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Journal of Advanced Research Jun 2024The Prime Editing (PE) system is a precise and versatile genome editing tool with great potential in plant breeding and plant synthetic biology. However, low PE...
INTRODUCTION
The Prime Editing (PE) system is a precise and versatile genome editing tool with great potential in plant breeding and plant synthetic biology. However, low PE efficiency severely restricts its application, especially in dicots. PE can introduce small tags to trace target protein or cis-element to regulate gene transcription which is an expertise superior to other gene editing tools. Owing to low efficiency, PE adaption in stably transformed Arabidopsis is lacking.
OBJECTIVES
This study aimed to investigate the issue of low PE efficiency in dicots and develop systematic solutions to improve it. Currently, PE in dicots is undetectable and inconsistent, and this study seeks to address it. Split PE into several parts showed better performance in some target sites in mammal cells. We plan to discover the optimal split PE combination in dicot.
METHODS
We conducted large-scale transformation experiments in dicot model plants Arabidopsis thaliana (At) and Nicotiana benthamiana (Nb) by Agrobacterium-mediated transformation with deep amplicon sequencing (0.2-0.5 million clean total reads).
RESULTS
The editing efficiency decreased upon using a fused reverse transcriptase (RT) or an extended pegRNA separately and further decreased dramatically when these were used together. With the help of the pol II strategy to express PE gRNA (pegRNA), we named the most effective split PE combination as a multi-modular assembled prime editing system (mPE). mPE exhibited improved precise editing efficiency on most gene sites with various editing types, ranging from 1.3-fold to 1288.5-fold and achieved PE on some sites that could not be edited by original PE2. Especially, mPE showed superiority for multi-base insertion with an average improvement of 197.9-fold.
CONCLUSION
The original PE architecture strongly inhibited the cleavage activity of Cas9. Split PE improved PE efficiency extensively and was in favor of introducing small insertions in dicot plants, indicating that different PE variants might have their own expertise.
PubMed: 38942381
DOI: 10.1016/j.jare.2024.06.021 -
Microbiology Resource Announcements Jun 2024sp. strain R1 was isolated from the stem of a tomato plant and exhibited antagonism with . The complete genome sequence of this bacterium consists of one 3,874,532 bp...
sp. strain R1 was isolated from the stem of a tomato plant and exhibited antagonism with . The complete genome sequence of this bacterium consists of one 3,874,532 bp circular chromosome and two plasmids.
PubMed: 38940606
DOI: 10.1128/mra.00242-24 -
Journal of Virological Methods Jun 2024Cucurbit leaf crumple virus (CuLCrV) is among the prominent viruses infecting cucurbits in the USA. Attainable procedures of virus inoculation to crops are prerequisite...
Cucurbit leaf crumple virus (CuLCrV) is among the prominent viruses infecting cucurbits in the USA. Attainable procedures of virus inoculation to crops are prerequisite for screening of resistance against the virus. Because mechanical (non-vector-mediated) infection by cucurbit leaf crumple virus (CuLCrV) is inefficient in economically important crops, screening for CuLCrV resistance is currently laborious and time-consuming using transmission by viruliferous whiteflies. We constructed an infectious partial tandem repeat construct of an isolate of CuLCrV from Georgia, USA, in the plant expression binary vector pCambia2300 and transformed it into Agrobacterium tumifaciens strain EHA105. Agroinfiltration of this construct into the abaxial surface of the leaves of common bean (Phaseolus vulgaris L.) produced a systemic infection characteristic of CuLCrV, although this approach was not successful for yellow squash. However, we report a very efficient and reproducible inoculation procedure established in squash when the leaves were injured with a microneedle and rubbed it with cell suspension harbouring the infectious viral construct.
PubMed: 38936512
DOI: 10.1016/j.jviromet.2024.114992 -
Microbiology Resource Announcements Jun 2024We report the complete genome sequence of strain EL101, isolated from the tree bark of in Kampar, Perak, Malaysia, obtained using Q20+ Nanopore Sequencing chemistry....
We report the complete genome sequence of strain EL101, isolated from the tree bark of in Kampar, Perak, Malaysia, obtained using Q20+ Nanopore Sequencing chemistry. The assembled genome has a total length of 5,324,685 bp, comprising a circular chromosome, a linear chromid, and two non-Ti circular plasmids.
PubMed: 38934609
DOI: 10.1128/mra.00298-24 -
Plants (Basel, Switzerland) Jun 2024As a type of cell-wall-relaxing protein that is widely present in plants, expansins have been shown to actively participate in the regulation of plant growth and...
As a type of cell-wall-relaxing protein that is widely present in plants, expansins have been shown to actively participate in the regulation of plant growth and responses to environmental stress. Wild soybeans have long existed in the wild environment and possess abundant resistance gene resources, which hold significant value for the improvement of cultivated soybean germplasm. In our previous study, we found that the wild soybean expansin gene is specifically transcribed in roots, and its transcription level significantly increases under salt and drought stress. To further identify the function of , in this study, we cloned the CDS sequence of this gene. The transcription pattern of in the roots of wild soybean under salt and drought stress was analyzed by qRT-PCR. Using an -mediated genetic transformation, we obtained soybean hairy roots overexpressing . Under 150 mM NaCl- and 100 mM mannitol-simulated drought stress, the relative growth values of the number, length, and weight of transgenic soybean hairy roots were significantly higher than those of the control group. We obtained the transcriptomes of transgenic and wild-type soybean hairy roots under normal growth conditions and under salt and drought stress through RNA sequencing. A transcriptomic analysis showed that the transcription of genes encoding expansins (EXPB family), peroxidase, H-transporting ATPase, and other genes was significantly upregulated in transgenic hairy roots under salt stress. Under drought stress, the transcription of expansin (EXPB/LB family) genes increased in transgenic hairy roots. In addition, the transcription of genes encoding peroxidases, calcium/calmodulin-dependent protein kinases, and dehydration-responsive proteins increased significantly. The results of qRT-PCR also confirmed that the transcription pattern of the above genes was consistent with the transcriptome. The differences in the transcript levels of the above genes may be the potential reason for the strong tolerance of soybean hairy roots overexpressing the gene under salt and drought stress. In conclusion, the expansin can be used as a valuable candidate gene for the molecular breeding of soybeans.
PubMed: 38931088
DOI: 10.3390/plants13121656 -
Plants (Basel, Switzerland) Jun 2024Abiotic stress significantly affects plant growth and has devastating effects on crop production. Drought stress is one of the main abiotic stressors. Actin is a major...
Abiotic stress significantly affects plant growth and has devastating effects on crop production. Drought stress is one of the main abiotic stressors. Actin is a major component of the cytoskeleton, and actin-depolymerizing factors (ADFs) are conserved actin-binding proteins in eukaryotes that play critical roles in plant responses to various stresses. In this study, we found that , an gene from the soybean , showed drastic upregulation under drought stress. Subcellular localization experiments in tobacco epidermal cells and tobacco protoplasts showed that GmADF13 was localized in the nucleus and cytoplasm. We characterized its biological function in transgenic and hairy root composite soybean plants. plants transformed with displayed a more robust drought tolerance than wild-type plants, including having a higher seed germination rate, longer roots, and healthy leaves under drought conditions. Similarly, -overexpressing (OE) soybean plants generated via the -mediated transformation of the hairy roots showed an improved drought tolerance. Leaves from OE plants showed higher relative water, chlorophyll, and proline contents, had a higher antioxidant enzyme activity, and had decreased malondialdehyde, hydrogen peroxide, and superoxide anion levels compared to those of control plants. Furthermore, under drought stress, OE activated the transcription of several drought-stress-related genes, such as , , , , and . Thus, is a positive regulator of the drought stress response, and it may play an essential role in plant growth under drought stress conditions. These results provide new insights into the functional elucidation of soybean . They may be helpful for breeding new soybean cultivars with a strong drought tolerance and further understanding how help plants adapt to abiotic stress.
PubMed: 38931083
DOI: 10.3390/plants13121651 -
Plants (Basel, Switzerland) Jun 2024The pathogenicity of grapevine geminivirus A (GGVA), a recently identified DNA virus, to grapevine plants remains largely unclear. Here, we report a new GGVA isolate...
Construction of an Infectious DNA Clone of Grapevine Geminivirus A Isolate GN and Its Biological Activity in Plants Analyzed Using an Efficient and Simple Inoculation Method.
The pathogenicity of grapevine geminivirus A (GGVA), a recently identified DNA virus, to grapevine plants remains largely unclear. Here, we report a new GGVA isolate (named GGVA) obtained from grapevine 'Queen Nina' plants with severe disease symptoms. The infectious clone of GGVA (pXT-GGVA) was constructed to investigate its pathogenicity. plants inoculated with GGVA by agroinfiltration displayed upward leaf curling and chlorotic mottling symptoms. A simple, quick, and efficient method for delivering DNA clones of GGVA into grapevine plants was developed, by which cells carrying pXT-GGVA were introduced into the roots of in vitro-grown 'Red Globe' grape plantlets with a syringe. By this method, all 'Red Globe' grape plants were systemically infected with GGVA, and the plants exhibited chlorotic mottling symptoms on their upper leaves and downward curling, interveinal yellowing, and leaf-margin necrosis symptoms on their lower leaves. Our results provide insights into the pathogenicity of GGVA and a simple and efficient inoculation method to deliver infectious viral clones to woody perennial plants.
PubMed: 38931032
DOI: 10.3390/plants13121601 -
Genes Jun 2024Small auxin-upregulated RNAs (), as the largest family of early auxin-responsive genes, play important roles in plant growth and development processes, such as auxin...
Small auxin-upregulated RNAs (), as the largest family of early auxin-responsive genes, play important roles in plant growth and development processes, such as auxin signaling and transport, hypocotyl development, and tolerance to environmental stresses. However, the functions of few genes are known in the root development of sweet potatoes. In this study, an gene was cloned and functionally analyzed. The IbSAUR36 protein was localized to the nucleus and plasma membrane. The transcriptional level of this gene was significantly higher in the pencil root and leaf.This gene was strongly induced by indole-3-acetic acid (IAA), but it was downregulated under methyl-jasmonate(MeJA) treatment. The promoter of contained the core -elements for phytohormone responsiveness. Promoter β-glucuronidase (GUS) analysis in showed that is highly expressed in the young tissues of plants, such as young leaves, roots, and buds. -overexpressing sweet potato roots were obtained by an efficient -mediated root transgenic system. We demonstrated that overexpression of promoted the accumulation of IAA, upregulated the genes encoding IAA synthesis and its signaling pathways, and downregulated the genes encoding lignin synthesis and JA signaling pathways. Taken together, these results show that plays an important role in adventitious root (AR) development by regulating IAA signaling, lignin synthesis, and JA signaling pathways in transgenic sweet potatoes.
Topics: Ipomoea batatas; Plant Roots; Plants, Genetically Modified; Indoleacetic Acids; Gene Expression Regulation, Plant; Plant Proteins; Plant Growth Regulators; Promoter Regions, Genetic; Cyclopentanes
PubMed: 38927696
DOI: 10.3390/genes15060760 -
Genes May 2024Broccoli, a popular international crop, is an important export vegetable in China. Broccoli is not only rich in protein, vitamins, and minerals but also has anticancer... (Review)
Review
Broccoli, a popular international crop, is an important export vegetable in China. Broccoli is not only rich in protein, vitamins, and minerals but also has anticancer and antiviral activities. Recently, an -mediated transformation system has been established and optimized in broccoli, and transgenic transformation and CRISPR-Cas9 gene editing techniques have been applied to improve broccoli quality, postharvest shelf life, glucoraphanin accumulation, and disease and stress resistance, among other factors. The construction and application of genetic transformation technology systems have led to rapid development in broccoli worldwide, which is also good for functional gene identification of some potential traits in broccoli. This review comprehensively summarizes the progress in transgenic technology and CRISPR-Cas9 gene editing for broccoli over the past four decades. Moreover, it explores the potential for future integration of digital and smart technologies into genetic transformation processes, thus demonstrating the promise of even more sophisticated and targeted crop improvements. As the field continues to evolve, these innovations are expected to play a pivotal role in the sustainable production of broccoli and the enhancement of its nutritional and health benefits.
Topics: Brassica; Gene Editing; CRISPR-Cas Systems; Plants, Genetically Modified
PubMed: 38927604
DOI: 10.3390/genes15060668 -
Biomolecules Jun 2024Ginseng ( C. A. Meyer) is an ancient and valuable Chinese herbal medicine, and ginsenoside, as the main active ingredient of ginseng, has received wide attention because...
Ginseng ( C. A. Meyer) is an ancient and valuable Chinese herbal medicine, and ginsenoside, as the main active ingredient of ginseng, has received wide attention because of its various pharmacological active effects. Cytochrome P450 is the largest family of enzymes in plant metabolism and is involved in the biosynthesis of terpenoids, alkaloids, lipids, and other primary and secondary plant metabolites. It is significant to explore more genes with unknown functions and reveal their roles in ginsenoside synthesis. In this study, based on the five genes screened in the pre-laboratory, through the correlation analysis with the content of ginsenosides and the analysis of the interactions network of the key enzyme genes for ginsenoside synthesis, we screened out those highly correlated with ginsenosides, , as the target gene from among the five genes. Methyl jasmonate-induced treatment of ginseng adventitious roots showed that the gene responded to methyl jasmonate induction and was involved in the synthesis of ginsenosides. The gene was cloned and the overexpression vector pBI121-PgCYP309 and the interference vector pART27-PgCYP309 were constructed. Transformation of ginseng adventitious roots by the -mediated method and successful induction of transgenic ginseng hairy roots were achieved. The transformation rate of ginseng hairy roots with overexpression of the gene was 22.7%, and the transformation rate of ginseng hairy roots with interference of the gene was 40%. Analysis of ginseng saponin content and relative gene expression levels in positive ginseng hairy root asexual lines revealed a significant increase in PPD, PPT, and PPT-type monomeric saponins Re and Rg2. The relative expression levels of and genes were also significantly increased. gene promotes the synthesis of ginsenosides, and it was preliminarily verified that gene can promote the synthesis of dammarane-type ginsenosides.
Topics: Panax; Cytochrome P-450 Enzyme System; Ginsenosides; Gene Expression Regulation, Plant; Plant Roots; Plant Proteins; Oxylipins; Acetates; Cyclopentanes
PubMed: 38927118
DOI: 10.3390/biom14060715