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Genomic deletions in Aureobasidium pullulans by an AMA1 plasmid for gRNA and CRISPR/Cas9 expression.Fungal Biology and Biotechnology Jun 2024Aureobasidium pullulans is a generalist polyextremotolerant black yeast fungus. It tolerates temperatures below 0 °C or salt concentrations up to 18%, among other...
BACKGROUND
Aureobasidium pullulans is a generalist polyextremotolerant black yeast fungus. It tolerates temperatures below 0 °C or salt concentrations up to 18%, among other stresses. A. pullulans genome sequencing revealed a high potential for producing bioactive metabolites. Only few molecular tools exist to edit the genome of A. pullulans, hence it is important to make full use of its potential. Two CRISPR/Cas9 methods have been proposed for the protoplast-based transformation of A. pullulans. These methods require the integration of a marker gene into the locus of the gene to be deleted, when the deletion of this gene does not yield a selectable phenotype. We present the adaptation of a plasmid-based CRISPR/Cas9 system developed in Aspergillus niger for A. pullulans to create deletion strains.
RESULTS
The A. niger CRISPR/Cas9 plasmid led to efficient genomic deletions in A. pullulans. In this study, strains with deletions ranging from 30 to 862 bp were obtained by using an AMA1 plasmid-based genome editing strategy.
CONCLUSION
The CRISPR/Cas9 transformation system presented in this study provides new opportunities for strain engineering of A. pullulans. This system allows expression of Cas9 and antibiotic resistance while being easy to adapt. This strategy could open the path to intensive genomic engineering in A. pullulans.
PubMed: 38824542
DOI: 10.1186/s40694-024-00175-4 -
Frontiers in Plant Science 2024Environmental stressors disrupt secretory protein folding and proteostasis in the endoplasmic reticulum (ER), leading to ER stress. The unfolded protein response (UPR)...
Protein disulfide isomerase-9 interacts with the lumenal region of the transmembrane endoplasmic reticulum stress sensor kinase, IRE1, to modulate the unfolded protein response in .
Environmental stressors disrupt secretory protein folding and proteostasis in the endoplasmic reticulum (ER), leading to ER stress. The unfolded protein response (UPR) senses ER stress and restores proteostasis by increasing the expression of ER-resident protein folding chaperones, such as protein disulfide isomerases (PDIs). In plants, the transmembrane ER stress sensor kinase, IRE1, activates the UPR by unconventionally splicing the mRNA encoding the bZIP60 transcription factor, triggering UPR gene transcription. The induced PDIs catalyze disulfide-based polypeptide folding to restore the folding capacity in the ER; however, the substrates with which PDIs interact are largely unknown. Here, we demonstrate that the PDI-M subfamily member, PDI9, modulates the UPR through interaction with IRE1. This PDI9-IRE1 interaction was largely dependent on Cys63 in the first dithiol redox active domain of PDI9, and Cys233 and Cys107 in the ER lumenal domain of IRE1A and IRE1B, respectively. and , PDI9 coimmunoprecipitated with IRE1A and IRE1B. Moreover, the PDI9:RFP and Green Fluorescence Protein (GFP):IRE1 fusions exhibited strong interactions as measured by fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer (FLIM-FRET) when coexpressed in mesophyll protoplasts. The UPR-responsive PDI9 promoter:mCherry reporter and the UPR-dependent splicing of the bZIP60 intron from the mRNA of the 35S::bZIP60-intron:GFP reporter were both significantly induced in the mutants, indicating a derepression and hyperactivation of UPR. The inductions of both reporters were substantially attenuated in the mutant. We propose a model in which PDI9 modulates the UPR through two competing activities: secretory protein folding and via interaction with IRE1 to maintain proteostasis in plants.
PubMed: 38817940
DOI: 10.3389/fpls.2024.1389658 -
Frontiers in Bioscience (Landmark... May 2024Oliver 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...
BACKGROUND
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 .
METHODS
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.
RESULTS
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%.
CONCLUSIONS
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.
Topics: Protoplasts; Eucommiaceae; Transfection; Green Fluorescent Proteins; Polyethylene Glycols
PubMed: 38812327
DOI: 10.31083/j.fbl2905187 -
The Plant Genome Jun 2024The clustered regularly interspaced short palindromic repeats (CRISPR) systems have been demonstrated to be the foremost compelling genetic tools for manipulating...
The clustered regularly interspaced short palindromic repeats (CRISPR) systems have been demonstrated to be the foremost compelling genetic tools for manipulating prokaryotic and eukaryotic genomes. Despite the robustness and versatility of Cas9 and Cas12a/b nucleases in mammalian cells and plants, their large protein sizes may hinder downstream applications. Therefore, investigating compact CRISPR nucleases will unlock numerous genome editing and delivery challenges that constrain genetic engineering and crop development. In this study, we assessed the archaeal miniature Un1Cas12f1 type-V CRISPR nuclease for genome editing in rice and tomato protoplasts. By adopting the reengineered guide RNA modifications ge4.1 and comparing polymerase II (Pol II) and polymerase III (Pol III) promoters, we demonstrated uncultured archaeon Cas12f1 (Un1Cas12f1) genome editing efficacy in rice and tomato protoplasts. We characterized the protospacer adjacent motif (PAM) requirements and mutation profiles of Un1Cas12f1 in both plant species. Interestingly, we found that Pol III promoters, not Pol II promoters, led to higher genome editing efficiency when they were used to drive guide RNA expression. Unlike in mammalian cells, the engineered Un1Cas12f1-RRA variant did not perform better than the wild-type Un1Cas12f1 nuclease, suggesting continued protein engineering and other innovative approaches are needed to further improve Un1Cas12f1 genome editing in plants.
Topics: Oryza; Solanum lycopersicum; Gene Editing; CRISPR-Cas Systems; Protoplasts; Genome, Plant
PubMed: 38807445
DOI: 10.1002/tpg2.20465 -
International Journal of Molecular... May 2024A homeobox transcription factor is a conserved transcription factor, ubiquitous in eukaryotes, that regulates the tissue formation of structure, cell differentiation,...
A homeobox transcription factor is a conserved transcription factor, ubiquitous in eukaryotes, that regulates the tissue formation of structure, cell differentiation, proliferation, and cancer. This study identified the homeobox transcription factor family and its distribution in var. at the whole genome level. It elucidated the gene structures and evolutionary characteristics of this family. Additionally, knockout experiments were carried out and the preliminary function of these transcription factors was studied. Through bioinformatics approaches, nine homeobox transcription factors () were identified in var. , and these contained HOX-conserved domains and helix-turn-helix secondary structures. Nine homeobox gene deletion mutants were obtained using the homologous recombinant gene knockout technique. Protoplast transformation was mediated by polyethylene glycol (PEG) and the transformants were identified using PCR. The knockouts of , , , , , , and genes resulted in a smaller growth diameter in var. . In contrast, the knockouts of the , , and genes inhibited the formation of conidia and led to a significant decrease in the pathogenicity. This study's results will provide insights for understanding the growth and development of var. . The pathogenic mechanism of the affected sugarcane will provide an essential theoretical basis for preventing and controlling sugarcane twisted leaf disease.
Topics: Saccharum; Homeodomain Proteins; Plant Diseases; Ascomycota; Transcription Factors; Plant Leaves; Phylogeny
PubMed: 38791383
DOI: 10.3390/ijms25105346 -
Journal of Separation Science May 2024Current techniques identifying herbal medicine species require marker labeling or lack systematical accuracy (expert authentication). There is an emerging interest in...
Current techniques identifying herbal medicine species require marker labeling or lack systematical accuracy (expert authentication). There is an emerging interest in developing an accurate and label-free tool for herbal medicine authentication. Here, a high-resolution microfluidic-based method is developed for identifying herbal species by protoplast subpopulations. Moso bamboo and henon bamboo are used as a model to be differentiated based on protoplast. Their biophysical properties factors are characterized to be 7.09 (± 0.39) × 10 V/m and 6.54 (± 0.26) × 10V/m, respectively. Their biophysical distributions could be distinguished by the Cramér-von Mises criterion with a 94.60% confidence level. The subpopulations of each were compared with conventional flow cytometry indicating the existence of subpopulations and the differences between the two species. The subsets divided by a biophysical factor of 8.05(± 0.51) × 10 V/m suggest good consistency with flow cytometry. The work demonstrated the possibility of microfluidics manipulation on protoplast for medication safety use taking advantage of dielectrophoresis. The device is promising in developing a reliable and accurate way of identifying herbal species with difficulties in authentication.
Topics: Protoplasts; Plant Leaves; Single-Cell Analysis; Flow Cytometry; Microfluidic Analytical Techniques; Microfluidics
PubMed: 38772720
DOI: 10.1002/jssc.202400120 -
Journal of Plant Physiology Aug 2024The oil palm (Elaeis guineensis) is emerging as the world's most important and prolific oilseed crop, celebrated for its impressive oil yield. However, the molecular...
The oil palm (Elaeis guineensis) is emerging as the world's most important and prolific oilseed crop, celebrated for its impressive oil yield. However, the molecular intricacies that govern lipid metabolism and fatty acid accumulation in oil palm fruits remain relatively underexplored. This study reveals a significant correlation between the expression of EgGRP2A, a transcription factor, and the expression of EgFATA in the oil palm. Yeast one-hybrid analysis and electrophoretic mobility shift assays (EMSA) reveal and confirm the binding interactions between EgGRP2A and the promoter region of EgFATA. Subsequent experiments in oil palm protoplasts show that transient overexpression of EgGRP2A leads to a marked upregulation of EgFATA expression. Conversely, downregulation of EgGRP2A in transgenic oil palm embryoids leads to a significant reduction in EgFATA expression. Metabolite profiling in the transgenic embryoids reveals a significant reduction in unsaturated fatty acids, particularly oleic acid. These findings promise profound insights into the regulatory orchestration of EgFATA and the synthesis of fatty acids, particularly oleic acid, in the oil palm. Furthermore, the results lay the foundation for future breeding and genetic improvement efforts aimed at increasing oleic acid content in oil palm varieties.
Topics: Arecaceae; Oleic Acid; Plant Proteins; Transcription Factors; Gene Expression Regulation, Plant; Plants, Genetically Modified
PubMed: 38772323
DOI: 10.1016/j.jplph.2024.154263 -
Nature Biotechnology May 2024The construction of synthetic gene circuits in plants has been limited by a lack of orthogonal and modular parts. Here, we implement a CRISPR (clustered regularly...
The construction of synthetic gene circuits in plants has been limited by a lack of orthogonal and modular parts. Here, we implement a CRISPR (clustered regularly interspaced short palindromic repeats) interference (CRISPRi)-based reversible gene circuit platform in plants. We create a toolkit of engineered repressible promoters of different strengths and construct NOT and NOR gates in Arabidopsis thaliana protoplasts. We determine the optimal processing system to express single guide RNAs from RNA Pol II promoters to introduce NOR gate programmability for interfacing with host regulatory sequences. The performance of a NOR gate in stably transformed Arabidopsis plants demonstrates the system's programmability and reversibility in a complex multicellular organism. Furthermore, cross-species activity of CRISPRi-based logic gates is shown in Physcomitrium patens, Triticum aestivum and Brassica napus protoplasts. Layering multiple NOR gates together creates OR, NIMPLY and AND logic functions, highlighting the modularity of our system. Our CRISPRi circuits are orthogonal, compact, reversible, programmable and modular and provide a platform for sophisticated spatiotemporal control of gene expression in plants.
PubMed: 38769424
DOI: 10.1038/s41587-024-02236-w -
Gene Sep 2024Polygalacturonase inhibitor protein (PGIP) restricts fungal growth and colonization and functions in plant immunity. Gray mold in cucumber is a common fungal disease...
Polygalacturonase inhibitor protein (PGIP) restricts fungal growth and colonization and functions in plant immunity. Gray mold in cucumber is a common fungal disease caused by Botrytis cinerea, and is widespread and difficult to control in cucumber (Cucumis sativus L.) production. In this study, Cucumis sativus polygalacturonase-inhibiting protein 2 (CsPGIP2) was found to be upregulated in response to gray mold in cucumber. CsPGIP2 was detected in the endoplasmic reticulum, cell membrane, and cell wall after transient transformation of protoplasts and tobacco. A possible interaction between Botrytis cinerea polygalacturonase 3 (BcPG3) and CsPGIP2 was supported by protein interaction prediction and BiFC analysis. Transgenic Arabidopsis plants expressing CsPGIP2 were constructed and exhibited smaller areas of gray mold infection compared to wild type (WT) plants after simultaneous inoculation. Evans blue dye (EBD) confirmed greater damage for WT plants, with more intense dyeing than for the transgenic Arabidopsis. Interestingly, compared to WT, transgenic Arabidopsis exhibited higher superoxide dismutase (AtSOD1) expression, antioxidant enzyme activities, lignin content, net photosynthetic rate (Pn), and photochemical activity. Our results suggest that CsPGIP2 stimulates a variety of plant defense mechanisms to enhance transgenic Arabidopsis resistance against gray mold disease.
Topics: Cucumis sativus; Arabidopsis; Plant Diseases; Botrytis; Disease Resistance; Plants, Genetically Modified; Plant Proteins; Gene Expression Regulation, Plant
PubMed: 38763363
DOI: 10.1016/j.gene.2024.148588 -
Bioelectrochemistry (Amsterdam,... Oct 2024It was previously reported that stress induces a cellular production of abscisic acid in plants, but no direct method shows the evidence. Here, an electrochemical...
It was previously reported that stress induces a cellular production of abscisic acid in plants, but no direct method shows the evidence. Here, an electrochemical microsensor involving an abscisic acid receptor PYL2 modified carbon fiber microelectrode was fabricated by self-assembly method, where the Cu combined with the histidine tag of PYL2 on the surface of microelectrode was used as the detection probe, the mediated reaction between Cu and ferricyanide realized the amplification responses and provided the microsensor with a high sensitivity for detection of abscisic acid with a detection limit of 0.8 nM. With use of this microsensor, an increase of extracellular abscisic acid from single rice protoplast induced by sulfate, osmotic and salinity stress was real-time monitored. Direct measurement of free extracellular abscisic acid in single plant cells might offer important new insights into its role in plants challenged by abiotic stresses.
Topics: Oryza; Abscisic Acid; Microelectrodes; Protoplasts; Plant Proteins; Biosensing Techniques; Copper; Electrochemical Techniques; Ferricyanides
PubMed: 38761493
DOI: 10.1016/j.bioelechem.2024.108733