, the source of cocoa, suffers significant losses to a variety of pathogens resulting in reduced incomes for millions of farmers in developing countries. Development of disease resistant cacao ...varieties is an essential strategy to combat this threat, but is limited by sources of genetic resistance and the slow generation time of this tropical tree crop. In this study, we present the first application of genome editing technology in cacao, using Agrobacterium-mediated transient transformation to introduce CRISPR/Cas9 components into cacao leaves and cotyledon cells. As a first proof of concept, we targeted the cacao
gene, a suppressor of the defense response. After demonstrating activity of designed single-guide RNAs (sgRNA)
, we used
to introduce a CRISPR/Cas9 system into leaf tissue, and identified the presence of deletions in 27% of
copies in the treated tissues. The edited tissue exhibited an increased resistance to infection with the cacao pathogen
and elevated expression of downstream defense genes. Analysis of off-target mutagenesis in sequences similar to sgRNA target sites using high-throughput sequencing did not reveal mutations above background sequencing error rates. These results confirm the function of NPR3 as a repressor of the cacao immune system and demonstrate the application of CRISPR/Cas9 as a powerful functional genomics tool for cacao. Several stably transformed and genome edited somatic embryos were obtained via
-mediated transformation, and ongoing work will test the effectiveness of this approach at a whole plant level.
•This study established a transient transformation system of tea plant leaves using antisense oligodeoxynucleotide (asODN) for gene suppression, by optimizing the concentration of AsDON and the ...number of days of injection.•The reliability of the gene suppression transient transformation system was verified by the gene expression level and metabolic level in theanine/anthocyanin biosynthesis pathways.•This study established a transient transformation system of tea plant leaves using agrobacterium for gene over-expression, by optimizing the injection solution concentrations, and sampling time post injection.•The reliability of the gene over-expression transient transformation system was verified by GFP fluorescence signal and protein level. The system was further proved by the gene expression level and metabolic level in theanine/anthocyanin biosynthesis pathways.•The over-expression transient transformation system was proved can be used to study the subcellular localization of tea plant proteins in vivo.
Tea, the most consumed non-alcohol beverage in the world, has many specialized secondary metabolites, and functional identification of their related genes is very important for the improvement of tea quality. However, a transient transformation system, which is considered as a valuable tool for gene function and protein subcellular localization, has not been established in tea plants. Here, we report a rapid and efficient system for transient transformation of tea plant leaves using antisense oligodeoxynucleotide (asODN) for gene suppression and Agrobacterium for gene over-expression. Specifically, we established the transient transformation system by analyzing different varieties, injection solution concentrations, and sampling time post injection, followed by green fluorescent protein (GFP) fluorescence signal and western blot analysis to confirm the reliability of the over-expression transient transformation system. Additionally, the suppression and over-expression effects of important genes relate to secondary metabolism were evaluated at gene expression and metabolic levels. Finally, a transcription factor was transiently over-expressed to determine its subcellular location by protoplasts successfully in the tea plant leaves. Our data indicate that this system is reliable and effective for studying gene function and protein localization in tea plant leaves, providing new insights into tea plant biology research.
Agrobacterium-mediated transformation is a key innovation for plant breeding, and routinely used in basic researches and applied biology. However, the transformation efficiency is often the limiting ...factor of this technique. In this study, we discovered that oxicam-type nonsteroidal anti-inflammatory drugs, including tenoxicam (TNX), increase the efficiency of Agrobacterium-mediated transient transformation. TNX treatment increased the transformation efficiency of Agrobacterium-mediated transformation of Arabidopsis thaliana mature leaves by agroinfiltration. The increase of efficiency by TNX treatment was not observed in dde2/ein2/pad4/sid2 quadruple mutant, indicating that TNX inhibits the immune system mediated by jasmonic acid, ethylene, and salicylic acid against to Agrobacterium. We also found that TNX-treatment is applicable for the transient expression and subcellular localization analysis of fluorescent-tagged proteins in Arabidopsis leaf cells. In addition, we found that TNX increases the efficiency of Agrobacterium-mediated transient transformation of Jatropha. Given that treatment with oxicam compounds is a simple and cost effective method, our findings will provide a new option to overcome limitations associated with Agrobacterium-mediated transformation of various plant species.
Siraitia grosvenorii (S. grosvenorii) is considered a promising economic crop due to its various bioactive compounds. Polyphenols, one of the main bioactive compounds in S. grosvenorii suspension ...cells, are highly beneficial for human health. Therefore, elucidating their biosynthetic regulatory mechanisms is crucial for the efficient production of these bioactive compounds. Previous studies on the regulation of plant bioactive components have more often focused on the role of protein-coding genes but overlooked non-coding genes. In this study, we provide a novel perspective on the role of long non-coding RNAs (lncRNAs) in regulating polyphenols biosynthesis in methyl jasmonate (125 µM)-treated S. grosvenorii cells. Firstly, metabolomics analysis revealed 70 differential metabolites mainly composed of polyphenols (phenolic acids and flavonoids) in the cells. The intracellular polyphenols content (maximum 41.12 mg g−1) increased synchronously with the upregulation of polyphenols biosynthetic enzyme activity. Secondly, transcriptomics analysis indicated 496 differentially-expressed lncRNAs and 3595 differentially-expressed mRNAs in the cells. Besides, 29 putative regulatory pairs between differentially-expressed lncRNAs and their target mRNAs were identified, which might affect plant signal transduction, polyphenols and their precursors biosynthesis, and related transport pathways, ultimately mediating the accumulation of polyphenols in the cells. Finally, using an efficient transient transformation strategy established in this study, an antisense-lncRNA of phenylalanine ammonia-lyase (lnc-PAL) was preliminarily proven to be a negative regulatory factor on polyphenols biosynthesis in S. grosvenorii. Therefore, our findings expanded the knowledge of lncRNAs in polyphenols biosynthesis, providing a theoretical basis for the efficient production of these compounds in S. grosvenorii cells.
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•Methyl jasmonate significantly enhanced the accumulation of polyphenols in Siraitia grosvenorii cells.•The primary polyphenols with significant difference were phenolic acids and flavonoids.•Numerous long non-coding RNAs associated with polyphenols biosynthesis were identified.•An efficient transient transformation system was established in Siraitia grosvenorii seedlings.•Lnc-PAL might regulate phenylalanine ammonia-lyase expression to mediate polyphenols biosynthesis.
•An efficient system for eggplant protoplast isolation was established.•An efficient transient transformation system for eggplant protoplast was developed.•The subcellular localization of several ...eggplant genes was verified using this system.•Bimolecular fluorescence complementation assay was identified by this system.•The transient transformation system was also suitable for dual-luciferase assays .
Protoplasts are widely used in transient expression systems that are a simple, robust, and powerful method for gene function verification. However, such a versatile tool has not been developed using eggplant. We here established an efficient protocol for protoplast isolation from plantlet leaves of eggplant by optimizing enzymatic conditions. This isolation required an optimal D-mannitol concentration (0.5 M), enzyme concentration (1.25% (w/v) cellulose and 0.4% (w/v) macerozyme), digestion time (3 h), and temperature (26 ℃). This protocol provided the highest yield (1.2 × 107/g fresh weight (FW)) and viability (96%) of protoplasts. Furthermore, the polyethylene glycol (PEG)-mediated transient transformation of eggplant protoplasts with the marker gene yellow fluorescent protein (YFP) was investigated by optimizing different crucial factors affecting transfection efficiency, including plasmid DNA amount, PEG concentration, and transfection duration. More than 50% transfection efficiency could be obtained with the optimized system. In addition, subcellular localization of several fusion proteins was verified, and the bimolecular fluorescence complementation assay was used to detect protein–protein interaction in this system. Dual-luciferase assays in eggplant protoplasts showed that the expression of anthocyanin biosynthesis-related structural genes (SmCHS and SmDFR) was activated by the transcription factor SmMYB1. Taken together, we developed a highly efficient and stable protocol for protoplast isolation and transfection in eggplant, thereby facilitating gene function characterization in molecular breeding.
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Agrobacterium tumefaciens is widely used as a versatile tool for development of stably transformed model plants and crops. However, the development of Agrobacterium based transient plant ...transformation methods attracted substantial attention in recent years. Transient transformation methods offer several applications advancing stable transformations such as rapid and scalable recombinant protein production and in planta functional genomics studies. Herein, we highlight Agrobacterium and plant genetics factors affecting transfer of T-DNA from Agrobacterium into the plant cell nucleus and subsequent transient transgene expression. We also review recent methods concerning Agrobacterium mediated transient transformation of model plants and crops and outline key physical, physiological and genetic factors leading to their successful establishment. Of interest are especially Agrobacterium based reverse genetics studies in economically important crops relying on use of RNA interference (RNAi) or virus-induced gene silencing (VIGS) technology. The applications of Agrobacterium based transient plant transformation technology in biotech industry are presented in thorough detail. These involve production of recombinant proteins (plantibodies, vaccines and therapeutics) and effectoromics-assisted breeding of late blight resistance in potato. In addition, we also discuss biotechnological potential of recombinant GFP technology and present own examples of successful Agrobacterium mediated transient plant transformations.
Grape (Vitis vinifera L.) berries are susceptible to quality deterioration after harvest. This study aimed to investigate the effect of folic acid (FA) treatment on the postharvest quality of table ...grape berries. Harvested ‘Kyoho’ (V. vinifera × V. labrusca L.) bunches were immersed in 1 mg L−1 FA solution for 15 min, then stored for sample collection and phenotype observation. Treatment with FA decreased the rates of berry abscission, water loss, and berry softening, reduced the accumulation of reactive oxygen species (ROS), and altered ROS metabolism-associated enzyme activities and gene expression levels. Transcriptome analysis revealed 507, 684, and 250 differentially expressed genes (DEGs) between FA-treated and non-treated berries at 3, 6, and 9 d after treatment, respectively. The DEGs were grouped into six modules according to a weighted correlation network analysis. A hub transcription factor, annotated as a WRKY transcription factor, was screened from the module associated with cell wall metabolism and identified as VvWRKY31. Transient transformation of grape berry slices revealed that overexpression of VvWRKY31 enhanced the relative expression level of VvRboh. The dual-luciferase assay showed that VvWRKY31 was capable of binding to VvRboh promoter. The present results provide insights into the potential molecular mechanism underlying FA-induced delay postharvest quality deterioration of grape berries. Specifically, FA inhibits expression of genes associated with ROS generation by reducing VvWRKY31 expression, thereby delaying quality deterioration induced by excessive accumulation of ROS. These results will be helpful in table grape breeding programs focused on delaying deterioration in postharvest quality.
•Folic acid affects ROS metabolism in postharvest grape.•VvWRKY31 is a hub transcription factor in cell wall metabolism.•VvWRKY31 promotes VvRboh expression by binding its promoter.
•Vacuum infiltration and sonication can improve the efficiency of transient transformation.•Over-expression of the StNHX1 gene improved the salt tolerance of pumpkin seedlings.•The lower epidermis of ...cotyledon was the optimal tissue for subcellular localization in pumpkin.•The CmPHD1-EGFP fusion protein was detected in the nucleus of cotyledon epidermal cells.
Currently, gene exploration and molecular breeding in pumpkin (Cucurbita moschata Duch.) is a challenging process. There are limited efficient stable transformation methods for pumpkin. Transient transformation is a promising tool for the study of gene function. Here, an efficient Agrobacterium-mediated transient transformation system was developed for gene function studies in pumpkin. In this system, the function of salt-tolerant gene StNHX1 was confirmed by over-expression in germinated seeds. The CmPHD1-EGFP fusion protein was detected in the nucleus of cotyledon epidermal cells. This system can be used to analyze gene function and protein subcellular localization in pumpkin. Its advantages are highly efficient, cost-effective and time-saving (∼14 days). It may play a key role in gene exploration and molecular breeding in pumpkin, especially in large-scale analyses.
Cotton fiber is the most important natural textile material in the world. Identification and functional characterization of genes regulating fiber development are fundamental for improving fiber ...quality and yield. However, stable cotton transformation is time-consuming, low in efficiency, and technically complex. Moreover, heterologous systems, such as
and tobacco, did not always work to elucidate the function of cotton fiber specifically expressed genes or their promoters. For these reasons, constructing a rapid transformation system using cotton fibers is necessary to study fiber's specifically expressed genes. In this study, we developed an easy and rapid
-mediated method for the transient transformation of genes and promoters in cotton fibers. First, we found that exogenous genes could be expressed in cotton fibers
using β-glucuronidase (GUS) and green fluorescence protein (GFP) as reporters. Second, parameters affecting transformation efficiency, including LBA4404
strain, 3 h infection time, and 2-day incubation time, were determined. Third, four different cotton genes that are specifically expressed in fibers were transiently transformed in cotton fibers, and the transcripts of these genes were detected ten to thousand times increase over the control. Fourth, GUS staining and activity analysis demonstrated that the activity profiles of
and
promoters in transformed fibers are similar to their native activity in developmental fibers. Furthermore, the transient transformation method was confirmed to be suitable for subcellular localization studies. In summary, the presented
-mediated transient transformation method is a fast, simple, and effective system for promoter characterization and protein expression in cotton fibers.
•Developed a highly efficient Agrobacterium-mediated transient expression system in apple fruit.•The system will be a rapid and high throughput technique for gene function analysis.•The texture of ...apple fruits significantly affect its infiltration efficiency.
Studies of gene function in apple fruits (Malus domestica) have been hindered by the long juvenile period of apple fruit trees. Transient gene expression is a relatively simple method for gene functional analyses, and has been broadly applied to several plant species; however, few studies have reported a method for systematic transient expression in apple fruits. Here, we developed a highly efficient and robust Agrobacterium-mediated transient expression system for apple fruit, which allows rapid analysis of diverse gene functions. Using a β-glucuronidase gene (GUS) as a reporter for Agrobacterium-mediated transformation, we found that the most suitable infiltration pressure for gene function assays in ‘Red Fuji’, ‘Granny Smith’ and ‘Royal Gala’ fruits was −90 kPa, but −70 kPa in ‘Golden Delicious’. The infiltration efficiency of ‘Golden Delicious’ was higher than that of the other apple cultivars. Transient silencing of MdMYB10, an anthocyanin biosynthesis regulator, altered anthocyanin content in ‘Red Fuji’, demonstrating that vacuum infiltration with Agrobacterium can be used for gene functional analysis. Also a significant positive correlation was found between microcrack density and infiltration efficiency. We conclude that this method represents a general tool for efficient and targeted transient regulation of gene expression in apple fruits.
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