Efficient genome editing in rice with miniature Cas12f variants.

Genome editing, particularly using the CRISPR/Cas system, has revolutionized biological research and crop improvement. Despite the widespread use of CRISPR/Cas9, it faces limitations such as PAM sequence requirements and challenges in delivering its large protein into plant cells. The hypercompact Cas12f, derived from Acidibacillus sulfuroxidans (AsCas12f), stands out due to its small size of only 422 amino acids and its preference for a T-rich motif, presenting advantageous features over SpCas9. However, its editing efficiency is extremely low in plants. Recent studies have generated two AsCas12f variants, AsCas12f-YHAM and AsCas12f-HKRA, demonstrating higher editing efficiencies in mammalian cells, yet their performance in plants remains unexplored. In this study, through a systematic investigation of genome cleavage activity in rice, we unveiled a substantial enhancement in editing efficiency for both AsCas12f variants, particularly for AsCas12f-HKRA, which achieved an editing efficiency of up to 53%. Furthermore, our analysis revealed that AsCas12f predominantly induces deletion in the target DNA, displaying a unique deletion pattern primarily concentrated at positions 12, 13, 23, and 24, resulting in deletion size mainly of 10 and 11 bp, suggesting significant potential for targeted DNA deletion using AsCas12f. These findings expand the toolbox for efficient genome editing in plants, offering promising prospects for precise genetic modifications in agriculture. Supplementary Information: The online version contains supplementary material available at 10.1007/s42994-024-00168-2.

Fabrication of Superhydrophobic Porous Brass by Chemical Dealloying for Efficient Emulsion Separation.

By taking advantage of typical dealloying and subsequent aging methods, a novel homogeneous porous brass with a micro/nano hierarchical structure was prepared without any chemical modification. The treatment of commercial brass with hot concentrated HCl solution caused preferential etching of Zn from Cu62Zn38 alloy foil, leaving a microporous skeleton with an average tortuous channel size of 1.6 µm for liquid transfer. After storage in the atmosphere for 7 days, the wettability of the dealloyed brass changed from superhydrophilic to superhydrophobic with a contact angle > 156° and sliding angle < 7°. The aging treatment enhanced the hydrophobicity of the brass by the formation of Cu2O on the surface. By virtue of the opposite wettability to water and oil, the aged brass separated surfactant-stabilized water-in-oil emulsions with separation efficiency of over 99.4% and permeate flux of about 851 L·m-2·h-1 even after recycling for 60 times. After 10 times of tape peeling or sandpaper abrasion, the aged brass maintained its superhydrophobicity, indicating its excellent mechanical stability. Moreover, the aged brass still retained its superhydrophobicity after exposure to high temperatures or corrosive solutions, displaying high resistance to extreme environments. The reason may be that the bicontinuous porous structure throughout the whole foil endows stable mechanical properties to tolerate extreme environments. This method should have a promising future in expanding the applications of alloys.

Progress in Electrochemical Immunosensors with Alkaline Phosphatase as the Signal Label.

Electrochemical immunosensors have shown great potential in clinical diagnosis, food safety, environmental protection, and other fields. The feasible and innovative combination of enzyme catalysis and other signal-amplified elements has yielded exciting progress in the development of electrochemical immunosensors. Alkaline phosphatase (ALP) is one of the most popularly used enzyme reporters in bioassays. It has been widely utilized to design electrochemical immunosensors owing to its significant advantages (e.g., high catalytic activity, high turnover number, and excellent substrate specificity). In this work, we summarized the achievements of electrochemical immunosensors with ALP as the signal reporter. We mainly focused on detection principles and signal amplification strategies and briefly discussed the challenges regarding how to further improve the performance of ALP-based immunoassays.

The protein phosphatase PC1 dephosphorylates and deactivates CatC to negatively regulate H2O2 homeostasis and salt tolerance in rice.

Catalase (CAT) is often phosphorylated and activated by protein kinases to maintain hydrogen peroxide (H2O2) homeostasis and protect cells against stresses, but whether and how CAT is switched off by protein phosphatases remains inconclusive. Here, we identified a manganese (Mn2+)-dependent protein phosphatase, which we named PHOSPHATASE OF CATALASE 1 (PC1), from rice (Oryza sativa L.) that negatively regulates salt and oxidative stress tolerance. PC1 specifically dephosphorylates CatC at Ser-9 to inhibit its tetramerization and thus activity in the peroxisome. PC1 overexpressing lines exhibited hypersensitivity to salt and oxidative stresses with a lower phospho-serine level of CATs. Phosphatase activity and seminal root growth assays indicated that PC1 promotes growth and plays a vital role during the transition from salt stress to normal growth conditions. Our findings demonstrate that PC1 acts as a molecular switch to dephosphorylate and deactivate CatC and negatively regulate H2O2 homeostasis and salt tolerance in rice. Moreover, knockout of PC1 not only improved H2O2-scavenging capacity and salt tolerance but also limited rice grain yield loss under salt stress conditions. Together, these results shed light on the mechanisms that switch off CAT and provide a strategy for breeding highly salt-tolerant rice.

Receptor-Like Cytoplasmic Kinase STK Confers Salt Tolerance in Rice.

BACKGROUND: Soil salinization is a major abiotic environmental stress factor threatening crop production throughout the world. Salt stress drastically affects the growth, development, and grain yield of rice (Oryza sativa L.), and the improvement of rice tolerance to salt stress is a desirable approach for meeting increasing food demand. Receptor-like cytoplasmic kinases (RLCKs) play essential roles in plant growth, development and responses to environmental stresses. However, little is known about their functions in salt stress. Previous reports have demonstrated that overexpression of an RLCK gene SALT TOLERANCE KINASE (STK) enhances salt tolerance in rice, and that STK may regulate the expression of GST (Glutathione S-transferase) genes. RESULTS: The expression of STK was rapidly induced by ABA. STK was highest expressed in the stem at the heading stage. STK was localized at the plasma membrane. Overexpression of STK in rice increased tolerance to salt stress and oxidative stress by increasing ROS scavenging ability and ABA sensitivity. In contrast, CRISPR/Cas9-mediated knockout of STK increased the sensitivity of rice to salt stress and oxidative stress. Transcriptome sequencing analysis suggested that STK increased the expression of GST genes (LOC_Os03g17480, LOC_Os10g38140 and LOC_Os10g38710) under salt stress. Reverse transcription quantitative PCR (RT-qPCR) suggested that four stress-related genes may be regulated by STK including OsABAR1, Os3BGlu6, OSBZ8 and OsSIK1. CONCLUSIONS: These findings suggest that STK plays a positive regulatory role in salt stress tolerance by inducing antioxidant defense and associated with the ABA signaling pathway in rice.

Transcriptome profiling of two super hybrid rice provides insights into the genetic basis of heterosis.

BACKGROUND: Heterosis is a phenomenon that hybrids show superior performance over their parents. The successful utilization of heterosis has greatly improved rice productivity, but the molecular basis of heterosis remains largely unclear. RESULTS: Here, the transcriptomes of young panicles and leaves of the two widely grown two-line super hybrid rice varieties (Jing-Liang-You-Hua-Zhan (JLYHZ) and Long-Liang-You-Hua-Zhan (LLYHZ)) and their parents were analyzed by RNA-seq. Transcriptome profiling of the hybrids revealed 1,778 ~ 9,404 differentially expressed genes (DEGs) in two tissues, which were identified by comparing with their parents. GO, and KEGG enrichment analysis showed that the pathways significantly enriched in both tissues of two hybrids were all related to yield and resistance, like circadian rhythm (GO:0,007,623), response to water deprivation (GO:0,009,414), and photosynthetic genes (osa00196). Allele-specific expression genes (ASEGs) were also identified in hybrids. The ASEGs were most significantly enriched in ionotropic glutamate receptor signaling pathway, which was hypothesized to be potential amino acid sensors in plants. Moreover, the ASEGs were also differentially expressed between parents. The number of variations in ASEGs is higher than expected, especially for large effect variations. The DEGs and ASEGs are the potential reasons for the formation of heterosis in the two elite super hybrid rice. CONCLUSIONS: Our results provide a comprehensive understanding of the heterosis of two-line super hybrid rice and facilitate the exploitation of heterosis in hybrid rice breeding with high yield heterosis.

Engineering of rice varieties with enhanced resistances to both blast and bacterial blight diseases via CRISPR/Cas9.

Rice blast and bacterial blight represent two of major diseases having devastating impact on the yield of rice in most rice-growing countries. Developments of resistant cultivars are the most economic and effective strategy to control these diseases. Here, we used CRISPR/Cas9-mediated gene editing to rapidly install mutations in three known broad-spectrum blast-resistant genes, Bsr-d1, Pi21 and ERF922, in an indica thermosensitive genic male sterile (TGMS) rice line Longke638S (LK638S). We obtained transgene-free homozygous single or triple mutants in T1 generations. While all single and triple mutants showed increased resistance to rice blast compared with wild type, the erf922 mutants displayed the strongest blast resistance similar with triple mutants. Surprisingly, we found that Pi21 or ERF922 single mutants conferred enhanced resistance to most of tested bacterial blight. Both resistances in mutants were attribute to the up-regulation of SA- and JA-pathway associated genes. Moreover, phenotypic analysis of these single mutants in paddy fields revealed that there were no trade-offs between resistances and main agricultural traits. Together, our study provides a rapid and effective way to generate rice varieties with resistance to both rice blast and bacterial blight.

Validating a segment on chromosome 7 of japonica for establishing low-cadmium accumulating indica rice variety.

Cadmium (Cd) contamination of rice is a serious food safety issue that has recently been gaining significant public attention. Therefore, reduction of Cd accumulation in rice grains is an important objective of rice breeding. The use of favourable alleles of Cd accumulating genes using marker-assisted selection (MAS) is theoretically feasible. In this study, we validated a segment covering OsHMA3-OsNramp5-OsNramp1 on chromosome 7 of japonica for establishing low-cadmium accumulating indica rice variety. The OsHMA3-OsNramp5-OsNramp1jap haplotype significantly decreased grain Cd concentration in middle-season indica genetic background. The improved 9311 carrying the OsHMA3-OsNramp5-OsNramp1jap haplotype with recurrent parent genome recovery of up to 91.6% resulted in approximately 31.8% decrease in Cd accumulation in the grain and with no penalty on yield. There is a genetic linkage-drag between OsHMA3-OsNramp5-OsNramp1 jap and the gene conditioning heading to days (HTD) in the early-season indica genetic background. Because the OsHMA3-OsNramp5-OsNramp1-Ghd7jap haplotype significantly increases grain Cd concentration and prolongs growth duration, the linkage-drag between OsHMA3-OsNramp5-OsNramp1 and Ghd7 should be broken down by large segregating populations or gene editing. A novel allele of OsHMA3 was identified from a wide-compatibility japonica cultivar, the expression differences of OsNramp1 and OsNramp5 in roots might contribute the Cd accumulating variation between japonica and indica variety.

The Receptor-Like Cytoplasmic Kinase STRK1 Phosphorylates and Activates CatC, Thereby Regulating H2O2 Homeostasis and Improving Salt Tolerance in Rice.

Salt stress can significantly affect plant growth and agricultural productivity. Receptor-like kinases (RLKs) are believed to play essential roles in plant growth, development, and responses to abiotic stresses. Here, we identify a receptor-like cytoplasmic kinase, salt tolerance receptor-like cytoplasmic kinase 1 (STRK1), from rice (Oryza sativa) that positively regulates salt and oxidative stress tolerance. Our results show that STRK1 anchors and interacts with CatC at the plasma membrane via palmitoylation. CatC is phosphorylated mainly at Tyr-210 and is activated by STRK1. The phosphorylation mimic form CatCY210D exhibits higher catalase activity both in vitro and in planta, and salt stress enhances STRK1-mediated tyrosine phosphorylation on CatC. Compared with wild-type plants, STRK1-overexpressing plants exhibited higher catalase activity and lower accumulation of H2O2 as well as higher tolerance to salt and oxidative stress. Our findings demonstrate that STRK1 improves salt and oxidative tolerance by phosphorylating and activating CatC and thereby regulating H2O2 homeostasis. Moreover, overexpression of STRK1 in rice not only improved growth at the seedling stage but also markedly limited the grain yield loss under salt stress conditions. Together, these results offer an opportunity to improve rice grain yield under salt stress.

Over-expression of a glutamate dehydrogenase gene, MgGDH, from Magnaporthe grisea confers tolerance to dehydration stress in transgenic rice.

MAIN CONCLUSION: Heterologous expression of a fungal NADP(H)-GDH gene ( MgGDH ) from Magnaporthe grisea can improve dehydration stress tolerance in rice by preventing toxic accumulation of ammonium. Glutamate dehydrogenase (GDH; EC 1.4.1.2 and EC 1.4.1.4) may act as a stress-responsive enzyme in detoxification of high intracellular ammonia and production of glutamate for proline synthesis under stress conditions. In present study, a fungal NADP(H)-GDH gene (MgGDH) from Magnaporthe grisea was over-expressed in rice (Oryza sativa L. cv. 'kitaake'), and the transgenic plants showed the improvement of tolerance to dehydration stress. The kinetic analysis showed that His-TF-MgGDH preferentially utilizes ammonium to produce L-glutamate. Moreover, the affinity of His-TF-MgGDH for ammonium was dramatically higher than that of His-TF-OsGDH for ammonium. Over-expressing MgGDH transgenic rice plants showed lower water-loss rate and higher completely close stomata than the wild-type plants under dehydration stress conditions. In transgenic plants, the NADP(H)-GDH activities were markedly higher than those in wild-type plants and the amination activity was significantly higher than the deamination activity. Compared with wild-type plants, the transgenic plants accumulated much less NH4 (+) but higher amounts of glutamate, proline and soluble sugar under dehydration stress conditions. These results indicate that heterologous expression of MgGDH can prevent toxic accumulation of ammonium and in return improve dehydration stress tolerance in rice.

Molecular cloning, characterization and function analysis of a GDH gene from Sclerotinia sclerotiorum in rice.

The full-length cDNA encoding a glutamate dehydrogenase (GDH) which catalyzes the reaction of reductive amination of α-oxoglutarate (α-OG) to glutamate (the anabolic activity) and the reverse reaction of oxidative deamination of glutamate (the catabolic activity) was isolated from Sclerotinia sclerotiorum, we designated it as SsGDH. Bioinformatics analysis revealed that SsGDH had a typical GDH spatial structure and extensive homology with other fungal or bacteria GDHs. To evaluate its function in rice, rice (Oryza sativa L. cv. 'kitaake') was transformed with SsGDH in a binary vector construct by Agrobacterium-mediated transformation. Transgenic rice plants showed that transcripts and proteins of SsGDH accumulated at higher levels and GDH enzymatic activity was obviously higher in transgenic rice plants compared with the non-transformant rice plants (CK), though phenotype including plant height, fresh weight and dry weight became slightly weaker compared with CK under 50, 500 and 5,000 µM nitrogen gradient nutrient solution treatment (NH4NO3 as a nitrogen source) after introducing SsGDH into rice. For enzymatic activity assay in vitro, recombinant His6-SsGDH protein was expressed in Escherichia coli BL21 (DE3) and purified by Ni-NTA agarose. Results suggested that recombinant His6-SsGDH protein had GDH activity using ammonium, α-OG, and L-glutamate separately as a substrate at two different concentrations, especially the affinity for ammonium was very high, and its Km value was only 0.28 ± 0.03 mM, indicating that SsGDH can assimilate more ammonium into rice. According to previous reports, transgenic plants expressing fungal or bacteria GDHs might show improved herbicide resistance. Basta resistance test showed that SsGDH expression in rice can significantly enhanced their tolerance to Basta than CK. In conclusion, our results may provide some clues for further investigation on nitrogen utilization via introducing exogenous GDHs from lower organisms into rice.

Construct hierarchical superhydrophobic silicon surfaces by chemical etching.

We present a simple approach for preparing hydrophobic silicon surfaces by constructing silicon nanowire arrays using Ag-assisted chemical etching without low-surface-energy material modification. The static and dynamic wetting properties of the nanostructured surfaces and their dependence on etching conditions were studied. It was revealed that the surface topologies of silicon nanowire arrays and their corresponding wetting properties could be tuned by varying the etching time. Under optimized etching conditions, superhydrophobic surfaces with an apparent contact angle larger than 150 degrees and a sliding angle smaller than 10 degrees were achieved due to the formation of a hierarchical structure. The origin of hydrophobic behavior was discussed based on Wenzel and Cassie models. In addition, the effects of surface modification of Si surface nanostructures on their hydrophobic characteristics were also investigated.

Inter- and intra-molecular H-bonds induced different nanostructures from a multi-H-bonding (MHB) amphiphile: nanofibers and nanodisks.

An MHB amphiphile, N-stearoyl-l-glutamic acid (C(18)-Glu), forms disk- and fiber-like nanostructures respectively in hydrophilic and hydrophobic environments due to the inter- and intra-molecular H-bonds.