Analysis of codon usage bias (CUB) in different species can reveal the patterns of genetic information transfer across those species. To better understand the characteristics of MYB10—a key regulator ...of anthocyanin biosynthesis—and identify the true (functional) MYB10 gene among the two candidates in Populus, we analysed the coding sequences of MYB10 genes in 10 different species using Codon W, CHIPS, CUSP, and CAI. Majority of the optimal amino acid codons of MYB10 genes ended with A/U, and GGA, UCA, GCA, AGA, and CCA were over-represented in all plant species studied. Among the two most promising MYB10 gene candidates in Populus, Potri.17G125700 shared a higher similarity of codon usage with MYB10 genes from other plant species, suggesting that it encodes the functional MYB10 in Populus. We verified this speculation by cloning both candidate MYB10 genes from Populus into vectors to produce transiently transformed seedlings. Colour phenotypes and anthocyanin content of the transiently transformed seedlings indicated that Potri.17G125700 encodes the true MYB10 transcription factor, which positively regulates anthocyanin accumulation in Populus. Furthermore, CUB analysis was used to select the most promising MYB12 candidate in Malus sp. (crabapple). Our results demonstrate the effectiveness of CUB analysis as a promising method to identify the functional gene from a set of candidates in long-living plants with complex genetics.
Protoplasts, derived from plant cells, exhibit remarkable totipotency and hold significant value across a wide spectrum of biological and biotechnological applications. These versatile applications ...encompass protein subcellular localization and interaction analysis, gene expression regulation, functional characterization, gene editing techniques, and single-cell sequencing. Protoplasts' usability stems from their inherent accessibility and their ability to efficiently incorporate exogenous genes. In this review, we provide a comprehensive overview, including details on isolation procedures and influencing factors, purification and viability assessment methodologies, and the utilization of the protoplast transient expression system. The aim is to provide a comprehensive overview of current applications and offer valuable insights into protoplast isolation and the establishment of transient expression systems in a diverse range of plant species, thereby serving as a valuable resource for the plant science community.
BACKGROUND: Transient gene expression via Agrobacterium-mediated DNA transfer offers a simple and fast method to analyze transgene functions. Although Arabidopsis is the most-studied model plant with ...powerful genetic and genomic resources, achieving highly efficient and consistent transient expression for gene function analysis in Arabidopsis remains challenging. RESULTS: We developed a highly efficient and robust Agrobacterium-mediated transient expression system, named AGROBEST (Agrobacterium-mediated enhanced seedling transformation), which achieves versatile analysis of diverse gene functions in intact Arabidopsis seedlings. Using β-glucuronidase (GUS) as a reporter for Agrobacterium-mediated transformation assay, we show that the use of a specific disarmed Agrobacterium strain with vir gene pre-induction resulted in homogenous GUS staining in cotyledons of young Arabidopsis seedlings. Optimization with AB salts in plant culture medium buffered with acidic pH 5.5 during Agrobacterium infection greatly enhanced the transient expression levels, which were significantly higher than with two existing methods. Importantly, the optimized method conferred 100% infected seedlings with highly increased transient expression in shoots and also transformation events in roots of ~70% infected seedlings in both the immune receptor mutant efr-1 and wild-type Col-0 seedlings. Finally, we demonstrated the versatile applicability of the method for examining transcription factor action and circadian reporter-gene regulation as well as protein subcellular localization and protein–protein interactions in physiological contexts. CONCLUSIONS: AGROBEST is a simple, fast, reliable, and robust transient expression system enabling high transient expression and transformation efficiency in Arabidopsis seedlings. Demonstration of the proof-of-concept experiments elevates the transient expression technology to the level of functional studies in Arabidopsis seedlings in addition to previous applications in fluorescent protein localization and protein–protein interaction studies. In addition, AGROBEST offers a new way to dissect the molecular mechanisms involved in Agrobacterium-mediated DNA transfer.
The
plant is the exclusive source of the valuable anticancer terpenoid indole alkaloids, vinblastine (VB) and vincristine (VC). The recent availability of transcriptome and genome resources for
...necessitates a fast and reliable method for studying gene function. In this study, we developed an
-mediated transient expression method to enable the functional study of genes rapidly
, conserving the compartmentalization observed in the VB and VC pathway. We focused on (1) improving the transformation method (syringe versus vacuum agroinfiltration) and cultivation conditions (seedling age,
density, and time point of maximum transgene expression), (2) improving transformation efficiency through the constitutive expression of the virulence genes and suppressing RNA silencing mechanisms, and (3) improving the vector design by incorporating introns, quantitative and qualitative reporter genes (luciferase and
genes), and accounting for transformation heterogeneity across the tissue using an internal control. Of all the parameters tested, vacuum infiltration of young seedlings (10-day-old, harvested 3 days post-infection) resulted in the strongest increase in transgene expression, at 18 - 57 fold higher than either vacuum or syringe infiltration of other seedling ages. Endowing the
strain with the mutated
or silencing suppressors within the same plasmid as the reporter gene further increased expression by 2 - 10 fold. For accurate measurement of promoter transactivation or activity, we included an internal control to normalize the differences in plant mass and transformation efficiency. Including the normalization gene (
luciferase) on the same plasmid as the reporter gene (firefly luciferase) consistently yielded a high signal and a high correlation between RLUC and FLUC. As proof of principle, we applied this approach to investigate the regulation of the
promoter with the well-known activator ORCA3 and repressor ZCT1. Our method demonstrated the quantitative assessment of both the activation and repression of promoter activity in
. Our efficient
-mediated seedling infiltration (EASI) protocol allows highly efficient, reproducible, and homogenous transformation of
cotyledons and provides a timely tool for the community to rapidly assess the function of genes
, particularly for investigating how transcription factors regulate terpenoid indole alkaloid biosynthesis.
CRISPR-based genome editing technologies continue to drive major advances in life sciences. A major challenge for realizing widespread use of genome editing in plants and agriculture is establishing ...methods that enable the rapid, comprehensive, and precise evaluation of editing technologies using transient methods. Here we report a new and rapid genome editing evaluation method using Agrobacterium infiltration techniques to enable broad-spectrum, simplistic, and precise assessments of genome editing efficiencies. We employed an anthocyanin marker to facilitate visual screenings of genome-edited cells for use in adult strawberry fruits as well as tomato fruits, cotton leaves, and sugar beet leaves. Using this method, we demonstrate the ability to quickly measure genome editing efficiencies mediated by SpCas9, LbCas12a, A3A-PBE, ABE8e, and PPE. This new method will allow researchers to rapidly and easily evaluate genome editing tools across a broad spectrum of plant species, further expediting the development of genome-edited agricultural crops.
The availability of a fast and controlled mitotic model system that could simplify the generation of genetic material and reduce the experimental time from months to days would largely benefit ...research in plant cell division. In this protocol, we propose the use of pavement cells of Nicotiana benthamiana leaves to study cell division, which is artificially induced by Agrobacterium-mediated transient overexpression of the transcription factor E2Fb. The cell division-inducing overexpression of E2Fb can be combined with the expression of fluorescent protein-tagged proteins of interest or with dyes, which could be visualized throughout the cell cycle under the microscope. This simple and affordable method enables the study of cell cycle regulation and cell division in plants, from genome replication to cell wall formation, in a fast and controlled manner, and can be used for functional studies when coupled with chemical inhibitors or reverse genetic approaches.
The Agrobacterium-mediated transient transformation, which proved effective in diverse plant species, has been widely applied for high-throughput gene function studies due to its simplicity, ...rapidity, and high efficiency. Despite the efforts have made on Artemisia annua transient expression, achieving high-throughput gene functional characterization basing on a fast and easy-manipulated transient transformation system in A. annua remains challenging. The first pair of true leaves of A. annua is an ideal candidate for Agrobacterium injection. EHA105 was the optimal strain that can be used for the development of the transient expression system. The supplementation of Triton X-100 at a concentration of 0.005% greatly improved the transient expression frequency. According to the histochemical beta-Glucuronidase (GUS) staining assay, high transient expression level of the reporter gene (GUS) maintained at least a week. Dual-luciferase (Dual-LUC) transient assays showed that the activity of cauliflower mosaic virus 35S (CaMV35S) promoter and its derivates varied between A. annua and tobacco. In A. annua, the CaMV35S promoter had comparable activity with double CaMV35S promoter, while in tobacco, CaMV35S exhibited approximately 50% activity of double CaMV35S promoter. Otherwise, despite the CaMV35S promoter and double CaMV35S promoter from GoldenBraid Kit 2.0 displayed high activity strength in tobacco, they demonstrated a very low activity in transiently expressed A. annua. The activity of UBQ10 promoter and endogenous UBQb promoter was investigated as well. Additionally, using our transient expression system, the transactivation of AaGSW1 and AaORA on AaCYP71AV1 promoter was confirmed. Dual-LUC assays demonstrated that AaHD8 activated the expression of two glandular secreting trichomes-specific lipid transfer protein genes AaLTP1 and AaLTP2, indicating that AaLTP1 and AaLTP2 might serve as downstream components of AaHD8-involved glandular trichome initiation and cuticle formation, as well as artemisinin secretion in A. annua. A simple, rapid, good-reproducibility, high-efficiency and low-cost transient transformation system in A. annua was developed. Our method offered a new way for gene functional characterization studies such as gene subcellular localization, promoter activity and transcription activation assays in A. annua, avoiding the aberrant phenotypes resulting from gene expression in a heterologous system.
Grape is an economically important crop but recalcitrant to
Agrobacterium
-mediated genetic transformation and in vitro regeneration. Here, we have developed a protocol for transient transformation ...of grapes by investigating the effects of explant pre-culture and duration of vacuum infiltration on transformation efficiency. Using sliced grape berries of “Shine-Muscat” (
Vitis labrusca
×
Vitis vinifera
) between the end of fruit expansion phase and the mature stage as explants, we firstly compared the effect of pre-culture explants into a susceptible state (incubation on Murashige and Skoog (MS) agar plate in the dark at 25 ± 1 °C for 48 h) with no pre-culture and then tested different vacuum infiltration times on transformation efficiency using
β-glucuronidase
(
GUS
) reporter system. Pre-culture increased the susceptibility of explants to the
agrobacteria
infection and increased transient transformation efficiency as assessed by histochemical GUS activity, with intense blue coloration compared with the faint staining observed in the non-susceptible explants. Using a Circulating Water Vacuum Pump system to facilitate
agrobacteria
entry into berry cells, we tested vacuum durations of 5, 10, and 15 min and observed that transformation efficiency increased with vacuum duration of infiltration. These results were confirmed by relative gene expression of
GUS
transgene as assessed by RT-qPCR and GUS activity assay. To further confirm the usefulness of our protocol, we transiently transformed grape berries with the hydrogen peroxide sensor gene
VvHPCA3
, and this was confirmed by gene expression analysis as well as increased sensitivity of the explants to hydrogen peroxide treatment. Overall, this study has resulted in a simple but efficient transient transformation protocol for grape berries and would be a valuable tool for the rapid testing of gene function and the study of key regulatory networks in this important crop.
Target leaf spot (TLS), which is caused by
(
), is one of the most important diseases in cucumber (
L.). Our previous research identified several
-responsive miRNAs in cucumber by high-throughput ...sequencing, including two known miRNAs and two novel miRNAs. The target genes of these miRNAs were related to secondary metabolism. In this study, we verified the interaction between these miRNAs and target genes by histochemical staining and fluorescence quantitative assays of GUS. We transiently expressed the candidate miRNAs and target genes in cucumber cotyledons to investigate the resistance to
. Transient expression of miR164d, miR396b, Novel-miR1, and Novel-miR7 in cucumber resulted in decreased resistance to
, while transient expression of
(inhibited by miR164d),
(inhibited by miR396b),
(inhibited by Novel-miR1), and
(inhibited by Novel-miR7) led to enhanced resistance to
. In addition, overexpression of
and
downregulated lignin synthesis, and overexpression of Novel-miR1 and Novel-miR7 also downregulated lignin synthesis, indicating that the regulation of
and
by Novel-miR1 and Novel-miR7 could affect lignin content. The tobacco rattle virus (TRV) induced short tandem target mimic (STTM)-miRNA silencing vector was successfully constructed, and target miRNAs were successfully silenced. The identification of disease resistance and lignin content showed that silencing candidate miRNAs could improve cucumber resistance to
.
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