Jihyeon Yu

Cooperation and Functional Diversification of Two Closely Related Galactolipase Genes for Jasmonate Biosynthesis

Jasmonic acid (JA) plays pivotal roles in diverse plant biological processes, including wound response. Chloroplast lipid hydrolysis is a critical step for JA biosynthesis, but the mechanism of this process remains elusive. We report here that DONGLE (DGL), a homolog of DEFECTIVE IN ANTHER DEHISCENCE1 (DAD1), encodes a chloroplast-targeted lipase with strong galactolipase and weak phospholipase A(1) activity. DGL is expressed in the leaves and has a specific role in maintaining basal JA content under normal conditions, and this expression regulates vegetative growth and is required for a rapid JA burst after wounding. During wounding, DGL and DAD1 have partially redundant functions for JA production, but they show different induction kinetics, indicating temporally separated roles: DGL plays a role in the early phase of JA production, and DAD1 plays a role in the late phase of JA production. Whereas DGL and DAD1 are necessary and sufficient for JA production, phospholipase D appears to modulate wound response by stimulating DGL and DAD1 expression.

WEREWOLF, a Regulator of Root Hair Pattern Formation, Controls Flowering Time through the Regulation of FT mRNA Stability

A key floral activator, FT, integrates stimuli from long-day, vernalization, and autonomous pathways and triggers flowering by directly regulating floral meristem identity genes in Arabidopsis (Arabidopsis thaliana). Since a small amount of FT transcript is sufficient for flowering, the FT level is strictly regulated by diverse genes. In this study, we show that WEREWOLF (WER), a MYB transcription factor regulating root hair pattern, is another regulator of FT. The mutant wer flowers late in long days but normal in short days and shows a weak sensitivity to vernalization, which indicates that WER controls flowering time through the photoperiod pathway. The expression and double mutant analyses showed that WER modulates FT transcript level independent of CONSTANS and FLOWERING LOCUS C. The histological analysis of WER shows that it is expressed in the epidermis of leaves, where FT is not expressed. Consistently, WER regulates not the transcription but the stability of FT mRNA. Our results reveal a novel regulatory mechanism of FT that is non cell autonomous.

In-situ observation of formation of nanosized TiO2 powder in chemical vapor condensation

Abstract The present study has attempted to in-situ observe the formation ofnanosized TiO2 powder during chemical vapor condensation process. For this purpose, the powders were sampled at various positions in the furnace using a quartz collecting rod of which surface acts as the powder condensation site. It was discussed in terms of size effect on the phase stability that the fine anatase particle transformed to the coarse rutile phase by growing in the high temperature region. Also the parabolic particle growth with the distance of reaction tube was explicated by computated residence time with solving temperature and gas velocity distribution numerically for different oxygen flow rate conditions.

The characteristics of nanosized TiO2 powders synthesized by chemical vapor condensation

Abstract The effect of the ratio of O 2 /He flow rate in the reactor on the characteristics of nanosized TiO 2 powder synthesized by chemical vapor condensation process was investigated under fixed conditions of supersaturation ratio, collision rate and residence time. As the ratio of O 2 /He flow rate increased, the particle size of TiO 2 powder almost remained unchanged but the agglomeration of nanoparticles enlarged in which the degree of agglomeration was defined as the ratio of particle size to crystallite size. Based upon the reaction of Ti(OR) 4 + 18O 2 →TiO 2 + 14H 2 O + 12CO 2 , it is presumed that the water vapor released from the reaction of precursor with oxygen gas is responsible for the particle agglomeration.

CUT-PCR: CRISPR-mediated, ultrasensitive detection of target DNA using PCR.

Circulating tumor DNA (ctDNA) has emerged as a tumor-specific biomarker for the early detection of various cancers. To date, several techniques have been devised to enrich the extremely small amounts of ctDNA present in plasma, but they are still insufficient for cancer diagnosis, especially at the early stage. Here, we developed a novel method, CUT (CRISPR-mediated, Ultrasensitive detection of Target DNA)-PCR, which uses CRISPR endonucleases to enrich and detect the extremely small amounts of tumor DNA fragments among the much more abundant wild-type DNA fragments by specifically eliminating the wild-type sequences. We computed that by using various orthologonal CRISPR endonucleases such as SpCas9 and FnCpf1, the CUT-PCR method would be applicable to 80% of known cancer-linked substitution mutations registered in the COSMIC database. We further verified that CUT-PCR together with targeted deep sequencing enables detection of a broad range of oncogenes with high sensitivity (<0.01%) and accuracy, which is superior to conventional targeted deep sequencing. In the end, we successfully applied CUT-PCR to detect sequences with oncogenic mutations in the ctDNA of colorectal cancer patients’ blood, suggesting that our technique could be adopted for diagnosing various types of cancer at early stages.

Photoautotrophic production of macular pigment in a Chlamydomonas reinhardtii strain generated by using DNA-free CRISPR-Cas9 RNP-mediated mutagenesis

Lutein and zeaxanthin are dietary carotenoids reported to be protective against age‐related macular degeneration. Recently, the green alga Chlamydomonas reinhardtii has received attention as a photosynthetic cell factory, but the potential of this alga for carotenoid production has not yet been evaluated. In this study, we selected the C. reinhardtii CC‐4349 strain as the best candidate among seven laboratory strains tested for carotenoid production. A knock‐out mutant of the zeaxanthin epoxidase gene induced by preassembled DNA‐free CRISPR‐Cas9 ribonucleoproteins in the CC‐4349 strain had a significantly higher zeaxanthin content (56‐fold) and productivity (47‐fold) than the wild type without the reduction in lutein level. Furthermore, we produced eggs fortified with lutein (2‐fold) and zeaxanthin (2.2‐fold) by feeding hens a diet containing the mutant. Our results clearly demonstrate the possibility of cost‐effective commercial use of microalgal mutants induced by DNA‐free CRISPR‐Cas9 ribonucleoproteins in algal biotechnology for the production of high‐value products.

Deletion of the chloroplast LTD protein impedes LHCI import and PSI–LHCI assembly in Chlamydomonas reinhardtii

The Chlamydomonas reinhardtii LTD null mutant generated by CRISPR–Cas9, displayed aberrant PSI–LHCI holocomplexes, suggesting that the LTD protein may selectively function in PSI–LHCI assembly in green microalgae

ID3 regulates the MDC1-mediated DNA damage response in order to maintain genome stability

MDC1 plays a critical role in the DNA damage response (DDR) by interacting directly with several factors including γ-H2AX. However, the mechanism by which MDC1 is recruited to damaged sites remains elusive. Here, we show that MDC1 interacts with a helix–loop–helix (HLH)-containing protein called inhibitor of DNA-binding 3 (ID3). In response to double-strand breaks (DSBs) in the genome, ATM phosphorylates ID3 at serine 65 within the HLH motif, and this modification allows a direct interaction with MDC1. Moreover, depletion of ID3 results in impaired formation of ionizing radiation (IR)-induced MDC1 foci, suppression of γ-H2AX-bound MDC1, impaired DSB repair, cellular hypersensitivity to IR, and genomic instability. Disruption of the MDC1–ID3 interaction prevents accumulation of MDC1 at sites of DSBs and suppresses DSB repair. Thus, our study uncovers an ID3-dependent mechanism of recruitment of MDC1 to DNA damage sites and suggests that the ID3–MDC1 interaction is crucial for DDR.MDC1 is a key component of the DNA damage response and interacts with several factors such as γ-H2AX. Here the authors show that MDC1 interacts with ID3, facilitating MDC1 recruitment to sites of damage and repair of breaks.

Web-based design and analysis tools for CRISPR base editing

Background As a result of its simplicity and high efficiency, the CRISPR-Cas system has been widely used as a genome editing tool. Recently, CRISPR base editors, which consist of deactivated Cas9 (dCas9) or Cas9 nickase (nCas9) linked with a cytidine or a guanine deaminase, have been developed. Base editing tools will be very useful for gene correction because they can produce highly specific DNA substitutions without the introduction of any donor DNA, but dedicated web-based tools to facilitate the use of such tools have not yet been developed. Results We present two web tools for base editors, named BE-Designer and BE-Analyzer. BE-Designer provides all possible base editor target sequences in a given input DNA sequence with useful information including potential off-target sites. BE-Analyzer, a tool for assessing base editing outcomes from next generation sequencing (NGS) data, provides information about mutations in a table and interactive graphs. Furthermore, because the tool runs client-side, large amounts of targeted deep sequencing data (>100MB) do not need to be uploaded to a server, substantially reducing running time and increasing data security. BE-Designer and BE-Analyzer can be freely accessed at http://www.rgenome.net/bedesigner/ and http://www.rgenome.net/be-analyzer/respectively Conclusion We develop two useful web tools to design target sequence (BE-Designer) and to analyze NGS data from experimental results (BE-Analyzer) for CRISPR base editors.

Author Correction: ID3 regulates the MDC1-mediated DNA damage response in order to maintain genome stability

This Article contains errors in Fig. 3, Fig. 4 and Fig. 7, for which we apologize. In Fig. 3, panel ‘b’, the 0.5 hour time point after Ku55933 treatment images were inadvertently replaced with duplicates of the 3 hour time point after Ku55933 treatment images in Fig. 3b. Additionally, in panel ‘b’, the 0.5 hour time point after Nu7026 treatment images were inadvertently replaced with duplicates of the 180 min time point after siMDC1 treatment images in Fig. 3d. In Fig. 4, panel ‘g’, RNF168 foci in U2OS cell images were inadvertently replaced with duplicates of RNF168 foci in HeLa cell images in Fig. 4f. In Fig. 7, panel ‘b’, the DAPI images 0.5 hours after IR under siID3 treatment were inadvertently replaced with DAPI images of a different field of view from the same experiment. Additionally, in panel ‘i’, the shID3 mock-treated GFP-ID3 cells image was inadvertently replace with duplications of the shID3 mock-treated GFP-ID3 cells image in Fig. 7g.