Fine-tuning rice heading date through multiplex editing of the regulatory regions of key genes by CRISPR-Cas9.

Heading date (or flowering time) is a key agronomic trait that affects seasonal and regional adaption of rice cultivars. An unoptimized heading date can either not achieve a high yield or has a high risk of encountering abiotic stresses. There is a strong demand on the mild to moderate adjusting the heading date in breeding practice. Genome editing is a promising method which allows more precise and faster changing the heading date of rice. However, direct knock out of major genes involved in regulating heading date will not always achieve a new germplasm with expected heading date. It is still challenging to quantitatively adjust the heading date of elite cultivars with best adaption for broader region. In this study, we used a CRISPR-Cas9 based genome editing strategy called high-efficiency multiplex promoter-targeting (HMP) to generate novel alleles at cis-regulatory regions of three major heading date genes: Hd1, Ghd7 and DTH8. We achieved a series of germplasm with quantitative variations of heading date by editing promoter regions and adjusting the expression levels of these genes. We performed field trials to screen for the best adapted lines for different regions. We successfully expanded an elite cultivar Ningjing8 (NJ8) to a higher latitude region by selecting a line with a mild early heading phenotype that escaped from cold stress and achieved high yield potential. Our study demonstrates that HMP is a powerful tool for quantitatively regulating rice heading date and expanding elite cultivars to broader regions.

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.

Ribonuclease H-like gene SMALL GRAIN2 regulates grain size in rice through brassinosteroid signaling pathway.

Grain size is a key agronomic trait that determines the yield in plants. Regulation of grain size by brassinosteroids (BRs) in rice has been widely reported. However, the relationship between the BR signaling pathway and grain size still requires further study. Here, we isolated a rice mutant, named small grain2 (sg2), which displayed smaller grain and a semi-dwarf phenotype. The decreased grain size was caused by repressed cell expansion in spikelet hulls of the sg2 mutant. Using map-based cloning combined with a MutMap approach, we cloned SG2, which encodes a plant-specific protein with a ribonuclease H-like domain. SG2 is a positive regulator downstream of GLYCOGEN SYNTHASE KINASE2 (GSK2) in response to BR signaling, and its mutation causes insensitivity to exogenous BR treatment. Genetical and biochemical analysis showed that GSK2 interacts with and phosphorylates SG2. We further found that BRs enhance the accumulation of SG2 in the nucleus, and subcellular distribution of SG2 is regulated by GSK2 kinase activity. In addition, Oryza sativa OVATE family protein 19 (OsOFP19), a negative regulator of grain shape, interacts with SG2 and plays an antagonistic role with SG2 in controlling gene expression and grain size. Our results indicated that SG2 is a new component of GSK2-related BR signaling response and regulates grain size by interacting with OsOFP19.

OsRE1 interacts with OsRIP1 to regulate rice heading date by finely modulating Ehd1 expression.

Heading date is a key agronomic trait affecting crop yield. In rice, Early heading date 1 (Ehd1) is an important B-type response regulator in determination of heading date. Although many regulatory factors of Ehd1 expression have been functionally characterized, the direct regulators of Ehd1 largely remain to be identified. Here, we identified a new regulator of Ehd1, OsRE1, that directly binds to the A-box motif in the Ehd1 promoter. Osre1 confers an early heading phenotype due to elevated expression levels of Ehd1. OsRE1 is a nucleus-localized bZIP transcription factor with a diurnal rhythmic expression pattern. Furthermore, we identified an OsRE1-interacting protein, OsRIP1, and demonstrated that OsRIP1 can repress the transcript expression of Ehd1 in an OsRE1-dependent manner. Our genetic data showed that OsRE1 and OsRIP1 may function upstream of Ehd1 in regulating heading date. Together, our results suggest that OsRE1 functions cooperatively with OsRIP1 to regulate heading date through finely modulating the expression of Ehd1. In addition, OsRE1 and OsRIP1 are two minor heading date regulators, which are more desirable for fine-tuning heading date to improve rice regional adaptability.

Transcriptional and post-transcriptional regulation of heading date in rice.

Rice is a facultative short day (SD) plant. In addition to serving as a model plant for molecular genetic studies of monocots, rice is a staple crop for about half of the world's population. Heading date is a critical agronomic trait, and many genes controlling heading date have been cloned over the last 2 decades. The mechanism of flowering in rice from recognition of day length by leaves to floral activation in the shoot apical meristem has been extensively studied. In this review, we summarise current progress on transcriptional and post-transcriptional regulation of heading date in rice, with emphasis on post-translational modifications of key regulators, including Heading date 1 (Hd1), Early heading date 1 (Ehd1), Grain number, plant height, and heading date7 (Ghd7). The contribution of heading date genes to heterosis and the expansion of rice cultivation areas from low-latitude to high-latitude regions are also discussed. To overcome the limitations of diverse genetic backgrounds used in heading date studies and to gain a clearer understanding of flowering in rice, we propose a systematic collection of genetic resources in a common genetic background. Strategies in breeding adapted cultivars by rational design are also discussed.

The rice PLATZ protein SHORT GRAIN6 determines grain size by regulating spikelet hull cell division.

Grain size is a major determinant of cereal grain yields; however, the relevant regulatory mechanisms controlling this trait have not been fully elucidated. The rice (Oryza sativa) mutant short grain6 (sg6) was identified based on its reduced grain length and weight. Here, we functionally characterized the role of SG6 in determining grain size through the regulation of spikelet hull cell division. SG6 encodes a previously uncharacterized plant AT-rich sequence and zinc-binding (PLATZ) protein that is ubiquitously localized throughout the cell and is preferentially expressed in the early developing panicles but not in the endosperm. The overexpression of SG6 resulted in significantly larger and heavier grains, as well as increased plant heights, which is consistent with its elevated spikelet hull cell division rate. Yeast two-hybrid analyses revealed that SG6 interacts with the core cell cycle machinery DP protein and several other putative cell division regulators, consistent with our transcriptomic analysis, which showed that SG6 activates the expression of many DNA replication and cell-cycle-related genes. These results confirm the crucial role of SG6 in determining grain size by regulating spikelet hull cell division and provide clues for understanding the functions of PLATZ family proteins and the network regulating cereal grain size.

Deficiency of mitochondrial outer membrane protein 64 confers rice resistance to both piercing-sucking and chewing insects in rice.

The brown planthopper (BPH) and striped stem borer (SSB) are the most devastating insect pests in rice (Oryza sativa) producing areas. Screening for endogenous resistant genes is the most practical strategy for rice insect-resistance breeding. Forty-five mutants showing high resistance against BPH were identified in a rice T-DNA insertion population (11,000 putative homozygous lines) after 4 years of large-scale field BPH-resistance phenotype screening. Detailed analysis showed that deficiency of rice mitochondrial outer membrane protein 64 (OM64) gene resulted in increased resistance to BPH. Mitochondrial outer membrane protein 64 protein is located in the outer mitochondrial membrane by subcellular localization and its deficiency constitutively activated hydrogen peroxide (H2 O2 ) signaling, which stimulated antibiosis and tolerance to BPH. The om64 mutant also showed enhanced resistance to SSB, a chewing insect, which was due to promotion of Jasmonic acid biosynthesis and related responses. Importantly, om64 plants presented no significant changes in rice yield-related characters. This study confirmed OM64 as a negative regulator of rice herbivore resistance through regulating H2 O2 production. Mitochondrial outer membrane protein 64 is a potentially efficient candidate to improve BPH and SSB resistance through gene deletion. Why the om64 mutant was resistant to both piercing-sucking and chewing insects via a gene deficiency in mitochondria is discussed.

Post-transcriptional regulation of Ghd7 protein stability by phytochrome and OsGI in photoperiodic control of flowering in rice.

Rice (Oryza sativa) is a facultative short-day (SD) plant, flowering early under SD and late under long-day (LD) conditions. Ghd7 is a major regulator of flowering time in rice, which strongly delays flowering under LD. Induction of Ghd7 expression by phytochromes has been shown to contribute to photoperiodic regulation of flowering in rice. Here, we show that Ghd7 also is regulated by phytochromes at a post-transcriptional level. We found that constitutive expression of Ghd7 delays flowering in the wild-type (WT) background, but not in the se5 mutant background (deficient in functional phytochromes) under LD and that Ghd7 protein fails to accumulate in the se5 mutant. We also found that co-expressing OsGIGANTEA (OsGI) with Ghd7 causes reduced accumulation of Ghd7 protein and partially suppresses the delayed flowering phenotype in the WT background, suggesting that phytochromes and OsGI play antagonist roles in regulating Ghd7 protein stability and flowering time. We show that OsPHYA, OsPHYB and OsGI could directly interact with Ghd7. Interestingly, OsPHYA and OsPHYB could inhibit the interaction between OsGI and Ghd7, thus helping to stabilize Ghd7 protein. Our results revealed a new level of Ghd7 regulation by phytochromes and OsGI in photoperiodic control of flowering in rice.

Global Analysis Reveals the Crucial Roles of DNA Methylation during Rice Seed Development.

Seed development is an important process of reproductive development and consists of embryo and endosperm development; both comprise several key processes. To determine and investigate the functions of the dynamic DNA methylome during seed development, we profiled the DNA methylation genome wide in a series of developmental stages of rice (Oryza sativa) embryo and endosperm by methylcytosine immunoprecipitation followed by Illumina sequencing. The results showed that embryo is hypermethylated predominantly around non-transposable element (TE) genes, short DNA-TEs, and short interspersed TEs compared with endosperm, and non-TE genes have the most diverse methylation status across seed development. In addition, lowly expressed genes are significantly enriched in hypermethylated genes, but not vice versa, confirming the crucial role of DNA methylation in suppressing gene transcription. Further analysis revealed the significantly decreased methylation at early developing stages (from 2 to 3 d after pollination), indicating a predominant role of demethylation during early endosperm development and that genes with a consistent negative correlation between DNA methylation change and expression change may be potentially directly regulated by DNA methylation. Interestingly, comparative analysis of the DNA methylation profiles revealed that both rice indica and japonica subspecies showed robust fluctuant profiles of DNA methylation levels in embryo and endosperm across seed development, with the highest methylation level at 6 d after pollination (2 d after pollination of endosperm in japonica as well), indicating that a complex and finely controlled methylation pattern is closely associated with seed development regulation. The systemic characterization of the dynamic DNA methylome in developing rice seeds will help us understand the effects and mechanism of epigenetic regulation in seed development.

CRISPR Primer Designer: Design primers for knockout and chromosome imaging CRISPR-Cas system.

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated system enables biologists to edit genomes precisely and provides a powerful tool for perturbing endogenous gene regulation, modulation of epigenetic markers, and genome architecture. However, there are concerns about the specificity of the system, especially the usages of knocking out a gene. Previous designing tools either were mostly built-in websites or ran as command-line programs, and none of them ran locally and acquired a user-friendly interface. In addition, with the development of CRISPR-derived systems, such as chromosome imaging, there were still no tools helping users to generate specific end-user spacers. We herein present CRISPR Primer Designer for researchers to design primers for CRISPR applications. The program has a user-friendly interface, can analyze the BLAST results by using multiple parameters, score for each candidate spacer, and generate the primers when using a certain plasmid. In addition, CRISPR Primer Designer runs locally and can be used to search spacer clusters, and exports primers for the CRISPR-Cas system-based chromosome imaging system.

Pollen semi-sterility1 encodes a kinesin-1-like protein important for male meiosis, anther dehiscence, and fertility in rice.

In flowering plants, male meiosis produces four microspores, which develop into pollen grains and are released by anther dehiscence to pollinate female gametophytes. The molecular and cellular mechanisms regulating male meiosis in rice (Oryza sativa) remain poorly understood. Here, we describe a rice pollen semi-sterility1 (pss1) mutant, which displays reduced spikelet fertility (~40%) primarily caused by reduced pollen viability (~50% viable), and defective anther dehiscence. Map-based molecular cloning revealed that PSS1 encodes a kinesin-1-like protein. PSS1 is broadly expressed in various organs, with highest expression in panicles. Furthermore, PSS1 expression is significantly upregulated during anther development and peaks during male meiosis. The PSS1-green fluorescent protein fusion is predominantly localized in the cytoplasm of rice protoplasts. Substitution of a conserved Arg (Arg-289) to His in the PSS1 motor domain nearly abolishes its microtubule-stimulated ATPase activity. Consistent with this, lagging chromosomes and chromosomal bridges were found at anaphase I and anaphase II of male meiosis in the pss1 mutant. Together, our results suggest that PSS1 defines a novel member of the kinesin-1 family essential for male meiotic chromosomal dynamics, male gametogenesis, and anther dehiscence in rice.

The RNA binding protein EHD6 recruits the m6A reader YTH07 and sequesters OsCOL4 mRNA into phase-separated ribonucleoprotein condensates to promote rice flowering.

N6-Methyladenosine (m6A) is one of the most abundant modifications of eukaryotic mRNA, but its comprehensive biological functionality remains further exploration. In this study, we identified and characterized a new flowering-promoting gene, EARLY HEADING DATE6 (EHD6), in rice. EHD6 encodes an RNA recognition motif (RRM)-containing RNA binding protein that is localized in the non-membranous cytoplasm ribonucleoprotein (RNP) granules and can bind both m6A-modified RNA and unmodified RNA indiscriminately. We found that EHD6 can physically interact with YTH07, a YTH (YT521-B homology) domain-containing m6A reader. We showed that their interaction enhances the binding of an m6A-modified RNA and triggers relocation of a portion of YTH07 from the cytoplasm into RNP granules through phase-separated condensation. Within these condensates, the mRNA of a rice flowering repressor, CONSTANS-like 4 (OsCOL4), becomes sequestered, leading to a reduction in its protein abundance and thus accelerated flowering through the Early heading date 1 pathway. Taken together, these results not only shed new light on the molecular mechanism of efficient m6A recognition by the collaboration between an RNA binding protein and YTH family m6A reader, but also uncover the potential for m6A-mediated translation regulation through phase-separated ribonucleoprotein condensation in rice.

The N6-methyladenosine binding proteins YTH03/05/10 coordinately regulate rice plant height.

N6-methyladenosine (m6A) is the most widely distributed and most abundant type of mRNA modification in eukaryotic. It provides a posttranscriptional level regulation of gene expression by regulating pre-mRNA splicing, mRNA degradation, or mRNA translational efficiency etc. The function of m6A modification is decoded by binding proteins that can specially bind to m6A. YT521-B homology (YTH) family proteins are the most important m6A-binding proteins in mammals and Arabidopsis. However, their roles in growth and development remain unknown. Here, we demonstrated that the YTH family proteins YTH03, YTH05 and YTH10 specifically bind to m6A-containing RNAs. Knockout of YTH03, YTH05 or YTH10 causes reduced plant height. Further research showed that simultaneously knockout of YTH03, YTH05 and YTH10 shows severe dwarf phenotype, suggesting these three genes regulate rice plant height in a functionally redundant manner. Additional transcriptome study showed that the reduced plant height of the yth03/05/10 triple mutant may be due to the blocked of diterpenoid and brassinolide synthesis pathway. Overall, we demonstrate that YTH03, YTH05 and YTH10 are all the m6A readers in rice and redundantly regulate rice plant height through the hormonal related pathway.

An ethylene response factor OsWR1 responsive to drought stress transcriptionally activates wax synthesis related genes and increases wax production in rice.

Increasing evidence has revealed the major enzymes-involved in Arabidopsis and maize wax/cutin synthesis; however, there is limited information about the genes-associated with wax/cutin synthesis in rice. Here we report the characterization of an ethylene response factor gene in rice. This rice wax synthesis regulatory gene 1 (OsWR1) is a homolog of Arabidopsis wax/cutin synthesis regulatory gene WIN1/SHN1. Transcript analysis showed that OsWR1 is induced by drought, abscisic acid and salt, and is predominantly expressed in leaves. Functional analyses indicated that overexpressing OsWR1 (Ox-WR1) improved while RNA interference OsWR1 rice (RI-WR1) decreased drought tolerance, consistent with water loss and cuticular permeability, suggesting that OsWR1-triggered drought response might be associated with cuticular characteristics. In addition, OsWR1 activated the expression of the genes-related to oxidative stress response and membrane stability. Gas chromatograph-mass spectrometry analysis further showed that OsWR1 modulated the wax synthesis through alteration of long chain fatty acids and alkanes, evidencing the regulation of OsWR1 in wax synthesis. Detection with real-time PCR amplification indicated that Ox-WR1 enhanced while RI-WR1 decreased the expression of wax/cutin synthesis related genes. Furthermore, OsWR1 physically interacted with the DRE and GCC box in the promoters of wax related genes OsLACS2 and OsFAE1'-L, indicating that OsWR1 at least directly modulates the expression of these genes. Thus our results indicate that OsWR1 is a positive regulator of wax synthesis related genes in rice, and this regulation, distinct from its homology regulator of WIN1/SHN1 in cutin synthesis, subsequently contributes to reduced water loss and enhanced drought tolerance.

Dual-fiber optic bioprobe system for triglyceride detection using surface plasmon resonance sensing and lipase-immobilized magnetic bead hydrolysis.

The rapid and accurate detection of triglyceride (TG) plays a valuable role in the prevention and control of dyslipidemia. In this paper, a novel method for TG detection using a dual-fiber optic bioprobe system, which can accurately detect different levels of TG concentration in serum, is proposed. The system employs disposable microprobe-type fiber optic surface plasmon resonance (SPR) biosensors for signal acquisition, providing high stability and portability while avoiding cross-contamination caused by repeated use. The proposed biosensor with a high sensitivity of 1.25 nm/(mg/mL) for TG detection in serum and a tiny diameter of 125 µm, was fabricated using a novel multimode fiber-single-mode fiber-reflector (MSR) structure, which has been scarcely ever reported to the best of our knowledge. In the process of TG detection, lipase-immobilized magnetic beads were introduced to specifically hydrolyze TG, and the relationship between the TG content and the SPR differential signal was obtained from dual-fiber optic bioprobe measurements of the TG sample before and after hydrolysis. The proposed method achieved TG detection in the concentration range of 0-8 mg/mL (including healthy and unhealthy levels of TG concentration in the human body). Additionally, the miniaturized fiber optic biosensors used in this work have the advantages of low sample consumption, high sensitivity, simple operation, label-free measurement, high selectivity, and low cost. This method provides a new pathway for rapid and reliable TG detection and has potential applications in medical research and clinical diagnosis.

A novel multimeric sCD19-streptavidin fusion protein for functional detection and selective expansion of CD19-targeted CAR-T cells.

BACKGROUND: CARs are engineered receptors comprising an immunoglobulin single-chain variable fragment (scFv) that identifies and binds to the target antigen, a transmembrane domain, and an intracellular T-cell signaling domain. CD19 is a B lineage-specific transmembrane glycoprotein and is expressed in more than 95% of B-cell malignancies. Streptavidin (SA) is a homo-tetrameric protein derived from Streptomyces avidinii, which can bind four biotin molecules with an extremely high affinity at a Kd value of 10-15 M. AIMS: The aim of the study is to generate a novel soluble multimeric fusion protein, sCD19-streptavidin (sCD19-SA) for functional detection and selective expansion of CD19-targeted CAR-T cells. METHODS: The fusion proteins CD19-SA was expressed in CHO cells and purified by use of Ni-nitrilotriacetic acid agarose beads. RESULTS: A novel fusion protein (sCD19-SA) was generated, consisting of the extracellular domain of human CD19 and the core region of SA, and could be used to functionally detect CD19-targeted CAR-T cells. Furthermore, this protein was demonstrated to form multimers to activate CAR-T cells to induce their selective expansion. Importantly, sCD19-SA-stimulated CD19-targeted CAR-T cells could improve antitumor effects in vivo. CONCLUSIONS: Our study has highlighted the potential of utilizing antigen-SA fusion proteins such as sCD19-SA for CAR-T therapy for the functional detection of CAR expression and selective expansion of CAR-T cells.

YTH Domain Proteins Play an Essential Role in Rice Growth and Stress Response.

As the most prevalent epi-transcriptional modification, m6A modifications play essential roles in regulating RNA fate. The molecular functions of YTH521-B homology (YTH) domain proteins, the most known READER proteins of m6A modifications, have been well-studied in animals. Although plants contain more YTH domain proteins than other eukaryotes, little is known about their biological importance. In dicot species Arabidopsis thaliana, the YTHDFA clade members ECT2/3/4 and CPSF30-L are well-studied and important for cell proliferation, plant organogenesis, and nitrate transport. More emphasis is needed on the biological functions of plant YTH proteins, especially monocot YTHs. Here we presented a detailed phylogenetic relationship of eukaryotic YTH proteins and clustered plant YTHDFC clade into three subclades. To determine the importance of monocot YTH proteins, YTH knockout mutants and RNAi-induced knockdown plants were constructed and used for phenotyping, transcriptomic analysis, and stress treatments. Knocking out or knocking down OsYTHs led to the downregulation of multicellular organismal regulation genes and resulted in growth defects. In addition, loss-of-function ythdfa mutants led to better salinity tolerance whereas ythdfc mutants were more sensitive to abiotic stress. Overall, our study establishes the functional relevance of rice YTH genes in plant growth regulation and stress response.

DHD4, a CONSTANS-like family transcription factor, delays heading date by affecting the formation of the FAC complex in rice.

Heading date (or flowering time) is one of the most important agronomic traits in rice, influencing its regional adaptability and crop yield. Many major-effect genes for rice heading date have been identified, but in practice they are difficult to be used for rice molecular breeding because of their dramatic effects on heading date. Genes with minor effects on heading date, which are more desirable for fine-tuning flowering time without significant yield penalty, were seldom reported. In this study, we identified a new minor-effect heading date repressor, Delayed Heading Date 4 (DHD4). The dhd4 mutant shows a slightly earlier flowering phenotype without a notable yield penalty compared with wild-type plants under natural long-day conditions. DHD4 encodes a CONSTANS-like transcription factor localized in the nucleus. Molecular, biochemical, and genetic assays show that DHD4 can compete with 14-3-3 to interact with OsFD1, thus affecting the formation of the Hd3a-14-3-3-OsFD1 tri-protein FAC complex, resulting in reduced expression of OsMADS14 and OsMADS15, and ultimately delaying flowering. Taken together, these results shed new light on the regulation of flowering time in rice and provide a promising target for fine-tuning flowering time to improve the regional adaptability of rice.

The pilot-scale preparation of the SA-hGM-CSF bi-functional fusion protein.

The granulocyte-macrophage colony-stimulating factor (GM-CSF) can be used to induce a powerful immune response. Based on the specific binding of biotin and streptavidin, SA-hGM-CSF was anchored on the surface of biotinylated tumor cells, which could enhance the anti-tumor effect of tumor cell vaccines in our previous reports, suggesting it would have potential clinical value. Preparation of the biologically active proteins in large-scale production is the basis of clinical application, however, only a small amount of biologically active protein was obtained according to previous studies. In this study, we researched the effects of various factors on the purification and simultaneous renaturation of SA-hGM-CSF fusion protein by single factor experiment and orthogonal experiment. Here, we developed a viable pilot-scale trial in the fermentation, purification, refolding and freeze-drying of SA-hGM-CSF proteins in order to efficiently obtain more biologically active proteins with high purity, which will lay the foundation for industrial production.