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PubMed2026

Characterization of oxidative status in maize protoplasts under temperature and saline-alkali stresses.

BACKGROUND: Protoplasts have emerged as a powerful model system in plant functional genomics, offering significant utility in functional gene analysis, protein interaction studies, and transient expression platforms for gene editing. Despite their versatility, inherent limitations restrict their broader application, highlighting the need for systematic investigations into their responses to abiotic stressors, such as temperature fluctuations and saline-alkali conditions (200 mM saline mixture: 170mM NaCl and 30mM Na2CO3, pH = 9.1). RESULTS: In this study, we comprehensively examined the effects of varying temperatures and saline-alkali stress on the integrity, viability, and reactive oxygen species (ROS) metabolism of maize protoplasts. Key markers of oxidative stress-including ROS accumulation, lipid peroxidation (measured as malondialdehyde, MDA), antioxidant enzyme activity (superoxide dismutase, SOD), and hydrogen peroxide (H2O2) levels-were quantified to assess the oxidative stress response. Protoplasts maintained at 4 °C demonstrated enhanced stability and antioxidant capacity, preserving cell viability and endogenous protein integrity for up to 16 h. Conversely, exposure to 37 °C significantly compromised protoplast viability, while incubation at 28 °C exerted minimal effects within 16 h. CONCLUSIONS: Our study investigated the effects of various temperature stresses and salt-alkali stress on maize protoplasts. The results demonstrated that both temperature and salt-alkali stress significantly impacted protoplast production, viability, and the expression of endogenous proteins. These findings not only characterize the redox response of maize protoplasts, but also provide guidance for protoplast isolation and other procedures: 4 °C is suitable for short-term maintenance, 25-28 °C for routine functional assays, and 37 °C should be avoided. These findings provide valuable insights into the stress responses of protoplasts and establish a foundation for future research aimed at improving plant stress tolerance through protoplast-based techniques.

Zea mays↗

PubMed2026

An effective method for isolation and regeneration of Solanum tuberosum mesophyll protoplasts for transgene-free genome editing.

An effective system for isolating and regenerating protoplasts is crucial for research in genome engineering. This study focused on refining a protocol for the isolation and regeneration of mesophyll protoplasts from the leaves of Solanum tuberosum cv. Kufri Jyoti. Key factors influencing protoplast yield and viability, such as dark pretreatment, pre-plasmolysis, enzyme concentrations, and osmoticum levels, were thoroughly assessed and optimized. The highest protoplast yield and viability were achieved with an enzyme mixture of 1.0% cellulase R-10 and 0.5% macerozyme R-10 after 16 h of incubation. Furthermore, culturing on a Murashige and Skoog-based medium (MSPI) without ammonium nitrate, enriched with an osmoticum concentration of 0.4 M and a carefully adjusted auxin-to-cytokinin ratio, successfully facilitated protoplast division, microcalli proliferation, and minicalli formation. Callus proliferation and shoot induction were accomplished on MS13K medium supplemented with naphthaleneacetic acid (NAA) and zeatin riboside. Root initiation and elongation were promoted on MS basal medium supplemented with indole-3-butyric acid (IBA) at 1 mg/L. The regenerated plantlets were subsequently acclimatized and hardened under controlled greenhouse conditions. This robust protoplast-to-plant protocol serves as a crucial resource for the introduction of ribonucleoprotein complexes into plant cells, facilitating accurate, transgene-free genome editing.

Callus induction↗

PubMed2025

Development of a highly efficient protoplast regeneration and transfection protocol for enhancing CRISPR genome editing of Brassica carinata.

Brassica carinata is an important oil crop with significant potential for food and industrial production. The application of the CRISPR/Cas9 genome editing tool in B. carinata could accelerate its breeding cycle. However, no efficient DNA-free gene editing method currently exists for this species. Protoplast-based CRISPR editing presents a promising solution, though it is often challenging for many crop species. In this study, we investigated several critical factors influencing in vitro shoot regeneration, including genotype, sugar type, selection and combination of plant growth regulators (PGRs), and culture duration on different media throughout various stages of protoplast development. As a result, we developed a highly efficient, five-stage protoplast regeneration protocol for B. carinata based on specific stages of protoplast development. Key findings of this study include the requirement for high concentrations of NAA and 2,4-D in the initial medium (MI) for cell wall formation, while a lower auxin concentration relative to cytokinin was necessary for active cell division (MII). For callus growth and shoot induction, a high cytokinin-to-auxin ratio was essential (MIII), and an even higher cytokinin-to-auxin ratio was optimal for shoot regeneration (MIV). For shoot elongation, low levels of BAP and GA3 were sufficient (MV). Our results also demonstrated that the duration of culture on different media and maintaining appropriate osmotic pressure at the early stages were crucial for successful protoplast regeneration. With this optimized protocol, we achieved an average regeneration frequency of up to 64% and a transfection efficiency of 40% using the GFP marker gene. This efficient protoplast regeneration protocol is now being employed for genome editing in our lab and is expected to significantly enhance the application of the CRISPR system in both basic research and the genetic improvement of B. carinata over the long term.

Brassica carinata↗

PubMed2026

An efficient woody plant protoplast platform enables transgene-free multiplex genome editing and rapid trait validation in pear.

Multiplex editing is crucial for analysing complex multiple-gene traits in woody plants, yet its application remains limited because of low transformation efficiency and lengthy regeneration cycles. To overcome these barriers, in this study we establish an efficient protoplast isolation protocol for pear (Pyrus) that employs 1.0% cellulase R10 and 0.4% macerozyme R10 with an 8.5 h digestion. Its broad applicability using different digestion times across seven other economically important woody plants is demonstrated. Coupling a 40% PEG-4000-mediated transfection regimen with DNA-free CRISPR/Cas9 ribonucleoprotein (RNP) delivery enables multiplex genome editing in isolated protoplasts. Using this platform, we simultaneously disrupted the key components of the chloroplast division apparatus ARC3, PARC6, and FtsZ2-1a in Pyrus bretschneideri and found that it consistently reproduced macro-chloroplast abnormalities, confirming effective multigene perturbation within a single cellular context. Notably, failure of chloroplast division activated chloroplast-to-nucleus retrograde signaling, as evidenced by the induction of the nuclear stress-response genes RBOHD and ZAT12, a concomitant surge in reactive oxygen species, and progression to severe cellular deformation. Thus, our study establishes a rapid, cross-genus protoplast-RNP workflow that enables DNA-free multiplex editing and accelerates genotype-to-phenotype analyses in woody perennials. The approach provides a practical foundation for functional genomics and supports advances in non-transgenic precision breeding of tree crops.

Protoplasts↗

PubMed2026

Optimization of protoplast based DNA isolation and genome analysis in a gamma-irradiated Aspergillus niger mutant strain.

Aspergillus niger is an important industrial fungus widely used for citric acid production and a range of biotechnological applications. In this study, a protoplast-based DNA isolation protocol was optimized for a gamma-irradiated A. niger AN-L103_M1 mutant strain, followed by whole-genome sequencing and functional genome analysis. Protoplast yield was strongly influenced by enzyme concentration and the molarity of the osmotic stabilizer. The highest yield was achieved at an enzyme concentration of 50 mg/mL (2.487 ± 0.04 × 10⁸ cells/mL) and 0.8 M KCl (2.550 ± 0.06 × 10⁸ cells/mL), with both factors showing significant effects (p < 0.0001) in GraphPad Prism 11.0.0. Whole-genome sequencing performed using an Illumina NovaSeq 6000 platform yielded a 37.06 Mb draft genome assembled into 537 contigs, with an N50 of 363,084 bp and a GC content of 48.2%. BUSCO 14 analysis showed high completeness (97.95% complete BUSCOs). Functional annotation and KEGG pathway mapping identified genes involved in glycolysis, the tricarboxylic acid cycle, and citrate biosynthesis, while biosynthetic gene cluster analysis revealed diverse potential for secondary metabolite production. These findings provide an optimized workflow for protoplast-based DNA isolation and genome-scale functional analysis in A. niger, proposing a basis for future comparative genomics, transformation studies, and experimentally validated metabolic engineering.

Aspergillus niger↗

PubMed2026

A streamlined protocol for small-scale protoplast generation and CRISPR/Cpf1-mediated genome editing in Fusarium oxysporum.

Fusarium oxysporum is a significant threat to agriculture and One Health, requiring advanced molecular tools for functional genomic analyses and biological control agent development. Existing gene-editing methods are hampered by costly protoplast preparation protocols and by CRISPR-Cas9 limitations, such as restricted protospacer adjacent motif (PAM) sequences and complex guide RNA requirements. We engineered an efficient CRISPR/Cpf1 system that overcomes these issues through three main innovations: small-scale protoplast generation using filter column-based methods that greatly reduce enzyme consumption while simplifying workflows, a CRISPR/Cpf1 system with shorter guide RNA design and staggered DNA cleavage to promote homologous recombination, and minimal homology arm strategies that significantly decrease cloning complexity. Extensive validation confirms successful gene targeting with molecular verification and functional analysis via standardized pathogenicity assays. This integrated platform offers affordable, accessible tools for systematic F. oxysporum research, enhancing fundamental understanding of plant-pathogen interactions and supporting high-throughput screening vital for agricultural biotechnology and biological agent development.

CRISPR/Cpf1↗

PubMed2026

Establishment of a CRISPR/Cas9-mediated system for targeted editing of the MFS gene in mint.

The key message of this study is that we established a CRISPR/Cas9-mediated genome-editing system for Mentha haplocalyx "738" by optimizing protoplast transient assay and screening effective regulatory elements. Targeted knockout of the MFS gene generated edited mint plants with reduced menthofuran content, offering a strategy for quality improvement of mint essential oil. The commercial value of mint (Mentha spp.) essential oil is often diminished by the presence of undesirable metabolites, notably menthofuran, which impairs flavor and raises safety concerns. This study aimed to develop a robust CRISPR/Cas9 gene editing system for mint 738 (Mentha haplocalyx "738") and apply it to disrupt the menthofuran synthase (MFS) gene, thereby redirecting metabolic flux to enhance oil quality. We established an optimized system for high-efficiency protoplast isolation and transient transformation from young mint leaves. Key parameters for enzymatic digestion (1.5% cellulase R10, 0.2% macerozyme R-10, 3 h) and PEG-mediated transformation (40% PEG6000, 0.4 M mannitol, 0.4 M CaCl₂) were systematically determined. Using this platform, we screened endogenous regulatory elements, identifying a truncated mint U6 promoter (HmU6.1-3P) and the tomato SlEF1α promoter as the most effective drivers for sgRNA and Cas9 expression, respectively. A CRISPR/Cas9 vector targeting the MFS gene was constructed and used for Agrobacterium-mediated stable transformation. The positive transgenic mint lines were obtained. Sequencing confirmed heritable mutations at the target sites within the MFS gene in multiple independent lines. The results revealed a substantial decrease in menthofuran content in the essential oil of the edited line #10 compared to the wild-type control, thereby demonstrating a viable strategy for improving mint essential oil quality through genome-editing.

CRISPR-Cas Systems↗

PubMed2024

Replication of single viruses across the kingdoms, Fungi, Plantae, and Animalia.

It is extremely rare that a single virus crosses host barriers across multiple kingdoms. Based on phylogenetic and paleovirological analyses, it has previously been hypothesized that single members of the family Partitiviridae could cross multiple kingdoms. Partitiviridae accommodates members characterized by their simple bisegmented double-stranded RNA genome; asymptomatic infections of host organisms; the absence of an extracellular route for entry in nature; and collectively broad host range. Herein, we show the replicability of single fungal partitiviruses in three kingdoms of host organisms: Fungi, Plantae, and Animalia. Betapartitiviruses of the phytopathogenic fungusRosellinia necatrix could replicate in protoplasts of the carrot (Daucus carota), Nicotiana benthamiana and Nicotiana tabacum, in some cases reaching a level detectable by agarose gel electrophoresis. Moreover, betapartitiviruses showed more robust replication than the tested alphapartitiviruses. One of the fungal betapartitiviruses, RnPV18, could persistently and stably infect carrot plants regenerated from virion-transfected protoplasts. Both alpha- and betapartitiviruses, although with different host preference, could replicate in two insect cell lines derived from the fall armyworm Spodoptera frugiperda and the fruit fly Drosophila melanogaster. Our results indicate the replicability of single partitiviruses in members of three kingdoms and provide insights into virus adaptation, host jumping, and evolution.

Animals↗

PubMed2026

Efficient CRISPR/Cas-SF01 genome editing tools with high editing efficiency in allotetraploid oilseed rape.

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas9 has been widely utilized for plant genome editing, but the protospacer adjacent motif (PAM) requirement limits its editing scope. CRISPR/Cas12i3 belongs to the type-VI Cas system that has gained extensive attention due to its smaller size and less restricted canonical TTN PAM sequence. In this study, we explored the newly developed Cas-SF01 system (Cas12i3 variant) for genome editing in oilseed rape. We established an efficient protoplast transformation system in oilseed rape to compare editing efficiency between Cas-SF01 and Cas9. Cas-SF01 shows cleavage activities at the tested 5'-TTN-3' PAM sites with editing outcomes sharing considerable similarities with the CRISPR-Cas9 system in protoplast. Cas-SF01 also induces high efficiency mutagenesis for multiple target sites in stable transformed oilseed rape lines, generating mutants with multilocular silique and male sterile phenotypes. Furthermore, Cas-SF01-derived cytosine base editors (CBEs) were developed to produce targeted C-to-T base edits. Compared to SpCas9, Cas-SF01 has an expanded PAM range and effectively recognizes TTN PAMs, which has substantially broadened the scope of editable sites within the rapeseed genome. No mutations were identified at the putative off-target sites among the edited plants. This study developed a robust, first-of-its-kind Cas12 system in the allotetraploid Brassica napus, expanding the scope of editing and enriching genome-editing toolkits for biological research and genetic improvement.

Brassica napus↗

PubMed2026

Genetic transformation of forage crops: comparative barriers, evidence, and emerging strategies.

Forage crops include phylogenetically and biologically distinct legumes and grasses, and their genetic transformation is constrained by different combinations of host response, DNA-delivery efficiency, regeneration competence, genotype dependence, and genome stability. This review critically compares evidence from forage legumes and forage grasses rather than treating these groups as a single transformation category. We evaluate Agrobacterium-mediated transformation, protoplast-based delivery, particle bombardment, CRISPR/Cas-enabled applications, developmental regulators (DRs), viral vectors, and nanomaterial-mediated delivery according to four practical outcomes: reproducibility across genotypes, recovery of regenerated plants, heritable transmission, and genetic stability. Direct evidence in forage crops shows that protocol performance is strongly species-, genotype-, explant-, and endpoint-dependent; efficiencies based on transient reporters or resistant callus therefore cannot be directly equated with stable, fertile events. DR-assisted regeneration has direct proof of concept in recalcitrant forage grasses, whereas stable nanomaterial-mediated transformation and virus-induced heritable editing remain unvalidated in forage crops. We conclude that current progress is best interpreted as the engineering of interacting delivery and regeneration constraints, not as a universal transition to genotype-independent transformation. Priority should be given to standardized outcome reporting, multi-genotype and inter-laboratory validation, controlled DR expression, and rigorous molecular and phenotypic assessment of regenerated plants.

Crops, Agricultural↗

PubMed2025

Efficient site-specific integration of kilobase-length DNA fragments in plant cells via Kp03 recombinase.

Targeted insertion of large DNA sequences into plant genomes remains a major challenge in synthetic biology. Here, we evaluate the large serine recombinase Kp03 for site-specific integration of DNA fragments in rice and Arabidopsis. In transient protoplast assays, Kp03 mediates efficient insertion of donor DNA up to 27.3 kilobases (kb), with plasmid integration efficiencies reaching 99.1% for fragments up to 3.4 kb. Truncation experiments reveal that a minimal 15-bp attB sequence is necessary for integration. As a proof of concept, Kp03 successfully incorporates a 3.4-kb donor DNA into the rice genome at a locus containing this minimal attB sequence. Moreover, in rice callus, combining Kp03 with the NM-PE genome editing system to install a 26-bp attB site enables targeted integration of a 3.4-kb donor at the desired genomic locus. These findings establish Kp03 as a versatile tool for plant genome engineering, with broad applications for synthetic biology.

Oryza↗

PubMed2026

Novel Mycoparasitic Mechanisms and Colonization Patterns on Poplar Revealed by GFP Tagging of the Biocontrol Fungus Clonostachys reniana.

Clonostachys rosea has long been the primary model for studying mycoparasitism within its genus; however, the potential of other species remains largely unexplored. In this study, we established a PEG-CaCl2-mediated protoplast transformation system for Clonostachys reniana. Our results demonstrate for the first time that this species is amenable to genetic manipulation and produces transformants with genetic stability, providing a reliable platform for functional genomic research in this fungus. Using green fluorescent protein tagging, we achieved stable transformants that retained wild-type physiological traits. Crucially, our data indicated that C. reniana utilizes a distinct mycoparasitic mechanism, which differs from the well-established sequential process of adhesion, coiling, and lysis seen in C. rosea. Confocal and scanning electron microscopy revealed that C. reniana, following initial coiling and invasive structure formation, penetrates the host hyphae of Botryosphaeria dothidea. It then grows longitudinally within the hyphal lumen, absorbing nutrients and eliminating the pathogen from the inside. Furthermore, C. reniana hyphae can colonize the intercellular spaces of the stem periderm in 84K poplar (Populus alba × P. glandulosa) while displaying a distinct tissue-specific behavior in the roots by forming a mantle on the root surface resembling that of ectomycorrhizae. These findings establish C. reniana as a highly promising secondary model species within the Clonostachys genus. By uncovering a novel "internal-consumption" mycoparasitic mode, this study expands our understanding of the ecological diversity of biocontrol fungi and provides a powerful genetic toolset for future functional genomic research.

Clonostachys reniana↗

PubMed2026

Whole-Genome Analysis of Bacillus Licheniformis Ali5 and Synthesis of Lichenysin via Genome Shuffling.

Whole-genome sequencing of Bacillus licheniformis Ali5 was performed via MGI-seq PE150 and Nanopore single-molecule real-time sequencing. The strain has a 4,114,664 bp circular genome encoding 4030 protein-coding genes. Functional annotation across NR, COG, GO, KEGG, CARD, BacMet, and CAZy databases identified 4025, 2812, 988, 1242, 72, 69, and 94 corresponding genes, respectively, and antiSMASH 6.0 revealed multiple antimicrobial biosynthetic gene clusters, including intact lichenysin and lichenicidin VK21 A1/A2 gene clusters. Three rounds of recursive protoplast fusion-based genome shuffling, paired with a dual-index screening system, significantly improved strain growth and lichenysin biosynthesis. Recombinants exhibited shortened lag phase, enhanced proliferation, improved stationary-phase stability, and higher diauxic peak biomass. PP3-176 and PP3-186 showed 4.6%-8.1% higher 12-h shake-flask titer and 3.1%-4.0% higher maximum titer than the parental average, with excellent fermentation stability. 1-L bioreactor validation confirmed strong scale-up potential. PP3-186 achieved 27.2% and 31.6% titer increases at 12 h and 20 h, while PP3-176 yielded 20.4% and 14.6% improvements with robust metabolic performance. This study validates genome shuffling as an effective strategy for enhancing lichenysin production, providing candidate strains and technical support for industrial application.

Bacillus licheniformis↗

PubMed2025

Using Callus as an Ex Vivo System for Chromatin Analysis.

Next-generation sequencing has revolutionized epigenetics research, enabling a comprehensive analysis of DNA methylation and histone modification profiles to explore complex biological systems at unprecedented depth. Deciphering the intricate epigenetic mechanisms that regulate gene activity presents significant challenges, including the issue of analyzing heterogeneous cell populations in bulk. Bulk analysis introduces bias and can obscure crucial information by averaging readouts from distinct cells. Various approaches have been developed to address this issue, such as cell-type-specific enrichment or single-cell sequencing techniques. However, the need for transgenic lines with fluorescent markers, along with technical challenges such as efficient protoplast isolation and low yield, limits their widespread adoption and use in multi-omic studies. This review discusses the pros and cons of these approaches, providing a valuable basis for selecting the most suitable strategy to minimize heterogeneity. We will also highlight the use of cotyledon-derived callus as an ex vivo system as a simple, accessible, and robust platform for enabling high-throughput multi-omic analyses.

Chromatin↗

PubMed2026

Genomic and functional characterization of sugar transporters reveals potential roles in sugar accumulation in a modern sugarcane cultivar.

Sugarcane (Saccharum spp.) is a globally important sugar crop whose productivity depends on efficient sugar transport from source to sink organs. However, systematic identification and functional characterization of sugar transporters (STs) in sugarcane cultivars remain limited. Here, we identified 190 non-redundant ST genes in sugarcane cultivar Guitang 42 (GT42) and phylogenetically classified them into nine groups within the Monosaccharide Transporter (MST), Sucrose Transporter (SUT), and Sugars Will Eventually be Exported Transporters (SWEET) families. Comparative evolutionary analysis revealed significant lineage-specific expansions in the PMT, STP subfamilies, and SWEET families compared to diploid and wild relatives, likely driven by polyploidization and intensive selection for sugar yield. Transcriptomic profiling across tissues and internode elongation stages demonstrated marked tissue-specific and developmental expression patterns. Yeast complementation assays confirmed the transport activity of candidate MSTs, SUTs and SWEETs, with confocal microscopy verifying their distinct subcellular localization at the plasma membrane, tonoplast, or endoplasmic reticulum. Furthermore, transient overexpression of several candidate transporters (ScSWEET4-T2, ScSWEET15, and ScTST4-T1) in Nicotiana benthamiana modulated soluble sugar accumulation, and their expression in sugarcane protoplasts activated key sugar-responsive marker genes (ScGPT2 and ScWIP4). Together, our study establishes a systematic genomic framework and identifies candidate functional transporters that govern sugar partitioning and storage, providing valuable genetic targets for molecular breeding and quality enhancement in sugarcane.

Functional characterization↗

PubMed2026

Engineered genomic attachment sites for site-specific recombinases enable high-efficiency integration in plants and human cells.

Here we developed a DNA-centric strategy for optimizing site-specific recombination by rationally engineering chimeric attachment sites. The high-activity att variants enhance Bxb1-mediated integration efficiency in human cells and plants. Among these att variants, the engineered attB(V111) site achieved 51.9% integration efficiency in HEK293T cells (1.7-fold versus wild-type attB) and 35.6% in rice protoplasts (4.4-fold versus wild-type attB). When paired with an engineered single protein mutant in the Bxb1 catalytic domain, the optimized system achieved targeted integration efficiencies of 31% for a CD19 chimeric antigen receptor cassette and 25% for an ornithine transcarbamylase expression cassette in human cells. In rice, these engineered variants enabled integration of a 5.8 kb herbicide-resistance cassette at a targeted genomic locus, with stable integration detected in 24% of regenerated plants. Oxford Nanopore-based long-read sequencing of edited plants reveals complete and precise insertion with high specificity. Propagation of edited seedlings to T1 plants confirms heritable editing to future generations. This approach provides a safe, broadly applicable approach for recombinase-based genome editing.

Journal Article↗

PubMed2025

CRISPR RNP-Mediated Transgene-Free Genome Editing in Plants: Advances, Challenges and Future Directions for Tree Species.

CRISPR ribonucleoprotein (RNP)-mediated genome editing offers a transgene-free platform for precise genetic modification in diverse herbaceous and tree species, including rice, wheat, apple, poplar, oil palm, rubber tree and grapevine. However, its application in woody plants faces distinct challenges, notably inefficient delivery and regeneration difficulties, particularly in species such as bamboo. While some of these issues also occur in herbaceous plants, they are often significantly more complex in woody species due to factors such as intricate cell wall architecture, widespread recalcitrant genotypes and inherent limitations of current delivery platforms. This review presents the first in-depth, critical re-evaluation of recent advancements in RNP-mediated editing in woody plants, highlighting these obstacles that warrant focused attention. Unlike plasmid-based CRISPR systems, RNP editing utilises Cas9/Cas12a protein-guide RNA complexes without integrating foreign DNA. This enables a DNA-free editing strategy that simplifies regulatory approval and minimises off-target effects due to the transient presence and rapid degradation of RNPs within plant cells. While PEG-mediated protoplast transfection and particle bombardment remain the primary reported methods for RNP delivery in trees, we evaluate promising alternative strategies such as lipofection, electroporation, cell-penetrating peptides and nanoparticle-based systems for targeted RNP delivery. Despite their promise, these advanced methods remain largely untested in woody species. Finally, we outline future research directions, including the development of tree-specific RNP delivery systems and regeneration protocols to enhance efficiency and minimise cytotoxicity. These innovations are essential for unlocking the full potential of RNP-mediated genome editing in long-lived tree species. This review provides a focused and timely roadmap for expanding the application of RNP technology across diverse woody plants.

Gene Editing↗

PubMed2025

Rice E3 ligase OsRFPH2-16 acts as a negative regulator to mediate the degradation of OsPIP1;1 under salt stress.

Soil salinity has a significant negative effect on rice productivity. We characterized the Oryza sativa RING Finger Protein H2-type-16 gene (OsRFPH2-16), which plays a negative role in response to salinity. The transcript levels of OsRFPH2-16 decreased under saline conditions. OsRFPH2-16 was expressed in the ER and tonoplasts of rice protoplasts. In addition, OsRFPH2-16 exhibited E3 ligase activity in an in vitro ubiquitination assay, whereas the mutant OsRFPH2-16C188A E3 ligase did not exhibit any activity. We constructed OsRFPH2-16-overexpressing (OX-2 and OX-4) and CRISPR/Cas9-mediated OsRFPH2-16-knockout (KO-4 and KO-16) plants and evaluated their salt responses. Under salt stress, OsRFPH2-16-knockout plants exhibited improved salt tolerance, characterized by low Na+ accumulation, high non-antioxidant content, and dynamic changes in the expression levels of Na+ transporter genes, compared with wild-type and OsRFPH2-16-overexpression plants. The aquaporin OsPIP1;1, an interacting partner, was identified using yeast two-hybridization, bimolecular fluorescence complementation, and pull-down assays. Degradation of OsPIP1;1 by the E3 ligase OsRFPH2-16 via the 26S proteasome system was confirmed through an in vitro degradation assay with the inhibitor MG132. These findings support that the E3 ligase functions as a negative regulator, leading to reduced Na+ accumulation in salt stress responses.

Oryza↗