in the highest birth rates with the lowest mutation rates. FokI-dCas9 presented well-balanced mutation and birth rates. Furthermore, we constructed a single all-in-one FokI-dCas9 vector targeting two different genomic loci, and validated its efficacy by blastocyst analysis, resulting in highly efficient simultaneous targeted mutagenesis.ConclusionsOur report offers several choices of researcher-friendly consolidated procedures for making CRISPR/Cas9-mediated knockout mice, with sophisticated construction systems for various types of CRISPR vectors, convenient preparation of in vitro fertilized or mated freeze-thawed oocytes, and an efficient method of mutant screening.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-015-0144-x) contains supplementary material, which is available to authorized users.

Phosphatidyl-inositol mannosides (PIM) are glycolipids unique to mycobacteria and other related bacteria that stimulate host immune responses and are implicated in mycobacteria pathogenicity. Here, we found that the FcRγ-coupled C-type lectin receptor DCAR (dendritic cell immunoactivating receptor; gene symbol Clec4b1) is a direct receptor for PIM. Mycobacteria activated reporter cells expressing DCAR, and delipidation of mycobacteria abolished this activity. Acylated PIMs purified from mycobacteria were identified as ligands for DCAR. DCAR was predominantly expressed in small peritoneal macrophages and monocyte-derived inflammatory cells in lungs and spleen. These cells produced monocyte chemoattractant protein-1 (MCP-1) upon PIM treatment, and absence of DCAR or FcRγ abrogated MCP-1 production. Upon mycobacterial infection, Clec4b1-deficient mice showed reduced numbers of monocyte-derived inflammatory cells at the infection site, impaired IFNγ production by T cells, and an increased bacterial load. Thus, DCAR is a critical receptor for PIM that functions to promote T cell responses against mycobacteria.

Current advances in producing genetically modified mice using genome-editing technologies have indicated the need for improvement of limiting factors including zygote collection for microinjection and their cryopreservation. Recently, we developed a novel superovulation technique using inhibin antiserum and equine chorionic gonadotropin to promote follicle growth. This method enabled the increased production of fertilized oocytes via in vitro fertilization compared with the conventional superovulation method. Here, we verify that the ultra-superovulation technique can be used for the efficient generation of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-mediated knockout mice by microinjection of plasmid vector or ribonucleoprotein into zygotes. We also investigated whether single-amino-acid-substituted mice and conditional knockout mice could be generated. Founder mice bearing base substitutions were generated more efficiently by co-microinjection of Cas9 protein, a guide RNA and single-stranded oligodeoxynucleotide (ssODN) than by plasmid microinjection with ssODN. The conditional allele was successfully introduced by the one-step insertion of an ssODN designed to carry an exon flanked by two loxP sequences and homology arms using a double-cut CRISPR-Cas9 strategy. Our study presents a useful method for the CRISPR-Cas9-based generation of genetically modified mice from the viewpoints of animal welfare and work efficiency.