Search Results (51)

Rice (Oryza sativa L.), a staple food for over half of the global population, plays a pivotal role in food security. Among its two primary groups, japonica and indica, temperate japonica varieties are particularly valued for their high-quality grain and culinary uses. Although some of these varieties are adapted to cooler climates, they often suffer from reduced productivity or increased disease susceptibility when cultivated in warmer productive environments. These limitations underscore the need for breeding programs to incorporate biotechnological tools that can enhance the adaptability and resilience of the plants. However, New Genomic Techniques (NGTs), including CRISPR-Cas9, require robust in vitro systems, which are still underdeveloped for temperate japonica genotypes. In this study, we developed a reproducible and adaptable protocol for protoplast isolation and regeneration from the temperate japonica cultivars ‘Ónix’ and ‘Platino’ using somatic embryos as the starting tissue. Protoplasts were isolated via enzymatic digestion (1.5% Cellulase Onozuka R-10 and 0.75% Macerozyme R-10) in 0.6 M AA medium over 18–20 h at 28 °C. Regeneration was achieved through encapsulation in alginate beads and coculture with feeder extracts in 2N6 medium, leading to embryogenic callus formation within 35 days. Seedlings were regenerated in N6R and N6F media and acclimatized under greenhouse conditions within three months. The isolated protoplast quality displayed viability rates of 70–99% within 48 h and supported transient PEG-mediated transfection with GFP. Additionally, the transient expression of a gene editing CRISPR-Cas9 construct targeting the DROUGHT AND SALT TOLERANCE (OsDST) gene confirmed genome editing capability. This protocol offers a scalable and genotype-adaptable system for protoplast-based regeneration and gene editing in temperate japonica rice, supporting the application of NGTs in the breeding of cold-adapted cultivars. Full article

LTR retrotransposons are widespread genomic elements that significantly impact genome structure and function. In Arabidopsis thaliana, the EVD LTR retrotransposon encodes a GAG protein essential for retrotransposon particle assembly. Here, we present a comprehensive analysis of the structural features, intracellular localization, and transcriptomic effects of the EVD GAG (evdGAG) protein. Using AlphaFold3, we identified canonical capsid (CA-NTD and CA-CTD) and nucleocapsid (NC) domains, with predicted disordered regions likely facilitating oligomerization. Transient expression of GFP-tagged evdGAG in protoplasts of A. thaliana and distant plant species (Nicotiana benthamiana and Helianthus annuus) revealed the formation of multiple large cytoplasmic aggregates resembling retrosomes, often localized near the nucleus. Stable overexpression of evdGAG in wild-type and ddm1 mutant backgrounds induced significant transcriptomic changes, including up-regulation of stress response and defense-related genes and downregulation of photosynthesis and chloroplast-associated pathways. Importantly, genes linked to stress granule formation were also up-regulated, suggesting a role for evdGAG in modulating cellular stress responses. Our findings provide novel insights into the cellular and molecular properties of plant retrotransposon GAG proteins and their influence on host gene expression. Full article

Protoplast-based transformation is a vital tool for genetic studies in fungi, yet no protoplast method existed for P. sclerotiorum-scaumcx01 before this study. Here, we optimized protoplast isolation, regeneration, and transformation efficiency. The highest protoplast yield (6.72 × 10⁶ cells/mL) was obtained from liquid mycelium after 12 h of enzymatic digestion at 28 °C using Lysing Enzymes, Yatalase, cellulase, and pectinase. Among osmotic stabilizers, 1 M MgSO₄ yielded the most viable protoplasts. Regeneration occurred via direct mycelial outgrowth and new protoplast formation, with a 1.02% regeneration rate. PEG-mediated transformation with a hygromycin resistance gene and GFP tagging resulted in stable GFP expression in fungal spores and mycelium over five generations. LC/MS-based metabolomic analysis revealed significant changes in glycerophospholipid metabolism, indicating lipid-related dynamics influenced by GFP tagging. Microscopy confirmed successful colonization of tomato roots by GFP-tagged scaumcx01, with GFP fluorescence observed in cortical tissues. Enzymatic (cellulase) seed pretreatment enhanced fungal colonization by modifying root surface properties, promoting plant–fungal interaction. This study establishes an efficient protoplast transformation system, reveals the metabolic impacts of genetic modifications, and demonstrates the potential of enzymatic seed treatment for enhancing plant–fungal interactions. Full article

Protoplasts are essential tools for genetic manipulation and functional genomics research in fungi. This study systematically optimized protoplast preparation conditions and examined transcriptional changes throughout the preparation and regeneration processes to elucidate the molecular mechanisms underlying the formation and regeneration of protoplasts in Lyophyllum decastes. The results indicated an optimal protoplast yield of 5.475 × 10⁶ cells/mL under conditions of fungal age at 10 days, digestion time of 2.25 h, enzyme concentration of 2%, and digestion temperature of 28 °C. The Z5 medium supplemented with L. decastes mycelial extract achieved a high regeneration rate of 2.86. RNA-seq analysis revealed 2432 differentially expressed genes (DEGs) during protoplast formation and 5825 DEGs during regeneration. Casein kinase I, cytochrome P450 (CYP52), and redox-regulated input receptor (PEX5) were significantly upregulated during the protoplast stage, while β-1,3-glucan synthase (SKN1), chitin synthase (CHS2), hydrophobin-1, and hydrophobin-2 showed significant upregulation during the protoplast regeneration phase. These findings provide a reference for the efficient preparation and regeneration of protoplasts and offer new insights into the molecular mechanisms of protoplast formation and cell wall regeneration in fungi. Full article

The efficient protoplast transient transformation system in plants is an important tool to study gene expression, metabolic pathways, and various mutagenic parameters, but it has not been established in Phelipanche aegyptiaca. As a root parasitic weed that endangers the growth of 29 species of plants in 12 families around the world, there is still no good control method for P. aegyptiaca. Even the parasitic mechanisms of P. aegyptiaca and the related genes regulating parasitism are not yet understood. In this study, by comparing the factors related to protoplast isolation and transfection, we developed the optimal protocol for protoplast isolation and transfection in Phelipanche aegyptiaca haustorium. The optimal protoplast yield and activity were 6.2 × 10⁶ protoplasts/g fresh weight [FW] and 87.85%, respectively, by using 0.5 mol/L mannitol, enzyme concentrations of 2.5% cellulase R-10 and 0.8% Macerozyme R-10 at 24 °C for 4 h. At the same time, transfection efficiency of protoplasts was up to 78.49% when using 30 μg plasmid, 40% polyethylene glycol (PEG) concentration, 24 °C incubation temperature, and 20 min transfection time. This is the first efficient protoplasts’ isolation and transient transformation system of Phelipanche aegyptiaca haustorium, laying a foundation for future studies on the gene function and mechanisms of haustorium formation in parasitic plants. Full article

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 Phoma sorghina var. saccharum 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 (PsHOX1–PsHOX9) were identified in P. sorghina var. saccharum, 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 PsHOX1, PsHOX2, PsHOX3, PsHOX4, PsHOX6, PsHOX8, and PsHOX9 genes resulted in a smaller growth diameter in P. sorghina var. saccharum. In contrast, the knockouts of the PsHOX3, PsHOX6, and PsHOX9 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 P. sorghina var. saccharum. The pathogenic mechanism of the affected sugarcane will provide an essential theoretical basis for preventing and controlling sugarcane twisted leaf disease. Full article

Filamentous fungi exhibit unparalleled potential as cell factories for protein production, owing to their adeptness in protein secretion and remarkable proficiency in post-translational modifications. This review delineates the role of filamentous fungi in bio-input technology across different generations and explores their capacity to generate secondary metabolites. Our investigation highlights filamentous fungi as frontrunners in the production of bioactive compounds, emphasizing the imperative nature of elucidating their metabolic repertoire. Furthermore, we delve into common strategies for genetic transformation in filamentous fungi, elucidating the underlying principles, advantages, and drawbacks of each technique. Taking a forward-looking approach, we explore the prospects of genome engineering, particularly the CRISPR-Cas9 technique, as a means to propel protein secretion in filamentous fungi. Detailed examination of the protein secretion pathways in these fungi provides insights into their industrial applications. Notably, extensive research within the scientific community has focused on Aspergillus and Trichoderma species for the industrial production of proteins and enzymes. This review also presents practical examples of genetic engineering strategies aimed at augmenting enzyme secretion in filamentous fungi for various industrial applications. These findings underscore the potential of filamentous fungi as versatile platforms for protein production and highlight avenues for future research and technological advancement in this field. Full article

The transcription factor WRKY53 of the model plant Arabidopsis thaliana is an important regulator of leaf senescence. Its expression, activity and degradation are tightly controlled by various mechanisms and feedback loops. Hydrogen peroxide is one of the inducing agents for WRKY53 expression, and a long-lasting intracellular increase in H₂O₂ content accompanies the upregulation of WRKY53 at the onset of leaf senescence. We have identified different antioxidative enzymes, including catalases (CATs), superoxide dismutases (SODs) and ascorbate peroxidases (APXs), as protein interaction partners of WRKY53 in a WRKY53-pulldown experiment at different developmental stages. The interaction of WRKY53 with these enzymes was confirmed in vivo by bimolecular fluorescence complementation assays (BiFC) in Arabidopsis protoplasts and transiently transformed tobacco leaves. The interaction with WRKY53 inhibited the activity of the enzyme isoforms CAT2, CAT3, APX1, Cu/ZuSOD1 and FeSOD1 (and vice versa), while the function of WRKY53 as a transcription factor was also inhibited by these complex formations. Other WRKY factors like WRKY18 or WRKY25 had no or only mild inhibitory effects on the enzyme activities, indicating that WRKY53 has a central position in this crosstalk. Taken together, we identified a new additional and unexpected feedback regulation between H₂O_2, the antioxidative enzymes and the transcription factor WRKY53. Full article

The Duboisia species, a group of plants native to Australia, have been historically valued for their pharmacological properties and have played a significant role in traditional medicine and pharmaceutical research. Persistent efforts are underway to enhance the efficacy of the active ingredient scopolamine, employing both conventional breeding methods and advanced biotechnology tools. The primary objective of this research was to establish a highly efficient method for isolating mesophyll protoplasts and facilitating their regeneration, thereby laying a robust foundation for the application of various advanced plant biotechnology tools in the pursuit of genetic enhancement. The mesophyll protoplast isolation process was developed for hybrid D. myoporoides × D. hopwoodii with careful optimisation of the following parameters: leaf strip size; incubation conditions; physical treatment; and enzyme concentration. The optimal parameters were combined in each individual step; the best enzyme concentration was determined to be 2% (w/v) cellulysin and 0.5% (w/v) macerase. Protoplast yield was found to be greatly affected by the enzyme concentrations. The isolated protoplasts were cultured at a density of 0.5 × 10⁵ to best sustain the highest cell division (33.2%) and a microcalli induction frequency of 17.9%. After 40 days of culture in a modified KM8P medium at 25 °C in darkness, visible microcalli were transferred to a solidified Murashige and Skoog (MS) medium with 1 mg L⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) for callus induction under a 16 h photoperiod. After 30 days of culture, compact organogenic calli were transferred into a solid MS medium with 6-benzylaminopurine (BA) alone or thidiazuron (TDZ) alone or in combination with BA or naphthalene acetic acid (NAA) for shoot regeneration. The maximum shoot regeneration frequency (63.3%) was observed in the medium with 1.5 mg L⁻¹ TDZ alone. For the first time, a reliable protoplast isolation and regeneration system from mesophyll cells was established for Duboisia with high protoplast viability, successful microcalli formation, and intact plant regeneration. This innovation will significantly contribute towards the genetic enhancement of the Duboisia species. Full article

Natural rubber is one of the most important industrial raw materials, and its biosynthesis is still a fascinating process that is still largely unknown. In this research, we studied Decaisnea insignis, a unique rubber-producing plant that is different from other rubber-producing species due to the presence of lactiferous canals in its pericarp. The present study aims to provide novel insights into the mechanisms underlying rubber accumulation and PCD by subjecting the Decaisnea insignis laticiferous canals to light microscopy, TUNEL assay, and DAPI staining, as well as viability analysis, cellular ultrastructure analysis, and molecular analysis using light microscopy, scanning electron microscopy, immunofluorescence labeling, transmission electron microscopy, and transcriptome sequencing. At the cellular level, the origin of small rubber particles in the laticiferous canals had no morphological correlation with other organelles, and these particles were freely produced in the cytosol. The volume of the rubber particles increased at the sunken and expanding stage, which were identified as having the characteristics of programmed cell death (PCD); meanwhile, plenty of the rubber precursors or rubber particles were engulfed by the vacuoles, indicating a vacuole-mediated autophagy process. The accumulation of rubber particles occurred after the degeneration of protoplasts, suggesting a close association between rubber biosynthesis and PCD. The molecular analysis revealed the expression patterns of key genes involved in rubber biosynthesis. The upstream genes DiIPP, DiFPP, and DiGGPPS showed a decreasing trend during fruit ripening, while DiHRT, which is responsible for rubber particle extension, exhibited the highest expression level during the rubber particle formation. Moreover, the transcription factors related to PCD, DiLSD1, and DiLOL2 showed a negative correlation with the expression pattern of DiHRT, thus exhibiting strict rules of sequential expression during rubber biosynthesis. Additionally, the expression trends of DiXCP1 and DiCEP1, which act as proteases during PCD, were positively correlated with DiGGPPS expression. In conclusion, the findings suggest that the autophagic PCD may play a crucial role in rubber accumulation in D. insignis. Further research is still needed to fully understand the complex regulatory network underlying rubber biosynthesis in plants. Full article

Prunus mume is a famous ornamental woody tree with colorful flowers. P. mume with yellow flowers is one of the most precious varieties. Regretfully, metabolites and regulatory mechanisms of yellow flowers in P. mume are still unclear. This hinders innovation of flower color breeding in P. mume. To elucidate the metabolic components and molecular mechanisms of yellow flowers, we analyzed transcriptome and metabolome between ‘HJH’ with yellow flowers and ‘ZLE’ with white flowers. Comparing the metabolome of the two varieties, we determined that carotenoids made contributions to the yellow flowers rather than flavonoids. Lutein was the key differential metabolite to cause yellow coloration of ‘HJH’. Transcriptome analysis revealed significant differences in the expression of carotenoid cleavage dioxygenase (CCD) between the two varieties. Specifically, the expression level of PmCCD4 was higher in ‘ZLE’ than that in ‘HJH’. Moreover, we identified six major transcription factors that probably regulated PmCCD4 to affect lutein accumulation. We speculated that carotenoid cleavage genes might be closely related to the yellow flower phenotype in P. mume. Further, the coding sequence of PmCCD4 has been cloned from the ‘HJH’ petals, and bioinformatics analysis revealed that PmCCD4 possessed conserved histidine residues, ensuring its enzymatic activity. PmCCD4 was closely related to PpCCD4, with a homology of 98.16%. Instantaneous transformation analysis in petal protoplasts of P. mume revealed PmCCD4 localization in the plastid. The overexpression of PmCCD4 significantly reduced the carotenoid content in tobacco plants, especially the lutein content, indicating that lutein might be the primary substrate for PmCCD4. We speculated that PmCCD4 might be involved in the cleavage of lutein in plastids, thereby affecting the formation of yellow flowers in P. mume. This work could establish a material and molecular basis of molecular breeding in P. mume for improving the flower color. Full article

Protoplasts are a versatile tool in plant biotechnology since they can be used for basic biological studies as well as for breeding strategies based on genome editing. An efficient protoplast isolation protocol is essential for conducting protoplast-based studies. To optimize the protoplast isolation protocol in cabbage (Brassica oleracea var. capitata L.), different enzyme solutions were tested for the isolation of leaf mesophyll protoplasts. In our experiments, the combination of 0.5% Cellulase Onozuka RS and 0.1% Macerozyme R-10 showed the best result. The optimized protocol proved suitable for the isolation of protoplasts from five different cabbage cultivars with yields ranging from 2.38 to 4.63 × 10⁶ protoplasts/g fresh weight (fw) and a viability of 93% or more. After three weeks in culture, protoplasts from all of the tested cultivars formed micro-calli, but further callus growth and shoot regeneration depended strongly on the genotype and regeneration protocol used. For shoot formation, 1 mg/L BAP in combination with auxin 0.2 mg/L NAA showed the best results with a regeneration of 23.5%. The results obtained will contribute to the development of different applications of cabbage protoplasts and facilitate the breeding process of this important horticultural crop. Full article

The purpose of this study was to investigate the relationship between lignan biosynthesis and programmed cell death (PCD) of ray parenchyma cells during the heartwood formation of Taiwania (Taiwania cryptomerioides Hayata). Since the PCD of ray parenchyma cells and the synthesis of lignans are the two main processes involved in the formation of heartwood, both of which need to be completed through gene regulation. Based on the results of genomics and bioinformatics analysis, that the PCD of tracheids are induced by genotoxic, and the PCD of ray parenchyma cells is induced by biological factors, such as fungi, bacteria, and viruses, which could induce oxidative stress. According to the results of time−of−flight secondary ion mass spectrometry (ToF−SIMS) analysis, lignans are produced in ray parenchyma cells, and the accumulation of savinin and its downstream lignans might be the cause of PCD in ray parenchyma cells. An in vitro experiment further confirmed that the accumulation of savinin could cause protoplasts of Taiwania’s xylem to produce taiwanin A, which is the marker of heartwood formation in Taiwania. Resulting in an increase in reactive oxygen species (ROS) content, which could induce oxidative stress in ray parenchyma cells and potentially lead to PCD. Based on these findings, we conclude that accumulation of savinin could be induced PCD of ray parenchyma cells in heartwood formation in Taiwania. Full article

In the fungus Fusarium fujikuroi, carotenoid production is up-regulated by light and down-regulated by the CarS RING finger protein, which modulates the mRNA levels of carotenoid pathway genes (car genes). To identify new potential regulators of car genes, we used a biotin-mediated pull-down procedure to detect proteins capable of binding to their promoters. We focused our attention on one of the proteins found in the screening, belonging to the High-Mobility Group (HMG) family that was named HmbC. The deletion of the hmbC gene resulted in increased carotenoid production due to higher mRNA levels of car biosynthetic genes. In addition, the deletion resulted in reduced carS mRNA levels, which could also explain the partial deregulation of the carotenoid pathway. The mutants exhibited other phenotypic traits, such as alterations in development under certain stress conditions, or reduced sensitivity to cell wall degrading enzymes, revealed by less efficient protoplast formation, indicating that HmbC is also involved in other cellular processes. In conclusion, we identified a protein of the HMG family that participates in the regulation of carotenoid biosynthesis. This is probably achieved through an epigenetic mechanism related to chromatin structure, as is frequent in this class of proteins. Full article

Chitin synthases (CHSs) are vital enzymes for the synthesis of chitin and play important and differential roles in fungal development, cell wall integrity, environmental adaptation, virulence, and metabolism in fungi. However, except for ChsC, a class III CHS, little is known about the functions of CHSs in Aspergillus niger, an important fungus that is widely applied in the fermentation industry and food processing, as well as a spoilage fungus of food and a human pathogen. This study showed the important functions of ChsA, a class II CHS, in A. niger using multi-phenotypic and transcriptional analyses under various conditions. The deletion of chsA led to severe defects in conidiation on different media and resulted in the formation of smaller and less compact pellets with less septa in hyphal cells during submerged fermentation. Compared with the WT, the ΔchsA mutants exhibited less chitin content, reduced growth under the stresses of cell wall-disturbing and oxidative agents, more released protoplasts, a thicker conidial wall, decreased production of amylases, pectinases, cellulases, and malic acid, and increased citric acid production. However, ΔchsA mutants displayed insignificant changes in their sensitivity to osmotic agents and infection ability on apple. These findings concurred with the alteration in the transcript levels and enzymatic activities of some phenotype-related genes. Conclusively, ChsA is important for cell wall integrity and mycelial morphology, and acts as a positive regulator of conidiation, cellular responses to oxidative stresses, and the production of malic acid and some enzymes, but negatively regulates the citric acid production in A. niger. Full article