Comprehensive analysis of MAPK gene family in upland cotton (Gossypium hirsutum) and functional characterization of GhMPK31 in regulating defense response to insect infestation.

KEY MESSAGE: The transcriptomic, phenotypic and metabolomic analysis of transgenic plants overexpressing GhMPK31 in upland cotton revealed the regulation of H2O2 burst and the synthesis of defensive metabolites by GhMPK31. Mitogen-activated protein kinases (MAPKs) are a crucial class of protein kinases, which play an essential role in various biological processes in plants. Upland cotton (G. hirsutum) is the most widely cultivated cotton species with high economic value. To gain a better understanding of the role of the MAPK gene family, we conducted a comprehensive analysis of the MAPK gene family in cotton. In this study, a total of 55 GhMPK genes were identified from the whole genome of G. hirsutum. Through an investigation of the expression patterns under diverse stress conditions, we discovered that the majority of GhMPK family members demonstrated robust responses to abiotic stress, pathogen stress and pest stress. Furthermore, the overexpression of GhMPK31 in cotton leaves led to a hypersensitive response (HR)-like cell death phenotype and impaired the defense capability of cotton against herbivorous insects. Transcriptome and metabolomics data analysis showed that overexpression of GhMPK31 enhanced the expression of H2O2-related genes and reduced the accumulation of defensive related metabolites. The direct evidence of GhMPK31 interacting with GhRBOHB (H2O2-generating protein) were found by Y2H, BiFC, and LCI. Therefore, we propose that the increase of H2O2 content caused by overexpression of GhMPK31 resulted in HR-like cell death in cotton leaves while reducing the accumulation of defensive metabolites, ultimately leading to a decrease in the defense ability of cotton against herbivorous insects. This study provides valuable insights into the function of MAPK genes in plant resistance to herbivorous insects.

Precise fine-turning of GhTFL1 by base editing tools defines ideal cotton plant architecture.

BACKGROUND: CRISPR/Cas-derived base editor enables precise editing of target sites and has been widely used for basic research and crop genetic improvement. However, the editing efficiency of base editors at different targets varies greatly. RESULTS: Here, we develop a set of highly efficient base editors in cotton plants. GhABE8e, which is fused to conventional nCas9, exhibits 99.9% editing efficiency, compared to GhABE7.10 with 64.9%, and no off-target editing is detected. We further replace nCas9 with dCpf1, which recognizes TTTV PAM sequences, to broaden the range of the target site. To explore the functional divergence of TERMINAL FLOWER 1 (TFL1), we edit the non-coding and coding regions of GhTFL1 with 26 targets to generate a comprehensive allelic population including 300 independent lines in cotton. This allows hidden pleiotropic roles for GhTFL1 to be revealed and allows us to rapidly achieve directed domestication of cotton and create ideotype germplasm with moderate height, shortened fruiting branches, compact plant, and early-flowering. Further, by exploring the molecular mechanism of the GhTFL1L86P and GhTFL1K53G+S78G mutations, we find that the GhTFL1L86P mutation weakens the binding strength of the GhTFL1 to other proteins but does not lead to a complete loss of GhTFL1 function. CONCLUSIONS: This strategy provides an important technical platform and genetic information for the study and creation of ideal plant architecture.

Construction of Host Plant Insect-Resistance Mutant Library by High-Throughput CRISPR/Cas9 System and Identification of A Broad-Spectrum Insect Resistance Gene.

Insects pose significant challenges in cotton-producing regions. Here, they describe a high-throughput CRISPR/Cas9-mediated large-scale mutagenesis library targeting endogenous insect-resistance-related genes in cotton. This library targeted 502 previously identified genes using 968 sgRNAs, generated ≈2000 T0 plants and achieved 97.29% genome editing with efficient heredity, reaching upto 84.78%. Several potential resistance-related mutants (10% of 200 lines) their identified that may contribute to cotton-insect molecular interaction. Among these, they selected 139 and 144 lines showing decreased resistance to pest infestation and targeting major latex-like protein 423 (GhMLP423) for in-depth study. Overexpression of GhMLP423 enhanced insect resistance by activating the plant systemic acquired resistance (SAR) of salicylic acid (SA) and pathogenesis-related (PR) genes. This activation is induced by an elevation of cytosolic calcium [Ca2+ ]cyt flux eliciting reactive oxygen species (ROS), which their demoted in GhMLP423 knockout (CR) plants. Protein-protein interaction assays revealed that GhMLP423 interacted with a human epidermal growth factor receptor substrate15 (EPS15) protein at the cell membrane. Together, they regulated the systemically propagating waves of Ca2+ and ROS, which in turn induced SAR. Collectively, this large-scale mutagenesis library provides an efficient strategy for functional genomics research of polyploid plant species and serves as a solid platform for genetic engineering of insect resistance.

The chromosome-scale reference genome of mirid bugs (Adelphocoris suturalis) genome provides insights into omnivory, insecticide resistance, and survival adaptation.

BACKGROUND: Adelphocoris suturalis (Hemiptera: Miridae) is a notorious agricultural pest, which causes serious economic losses to a diverse range of agricultural crops around the world. The poor understanding of its genomic characteristics has seriously hindered the establishment of sustainable and environment-friendly agricultural pest management through biotechnology and biological insecticides. RESULTS: Here, we report a chromosome-level assembled genome of A. suturalis by integrating Illumina short reads, PacBio, 10x Chromium, and Hi-C mapping technologies. The resulting 1.29 Gb assembly contains twelve chromosomal pseudomolecules with an N50 of 1.4 and 120.6 Mb for the contigs and scaffolds, respectively, and carries 20,010 protein-coding genes. The considerable size of the A. suturalis genome is predominantly attributed to a high amount of retrotransposons, especially long interspersed nuclear elements (LINEs). Transcriptomic and phylogenetic analyses suggest that A. suturalis-specific candidate effectors, and expansion and expression of gene families associated with omnivory, insecticide resistance and reproductive characteristics, such as digestion, detoxification, chemosensory receptors and long-distance migration likely contribute to its strong environmental adaptability and ability to damage crops. Additionally, 19 highly credible effector candidates were identified and transiently overexpressed in Nicotiana benthamiana for functional assays and potential targeting for insect resistance genetic engineering. CONCLUSIONS: The high-quality genome of A. suturalis provides an important genomic landscape for further investigations into the mechanisms of omnivory, insecticide resistance and survival adaptation, and for the development of integrated management strategies.

Establishment of insomnia model of chronic unpredictable stress in rats.

It is well known that stressful situation is one of the important factors causing insomnia, however, the underlying mechanism is still elusive. Therefore, the establishment of a suitable animal model of stress insomnia will be of great help to solve this problem. In this study, by combining with chronic unpredictable stress (multitude of stressors) and sleep deprivation, we attempted to establish a rat model of stress insomnia. It was observed that rats with stress insomnia showed significant weight loss, and less sleep quality in pentobarbital sodium induced sleep test and electroencephalogram detection. Moreover, rats with stress insomnia showed greater depression and anxiety detected by forced swimming, sucrose preference test and open field. Since oxidative stress has been reported to be involved in insomnia, we further evaluated the production of oxidative stress and found that the levels of lipid peroxidation product malondialdehyde (MDA) in liver, serum total bilirubin and urine biopyrrin were all significantly increased in rats with stress insomnia. In addition, we also found that the memory of these rats with stress insomnia was also obviously reduced in water maze. Taken together, these results demonstrate that the emotional behaviors, memory, oxidative and metabolism of the rats were all significantly changed after modeling, indicating a rat model of stress insomnia was successful establishment, and this animal model will provide basis to further explore the underlying mechanism of chronic stress in insomnia.

Single-cell resolution analysis reveals the preparation for reprogramming the fate of stem cell niche in cotton lateral meristem.

BACKGROUND: Somatic embryogenesis is a major process for plant regeneration. However, cell communication and the gene regulatory network responsible for cell reprogramming during somatic embryogenesis are still largely unclear. Recent advances in single-cell technologies enable us to explore the mechanism of plant regeneration at single-cell resolution. RESULTS: We generate a high-resolution single-cell transcriptomic landscape of hypocotyl tissue from the highly regenerable cotton genotype Jin668 and the recalcitrant TM-1. We identify nine putative cell clusters and 23 cluster-specific marker genes for both cultivars. We find that the primary vascular cell is the major cell type that undergoes cell fate transition in response to external stimulation. Further developmental trajectory and gene regulatory network analysis of these cell clusters reveals that a total of 41 hormone response-related genes, including LAX2, LAX1, and LOX3, exhibit different expression patterns in the primary xylem and cambium region of Jin668 and TM-1. We also identify novel genes, including CSEF, PIS1, AFB2, ATHB2, PLC2, and PLT3, that are involved in regeneration. We demonstrate that LAX2, LAX1 and LOX3 play important roles in callus proliferation and plant regeneration by CRISPR/Cas9 editing and overexpression assay. CONCLUSIONS: This study provides novel insights on the role of the regulatory network in cell fate transition and reprogramming during plant regeneration driven by somatic embryogenesis.

Compound Qiying Granules alleviates diabetic peripheral neuropathy by inhibiting endoplasmic reticulum stress and apoptosis.

BACKGROUND: Diabetic peripheral neuropathy (DPN) is a major complication of diabetes. This study aimed to investigate the therapeutic effects and molecular mechanisms of Compound Qiying Granules (CQYG) for DPN. METHODS: Rats and RSC96 cells of DPN models were established to evaluate the therapeutic effects of CQYG. Then the morphology and apoptotic changes of sciatic nerves were detected. Further, tandem mass tag based quantitative proteomics technology was used to identify differentially expressed proteins (DEPs) and the underlying molecular mechanisms. Protein expression of key signaling pathways was also detected. RESULTS: CQYG treatment significantly improved blood glucose and oxidative stress levels, and further reduced nerve fiber myelination lesions, denervation, and apoptosis in DPN rats. Further, 2176 DEPs were found in CQYG treated DPN rats. Enrichment analysis showed that protein processing in the endoplasmic reticulum (ER), and apoptosis were all inhibited after CQYG treatment. Next, CQYG treatment reduced inflammatory factor expression, mitochondrial damage, and apoptosis in RSC96 cells which induced by high glucose. Transmission electron microscopy results found that CQYG treatment improved the morphology of nerve myelin, mitochondria, and ER. CQYG treatment decreased ER stress and apoptosis pathway proteins that were highly expressed in DPN models. In addition, we also predicted the potential targets of CQYG in DEPs. CONCLUSIONS: CQYG exerts neuroprotective effects in experimental diabetic neuropathy through anti-ER stress and anti-apoptosis.

A near-complete genome assembly of Catharanthus roseus and insights into its vinblastine biosynthesis and high susceptibility to the Huanglongbing pathogen.

This study reports the assembly of a near-complete genome of Catharanthus roseus, consisting of 561.7 Mb scaffolded into 8 pseudochromosomes with a contig N50 of 24.7 Mb and a scaffold N50 of 71.1 Mb. The assembly enables the construction of a gene regulatory network of the vinblastine biosynthetic pathway and provides insights into the high susceptibility of C. roseus to the Huanglongbing pathogen.

Knowledge Tensor-Aided Breast Ultrasound Image Assistant Inference Framework.

Breast cancer is one of the most prevalent cancers in women nowadays, and medical intervention at an early stage of cancer can significantly improve the prognosis of patients. Breast ultrasound (BUS) is a widely used tool for the early screening of breast cancer in primary care hospitals but it relies heavily on the ability and experience of physicians. Accordingly, we propose a knowledge tensor-based Breast Imaging Reporting and Data System (BI-RADS)-score-assisted generalized inference model, which uses the BI-RADS score of senior physicians as the gold standard to construct a knowledge tensor model to infer the benignity and malignancy of breast tumors and axes the diagnostic results against those of junior physicians to provide an aid for breast ultrasound diagnosis. The experimental results showed that the diagnostic AUC of the knowledge tensor constructed using the BI-RADS characteristics labeled by senior radiologists achieved 0.983 (95% confidential interval (CI) = 0.975-0.992) for benign and malignant breast cancer, while the diagnostic performance of the knowledge tensor constructed using the BI-RADS characteristics labeled by junior radiologists was only 0.849 (95% CI = 0.823-0.876). With the knowledge tensor fusion, the AUC is improved to 0.887 (95% CI = 0.864-0.909). Therefore, our proposed knowledge tensor can effectively help reduce the misclassification of BI-RADS characteristics by senior radiologists and, thus, improve the diagnostic performance of breast-ultrasound-assisted diagnosis.

CD74 Interacts with Proteins of Enterovirus D68 To Inhibit Virus Replication.

Enterovirus D68 (EV-D68) is a member of the species Enterovirus D in the genus Enterovirus of the family Picornaviridae. As an emerging non-polio enterovirus, EV-D68 is widely spread all over the world and causes severe neurological and respiratory illnesses. Although the intrinsic restriction factors in the cell provide a frontline defense, the molecular nature of virus-host interactions remains elusive. Here, we provide evidence that the major histocompatibility complex class II chaperone, CD74, inhibits EV-D68 replication in infected cells by interacting with the second hydrophobic region of 2B protein, while EV-D68 attenuates the antiviral role of CD74 through 3Cpro cleavage. 3Cpro cleaves CD74 at Gln-125. The equilibrium between CD74 and EV-D68 3Cpro determines the outcome of viral infection. IMPORTANCE As an emerging non-polio enterovirus, EV-D68 is widely spread all over the world and causes severe neurological and respiratory illnesses. Here, we report that CD74 inhibits viral replication in infected cells by targeting 2B protein of EV-D68, while EV-D68 attenuates the antiviral role of CD74 through 3Cpro cleavage. The equilibrium between CD74 and EV-D68 3Cpro determines the outcome of viral infection.

Genetic dissection of protein and starch during wheat grain development using QTL mapping and GWAS.

Protein, starch, and their components are important for wheat grain yield and end-products, which are affected by wheat grain development. Therefore, QTL mapping and a genome-wide association study (GWAS) of grain protein content (GPC), glutenin macropolymer content (GMP), amylopectin content (GApC), and amylose content (GAsC) were performed on wheat grain development at 7, 14, 21, and 28 days after anthesis (DAA) in two environments using a recombinant inbred line (RIL) population of 256 stable lines and a panel of 205 wheat accessions. A total of 29 unconditional QTLs, 13 conditional QTLs, 99 unconditional marker-trait associations (MTAs), and 14 conditional MTAs significantly associated (p < 10-4) with four quality traits were found to be distributed on 15 chromosomes, with the phenotypic variation explained (PVE) ranging from 5.35% to 39.86%. Among these genomic variations, three major QTLs [QGPC3B, QGPC2A, and QGPC(S3|S2)3B] and SNP clusters on the 3A and 6B chromosomes were detected for GPC, and the SNP TA005876-0602 was stably expressed during the three periods in the natural population. The QGMP3B locus was detected five times in three developmental stages in two environments with 5.89%-33.62% PVE, and SNP clusters for GMP content were found on the 3A and 3B chromosomes. For GApC, the QGApC3B.1 locus had the highest PVE of 25.69%, and SNP clusters were found on chromosomes 4A, 4B, 5B, 6B, and 7B. Four major QTLs of GAsC were detected at 21 and 28 DAA. Most interestingly, both QTL mapping and GWAS analysis indicated that four chromosomes (3B, 4A, 6B, and 7A) were mainly involved in the development of protein, GMP, amylopectin, and amylose synthesis. Of these, the wPt-5870-wPt-3620 marker interval on chromosome 3B seemed to be most important because it played an important role in the synthesis of GMP and amylopectin before 7 DAA, in the synthesis of protein and GMP from 14 to 21 DAA, and in the development of GApC and GAsC from 21 to 28 DAA. Using the annotation information of IWGSC Chinese Spring RefSeq v1.1 genome assembly, we predicted 28 and 69 candidate genes for major loci from QTL mapping and GWAS, respectively. Most of them have multiple effects on protein and starch synthesis during grain development. These results provide new insights and information for the potential regulatory network between grain protein and starch synthesis.

Establishment of a chronic insomnia rat model of sleep fragmentation using unstable platforms surrounded by water.

Chronic fragmented sleep is a very common type of insomnia that affects the daily lives of numerous people around the world. However, its pathogenesis is not very clear and a corresponding rat model has not been reported for this purpose at present. The present study aimed to establish a rat model of chronic insomnia with sleep fragmentation using self-made multiple strings of unstable platforms surrounded by shallow water. During the establishment of the models, changes in body weight and differences in food and water intake in the daytime and at night were acquired. The rat models were assessed using several tests, including the Morris water maze test, pentobarbital sodium-induced sleep, infrared monitoring and electroencephalogram/electromyography during sleep. The expression levels of certain inflammatory factors and orexin A were detected in the serum and brain tissues using ELISAs, immunohistochemistry and immunofluorescence. The expression levels of orexin 1 receptor (orexin 1r) were also detected in the brain. Polysomnography indicated that the model rats were successfully prepared with reduced non-rapid eye movement (non-REM) sleep in the daytime, which was increased at night, and considerably lower REM duration during the day and night. The number of instances of sleep arousals were also increased in the day and at night, and the average duration of each sleep bout was decreased in the daytime. The body weights of the model rats increased at a normal rate. However, the reduction of body weight in the daytime and increased in body weight at night were significantly less than those of the control rats. The daytime food and water consumption of the model rats increased significantly compared with that of the control rats, but was similar to that of the control group at night. The Morris water maze test indicated that the model rats were slow to learn to escape the platforms and performed a lower number of target crossings. The pentobarbital-induced sleep experiment confirmed that the model rats exhibited a longer sleep latency and shorter sleep time. The serum IL-1ß, IL-6, TNF-α and orexin A levels of the model rats were significantly increased, whereas their serum IL-10 levels were significantly decreased compared with those of the control rats. The expression levels of IL-1ß, IL-6, orexin A and orexin 1r in the brain tissues of the model rats were also significantly increased. In conclusion, these data indicate that learning and memory function, sleep time, arousal times, diurnal and nocturnal body weight changes, food and water intake, and expression levels of the specific inflammatory factors orexin A and orexin 1r were altered in the model rats. This suggests the chronic insomnia rat model with sleep fragmentation was successfully established using multiple strings of unstable platforms surrounded by water.

Contrast-enhanced US with Perfluorobutane to Diagnose Small Lateral Cervical Lymph Node Metastases of Papillary Thyroid Carcinoma.

Background Although metastasis of lateral cervical lymph nodes (LNs) is common in papillary thyroid carcinoma (PTC), correctly diagnosing small metastatic LNs with US remains challenging. The use of contrast-enhanced US (CEUS), especially the postvascular phase of CEUS with perfluorobutane contrast material, might contribute to improved diagnosis of metastatic LNs in PTC. Purpose To assess the diagnostic value of the postvascular phase of CEUS with perfluorobutane in suspicious small lateral cervical LNs (short-axis diameter ≤8 mm) in patients with PTC. Materials and Methods This single-center study prospectively enrolled consecutive participants with confirmed PTC and suspicious LNs at US from October 2020 to October 2021. All participants underwent CEUS, 1 week before biopsy or surgery, with intravenous perfluorobutane contrast material to visualize the LNs in the vascular phase (5-60 seconds after injection) and postvascular phase (10-30 minutes after injection). The reference standard was cytologic and surgical histologic assessment of the LNs. The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of sonographic features were calculated, and the diagnostic performance of US, CEUS, and the combination of postvascular phase and US features was assessed using multivariable logistic regression models. Results A total of 135 participants (median age, 36 years [IQR, 30-46 years]; 100 women) with 161 suspicious LNs at US were evaluated, including 67 metastatic and 94 benign LNs. The specificity of perfusion defect as a sonographic feature in the vascular phase reached 96% (90 of 94 LNs), and the negative predictive value of non-isoenhancement (ie, hypoenhancement, partial enhancement, and no enhancement) in the postvascular phase reached 100% (83 of 83 LNs). The area under the receiver operating characteristic curve (AUC) of the combination of postvascular phase and US features was 0.94 (95% CI: 0.89, 0.97), significantly higher than that of US features alone (AUC, 0.73; 95% CI: 0.65, 0.79; P < .001). Conclusion The postvascular phase of CEUS with perfluorobutane demonstrated excellent performance for diagnosing suspicious small lateral cervical LNs in participants with PTC. Published under a CC BY 4.0 license Supplemental material is available for this article. See also the editorial by Gunabushanam in this issue.

Activated carbon enhanced traditional activated sludge process for chemical explosion accident wastewater treatment.

With the development of industries, explosion accidents occur frequently during production, transportation, usage and storage of hazard chemicals. It remained challenging to efficiently treat the resultant wastewater. As an enhancement of traditional process, the activated carbon-activated sludge (AC-AS) process has a promising potential in treating wastewater with high concentrations of toxic compounds, chemical oxygen demand (COD) and ammonia nitrogen (NH4+-N), etc. In this paper, activated carbon (AC), activated sludge (AS) and AC-AS were used to treat the wastewater produced from an explosion accident in the Xiangshui Chemical Industrial Park. The removal efficiency was assessed by the removal performances of COD, dissolved organic carbon (DOC), NH4+-N, aniline and nitrobenzene. Increased removal efficiency and shortened treatment time were achieved in the AC-AS system. To achieve the same COD, DOC and aniline removal (90%), the AC-AS system saved 30, 38 and 58 h compared with the AS system, respectively. The enhancement mechanism of AC on the AS was explored by metagenomic analysis and three-dimensional excitation-emission-matrix spectra (3DEEMs). More organics, especially aromatic substances were removed in the AC-AS system. These results showed that the addition of AC promoted the microbial activity in pollutant degradation. Bacteria, such as Pyrinomonas, Acidobacteria and Nitrospira and genes, such as hao, pmoA-amoA, pmoB-amoB and pmoC-amoC, were found in the AC-AS reactor, which might have played important roles in the degradation of pollutants. To sum up, AC might have enhanced the growth of aerobic bacteria which further improved the removal efficiency via the combined effects of adsorption and biodegradation. The successful treatment of Xiangshui accident wastewater using the AC-AS demonstrated the potential universal characteristics of the process for the treatment of wastewater with high concentration of organic matter and toxicity. This study is expected to provide reference and guidance for the treatment of similar accident wastewaters.

The complex genome and adaptive evolution of polyploid Chinese pepper (Zanthoxylum armatum and Zanthoxylum bungeanum).

Zanthoxylum armatum and Zanthoxylum bungeanum, known as 'Chinese pepper', are distinguished by their extraordinary complex genomes, phenotypic innovation of adaptive evolution and species-special metabolites. Here, we report reference-grade genomes of Z. armatum and Z. bungeanum. Using high coverage sequence data and comprehensive assembly strategies, we derived 66 pseudochromosomes comprising 33 homologous phased groups of two subgenomes, including autotetraploid Z. armatum. The genomic rearrangements and two whole-genome duplications created large (~4.5 Gb) complex genomes with a high ratio of repetitive sequences (>82%) and high chromosome number (2n = 4x = 132). Further analysis of the high-quality genomes shed lights on the genomic basis of involutional reproduction, allomones biosynthesis and adaptive evolution in Chinese pepper, revealing a high consistent relationship between genomic evolution, environmental factors and phenotypic innovation. Our study provides genomic resources and new insights for investigating diversification and phenotypic innovation in Chinese pepper, with broader implications for the protection of plants under severe environmental changes.

Differentiation of benign and malignant breast lesions using diffusion-weighted imaging with a fractional-order calculus model.

PURPOSE: To assess the feasibility of using three diffusion parameters (D, ß, and µ) derived from fractional-order calculus (FROC) diffusion model for improving the differentiation between benign and malignant breast lesions. METHOD: In this prospective study, 103 patients with breast lesions were enrolled. All subjects underwent diffusion-weighted imaging (DWI) with 12b values. Inter-observer agreement with respect to quantification of parameters by two radiologists was assessed using intraclass coefficient. Conventional apparent diffusion coefficient (ADC) and three FROC model parameters D, ß, and µ were compared between the benign lesion and malignant lesion groups using the Mann-Whitney U test. Then, a comprehensive prediction model was created by using binary logistic regression. Receiver operating characteristic (ROC) curve analysis was performed to evaluate the diagnostic performance of the parameters using histopathological diagnosis as the reference standard. RESULTS: The FROC parameters and ADC all exhibited significant differences between benign lesions and malignant lesions (P＜0.001). Among the individual parameters, the sensitivity of µ was higher than ADC (95.92% for µ vs 91.84% for ADC), and the specificity of ß was higher than ADC (72.22% for ß vs 70.37% for ADC). The combination of ADC and FROC parameters (D and ß) generated the largest area under the ROC curve (0.841) when compared with individual parameters, indicating an improved performance for differentiating benign lesions from malignant lesions. CONCLUSIONS: This study demonstrated the feasibility of using the FROC diffusion model to improve the accuracy of identifying malignant breast lesions.

CRISPR/Cas Genome Editing Technologies for Plant Improvement against Biotic and Abiotic Stresses: Advances, Limitations, and Future Perspectives.

Crossbreeding, mutation breeding, and traditional transgenic breeding take much time to improve desirable characters/traits. CRISPR/Cas-mediated genome editing (GE) is a game-changing tool that can create variation in desired traits, such as biotic and abiotic resistance, increase quality and yield in less time with easy applications, high efficiency, and low cost in producing the targeted edits for rapid improvement of crop plants. Plant pathogens and the severe environment cause considerable crop losses worldwide. GE approaches have emerged and opened new doors for breeding multiple-resistance crop varieties. Here, we have summarized recent advances in CRISPR/Cas-mediated GE for resistance against biotic and abiotic stresses in a crop molecular breeding program that includes the modification and improvement of genes response to biotic stresses induced by fungus, virus, and bacterial pathogens. We also discussed in depth the application of CRISPR/Cas for abiotic stresses (herbicide, drought, heat, and cold) in plants. In addition, we discussed the limitations and future challenges faced by breeders using GE tools for crop improvement and suggested directions for future improvements in GE for agricultural applications, providing novel ideas to create super cultivars with broad resistance to biotic and abiotic stress.

Conditional QTL mapping for seed germination and seedling traits under salt stress and candidate gene prediction in wheat.

Breeding new wheat varieties with salt resistance is one of the best ways to solve a constraint on the sustainability and expansion of wheat cultivation. Therefore, understanding the molecular components or genes related to salt tolerance must contribute to the cultivation of salt-tolerant varieties. The present study used a recombinant inbred line (RIL) population to genetically dissect the effects of different salt stress concentrations on wheat seed germination and seedling traits using two quantitative trait locus (QTL) mapping methods. A total of 31 unconditional and 11 conditional QTLs for salt tolerance were identified on 11 chromosomes explaining phenotypic variation (PVE) ranging from 2.01 to 65.76%. Of these, 15 major QTLs were found accounting for more than 10% PVE. QTL clusters were detected on chromosomes 2A and 3B in the marker intervals 'wPt-8328 and wPt-2087' and 'wPt-666008 and wPt-3620', respectively, involving more than one salt tolerance trait. QRdw3B and QSfw3B.2 were most consistent in two or more salt stress treatments. 16 candidate genes associated with salt tolerance were predicted in wheat. These results could be useful to improve salt tolerance by marker-assisted selection (MAS) and shed new light on understanding the genetic basis of salt tolerance in wheat.

Highly Efficient Genome Editing Using Geminivirus-Based CRISPR/Cas9 System in Cotton Plant.

Upland cotton (Gossypium hirsutum), an allotetraploid, contains At- and Dt- subgenome and most genes have multiple homologous copies, which pose a huge challenge to investigate genes' function due to the functional redundancy. Therefore, it is of great significance to establish effective techniques for the functional genomics in cotton. In this study, we tested two novel genome editing vectors and compared them with the CRISPR/Cas9 system (pRGEB32-GhU6.7) developed in our laboratory previously. In the first new vector, the sgRNA transcription unite was constructed into the replicon (LIR-Donor-SIR-Rep-LIR) of the bean yellow dwarf virus (BeYDV) and named as pBeYDV-Cas9-KO and in the second vector, the ubiquitin promoter that drives Cas9 protein was replaced with a constitutive CaMV 35S promoter and defined as pRGEB32-35S. The results from transgenic cotton calli/plants revealed that pBeYDV-Cas9-KO vector showed the highest editing efficiency of GhCLA1 in At and Dt subgenomes edited simultaneously up to 73.3% compared to the 44.6% of pRGEB32-GhU6.7 and 51.2% of pRGEB32-35S. The editing efficiency of GhCLA1 in At and Dt subgenome by pBeYDV-Cas9-KO was 85.7% and 97.2%, respectively, whereas the efficiency by pRGEB32-GhU6.7 and pRGEB32-35S vectors was 67.7%, 86.5%, 84%, and 87.2%, respectively. The editing profile of pBeYDV-Cas9-KO was mainly composed of fragment deletion, accounting for 84.0% and ranging 1-10 bp in length. The main editing sites are located at positions 11-17 upstream of PAM site. The off-target effects were not detected in all potential off-target sites. Taken together, the pBeYDV-Cas9-KO system has high editing efficiency and specificity with wide editing range than the traditional CRISPR/Cas9 system, which provides a powerful tool for cotton functional genomics research and molecular breeding.