A natural variation in OsDSK2a modulates plant growth and salt tolerance through phosphorylation by SnRK1A in rice.

Plants grow rapidly for maximal production under optimal conditions; however, they adopt a slower growth strategy to maintain survival when facing environmental stresses. As salt stress restricts crop architecture and grain yield, identifying genetic variations associated with growth and yield responses to salinity is critical for breeding optimal crop varieties. OsDSK2a is a pivotal modulator of plant growth and salt tolerance via the modulation of gibberellic acid (GA) metabolism; however, its regulation remains unclear. Here, we showed that OsDSK2a can be phosphorylated at the second amino acid (S2) to maintain its stability. The gene-edited mutant osdsk2aS2G showed decreased plant height and enhanced salt tolerance. SnRK1A modulated OsDSK2a-S2 phosphorylation and played a substantial role in GA metabolism. Genetic analysis indicated that SnRK1A functions upstream of OsDSK2a and affects plant growth and salt tolerance. Moreover, SnRK1A activity was suppressed under salt stress, resulting in decreased phosphorylation and abundance of OsDSK2a. Thus, SnRK1A preserves the stability of OsDSK2a to maintain plant growth under normal conditions, and reduces the abundance of OsDSK2a to limit growth under salt stress. Haplotype analysis using 3 K-RG data identified a natural variation in OsDSK2a-S2. The allele of OsDSK2a-G downregulates plant height and improves salt-inhibited grain yield. Thus, our findings revealed a new mechanism for OsDSK2a stability and provided a valuable target for crop breeding to overcome yield limitations under salinity stress.

Decoding the transcriptional heterogeneity, differentiation lineage, clinical significance in tissue-resident memory CD8 T cell of the small intestine by single-cell analysis.

Resident memory T (Trm) cells which are specifically located in non-lymphoid tissues showed distinct phenotypes and functions compared to circulating memory T cells and were vital for the initiation of robust immune response within tissues. However, the heterogeneity in the transcriptional features, development pathways, and cancer response of Trm cells in the small intestine was not demonstrated. Here, we integrated scRNA-seq and scTCR-seq data pan-tissue T cells to explore the heterogeneity of Trm cells and their development pathways. Trm were enriched in tissue-specific immune response and those in the DUO specially interacted with B cells via TNF and MHC-I signatures. T cell lineage analyses demonstrated that Trm might be derived from the T_CD4/CD8 subset within the same organ or migrated from spleen and mesenteric lymph nodes. We compared the immune repertoire of Trm among organs and implied that clonotypes in both DUO and ILE were less expanded and hydrophilic TRB CDR3s were enriched in the DUO. We further demonstrated that Trm in the intestine infiltrated the colorectal cancer and several effector molecules were highly expressed. Finally, the TCGA dataset of colorectal cancer implied that the infiltration of Trm from the DUO and the ILE was beneficial for overall survival and the response to immune checkpoint blockade.

Structures suggest an approach for converting weak self-peptide tumor antigens into superagonists for CD8 T cells in cancer.

Tumors frequently express unmutated self-tumor-associated antigens (self-TAAs). However, trial results using self-TAAs as vaccine targets against cancer are mixed, often attributed to deletion of T cells with high-affinity receptors (TCRs) for self-TAAs during T cell development. Mutating these weak self-TAAs to produce higher affinity, effective vaccines is challenging, since the mutations may not benefit all members of the broad self-TAA-specific T cell repertoire. We previously identified a common weak murine self-TAA that we converted to a highly effective antitumor vaccine by a single amino acid substitution. In this case the modified and natural self-TAAs still raised very similar sets of CD8 T cells. Our structural studies herein show that the modification of the self-TAA resulted in a subtle change in the major histocompatibility complex I-TAA structure. This amino acid substitution allowed a dramatic conformational change in the peptide during subsequent TCR engagement, creating a large increase in TCR affinity and accounting for the efficacy of the modified self-TAA as a vaccine. These results show that carefully selected, well-characterized modifications to a poorly immunogenic self-TAA can rescue the immune response of the large repertoire of weakly responding natural self-TAA-specific CD8 T cells, driving them to proliferate and differentiate into functional effectors. Subsequently, the unmodified self-TAA on the tumor cells, while unable to drive this response, is nevertheless a sufficient target for the CD8 cytotoxic effectors. Our results suggest a pathway for more efficiently identifying variants of common self-TAAs, which could be useful in vaccine development, complementing other current nonantigen-specific immunotherapies.

The Ubiquitin-Binding Protein OsDSK2a Mediates Seedling Growth and Salt Responses by Regulating Gibberellin Metabolism in Rice.

UBL-UBA (ubiquitin-like-ubiquitin-associated) proteins are ubiquitin receptors and transporters in the ubiquitin-proteasome system that play key roles in plant growth and development. High salinity restricts plant growth by disrupting cellular metabolism, but whether UBL-UBA proteins are involved in this process is unclear. Here, we demonstrate that the UBL-UBA protein OsDSK2a (DOMINANT SUPPRESSOR of KAR2) mediates seedling growth and salt responses in rice (Oryza sativa). Through analysis of osdsk2a, a mutant with retarded seedling growth, as well as in vitro and in vivo assays, we demonstrate that OsDSK2a combines with polyubiquitin chains and interacts with the gibberellin (GA)-deactivating enzyme ELONGATED UPPERMOST INTERNODE (EUI), resulting in its degradation through the ubiquitin-proteasome system. Bioactive GA levels were reduced, and plant growth was retarded in the osdsk2a mutant. By contrast, eui mutants displayed increased seedling growth and bioactive GA levels. OsDSK2a levels decreased in plants under salt stress. Moreover, EUI accumulated under salt stress more rapidly in osdsk2a than in wild-type plants. Thus, OsDSK2a and EUI play opposite roles in regulating plant growth under salt stress by affecting GA metabolism. Under salt stress, OsDSK2a levels decrease, thereby increasing EUI accumulation, which promotes GA metabolism and reduces plant growth.

A comparative study of modern total ankle replacement and ankle arthrodesis for ankle osteoarthritis at different follow-up times: a systematic review and meta-analysis.

PURPOSE: Total ankle replacement (TAR) or ankle arthrodesis (AA) is the main surgical treatment for end-stage ankle osteoarthritis. However, the therapeutic effect of the two surgical procedures at different follow-up times remains controversial. The purpose of this meta-analysis is to compare the short-term, medium-term, and long-term safety and efficiency of the two modern surgical treatments. METHODS: We conducted a comprehensive search in PubMed, EMBASE, Cochrane library databases, Web of Science, and Scopus. The main results were the patient's reported outcome measure (PROM) score, satisfaction, complications, reoperation, and surgery success rate. Different follow-up times and implant designs were used to evaluate the source of heterogeneity. We used a fixed effects model for meta-analysis and I2 statistic for evaluating heterogeneity. RESULTS: Thirty-seven comparative studies were included. In the short term, TAR significantly improved clinical scores (AOFAS score: WMD = 7.07, 95% Cl: 0.41-13.74, I2 = 0.0%; SF-36 PCS score: WMD = 2.40, 95% Cl: 2.22-2.58, I2 = 0.0%; SF-36 MCS score: WMD = 0.40, 95% Cl: 0.22-0.57, I2 = 0.0%; VAS for pain: WMD = - 0.50, 95% Cl: - 0.56-0.44, I2 = 44.3%) and had the lower incidence of revision (RR = 0.43, 95% CI: 0.23-0.81, I2 = 0.0%) and complications (RR = 0.67, 95% Cl: 0.50-0.90, I2 = 0.0%). In the medium term, there were still higher improvements in both the clinical scores (SF-36 PCS score: WMD = 1.57, 95% Cl: 1.36-1.78, I2 = 20.9%; SF-36 MCS score: WMD = 0.81, 95% Cl: 0.63-0.99, I2 = 48.8%) and the patient satisfaction (RR = 1.24, 95% Cl: 1.08-1.41, I2 = 12.1%) in the TAR group, but its total complications rate (RR = 1.84, 95% Cl: 1.26-2.68, I2 = 14.9%) and revision rate (RR = 1.58, 95% CI: 1.17-2.14, I2 = 84.6%) were significantly higher than that of the AA group. In the long term, there was no significant difference in clinical score and satisfaction, and a higher incidence of revision (RR = 2.32, 95% Cl: 1.70-3.16, I2 = 0.0%) and complications (RR = 3.18, 95% Cl: 1.69-5.99, I2 = 0.0%) was observed in TAR than in AA. The result of the third-generation design subgroup was consistent with that of the above pooled results. CONCLUSION: TAR had advantages over AA in the short term due to better performance in terms of PROMs, complications, and reoperation rates, but its complications become a disadvantage in the medium term. In the long term, AA seems to be favored because of lower complications and revision rates, although there is no difference in clinical scores.

Preparation and cytotoxicity evaluation of folic acid-modified YF8-OA self-assembled lipid prodrug nanoparticles.

This study aimed to improve the use of YF8, a matrine derivative obtained through chemical transformation of matrine extracted from Sophora alopecuroides. YF8 has demonstrated improved cytotoxicity compared to matrine, but its hydrophobic nature hinders its application. To overcome this, the lipid prodrug YF8-OA was synthesized by linking oleic acid (OA) to YF8 through an ester bond. Although YF8-OA could self-assemble into unique nanostructures in water, it was not sufficiently stable. To enhance the stability of YF8-OA lipid prodrug nanoparticles (LPs), we employed the strategy of PEGylation using DSPE-mPEG2000 or DSPE-mPEG2000 conjugated with folic acid (FA). This resulted in the formation of uniform spherical nanoparticles with greatly improved stability and a maximum drug load capacity upto 58.63%. Cytotoxicity was evaluated in A549, HeLa, and HepG2 cell lines. The results showed that in HeLa cells, the IC50 value of YF8-OA/LPs with FA-modified PEGylation was significantly lower than that of YF8-OA/LPs modified by PEGylation alone. However, no significant enhancement was observed in A549 and HepG2 cells. In conclusion, the lipid prodrug YF8-OA can form nanoparticles in aqueous solution to address its poor water solubility. Modification with FA resulted in further enhanced cytotoxicity, providing a potential avenue for exerting the antitumor activity of matrine analogs.

Activating receptor KIR2DS2 bound to HLA-C1 reveals the novel recognition features of activating receptor.

Killer cell immunoglobulin-like receptors (KIRs) are important receptors for regulating the killing of virus-infected or cancer cells of natural killer (NK) cells. KIR2DS2 can recognize peptides derived from hepatitis C virus (HCV) or global flaviviruses (such as dengue and Zika) presented by HLA-C*0102 to activate NK cells, and has shown promising results when used for cancer immunotherapy. Here, we present the complex structure of KIR2DS2 with HLA-C*0102 at a resolution of 2·5Å. Our structure reveals that KIR2DS2 can bind with HLA-C*0102 and HLA-A*1101 in two different directions. Moreover, Tyr45 (in activating receptor KIR2DS2) and Phe45 (in inhibitory KIRs) distinguish the two different binding models and binding affinity between activating KIRs and inhibitory KIRs. The conserved 'AT' motif of the peptide mediates recognition and determines the peptide specificity of recognition. These structural characteristics shed light on how KIRs activate NK cells and can provide a molecular basis for immunotherapy by NK cells.

Genome editing with type II-C CRISPR-Cas9 systems from Neisseria meningitidis in rice.

Two type II-C Cas9 orthologs (Nm1Cas9 and Nm2Cas9) were recently identified from Neisseria meningitidis and have been extensively used in mammalian cells, but whether these NmCas9 orthologs or other type II-C Cas9 proteins can mediate genome editing in plants remains unclear. In this study, we developed and optimized targeted mutagenesis systems from NmCas9s for plants. Efficient genome editing at the target with N4 GATT and N4 CC protospacer adjacent motifs (PAMs) was achieved with Nm1Cas9 and Nm2Cas9 respectively. These results indicated that a highly active editing system could be developed from type II-C Cas9s with distinct PAM preferences, thus providing a reliable strategy to extend the scope of genome editing in plants. Base editors (BEs) were further developed from the NmCas9s. The editing efficiency of adenine BEs (ABEs) of TadA*-7.10 and cytosine BEs (CBEs) of rat APOBEC1 (rAPO1) or human APOBEC3a (hA3A) were extremely limited, whereas ABEs of TadA-8e and CBEs of Petromyzon marinus cytidine deaminase 1 (PmCDA1) exhibited markedly improved performance on the same targets. In addition, we found that fusion of a single-stranded DNA-binding domain from the human Rad51 protein enhanced the base editing capability of rAPO1-CBEs of NmCas9s. Together, our results suggest that the engineering of NmCas9s or other type II-C Cas9s can provide useful alternatives for crop genome editing.

The Receptor Binding Domain of SARS-CoV-2 Lambda Variant Has a Better Chance Than the Delta Variant in Evading BNT162b2 COVID-19 mRNA Vaccine-Induced Humoral Immunity.

The SARS-CoV-2 Delta and Lambda variants had been named variants of concern (VOC) and variants of interest (VOI), respectively, by the World Health Organization (WHO). Both variants have two mutations in the spike receptor binding domain (RBD) region, with L452R and T478K mutations in the Delta variant, and L452Q and F490S mutations in the Lambda variant. We used surface plasmon resonance (SPR)-based technology to evaluate the effect of these mutations on human angiotensin-converting enzyme 2 (ACE2) and Bamlanivimab binding. The affinity for the RBD ligand, ACE2, of the Delta RBD is approximately twice as strong as that of the wild type RBD, an increase that accounts for the increased infectivity of the Delta variant. On the other hand, in spite of its amino acid changes, the Lambda RBD has similar affinity to ACE2 as the wild type RBD. The protective anti-wild type RBD antibody Bamlanivimab binds very poorly to the Delta RBD and not at all to the Lambda RBD. Nevertheless, serum antibodies from individuals immunized with the BNT162b2 vaccine were found to bind well to the Delta RBD, but less efficiently to the Lambda RBD in contrast. As a result, the blocking ability of ACE2 binding by serum antibodies was decreased more by the Lambda than the Delta RBD. Titers of sera from BNT162b2 mRNA vaccinated individuals dropped 3-fold within six months of vaccination regardless of whether the target RBD was wild type, Delta or Lambda. This may account partially for the fall off with time in the protective effect of vaccines against any variant.

Development of an efficient plant dual cytosine and adenine editor.

An enhanced CDA-like (eCDAL) was established from Japanese lamprey CDA1-like 4 to achieve a high editing frequency in a broad region as a C-terminal cytosine base editors (CT-CBE). Then, a novel plant dual-base editor version 1(pDuBE1) was developed by integrating TadA-8e into eCDAL. The editing efficiency of pDuBE1 could reach to 87.6%, with frequencies of concurrent A-to-G and C-to-T conversions as high as 49.7% in stably transformed plant cells. Our results showed that pDuBE1 could mediate robust dual editing in plant genome, providing a powerful manipulation tool for precise crop breeding and screening platforms for in planta direct evolution.

MnS@N,S Co-Doped Carbon Core/Shell Nanocubes: Sulfur-Bridged Bonds Enhanced Na-Storage Properties Revealed by In Situ Raman Spectroscopy and Transmission Electron Microscopy.

Rational structure and morphology design are of great significance to realize excellent Na storage for advanced electrode materials in sodium-ion batteries (SIBs). Herein, a cube-like core/shell composite of single MnS nanocubes (≈50 nm) encapsulated in N, S co-doped carbon (MnS@NSC) with strong CSMn bond interactions is successfully prepared as outstanding anode material for SIBs. The carbon shell significantly restricts the expansion of the MnS volume in successive sodiation/desodiation processes, as demonstrated by in situ transmission electron microscopy (TEM) of one single MnS@NSC nanocube. Moreover, the in situ generated CSMn bonds between the MnS core and carbon shell play a significant role in improving the Na-storage stability and reversibility of MnS@NSC, as revealed by in situ Raman and TEM. As a result, MnS@NSC exhibits a high reversible specific capacity of 594.2 mAh g-1 at a current density of 100 mA g-1 and an excellent rate performance. It also achieves a remarkable cycling stability of 329.1 mAh g-1 after 3000 charge/discharge cycles at 1 A g-1 corresponding to a low capacity attenuation rate of 0.0068% per cycle, which is superior to that of pristine MnS and most of the reported Mn-based anode materials in SIBs.

The activation of OsEIL1 on YUC8 transcription and auxin biosynthesis is required for ethylene-inhibited root elongation in rice early seedling development.

Rice is an important monocotyledonous crop worldwide; it differs from the dicotyledonous plant Arabidopsis in many aspects. In Arabidopsis, ethylene and auxin act synergistically to regulate root growth and development. However, their interaction in rice is still unclear. Here, we report that the transcriptional activation of OsEIL1 on the expression of YUC8/REIN7 and indole-3-pyruvic acid (IPA)-dependent auxin biosynthesis is required for ethylene-inhibited root elongation. Using an inhibitor of YUC activity, which regulates auxin biosynthesis via the conversion of IPA to indole-3-acetic acid (IAA), we showed that ethylene-inhibited primary root elongation is dependent on YUC-based auxin biosynthesis. By screening phenotypes of seedling primary root from mutagenesis libraries following ethylene treatment, we identified a rice ethylene-insensitive mutant, rein7-1, in which YUC8/REIN7 is truncated at its C-terminus. Mutation in YUC8/REIN7 reduced auxin biosynthesis in rice, while YUC8/REIN7 overexpression enhanced ethylene sensitivity in the roots. Moreover, YUC8/REIN7 catalyzed the conversion of IPA to IAA, truncated version at C-terminal end of the YUC8/REIN7 resulted in significant reduction of enzymatic activity, indicating that YUC8/REIN7 is required for IPA-dependent auxin biosynthesis and ethylene-inhibited root elongation in rice early seedlings. Further investigations indicated that ethylene induced YUC8/REIN7 expression and promoted auxin accumulation in roots. Addition of low concentrations of IAA rescued the ethylene response in the rein7-1, strongly demonstrating that ethylene-inhibited root elongation depends on IPA-dependent auxin biosynthesis. Genetic studies revealed that YUC8/REIN7-mediated auxin biosynthesis functioned downstream of OsEIL1, which directly activated the expression of YUC8/REIN7. Thus, our findings reveal a model of interaction between ethylene and auxin in rice seedling primary root elongation, enhancing our understanding of ethylene signaling in rice.

Substrate analog interaction with MCR-1 offers insight into the rising threat of the plasmid-mediated transferable colistin resistance.

Colistin is considered a last-resort antibiotic against most gram-negative bacteria. Recent discoveries of a plasmid-mediated, transferable mobilized colistin-resistance gene ( mcr-1) on all continents have heralded the imminent emergence of pan-drug-resistant superbacteria. The inner-membrane protein MCR-1 can catalyze the transfer of phosphoethanolamine (PEA) to lipid A, resulting in colistin resistance. However, little is known about the mechanism, and few drugs exist to address this issue. We present crystal structures revealing the MCR-1 catalytic domain (cMCR-1) as a monozinc metalloprotein with ethanolamine (ETA) and d-glucose, respectively, thus highlighting 2 possible substrate-binding pockets in the MCR-1-catalyzed PEA transfer reaction. Mutation of the residues involved in ETA and d-glucose binding impairs colistin resistance in recombinant Escherichia coli containing full-length MCR-1. Partial analogs of the substrate are used for cocrystallization with cMCR-1, providing valuable information about the family of PEA transferases. One of the analogs, ETA, causes clear inhibition of polymyxin B resistance, highlighting its potential for drug development. These data demonstrate the crucial role of the PEA- and lipid A-binding pockets and provide novel insights into the structure-based mechanisms, important drug-target hot spots, and a drug template for further drug development to combat the urgent, rising threat of MCR-1-mediated antibiotic resistance.-Wei, P., Song, G., Shi, M., Zhou, Y., Liu, Y., Lei, J., Chen, P., Yin, L. Substrate analog interaction with MCR-1 offers insight into the rising threat of the plasmid-mediated transferable colistin resistance.

Functional and structural characterization of a ß-glucosidase involved in saponin metabolism from intestinal bacteria.

Saponins are natural glycosides widely used in medicine and the food industry. Although saponin metabolism in human is dependent on intestinal microbes, few involving bacteria enzymes have been identified. We cloned BlBG3, a GH3 ß-glucosidase from Bifidobacterium longum, from human stool. We found that BlBG3 catalyzes the hydrolysis of glycoside furostanol and ginsenoside Rb1 at higher efficiency than other microbial ß-glucosidases. Structural analysis of BlBG3 in complex with d-glucose revealed its three unique loops, which form a deep pocket and participate in substrate binding. To understand how substrate is bound to the pocket, molecular docking was performed and the binding interactions of protobioside with BlBG3 were revealed. Mutational study suggested that R484 and H642 are critical for enzymatic activity. Our study presents the first structural and functional analysis of a saponin-processing enzyme from human microbiota.

Isolation and identification of five cold-inducible promoters from Oryza sativa.

MAIN CONCLUSION: Five promoters of the cold-inducible rice genes were isolated. The quantitative and qualitative expression analyses in the high generation transgenic rice suggest that the genes are stably induced by low temperature. Cold-inducible promoters are highly desirable for stress-inducible gene expression in crop genetic engineering. In this study, five rice genes, including OsABA8ox1, OsMYB1R35, OsERF104, OsCYP19-4, and OsABCB5, were found to be transcriptionally induced by cold stress. The promoters of these five genes were isolated, and their activities were identified in various tissues of transgenic rice plants at different growth stages both before and after cold stress. Histochemical staining, quantitative fluorescence assays, and GUSplus gene expression assays in corresponding promoter-GUSplus transgenic rice plants confirmed that the five promoters were cold-inducible with different expression patterns and strengths. The OsABA8ox1 and OsERF104 promoters had very low background expression; in contrast, the OsMYB1R35 promoter had higher basal activity in the roots, and OsCYP19-4 promoter activity was preferentially high in leaves and flowers of untreated transgenic lines. The OsABCB5 promoter had the highest basal activity among the five promoters. After cold induction, the activities of the OsABA8ox1, OsMYB1R35, and OsABCB5 promoters were high in both roots and leaves, slightly lower than that of the constitutively expressed OsActin1 promoter but comparable to that of the AtRD29A promoter. During the cold treatment time course, the activities of OsABA8ox1 and OsABCB5 promoters were quickly up-regulated in the early period and peaked at 24 h, after which the induction level gradually decreased until 48 h. The activities of the OsMYB1R35 and OsCYP19-4 promoters increased under stress in a time-dependent manner, while OsERF104 promoter activity began to increase at 4 h and then decreased strongly. Furthermore, activities' analysis in T3, T4, and T5 homozygous progeny of single-copy plants revealed that five promoters maintained their activities at comparable levels with no evidence of silencing under cold stress. Overall, the five cold-inducible rice promoters described herein could potentially be used in crop biotechnology.

Isolation of five rice nonendosperm tissue-expressed promoters and evaluation of their activities in transgenic rice.

Using promoters expressed in nonendosperm tissues to activate target genes in specific plant tissues or organs with very limited expression in the endosperm is an attractive approach in crop transgenic engineering. In this article, five putative nonendosperm tissue-expressed promoters were cloned from the rice genome and designated POsNETE1 , POsNETE2 , POsNETE3 , POsNETE4 and POsNETE5 . By qualitatively and quantitatively examining GUSplus reporter gene expression in transgenic rice plants, POsNETE1 -POsNETE5 were all found to be active in the roots, leaves, stems, sheaths and panicles but not in the endosperm of plants at different developmental stages. In addition, POsNETE2 , POsNETE4 and POsNETE5 were also inactive in rice embryos. Among these promoters, POsNETE4 and POsNETE5 exhibited higher activities in all of the tested tissues, and their activities in stems, leaves, roots and sheaths were higher than or comparable to those of the rice Actin1 promoter. We also progressively monitored the activities of POsNETE1 -POsNETE5 in two generations of single-copy lines and found that these promoters were stably expressed between generations. Transgenic rice was produced using POsNETE4 and POsNETE5 to drive a modified Bt gene, mCry1Ab. Bt protein expressed in the tested plants ranged from 1769.4 to 4428.8 ng/g fresh leaves, whereas Bt protein was barely detected in the endosperm. Overall, our study identified five novel nonendosperm tissue-expressed promoters that might be suitable for rice genetic engineering and might reduce potential social concern regarding the safety of GMO crops.

Generation of targeted mutant rice using a CRISPR-Cpf1 system.

CRISPR-Cpf1 is a newly identified CRISPR-Cas system, and Cpf1 was recently engineered as a molecular tool for targeted genome editing in mammalian cells. To test whether the engineered CRISPR-Cpf1 system could induce the production of rice mutants, we selected two genome targets in the OsPDS and OsBEL genes. Our results show that both targets could be efficiently mutated in transgenic rice plants using CRISPR-Cpf1. We found that pre-crRNAs with a full-length direct repeat sequence exhibited considerably increased efficiencies compared with mature crRNAs. In addition, the specificity and transmission of the mutation were investigated, and the behaviours of crRNA-Cpf1-induced plant targeted genome mutagenesis were assessed. Taken together, our results indicate that CRISPR-Cpf1 expression via stable transformation can efficiently generate specific and heritable targeted mutations in rice and thereby constitutes a novel and important approach to specific and precise plant genome editing.

Identification of a regulatory element responsible for salt induction of rice OsRAV2 through ex situ and in situ promoter analysis.

Salt is a major environmental stress factor that can affect rice growth and yields. Recent studies suggested that members of the AP2/ERF domain-containing RAV (related to ABI3/VP1) TF family are involved in abiotic stress adaptation. However, the transcriptional response of rice RAV genes (OsRAVs) to salt has not yet been fully characterized. In this study, the expression patterns of all five OsRAVs were examined under salt stress. Only one gene, OsRAV2, was stably induced by high-salinity treatment. Further expression profile analyses indicated that OsRAV2 is transcriptionally regulated by salt, but not KCl, osmotic stress, cold or ABA (abscisic acid) treatment. To elucidate the regulatory mechanism of the stress response at the transcriptional level, we isolated and characterized the promoter region of OsRAV2 (P OsRAV2 ). Transgenic analysis indicated that P OsRAV2 is induced by salt stress but not osmotic stress or ABA treatment. Serial 5' deletions and site-specific mutations in P OsRAV2 revealed that a GT-1 element located at position -664 relative to the putative translation start site is essential for the salt induction of P OsRAV2 . The regulatory function of the GT-1 element in the salt induction of OsRAV2 was verified in situ in plants with targeted mutations generated using the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) system. Taken together, our results indicate that the GT-1 element directly controls the salt response of OsRAV2. This study provides a better understanding of the putative functions of OsRAVs and the molecular regulatory mechanisms of plant genes under salt stress.

Using an organic radical precursor as an electron injection material for efficient and stable organic light-emitting diodes.

Materials with strong reducibility have been used as electron injection layers (EILs) to lower the work function of cathodes and reduce the driving voltage of organic light-emitting diodes (OLEDs). However, the most prominent electron injection materials presented so far are high-temperature-evaporable inorganic salts based on alkaline metals, which suffer from a high tendency of metal diffusion throughout the organic layer and thus reduce the device efficiency and stability. Here, we introduce a new kind of EIL based on a stable precursor of a strongly reducing organic radical. By using an organic precursor, we are able to take the advantage of the low-evaporation-temperature and avoid the problem of metal diffusion, thus improving the device efficiency and stability. Ultraviolet photoelectron spectroscopy (UPS) study indicates that inserting a thin layer of organic radical between the electron transport layer and cathode could greatly reduce the electron injection barrier due to the strong interaction of radical with cathode and the electron transporting material. As a result, OLEDs with an organic radical as the EIL showed a 25.2% higher efficiency and 2.2 times longer lifetime than the control device with conventional LiF as the EIL.