In a previous study, we reported a transient transformation system using repeated screening for hygromycin B (Hyg) resistance in the basidiomycete
Ceriporiopsis subvermispora
. In the present study, ...by combining this technique with CRISPR/Cas9, we demonstrated successful marker-free genome editing in
Pleurotus ostreatus
, which is one of the most economically important cultivated mushrooms as well as a model white-rot fungus. At first, transformant selection mediated by the transient expression of marker genes was demonstrated using a plasmid harboring the Hyg resistance gene (
hph
) in
P. ostreatus
. Then, genome editing of
fcy1
, which confers 5-fluorocytosine (5-FC) resistance to the host cell, was performed by the transient expression of Cas9, gRNA, and
hph
and strains with 5-FC resistance and Hyg sensitivity were isolated. Additionally, genome editing of
fcy1
in these strains was confirmed by Sanger sequencing. To our knowledge, this is the first report of marker-free genome editing through the transient expression of Cas9, gRNA, and
hph
in agaricomycetes, which opens the door for repeated genome editing in these fungi.
Phosphofructokinase plays an essential role in sugar metabolism in plants. Plants possess two types of phosphofructokinase proteins for phosphorylation of fructose-6-phosphate, the ...pyrophosphate-dependent fructose-6-phosphate phosphotransferase (PFP), and the ATP-dependent phosphofructokinase (PFK). Until now, the gene evolution, expression patterns, and functions of phosphofructokinase proteins were unknown in pear. In this report, 14 phosphofructokinase genes were identified in pear. The phylogenetic tree indicated that the phosphofructokinase gene family could be grouped into two subfamilies, with 10 genes belonging to the PbPFK subfamily, and 4 genes belonging to the PbPFP subfamily. Conserved motifs and exon numbers of the phosphofructokinase were found in pear and other six species. The evolution analysis indicated that WGD/Segmental and dispersed duplications were the main duplication models for the phosphofructokinase genes expansion in pear and other six species. Analysis of cis-regulatory element sequences of all phosphofructokinase genes identified light regulation and the MYB binding site in the promoter of all pear phosphofructokinase genes, suggesting that phosphofructokinase might could be regulated by light and MYB transcription factors (TFs). Gene expression patterns revealed that PbPFP1 showed similar pattern with sorbitol contents, suggesting important contributions to sugar accumulation during fruit development. Further functional analysis indicated that the phosphofructokinase gene PbPFP1 was localized on plasma membrane compartment, indicating that PbPFP1 had function in plasma membrane. Transient transformation of PbPFP1 in pear fruits led to significant increases of fructose and sorbitol compared to controls. Overall, our study provides important insights into the gene expression patterns and important potential functions of phosphofructokinase for sugar accumulation in pear fruits, which will help to enrich understanding of sugar-related bio-pathways and lay the molecular basis for fruit quality improvement.
•14 phosphofructokinases were identified in pear, and grouped into two subfamilies.•WGD/Segmental and dispersed duplications were the main duplication models in pear.•The motifs and exon numbers of phosphofructokinases were conserved in pear.•PbPFP1 is a membrane protein that contributes to the sugar accumulation in pear.
Members of the
genus are fascinating plants for many biologists as they are the smallest flowering plants on Earth and exhibit a reduced body plan that is of great interest to developmental ...biologists. There has also been recent interest in the use of these species for bioenergy or biorefining. Molecular and developmental studies have been limited in
species due to the high genome complexity and uncertainties regarding the stable genetic transformation. In this manuscript we present new protocols for both stable and transient genetic transformation for
using
. For the transient transformation, we used
fronds whereas we used clusters for the stable transformation. As proof of concept we transformed two synthetic promoter constructs driving expression of the GUS marker gene, that have previously been used to monitor auxin and cytokinin output in a variety of species. Using these approaches we obtained a Transformation Efficiency (TE) of 0.14% for the stable transformation and 21.8% for the transient transformation. The efficiency of these two methods of transformation are sufficient to allow future studies to investigate gene function. This is the first report for successful stable transformation of
.
Tripogon loliiformis, an Australian native resurrection grass having an abundant gene pool for combating desiccation, can be the putative model system for functional characterization of stress ...tolerance genes due to its diploid genome and being a monocotyledonous plant and member of the grass family (Poaceae), like many important cereal crops. For developing callus mediated regeneration from mature grains of Tripogon, Murashige and Skoog medium containing growth regulator, 2,4-dichlorophenoxyacetic acid (2,4-D) at 1.0mgL−1 was the optimum concentration for induction and proliferation of healthy cream calli. Successful regeneration of shoots from callus clumps in MS medium supplemented with 2.0mgL−1 6-benzylaminopurine (BAP) and 0.5mgL−1 α-naphthalene acetic acid (NAA) was obtained from 2 consecutive rounds subculturing of the calli at 3 weeks interval. In addition, rooting needed another 2 rounds within the same media with 2.0mgL−1 BAP but with 0.25mgL−1 NAA. The transient expression of UidA gene at 3 days after Tripogon callus transformation, performed with Agrobacterium tumefaciens strains AGL1 and LBA4404 following rice and Brachypodium distachyon transformation protocols, indicates successful Agrobacterium infection and gene delivery in calli. A stable transformation system for Tripogon loliiformis can be developed near future following the protocols in this study.
Agrobacterium tumefaciens‐mediated transient transformation has been a useful procedure for characterization of proteins and their functions in plants, including analysis of protein–protein ...interactions. Agrobacterium‐mediated transient transformation of Nicotiana benthamiana by leaf infiltration has been widely used due to its ease and high efficiency. However, in Arabidopsis this procedure has been challenging. Previous studies suggested that this difficulty was caused by plant immune responses triggered by perception of Agrobacterium. Here, we report a simple and robust method for Agrobacterium‐mediated transient transformation in Arabidopsis. AvrPto is an effector protein from the bacterial plant pathogen Pseudomonas syringae that suppresses plant immunity by interfering with plant immune receptors. We used transgenic Arabidopsis plants that conditionally express AvrPto under the control of a dexamethasone (DEX)‐inducible promoter. When the transgenic plants were pretreated with DEX prior to infection with Agrobacterium carrying a β‐glucuronidase (GUS, uidA) gene with an artificial intron and driven by the CaMV 35S promoter, transient GUS expression was dramatically enhanced compared to that in mock‐pretreated plants. This transient expression system was successfully applied to analysis of the subcellular localization of a cyan fluorescent protein (CFP) fusion and a protein–protein interaction in Arabidopsis. Our findings enable efficient use of Agrobacterium‐mediated transient transformation in Arabidopsis thaliana.
Beside the well known nutritional and health benefits, strawberry (FragariaXananassa) crop draws increasing attention as plant model system for the Rosaceae family, due to the short generation time, ...the rapid in vitro regeneration, and to the availability of the genome sequence of F.Xananassa and F. vesca species. In the last years, the use of high-throughput sequence technologies provided large amounts of molecular information on the genes possibly related to several biological processes of this crop. Nevertheless, the function of most genes or gene products is still poorly understood and needs investigation. Transient transformation technology provides a powerful tool to study gene function in vivo, avoiding difficult drawbacks that typically affect the stable transformation protocols, such as transformation efficiency, transformants selection, and regeneration. In this review we provide an overview of the use of transient expression in the investigation of the function of genes important for strawberry fruit development, defense and nutritional properties. The technical aspects related to an efficient use of this technique are described, and the possible impact and application in strawberry crop improvement are discussed.
Summary
In recent years, lipid droplets have emerged as dynamic organelles rather than inactive storage sites for triacylglycerol. The number of proteins known to be associated with lipid droplets ...has increased, but remains small in comparison with those found with other organelles. Also the mechanisms of how lipid droplets are recognized and bound by proteins need deeper investigation. Here, we present a fast, simple and inexpensive approach to assay proteins for their association with lipid droplets in vivo that can help to screen protein candidates or mutated variants of proteins for their association in an efficient manner. For this, a system to transiently transform Nicotiana tabacum pollen grains was used because these naturally contain lipid droplets. We designed vectors for fast cloning of genes as fusions with either mVenus or mCherry. This allowed us to assay colocalization with lipid droplets stained with Nile Red and Bodipy 505/515, respectively. We successfully tested our system not only for proteins from Arabidopsis thaliana, but also for proteins from the moss Physcomitrella patens and the alga Chlamydomonas reinhardtii. The small size of the vector used allows easy exchange of codons by site‐directed mutagenesis. We used this to show that two proline residues in the proline knot of a caleosin are not essential for the binding of lipid droplets. We also demonstrated that peroxisomes are not associated with the lipid droplets in tobacco pollen tubes, which reduces the risk of false interpretation of microscopic data in our system.
Significance Statement
Only a few lipid droplet‐associated proteins are known, and their binding mechanisms are mostly unclear. Here we show that transient expression of constructs in tobacco pollen tubes, which naturally contain lipid droplets, can be used to test protein localization to lipid droplets.
Chagas disease—caused by the parasite
Trypanosoma cruzi
—is a neglected tropical disease for which available drugs are not fully effective in the chronic stage and a vaccine is not available yet. ...Microalgae represent a promising platform for the production and oral delivery of low-cost vaccines. Herein, we report a vaccine prototype against
T. cruzi
produced in a microalgae platform, based on the candidate antigen Tc24 with a C terminus fusion with the Co1 peptide (Tc24:Co1 vaccine prototype). After modeling the tertiary structure, in silico studies suggested that the chimeric protein is antigenic, not allergenic, and molecular docking indicated binding with Toll-like receptors 2 and 4. Thus, Tc24:Co1 was expressed in the marine microalga
Schizochytrium
sp., and Western blot confirmed the expression at 48 h after induction, with a yield of 632 µg/L of algal culture (300 μg/g of lyophilized algal cells) as measured by the enzyme-linked immunosorbent assay (ELISA). Upon oral administration of whole-cell
Schizochytrium
sp. expressing Tc24:Co1 (7.5 µg or 15 µg of Tc24:Co1 doses) in mice, specific serum IgG and intestinal mucosa IgA responses were detected in addition to an increase in serum Th1/Th2 cytokines. In conclusion,
Schizochytrium
sp.-expressing Tc24:Co1 is a promising oral vaccine prototype to be evaluated in an animal model of
Trypanosoma cruzi
infection.
Graphical Abstract
Oliver is a unique high-quality natural rubber tree species and rare medicinal tree species in China. The rapid characterization of
gene function has been severely hampered by the limitations of ...genetic transformation methods and breeding cycles. The polyethylene glycol (PEG)-mediated protoplast transformation system is a multifunctional and rapid tool for the analysis of functional genes
, but it has not been established in
.
In this study, a large number of highly active protoplasts were isolated from the stems of
seedlings by enzymatic digestion, and green fluorescent protein expression was facilitated using a PEG-mediated method.
Optimal enzymatic digestion occurred when the enzyme was digested for 10 h in an enzymatic solution containing 2.5% Cellulase R-10 (w/v), 0.6% Macerozyme R-10 (w/v), 2.5% pectinase (w/v), 0.5% hemicellulase (w/v), and 0.6 mol/L mannitol. The active protoplast yield under this condition was 1.13 × 106 protoplasts/g fresh weight, and the protoplast activity was as high as 94.84%.
This study established the first protoplasm isolation and transient transformation system in hard rubber wood, which lays the foundation for subsequent functional studies of
genes to achieve high-throughput analysis, and provides a reference for future gene function studies of medicinal and woody plants.
Phaseolus vulgaris is one of the most extensively studied model legumes in the world. The P. vulgaris genome sequence is available; therefore, the need for an efficient and rapid transformation ...system is more imperative than ever. The functional characterization of P. vulgaris genes is impeded chiefly due to the non-amenable nature of Phaseolus sp. to stable genetic transformation. Transient transformation systems are convenient and versatile alternatives for rapid gene functional characterization studies. Hence, the present work focuses on standardizing methodologies for protoplast isolation from multiple tissues and transient transformation protocols for rapid gene expression analysis in the recalcitrant grain legume P. vulgaris.
Herein, we provide methodologies for the high-throughput isolation of leaf mesophyll-, flower petal-, hypocotyl-, root- and nodule-derived protoplasts from P. vulgaris. The highly efficient polyethylene glycol-mannitol magnesium (PEG-MMG)-mediated transformation of leaf mesophyll protoplasts was optimized using a GUS reporter gene. We used the P. vulgaris SNF1-related protein kinase 1 (PvSnRK1) gene as proof of concept to demonstrate rapid gene functional analysis. An RT-qPCR analysis of protoplasts that had been transformed with PvSnRK1-RNAi and PvSnRK1-OE vectors showed the significant downregulation and ectopic constitutive expression (overexpression), respectively, of the PvSnRK1 transcript. We also demonstrated an improved transient transformation approach, sonication-assisted Agrobacterium-mediated transformation (SAAT), for the leaf disc infiltration of P. vulgaris. Interestingly, this method resulted in a 90 % transformation efficiency and transformed 60-85 % of the cells in a given area of the leaf surface. The constitutive expression of YFP further confirmed the amenability of the system to gene functional characterization studies.
We present simple and efficient methodologies for protoplast isolation from multiple P. vulgaris tissues. We also provide a high-efficiency and amenable method for leaf mesophyll transformation for rapid gene functional characterization studies. Furthermore, a modified SAAT leaf disc infiltration approach aids in validating genes and their functions. Together, these methods help to rapidly unravel novel gene functions and are promising tools for P. vulgaris research.
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