Search Results (411)

The modulation of plant responses to abscisic acid (ABA) and/or abiotic stresses can be manipulated by the expression of ABA-responsive genes, which is affected by phytohormone ABA. While some ABA-responsive genes have been shown to regulate plant responses to ABA and/or abiotic stresses, the functions of numerous ABA-responsive genes remain unknown. Therefore, characterizing these unstudied genes would provide a practical way to identify novel regulators of plant adaptations to ABA and/or abiotic stresses. Here, we characterized four closely related unstudied ABA-responsive genes in Arabidopsis thaliana, named Arabidopsis thaliana ABA-up regulated genes (AtAUGs). We found that ABA treatment induces AtAUGs expression level, and our results in transfected protoplasts show that AtAUGs exhibit nucleus localization and downregulate the co-transfected reporter expression level. The results of ABA sensitivity assays, including seed germination, cotyledon greening, and root extension assay show that transgenic plants overexpressing AtAUGs had increased sensitivity, but ataugs mutants generated by isolating T-DNA insertion lines or through CRISPR/Cas9 gene-editing of AtAUGs had decreased sensitivity; in addition, the greatest decrease in ABA sensitivity was observed in the ataug1 ataug2 ataug3 ataug4 (ataug1234) quadruple mutants. The qRT-PCR results show that the expression levels of several Type 2C Protein Phosphatase (PP2C) genes, the key negative regulator genes of ABA signaling including PP2CA, Hypersensitive to ABA 1 (HAB1), HAB2, Highly ABA-Induced PP2C protein 3 (HAI3), ABA-Hypersensitive Germination 1 (AHG1), and ABA Insensitive 2 (ABI2) decreased in 35S:AtAUGs transgenic plants, but increased in the ataug1234 quadruple mutants. Taken together, these results suggest that AtAUGs are ABA-responsive genes, and AtAUGs positively regulate ABA responses in a redundant manner, by downregulating the expression of crucial negative regulator genes in ABA signaling. Full article

Broccoli (Brassica oleracea var. italica) is an important crop valued for its nutritional and health-promoting properties, yet its biotechnological improvement is limited by low effectivity and genotype-dependent transformation protocols. The absence of reliable transient expression systems further constrains functional genomics and genome-editing applications. Here, we optimized regeneration and transformation protocols for different broccoli genotypes. Endoreduplication patterns in young tissues were analyzed by flow cytometry to identify suitable explants, and combinations of plant growth regulators were tested to develop an efficient organogenic medium. Stable transformation was achieved via Agrobacterium tumefaciens using nptII and eGFP markers. Cotyledons and hypocotyls up to day 7 showed similar endoreduplication patterns, with abundant 2n cells, but hypocotyls exhibited higher regeneration capacity. The optimized medium supported efficient organogenesis while maintaining diploidy. Transformation efficiency reached 10.4% in ‘S1’ and 2.8% in ‘Naxos’, highlighting genotype dependence. In parallel, a transient expression system was established using cotyledon-derived protoplasts and electroporation-mediated DNA delivery. GFP expression was confirmed through fluorescence microscopy, confocal imaging, and Western blotting. These protocols provide a robust toolkit for broccoli genetic manipulation, facilitating molecular biology studies in the native plant, functional genomics and genome-editing strategies, including CRISPR-based approaches. Full article

Plants have a remarkable ability to regenerate tissues and organs from single cells, a property that underpins in vitro protoplast regeneration. Efficient protoplast-to-plant regeneration remains a major bottleneck for genome engineering in many crop species, including broccoli (Brassica oleracea var. italica). In this study, we established and optimized a regeneration system for broccoli cv. Claremont by evaluating enzyme composition, light quality, and culture media at successive stages of development. Among the tested enzyme mixtures, 1.5% Cellulase R-10 combined with 0.4% Macerozyme R-10 yielded the highest protoplast viability and recovery. Alginate-embedded protoplasts were cultured under control (dark), blue, and red + far-red LED illumination. Red + far-red treatment significantly enhanced microcolony formation, plating efficiency, and shoot regeneration compared with blue light, whereas blue illumination consistently reduced regenerative performance. The inclusion of activated charcoal in the regeneration medium further increased shoot production. The generalized linear model analyses identified light quality as a significant predictor of both shoot number and regeneration. To our knowledge, this study provides one of the first demonstrations of LED-assisted enhancement of protoplast regeneration in broccoli. The optimized protocol enables whole-plant recovery within approximately 5 months and offers a practical platform for CRISPR-based genome editing and advanced breeding applications in B. oleracea. Full article

Fusarium circinatum Nirenberg & O’Donnell (the causal agent of Pine Pitch Canker, PPC), one of the most devastating threats to pine forests and nurseries worldwide, induces canker disease on a wide range of pine species. However, its status as a quarantined pathogen and the scarcity of reliable genetic manipulation tools have long impeded in-depth genomic research on this fungus, and the infection mechanisms of F. circinatum remain an urgent area for investigation. A key approach to expounding its pathogenicity is to perform gene editing on candidate genes, which requires an efficient transformation system. Protoplast-mediated transformation is a critical means for investigating plant-pathogen interactions. During the course of this study, we constructed a PEG/CaCl₂-mediated protoplast transformation method for F. circinatum. Following systematic optimization of transformation conditions, strain A015-1 was selected as the model organism. The optimal enzymolysis system consisted of 5 mg/mL Lysing enzymes, 12.5 mg/mL Driselase, and 7.5 mg/mL Snailase, with incubation at 30 °C for 3 h under shaking at 80 rpm. All positive transformants exhibited strong green fluorescent signals. A total of 31 transformants were obtained after hygromycin B (HPH) selection, and PCR verification confirmed successful amplification of the gfp and hph gene fragments in 30 transformants, corresponding to a positive rate of 96%. Transformation efficiency was calculated as the number of PCR-positive transformants per microgram of plasmid DNA, yielding an efficiency of 1 transformant/μg plasmid DNA under optimal conditions. Furthermore, no significant differences were observed in vegetative growth, development, or pathogenicity between the transformants and the wild-type (WT) strain. In addition, Green fluorescent protein (GFP) was efficiently transformed into F. circinatum protoplasts and functionally expressed. Collectively, this study successfully established a stable transformation system for F. circinatum, providing a foundational platform for analyzing virulence-related functional genes involved in host infection and deciphering the molecular mechanisms underlying the pathogen’s colonization of pine hosts. Full article

This study aims to construct a nattokinase (NK) high-yielding strain using the multiple-scale breeding method. First, an NK-producing strain Bacillus subtilis A-1 was isolated from fermented soybean, which produces 254 FU/mL of NK. Subsequently, ARTP mutagenesis was employed to screen high-yield mutants with resistance to rifampicin (i.e., strain R-F7), kanamycin (i.e., strain K-E11), and gentamicin (i.e., strain G-D5), and the resulted strains showed NK activity increases of 113.78%, 76.38%, and 62.99%, respectively. Moreover, a fusion strain C-D7 with resistant to the above three antibiotics (i.e., rifampicin, kanamycin, and gentamicin) was obtained by protoplast fusion, which produced 610 FU/mL of NK and represents a 140.16% higher that of strain A-1. The fermenting property of strain C-D7 was also done in a 5-L bioreactor, and results indicated that strain C-D7 produced 1020 ± 35 FU/mL of NK under a two-stage pH control strategy and a two-step feeding strategy. To elucidate the genetic basis for the high-yield phenotype of C-D7. comparative whole-genome analysis was performed between C-D7 and the parental strain A-1. The results revealed that C-D7 harbors specific mutations across multiple functional categories, primarily in genes related to transcription, translation, global regulation, as well as metabolism and secretion. The biological processes affected by these mutations show a strong correlation with the high-yield trait, suggesting their potential collective role in contributing to the observed increase in nattokinase production. Lastly, ituD and srfAC were knocked out to reduce foam during fermentation, thus reducing the use of antifoaming agents and mitigating the negative effects on cell growth. In a word, a genetically stable, high-yield, and low-foaming Bacillus subtilis strain C-D7-ΔDouble was constructed in this study, which provides a core microbial resource and process foundation for the low-cost industrial production of nattokinase. Full article

The Solanaceae family includes some of the most economically and agronomically important crops, such as tomato, potato, pepper and eggplant. Recently, CRISPR/Cas-based genome editing has emerged as a powerful tool for functional genomics and crop improvement, enabling precise and efficient genetic modifications. This review provides an overview of CRISPR/Cas-mediated genome editing technologies and their applications in the major cultivated Solanaceae crops. The use of CRISPR/Cas9 systems for targeted gene knockout and knock-in approaches is described, together with advances in precision editing strategies such as base editing and prime editing, which allow precise nucleotide substitutions and small sequence changes. The expanding CRISPR toolbox is further explored through alternative Cas proteins, such as Cas12a and Cas13 with distinct targeting features and potential applications. Emerging delivery strategies, including ribonucleoprotein-mediated editing in protoplasts, virus-induced gene editing (VIGE), de novo induction of meristems and genome editing by grafting, represent promising approaches to generate transgene-free edited plants. In addition, the current status of field trials involving genome-edited Solanaceae crops in Europe is outlined, considering the regulatory landscape and legislative requirements for their release in the environment. Despite regulatory constraints, some genome-edited crops have reached the market, highlighting their potential to contribute to sustainable agriculture and crop improvement. Full article

Aspergillus fijiensis is an industrially important filamentous fungus, whose genetic analysis has been limited by the absence of species-specific tools. This study establishes an optimized CRISPR–Cas9 genome editing platform for A. fijiensis, from protoplast preparation to DNA repair pathway engineering. Antibiotic screening first identified hygromycin B and 5-FOA (5-fluoroorotic acid) as effective positive and counter-selection markers. A high-efficiency protoplast regeneration protocol was developed depending on specific osmotic stabilization and mycelial competence. Evaluation of a plasmid-based CRISPR system revealed that while autonomous replication was feasible, gene editing was constrained by low efficiency and a predominant bias toward NHEJ (non-homologous end joining). We implemented a Cas9–sgRNA RNP (ribonucleoprotein) delivery approach, with RNP delivery alone producing frequent indels. However, targeted integration remained inefficient when using conventional MMEJ (Microhomology-mediated end joining) donors. By employing donors containing short (5 bp) microhomology arms between cleavage sites, we effectively engaged the MMEJ pathway, enabling precise insertions and large-fragment deletions in 92% of the analyzed transformants. Donor templates containing minimal 5 bp microhomology sequences could effectively shift the predominant repair pathway from NHEJ to MMEJ. These findings demonstrate that MMEJ is the superior pathway with a unique mechanism for genome engineering in A. fijiensis, providing a versatile toolkit for unlocking the biotechnological potential of this recalcitrant species and a successful paradigm for establishing genetic systems in other species. Full article

Plant pathogens are a major cause of crop yield loss, making disease resistance breeding crucial for crop improvement. Plants have evolved innate immune systems, mediated by immune-related genes such as nucleotide-binding site leucine-rich repeat (NLR), pattern-recognition receptors (PRR) and susceptibility genes, which are essential resources for breeding disease-resistant plants. To identify immunity genes, extensive genetic approaches that examine the association between resistance phenotypes and genomic regions have been applied with great success. While genetic methods remain important for identifying immunity genes, novel strategies that rely on functional rather than genetic association with disease resistance offer unique advantages. For example, mutagenesis with R gene enrichment sequencing (MutRenSeq) enabled the identification of wheat resistance genes Sr22 and Sr45 by comparing the NLRomes of resistant and susceptible lines while single-cell RNA sequencing resolved cell-type-specific responses to pathogen infection and revealed ZmChit7, especially in maize epidermal and guard cells. These approaches reach beyond existing natural variation, can accelerate experimental timelines, reduce the experimental scale, and provide mechanistic insights into pathogen resistance. This review discusses emerging techniques that generate focused candidate immunity gene lists or accelerate their validation, as both are required to identify causal variants for resistance breeding. We consider advances in RenSeq-derived methods, spatial omics, proximity labelling, computational prediction, Clustered regularly interspaced short palindromic repeats (CRISPR) screens, and cell death assays. These approaches are reshaping resistance breeding pipelines beyond association-based discovery. By discussing the strengths and limitations of these emerging methods and their combinations, we outline current opportunities and future directions to help plant pathologists to more effectively identify and validate plant immunity genes. Full article

Low temperature is a major abiotic stress that affects maize across its entire growth cycle, with the germination stage being particularly sensitive. To investigate the genetic basis of early-stage cold tolerance, we used quantitative trait locus mapping and identified ZmbHLH30 as a candidate gene regulating maize responses to low temperature. The ZmbHLH30 protein is localized in the cytoplasm of maize protoplasts, and ZmbHLH30 promoter drives β-glucuronidase (GUS) expression in Arabidopsis thaliana leaves. The promoter region of ZmbHLH30 contains multiple environmental stress-responsive elements, including motifs associated with cold and auxin responses. Overexpression of ZmbHLH30 significantly enhanced cold tolerance at the germination, bud, and seedling stages, with the strongest effect observed during germination, where the cold-tolerance D-value increased by 0.366 relative to the control. In contrast, CRISPR/Cas9 knockout lines showed a 0.399 decrease in D-value. Under cold stress, ZmbHLH30 expression was markedly induced in overexpression lines but suppressed in knockout lines. Integrated transcriptomic and metabolomic analyses further identified ZmbHLH30 as a key regulator of cold tolerance in maize. Full article

Ophiocordyceps xuefengensis is an important medicinal fungus with considerable pharmaceutical and economic value. However, its industrial and scientific utilization has been severely limited by the lack of an efficient genetic transformation system, largely due to limited genomic information and wild growth. In this study, we established an efficient and stable plasmid transformation system within O. xuefengensis protoplasts mediated by PEG. To overcome low protoplast yield and transformation efficiency, key factors influencing protoplast preparation including enzyme composition and concentration, fungal age, and digestion conditions were systematically optimized. The optimal protocol involved digesting 4-day-old mycelia with a mixture of 1.5% lywallzyme 1 and 1.5% snailase at 34 °C and 130 rpm for 3.5 h, yielding at least 9.42 × 10⁷ CFU/mL protoplasts. Protoplast regeneration was significantly enhanced in PY medium supplemented with 0.6 M mannitol. Under these optimized conditions, a transformation efficiency of 45.5% was achieved, with stable plasmid integration confirmed over four successive generations. Furthermore, the transformation system was successfully applied to functional gene characterization by driving exogenous gene expression using the endogenous gpd1 promoter. This study provides a foundational platform for functional gene analysis and paves the way for further elucidation of growth and development mechanisms and metabolic engineering in O. xuefengensis. Full article

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system is a simple and powerful tool, which enables gene knockout or insertion of new gene cassettes. This method has been applied to various plants and is used for crop improvement. Cultivated strawberry (Fragaria× ananassa), a member of the Rosaceae family, is a high-value horticultural crop. However, its complex octoploid genome poses challenges for precise genome editing in polyploids. This study aimed to establish a protoplast-based, DNA-free genome-editing approach in the cultivated octoploid strawberry. We optimized the culture conditions and enzyme combinations to enable efficient protoplast isolation from the fully developed leaves. The highest protoplast yield was obtained with a Murashige and Skoog medium containing 1% sucrose and 2 mg/L 6-benzylaminopurine (BA), along with enzymatic digestion using 2% Viscozyme, 1% Celluclast, and 1% Pectinex. Transient transfection conditions were optimized using a green fluorescence protein (GFP) plasmid with the highest expression efficiency (up to 52.5%) observed using 40% PEG 4000 and 20 min incubation. Under these conditions, Cas9 ribonucleoproteins (RNPs) targeting the FaPDS and FaPG1 genes were introduced, and guide RNA (gRNA) screening was conducted by targeted deep sequencing. In conclusion, this study successfully demonstrated protoplast isolation and DNA-free CRISPR/Cas9 genome editing in cultivated strawberry. The optimized protoplast-based system provides a valuable platform for functional genomics and molecular breeding efforts in octoploid strawberries. Full article

The remarkable plasticity of plants is best exemplified by the capacity of their somatic cells to regenerate entire organs or the organism itself. The molecular and cellular events underlying this ability are complex and multifaceted. The initial phase leading to cell cycle reactivation is often called dedifferentiation. This process is triggered either by wounding or exogenous hormone application. In this opinion paper, I propose that the dedifferentiation of mature somatic cells is a two-step process. It involves a transition into a transient senescence-like state induced by stress and/or signals emanating from dying cells. This state entails the loss of genetic information required for cell differentiation, resulting in a critical cellular condition. In the absence of subsequent proliferative signals, dedifferentiating (senescing) cells become committed to programmed cell death. Exogenous and/or endogenous plant hormones, such as auxin and cytokinin, might override this pathway. This rescue step, in most cases, activates cell divisions to replace lost cells/tissues. If cell division is maintained, it may result in callus formation. A callus is not an undifferentiated, homogeneous mass of cells. It is an unorganised tissue with at least some cells having ground-tissue-like molecular identity and high developmental potential. A callus might also form from pre-existing competent cell populations, e.g., pericycle cells, with no senescence-like intermitting state. It is discussed whether this “one-step” callus-formation pathway can be considered dedifferentiation. Full article

CRISPR-Cas9 technology has opened new perspectives in genome editing of clonally, asexually propagated and polyploid plants by enabling multiple allelic gene edits. Traditional Agrobacterium- and particle bombardment-mediated transformations, which rely on integration of gene-editing transgene cassettes, have been efficiently applied to several plants; however, concerns about the acceptability of resultant edited transgenic genotypes make these methods less attractive for vegetatively propagated crops. We leveraged and optimized the CRISPR-Cas9/sgRNA-RNPs system for delivery into protoplasts of the hexaploid sweetpotato cultivar PI-318846, targeting eukaryotic translation initiation factor isoform 4E genes to enhance resistance to SPFMV potyviruses. To evaluate the efficiency of pre-assembled Cas9/sgRNA-RNP in sweetpotato transfection, single guide RNAs were designed to target putative host susceptibility genes: IbeIF4E, IbeIF(iso)4E, and IbCBP. Freshly isolated leaf protoplasts were subjected to CRISPR-CAS9-RNP PEG-mediated transfection under different parameters. Sweetpotato regenerants screened using PCR-RE-T7 assay, sequencing, and Inference CRISPR Edit analyses of target-site amplicons revealed the most efficient editing conditions utilizing 25% PEG with a 3:1 (15 µg:45 µg) ratio of Cas9/sgRNA-RNP for 25 min and 48 h incubation period. Different allelic InDels were obtained with editing efficiencies of 10–20% in regenerated plantlets, demonstrating that PEG-mediated CRISPR-RNP transfection system is key for advancing DNA-free editing tools in polyploid and vegetatively propagated crops. Full article

Sporothrix species are thermally dimorphic fungi responsible for sporotrichosis, a globally prevalent subcutaneous mycosis and an emerging zoonotic threat, particularly in South America. The high virulence of Sporothrix brasiliensis and its efficient transmission from cats to humans have intensified recent outbreaks, underscoring the importance of understanding the pathogenic mechanisms. While several putative virulence factors have been identified, such as melanin production, cell wall remodeling, extracellular vesicles, and thermotolerance, functional studies remain hampered by limited molecular tools. Recent advances, including random mutagenesis, protoplast-mediated transformation, Agrobacterium tumefaciens-mediated transformation, RNA interference and CRISPR/Cas9-based genome editing, are changing this landscape. These methods have enabled the functional validation of key virulence factors and the investigation of gene function in both environmental and clinical strains. In this review, we summarize the genetic toolbox available for Sporothrix, outline current challenges, and discuss how these strategies are reshaping the study of fungal virulence and host–pathogen interactions. Full article

Eucommia ulmoides, a tree species native to China, holds considerable medicinal, ecological, and industrial importance. However, the absence of an efficient and stable genetic transformation system poses significant challenges to gene function studies and molecular breeding in E. ulmoides. Protoplasts, which lack cell walls, serve as effective receptors for transient transformation and are thus ideal for genetic engineering research. In this study, the optimal conditions for callus induction were identified, and formation of the embryogenic callus was confirmed by histological analysis. Furthermore, we developed an efficient protoplast isolation and PEG-mediated transient transformation system using suitable embryogenic callus as the starting material. Our findings revealed that the optimal medium for inducing embryogenic callus was B5 + 1.5 mg/L 6-BA + 0.5 mg/L NAA + 30 g/L sucrose + 7 g/L agar (pH = 5.8). In this medium, the induction rate of callus achieved 97.50%, and the rate of embryogenic callus formation was 86.30%. For protoplast isolation, the best conditions involved enzymatic digestion with 1.5% cellulase R-10 and 1.0% macerozyme R-10 at an osmotic pressure of 0.6 M for 4 h, resulting in 1.82 × 10⁶ protoplasts/g FW with 91.13% viability. The highest transfection efficiency (53.23%) was attained when protoplasts were cultured with 10 µg of plasmid and 40% PEG4000 for 20 min. This study successfully established a stable and efficient system for protoplast isolation and transient transformation in E. ulmoides, offering technical support for exploring somatic hybridisation and transient gene expression in this species. Full article