Search Results (64)

Fusarium crown rot (FCR), caused by Fusarium pseudograminearum, is a devastating wheat disease leading to significant yield losses worldwide. However, the pathogenic mechanism of F. pseudograminearum and its resistance to fungicides remain poorly understood. In this study, we identified a hypothetical gene encoding GPI-anchored protein, designated FpPer1, by screening a T-DNA insertion mutant library of F. pseudograminearum for tebuconazole resistance. The ΔFpper1 mutant exhibited increased sensitivity to the triazole antifungal drugs and fludioxonil. Additionally, the deletion of FpPER1 impaired fungal growth, conidiation, and pathogenicity in barley leaves and wheat coleoptiles. Furthermore, the ΔFpper1 mutant displayed enhanced susceptibility to various environmental stresses, including NaCl, CR, sorbitol, H₂O₂, and SDS. The mutant also showed reduced penetration peg formation and impaired reactive oxygen species (ROS) scavenging ability during infection. Subcellular localization analysis revealed that FpPer1-GFP co-localized with the endoplasmic reticulum (ER) marker RFP-HDEL in both conidia and hyphae, indicating its localization in the ER. In summary, our findings demonstrate that FpPER1 plays an important role in pathogenicity and fungicide resistance in F. pseudograminearum. This study not only provides a theoretical foundation for understanding fungal virulence mechanisms but also offers practical insights for developing novel fungicide strategies. Full article

Welsh onion (Allium fistulosum L.), a globally significant vegetable, flavoring agent, and phytomedicine resource, has remained unavailable with established transient expression platforms for functional genomic investigations. To address this critical methodological limitation, we present systematically optimized protocols for both Agrobacterium-mediated hairy root transformation and protoplast transient expression systems, achieving significant advances in transformation efficiency for this species. Through systematic optimization of key parameters, including Agrobacterium rhizogenes (A. rhizogenes) strain selection (with Ar.Qual demonstrating superior performance), explant type efficacy, bacterial suspension optical density (OD₆₀₀ = 0.3), and acetosyringone induction concentration (100 μM), we established a highly efficient stem disc infection methodology, achieving 88.75% hairy root induction efficiency. Subsequent optimization of protoplast isolation protocols identified the optimal enzymatic digestion conditions: 6-h dark digestion of young leaves using 1.0% (w/v) Cellulase R-10, 0.7% (w/v) Macerozyme R-10, and 0.4 M mannitol, yielding 3.3 × 10⁶ viable protoplasts g⁻¹ FW with 90% viability. System functionality validation through PEG-mediated transient transformation demonstrated successful green fluorescent protein (GFP) reporter gene expression, confirmed by fluorescence microscopy. As the first documented transient expression platforms for Welsh onion, these protocols enable essential molecular investigations, including in planta promoter activity profiling, subcellular protein localization, and CRISPR-based genome-editing validation. This methodological breakthrough overcomes previous technical constraints in Welsh onion molecular biology, providing critical tools for accelerated gene functional characterization in this agriculturally important species. Full article

Interactions between chemical drugs and their target proteins are fundamental to drug screening and precision therapy in modern clinical medicine. However, elucidating these interactions within living cells remains challenging due to the limited availability of efficient detection methods. Despite substantial efforts, technical limitations still impede the identification of direct interactors. In this study, we present a simple method to detect the binding between a chemical drug and its target proteins in live cells. This approach utilizes a self-labeling protein (SLP) tag system, specifically HaloTag which is a modified haloalkane dehalogenase, combined with spatially localized expression of the SLP. To implement this system, dasatinib was conjugated to a HaloTag ligand, and the HaloTag protein was expressed in specific intracellular compartments, such as endosomes or F-actin structures. Upon treatment of cells with the HaloTag ligand-conjugated dasatinib, green fluorescent protein (GFP)-fused cytoplasmic dasatinib target proteins were observed to co-localize with the HaloTag at these subcellular structures, thereby indicating direct drug–target binding. This method provides a good spatial resolution with a high signal-to-noise ratio and low false-positive signals across a high background and false-positive/false-negative signals from endogenous proteins and/or non-specific binding. In this context, we believe that our method is a useful platform for visualizing the binding between chemical drugs and their cytoplasmic targets within living systems. Full article

The R2R3-MYB family of transcription factors (TFs) plays a crucial role in cell specification and secondary metabolism regulation during plant development. In Arabidopsis, MS188, a typical R2R3-MYB protein, is essential for tapetal development and pollen wall formation. However, the nuclear localization sequence (NLS) responsible for directing MS188 into the nucleus has not been fully elucidated. In this study, the subcellular localization of the NLS-containing proteins was determined by GFP tagging in tobacco leaves, and three NLS regions within MS188 were identified: two located at the N-terminus of R2-MYB and one at the C-terminus of R3-MYB. We further narrowed the NLSs located at amino acids (AAs) 12–15, 18–22, and 96–107 via point mutation analysis. Combined with the cytoplasmic protein FBA6, these NLSs fusion proteins could localize in the nucleus. Importantly, the proteins with mutations in AAs 18–22 exhibited completely cytoplasmic signals, whereas other mutated sites partially abolished the nuclear signals. These findings suggest that the NLS at AAs 18–22 is sufficient for nuclear localization. To confirm the NLS functions in vivo, we constructed the vectors including the MS188 gene without the NLS sites, which failed to complement the male sterile phenotype of ms188. We also searched the highly conserved NLSs in other R2R3-MYB TFs and showed they are required for nuclear localization. Collectively, these findings revealed the specific NLS regions within R2R3-MYB transcription factors and highlighted their critical role for subcellular localization in plant developmental regulation. Full article

Areca palm (Areca catechu L.) is an economically significant crop in tropical and subtropical regions. However, an efficient transformation and gene editing system for genetic improvement has still not been established. In this study, protoplasts were isolated from juvenile leaves, followed by PEG-mediated transformation and gene editing targeting the areca palm AcPDS via the CRISPR/Cas9 system. High yield (9.08 × 10⁶ cells/g FW) and viability (91.57%) protoplasts were isolated successfully by digestion for 5 h in an enzyme solution. Transformation efficiency (11.85%) was obtained through PEG-mediated transformation (incubation for 30 min in the mixture containing 40% PEG-4000, 400 mM CaCl₂, 30 µg of plasmid DNA, and 100 µL of protoplasts). Furthermore, subcellular localization was established by the cotransformation of GFP and pNLS-mCherry in the protoplasts. Moreover, the editing efficiency (2.82%) of AcPDS using the CRISPR/Cas9 system was detected by Hi-TOM sequencing. This study established an efficient transformation and gene editing system based on protoplasts in areca palm, which will be beneficial for gene function verification and genetic improvement in areca palm and other tropical palm crops. Full article

Raspberry is a berry whose fruit is not tolerant to storage; breeding varieties with extended storage time and high comprehensive quality are significant for raspberries in cold regions. 1-Aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) is a limiting enzyme in the ethylene synthesis process, which plays essential roles in fruit ripening and softening in plants. In this study, the RiACS1 gene in raspberry (Rubus idaeus L.) variety ‘Polka’ was cloned. The RiACS1 gene overexpression vector was constructed and transformed into tomato plants using the Agrobacterium tumefaciens infection method to verify its function in their reproductive development. The RiACS1 gene, with a total length of 1476 bp, encoded a protein with 491 amino acids. The subcellular localization analysis of the RiACS1 protein in the tobacco transient expression system revealed that the RiACS1-GFP fusion protein was mainly located in the nucleus. Compared with the control, the flowering time and fruit color turning time of transgenic strains were advanced, and the fruit hardness was reduced. Overexpression of RiACS1 increased the activity of ACC synthase, ethylene release rate, and respiration rate during the transchromic phase. It changed the substance content, increased the content of vitamin C and anthocyanin in the fruit ripening process, and decreased the content of chlorophyll and titrable acid at the maturity stage. In addition, RiACS1 increased the relative expression levels of ethylene synthesis-related genes such as SlACS4, SlACO3, and SlACO1 in the fruit ripening process, while it decreased the expression levels of SlACS2 at the maturity stage. These results suggested that the RiACS1 gene could promote early flowering and fruit ripening in tomato plants. This study provided a basis for further modifying raspberry varieties using molecular biology techniques. Full article

Salt stress significantly impairs plant growth, presenting a challenge to agricultural productivity. Exploring the regulatory mechanisms underlying salt stress responses is critically important. Here, we identified a significant role for the maize LESION-SIMULATING DISEASE transcription factor, ZmLSD1, in enhancing salt stress response. Subcellular localization analysis indicated that ZmLSD1-GFP was localized in the nucleus in the maize protoplast. Overexpressing ZmLSD1 in maize obviously enhanced the tolerance of plants to salt stress. Physiological analysis indicated that overexpressed ZmLSD1 in maize could mitigate the accumulation of H₂O₂ and MDA content exposed to salt stress. RNA-seq and qPCR-PCR analyses showed that ZmLSD1 positively regulated ZmWRKY29 expression. ChIP-qPCR and EMSA experiments demonstrated that ZmLSD1 could directly bind to the promoter of ZmWRKY29 through the GTAC motif both in vitro and in vivo. Overall, our findings suggest that ZmLSD1 plays a positive role in enhancing the tolerance of maize to salt by affecting ZmWRKY29 expression. Full article

Passion fruit is a valued tropical fruit crop that faces environment-related growth strains. TCP genes are important for both growth modulation and stress prevention in plants. Herein, we systematically analyzed the TCP gene family in passion fruit, recognizing 30 members. Genes exhibiting closer phylogenetic relationships exhibited similar protein and gene structures. Gene members of the TCP family showed developmental-stage- or tissue-specific expression profiles during the passion fruit life cycle. Transcriptome data also demonstrated that many PeTCPs showed induced expression in response to hormonal treatments and cold, heat, and salt stress. Based on transcriptomics data, eight candidate genes were chosen for preferential gene expression confirmation under cold stress conditions. The qRT-PCR assays suggested PeTCP15/16/17/19/23 upregulation, while PeTCP1/11/25 downregulation after cold stress. Additionally, TCP19/20/29/30 exhibited in silico binding with cold-stress-related miRNA319s. GFP subcellular localization assays exhibited PeTCP19/1 were localized at the nucleus. This study will aid in the establishment of novel germplasm, as well as the further investigation of the roles of PeTCPs and their cold stress resistance characteristics. Full article

Effectors are considered to be virulence factors secreted by pathogens, which play an important role during host-pathogen interactions. In this study, the candidate effector Pt9226 was cloned from genomic DNA of Puccinia triticina (Pt) pathotype THTT, and there were six exons and five introns in the 877 bp sequence, with the corresponding open reading frame of 447 bp in length, encoding a protein of 148 amino acids. There was only one polymorphic locus of I142V among the six Pt pathotypes analyzed. Bioinformatics analysis showed that Pt9226 had 96.46% homology with the hypothetical putative protein PTTG_26361 (OAV96349.1) in the Pt pathotype BBBD. RT-qPCR analyses showed that the expression of Pt9226 was induced after Pt inoculation, with a peak at 36 hpi, which was 20 times higher than the initial expression at 0 hpi, and another high expression was observed at 96 hpi. No secretory function was detected for the Pt9226-predicted signal peptide. The subcellular localization of Pt9226^Δsp-GFP was found to be multiple, localized in the tobacco leaves. Pt9226 could inhibit programmed cell death (PCD) induced by BAX/INF1 in tobacco as well as DC3000-induced PCD in wheat. The transient expression of Pt9226 in 26 wheat near-isogenic lines (NILs) by a bacterial type III secretion system of Pseudomonas fluorescens EtHAn suppressed callose accumulation triggered by Ethan in wheat near-isogenic lines TcLr15, TcLr25, and TcLr30, and it also suppressed the ROS accumulation in TcLr15. RT-qPCR analysis showed that the expression of genes coded for pathogenesis-related protein TaPR1, TaPR2, and thaumatin-like protein TaTLP1, were suppressed, while the expression of PtEF-1α was induced, with 1.6 times at 72 h post inoculation, and TaSOD was induced only at 24 and 48 h compared with the control, when the Pt pathotype THTT was inoculated on a transient expression of Pt9226 in wheat TcLr15. Combining all above, Pt9226 acts as a virulence effector in the interaction between the Pt pathotype THTT and wheat. Full article

Glutathione S-transferases (GSTs) are members of a protein superfamily with diverse physiological functions, including cellular detoxification and protection against oxidative damage. However, there is limited research on GSTs responding to cadmium (Cd) stress. This study classified 46 GST genes in Dendrobium officinale (D. officinale) into nine groups using model construction and domain annotation. Evolutionary analysis revealed nine subfamilies with diverse physical and chemical properties. Prediction of subcellular localization revealed that half of the GST members were located in the cytoplasm. According to the expression analysis of GST family genes responding to Cd stress, DoGST5 responded significantly to Cd stress. Transient expression of DoGST5-GFP in tobacco leaves revealed that DoGST5 was localized in the cytoplasm. DoGST5 overexpression in Arabidopsis enhanced Cd tolerance by reducing Cd-induced H₂O₂ and O₂⁻ levels. These findings demonstrate that DoGST5 plays a critical role in enhancing Cd tolerance by balancing reactive oxygen species (ROS) levels, offering potential applications for improving plant adaptability to heavy metal stress. Full article

An Arabidopsis sterol mutant, smt2 smt3, defective in sterolmethyltransferase2 (SMT2), exhibits severe growth abnormalities. The loss of C-24 ethyl sterols, maintaining the biosynthesis of C-24 methyl sterols and brassinosteroids, suggests specific roles of C-24 ethyl sterols. We characterized the subcellular localizations of fluorescent protein-fused sterol biosynthetic enzymes, such as SMT2-GFP, and found these enzymes in the endoplasmic reticulum during interphase and identified their movement to the division plane during cytokinesis. The mobilization of endoplasmic reticulum-localized SMT2-GFP was independent of the polarized transport of cytokinetic vesicles to the division plane. In smt2 smt3, SMT2-GFP moved to the abnormal division plane, and unclear cell plate ends were surrounded by hazy structures from SMT2-GFP fluorescent signals and unincorporated cellulose debris. Unusual cortical microtubule organization and impaired cytoskeletal function accompanied the failure to determine the cortical division site and division plane formation. These results indicated that both endoplasmic reticulum membrane remodeling and cytokinetic vesicle transport during cytokinesis were impaired, resulting in the defects of cell wall generation. The cell wall integrity was compromised in the daughter cells, preventing the correct determination of the subsequent cell division site. We discuss the possible roles of C-24 ethyl sterols in the interaction between the cytoskeletal network and the plasma membrane. Full article

The auxin/indoleacetic acid (Aux/IAA) family plays a central role in regulating gene expression during auxin signal transduction. Nonetheless, there is limited knowledge regarding this gene family in sugarcane. In this study, 92 members of the IAA family were identified in Saccharum spontaneum, distributed on 32 chromosomes, and classified into three clusters based on phylogeny and motif compositions. Segmental duplication and recombination events contributed largely to the expansion of this superfamily. Additionally, cis-acting elements in the promoters of SsIAAs involved in plant hormone regulation and stress responsiveness were predicted. Transcriptomics data revealed that most SsIAA expressions were significantly higher in stems and basal parts of leaves, and at nighttime, suggesting that these genes might be involved in sugar transport. QRT-PCR assays confirmed that cold and salt stress significantly induced four and five SsIAAs, respectively. GFP-subcellular localization showed that SsIAA23 and SsIAA12a were localized in the nucleus, consistent with the results of bioinformatics analysis. In conclusion, to a certain extent, the functional redundancy of family members caused by the expansion of the sugarcane IAA gene family is related to stress resistance and regeneration of sugarcane as a perennial crop. This study reveals the gene evolution and function of the SsIAA gene family in sugarcane, laying the foundation for further research on its mode of action. Full article

This study systematically investigated the physiological and molecular responses of the wheat mutant ‘XC-MU201’ under high-temperature stress through comprehensive transcriptome analysis and physiological measurements. RNA sequencing of 21 samples across seven different treatment groups revealed, through Weighted Gene Co-expression Network Analysis (WGCNA), 13 modules among 9071 genes closely related to high-temperature treatments. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed significant enrichment of lignin biosynthesis-related modules under high-temperature conditions, especially at the H-10DAT, H-20DAT, and H-30DAT time points. Experimental results demonstrated a significant increase in lignin content in high-temperature-treated samples, confirmed by tissue staining methods, indicating wheat’s adaptation to heat damage through lignin accumulation. The phenylalanine ammonia-lyase gene (TaPAL33) was significantly upregulated under high-temperature stress, peaking at H-30DAT, suggesting its critical role in cellular defense mechanisms. Overexpression of TaPAL33 in the wheat variety ‘Xinchun 11’ enhanced lignin synthesis but inhibited growth. Subcellular localization of GFP-labeled TaPAL33 in tobacco cells showed its distribution mainly in the cytoplasm and cell membrane. Transgenic wheat exhibited higher PAL enzyme activity, enhanced antioxidant defense, and reduced oxidative damage under high-temperature stress, outperforming wild-type wheat. These results highlight TaPAL33’s key role in improving wheat heat tolerance and provide a genetic foundation for future research and applications. Full article

Thaumatin-like proteins (TLPs) in plants are involved in diverse biotic and abiotic stresses, including antifungal activity, low temperature, drought, and high salinity. However, the roles of the TLP genes are rarely reported in early flowering. Here, the TLP gene family was identified in P. trichocarpa. The 49 PtTLP genes were classified into 10 clusters, and gene structures, conserved motifs, and expression patterns were analyzed in these PtTLP genes. Among 49 PtTLP genes, the PtTLP6 transcription level is preferentially high in stems, and GUS staining signals were mainly detected in the phloem tissues of the PtTLP6pro::GUS transgenic poplars. We generated transgenic Arabidopsis plants overexpressing the PtTLP6 gene, and its overexpression lines showed early flowering phenotypes. However, the expression levels of main flowering regulating genes were not significantly altered in these PtTLP6-overexpressing plants. Our data further showed that overexpression of the PtTLP6 gene led to a reactive oxygen species (ROS) burst in Arabidopsis, which might advance the development process of transgenic plants. In addition, subcellular localization of PtTLP6-fused green fluorescent protein (GFP) was in peroxisome, as suggested by tobacco leaf transient transformation. Overall, this work provides a comprehensive analysis of the TLP gene family in Populus and an insight into the role of TLPs in woody plants. Full article

Accurate determination of protein localization, levels, or protein−protein interactions is pivotal for the study of their function, and in situ protein labeling via homologous recombination has emerged as a critical tool in many organisms. While this approach has been refined in various model fungi, the study of protein function in most plant pathogens has predominantly relied on ex situ or overexpression manipulations. To dissect the molecular mechanisms of development and infection for Verticillium dahliae, a formidable plant pathogen responsible for vascular wilt diseases, we have established a robust, homologous recombination-based in situ protein labeling strategy in this organism. Utilizing Agrobacterium tumefaciens-mediated transformation (ATMT), this methodology facilitates the precise tagging of specific proteins at their C-termini with epitopes, such as GFP and Flag, within the native context of V. dahliae. We demonstrate the efficacy of our approach through the in situ labeling of VdCf2 and VdDMM2, followed by subsequent confirmation via subcellular localization and protein-level analyses. Our findings confirm the applicability of homologous recombination for in situ protein labeling in V. dahliae and suggest its potential utility across a broad spectrum of filamentous fungi. This labeling method stands to significantly advance the field of functional genomics in plant pathogenic fungi, offering a versatile and powerful tool for the elucidation of protein function. Full article