Search Results (36)

Plant U-box (PUB) E3 ubiquitin ligases have undergone significant expansion compared to their fungal and animal counterparts. These E3 ligases play critical roles in diverse biological processes, including responses to biotic and abiotic stresses. However, systematic identification of PUB genes in cucumber (Cucumis sativus L.) has been lacking, and their expression and functional characterization remain largely unexplored. Leveraging the recently released near-complete cucumber genome, we identified 53 putative PUB proteins classified into eight distinct groups based on domain architecture. The molecular weights of CsPUBs range from 26 to 166 kilodaltons (kDa). Exon numbers in CsPUB genes vary substantially, with CsPUB48 containing a maximum of 17 exons, while 18 CsPUB genes harbor only a single exon. Chromosomal distribution of CsPUBs is uneven, with Chr 3 harboring the highest density (12 genes) and Chr 7 the lowest (1 gene). Notably, tandem duplications (e.g., CsPUB29-CsPUB36 and CsPUB18-CsPUB49) and seven collinear gene pairs were identified, suggesting evolutionary diversification. Promoter regions of CsPUBs are enriched with cis-regulatory elements linked to plant growth and development, phytohormone, stress responses, light, and so on, implying their regulatory roles in various biological processes. Expression profiling revealed tissue-specific patterns and differential regulation of multiple CsPUBs under stress conditions. Subcellular localization studies demonstrated that CsPUBs target diverse organelles, with some localizing to punctate structures potentially representing uncharacterized compartments. Collectively, this systematic analysis establishes a comprehensive framework for understanding particular CsPUB functions. Full article

Tomato brown rugose fruit virus (ToBRFV) is an important emerging virus that poses a serious threat to the global agricultural economy. Ubiquitination is one of the key post-translational protein modification types in plant responses to biotic stress, but the extent to which ToBRFV infection alters the overall ubiquitination status has not been reported. This study conducted integrated ubiquitome and proteome analyses of Nicotiana benthamiana leaves infected with ToBRFV and identified differentially ubiquitinated proteins. A total of 346 lysine sites on 302 identified proteins were found to be affected, with 260 sites exhibiting upregulated ubiquitination levels in 224 proteins and 86 sites showing downregulated ubiquitination levels in 80 proteins. The differentially ubiquitinated proteins were primarily localized in the cytoplasm (29%), nucleus (18%), plasma membrane (8.9%), mitochondria (5.1%), and chloroplasts (4.6%). Fourteen conserved ubiquitination motifs, including ENNNK, ENNK, SK, and KNG, were identified. Furthermore, enrichment analysis indicated that ToBRFV infection induces an increase in the ubiquitination levels of proteins associated with ion transport, MAPK signaling pathways, and plant hormone signal transduction, while the ubiquitination levels of proteins related to carbon metabolism and secondary metabolite synthesis decreased. Functional analysis of the three differentially ubiquitinated proteins revealed that a RING/U-box superfamily protein negatively regulates ToBRFV infection. Our work provides the first systematic analysis of the ubiquitination profile in N. benthamiana leaves following ToBRFV infection, providing important resources for further studies on the regulatory mechanisms of ubiquitination in plant responses to ToBRFV. Full article

The U-box E3 ubiquitin ligase (PUB) gene family plays an important role in regulating plant responses to abiotic stress. Poncirus trifoliata (trifoliate orange), a citrus rootstock with notable cold, drought, and salt tolerance, serves as an excellent model for studying stress-responsive genes. In this study, a total of 47 PUB genes (PtrPUBs) were identified in the trifoliate orange genome. Chromosomal distribution analysis indicated that PtrPUB genes were unevenly distributed across nine trifoliate orange chromosomes. Phylogenetic tree analysis indicated that 170 PUB proteins from trifoliate orange, Arabidopsis thaliana, and tomato were clustered into five subfamilies. Gene structure, conserved domain, and motif analyses revealed diverse exon–intron and motif organizations of PtrPUB genes, suggesting potential functional differentiation among PtrPUBs. Cis-acting analysis indicated that the promoters of PtrPUB genes harbor elements related to hormone signaling (ABA, MeJA), drought stress, and low-temperature responses. Transcriptomic data and qRT-PCR results suggested that PtrPUB genes are responsive to ABA and dehydration treatments. This study provides a foundation for understanding the functional roles of PUB genes in trifoliate orange and offers insights for improving stress tolerance in citrus breeding programs. Full article

Pathogens deploy various molecular mechanisms to overcome host defenses, among which glycoside hydrolases (GHs) play a critical role as virulence factors. Understanding the functional roles of these enzymes is essential for uncovering pathogen–host interactions and developing strategies for disease management. Fusarium wilt has occurred in the main Piper nigrum cultivation regions, which seriously affects the yield and quality of P. nigrum. Here, we identified and characterized FsGH28c, a GH28 family member in Fusarium solani. Its expression was significantly upregulated during the infection of black pepper (Piper nigrum) roots by F. solani cv. WN-1, indicating its potential role in pathogenicity. FsGH28c elicited cell death in Nicotiana benthamiana and modulated the expression of genes related to pathogenesis. FsGH28c exerts a positive influence on the pathogenicity of F. solani. The knockout of FsGH28c mutant strains markedly attenuated F. solani ’s virulence in black pepper plants. The knockout mutant strains decrease the ability of F. solani to utilize carbon sources. The FsGH28c deletion did not affect mycelial growth on PDA but did impact spore development. We identified a U-box protein, PnPUB35, interacting with FsGH28c using yeast two-hybrid and bimolecular fluorescence complementation assays. PnPUB35 conferred enhanced resistance to F. solani in black pepper through positive regulation. These findings suggest that FsGH28c may function as a virulence factor by modulating host immune responses through its interaction with PnPUB35. Full article

Plant U-box E3 ubiquitin ligases (PUBs) serve as crucial regulators of protein degradation and are fundamentally involved in plant developmental processes and stress response mechanisms. Despite their well-characterized roles in model plant species, the PUB gene family in the hybrid poplar (Populus alba × P. tremula var. glandulosa) remains poorly understood. By conducting a comprehensive genome-wide analysis, we identified 152 PUB genes in poplar and phylogenetically classified them into five distinct clades based on a comparative analysis with Arabidopsis thaliana and tomato PUB homologs. The structural characterization revealed that numerous PagPUB proteins possess additional functional domains, including ARM and WD40 repeats, which are indicative of potential functional diversification. Genomic distribution and synteny analyses demonstrated that the expansion of the PUB gene family predominantly resulted from whole-genome duplication (WGD) events, with evolutionary constraint analyses (Ka/Ks ratios < 1) suggesting strong purifying selection. An examination of the promoter region uncovered an abundance of stress-responsive cis-elements, particularly ABRE and MYB binding sites associated with abiotic stress and hormonal regulation. Transcriptome profiling demonstrated both tissue-specific expression patterns and dynamic regulation under diverse stress conditions, including drought, salinity, temperature extremes, and pathogen infection. Our findings provide the first systematic characterization of the PUB gene family in poplar and establish a valuable framework for elucidating their evolutionary history and functional significance in environmental stress adaptation. Full article

The ubiquitination and degradation of proteins are widely involved in plant biotic and abiotic stress responses. E3 ubiquitin ligases play an important role in the ubiquitination of specific proteins. In this study, we identified the function of a U-box E3 ubiquitin ligase gene OsPUB57 in rice. Expression analyses revealed that OsPUB57 was mainly expressed in the aboveground part of rice. Drought, salt, cold, JA (jasmonic acid), PAMPs (pathogen-associated molecular patterns) or Magnaportheoryzae treatment could significantly suppress the expression of OsPUB57 in rice. Compared with wild-type plants, OsPUB57-overexpressing plants showed a decrease in resistance to M. oryzae, while the mutant plants exhibited an enhancement of M. oryzae resistance. The expression level detection indicated that OsPUB57 negatively regulates rice blast resistance, probably by down-regulating the expression of the defense-related genes OsPR1a and OsAOS2. This study provides a candidate gene for the genetic improvement of rice blast resistance. Full article

Maize lethal necrosis (MLN) is a significant threat to food security in Sub-Saharan Africa (SSA), with limited commercial inbred lines displaying tolerance. This study analyzed the transcriptomes of four commercially used maize inbred lines and a non-adapted inbred line, all with varying response levels to MLN. RNA-Seq revealed differentially expressed genes in response to infection by maize chlorotic mottle virus (MCMV) and sugarcane mosaic virus (SCMV), the causative agents of MLN. Key findings included the identification of components of the plant innate immune system, such as differentially regulated R genes (mainly LRRs), and activation/deactivation of virus resistance pathways, including RNA interference (RNAi) via Argonaute (AGO), Dicer-like proteins, and the ubiquitin–proteasome system (UPS) via RING/U-box and ubiquitin ligases. Genes associated with redox signaling, WRKY transcription factors, and cell modification were also differentially expressed. Additionally, the expression of translation initiation and elongation factors, eIF4E and eIF4G, correlated with the presence of MLN viruses. These findings provide valuable insights into the molecular mechanisms of MLN resistance and highlight potential gene candidates for engineering or selecting MLN-resistant maize germplasm for SSA. Full article

The ubiquitin-proteasome system (UPS) is a key protein degradation pathway in eukaryotes, in which E3 ubiquitin ligases mediate protein ubiquitination, directly or indirectly targeting substrate proteins to regulate various biological processes, including plant growth, hormone signaling, immune responses, and adaptation to abiotic stress. In this study, we identified plant U-box protein 51 in Solanum tuberosum (StPUB51) as an E3 ubiquitin ligase through transcriptomic analysis, and used it as a candidate gene for gene-function analysis. Quantitative real-time PCR (qRT-PCR) was used to examine StPUB51 expression across different tissues, and its expression patterns under simulated drought stress induced by polyethylene glycol (PEG 6000) were assessed. Transgenic plants overexpressing StPUB51 and plants with down-regulated StPUB51 expression were generated to evaluate drought tolerance. The activities of key antioxidant enzymes-superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) as well as malondialdehyde (MDA) content in transgenic plants’ leaves were measured under drought conditions. Protein–protein interactions involving StPUB51 were explored via yeast two-hybrid (Y2H) screening, with interaction verification by bimolecular fluorescence complementation (BiFC). StPUB51 was predominantly expressed in stems, with lower expression observed in tubers, and its expression was significantly upregulated in response to 20% PEG-6000 simulated drought. Subcellular localization assays revealed nuclear localization of the StPUB51 protein. Under drought stress, StPUB51-overexpressing plants exhibited enhanced SOD, POD, and CAT activities and reduced MDA levels, in contrast to plants with suppressed StPUB51 expression. Y2H and BiFC analyses identified two interacting proteins, StSKP2A and StGATA1, which may be functionally linked to StPUB51. Collectively, these findings suggest that StPUB51 plays a positive regulatory role in drought tolerance, enhancing resilience in potato growth and stress adaptation. Full article

This study explores the extraction and characterization of proteolytic enzymes from brewer’s spent grain (BSG) and their potential as sustainable coagulants in the dairy industry. BSG samples from various beer types (Blonde Ale, IPA, Kölsch, Honey, and Porter) were obtained from two artisanal breweries in Mar del Plata, Argentina. Optimization of caseinolytic activity (CA) and protein extraction was conducted using a Plackett–Burman design, followed by a Box–Behnken design. Optimal protein concentration was achieved at intermediate pH and high temperature, while CA peaked at pH 8.0. The specific caseinolytic activity (SCA) varied among the extracts, with BSG3 showing the highest activity (99.6 U mg⁻¹) and BSG1 the lowest (60.4 U mg⁻¹). Protease inhibitor assays suggested the presence of aspartic, serine, metallo, and cysteine proteases. BSG3 and BSG4 showed the highest hydrolysis rates for α-casein (70% and 78%). For κ-casein, BSG1, BSG2, and BSG3 demonstrated moderate activity (56.5%, 49%, and 55.8), while BSG4 and BSG5 exhibited the lowest activity. Additionally, the milk-clotting activity (MCA) of BSG extracts was comparable to plant-based coagulants like Cynara cardunculus and Ficus carica. These findings highlight the potential of BSG-derived proteases as alternative coagulants for cheese production, offering a sustainable link between the brewing and dairy industries. Full article

E3 ubiquitin ligases known as plant U-box (PUB) proteins regulate a variety of aspects of plant growth, development, and stress response. However, the functions and characteristics of the PUB gene family in alfalfa remain unclear. This work involved a genome-wide examination of the alfalfa U-box E3 ubiquitin ligase gene. In total, 210 members were identified and divided into five categories according to their homology with the members of the U-box gene family in Arabidopsis thaliana. The phylogenetic analysis, conserved motifs, chromosomal localization, promoters, and regulatory networks of this gene were investigated. Chromosomal localization and covariance analyses indicated that the MsPUB genes expanded MsPUB gene family members through gene duplication events during evolution. MsPUB genes may be involved in the light response, phytohormone response, growth, and development of several biological activities, according to cis-acting element analysis of promoters. In addition, transcriptome analysis and expression analysis by qRT-PCR indicated that most MsPUB genes were significantly upregulated under cold stress, drought stress, and salt stress treatments. Among them, MsPUBS106 and MsPUBS185 were significantly and positively correlated with cold resistance in alfalfa. MsPUBS110, MsPUBS067, MsPUBS111 and MsPUB155 were comprehensively involved in drought stress, low temperature, and salt stress resistance. All things considered, these discoveries offer fresh perspectives on the composition, development, and roles of the PUB gene family in alfalfa. They also provide theoretical guidance for further investigations into the mechanisms regulating the development, evolution, and stress tolerance of MsPUB. Full article

The common bean (Phaseolus vulgaris L.) is an economically important food crop grown worldwide; however, its production is affected by various environmental stresses, including cold, heat, and drought stress. The plant U-box (PUB) protein family participates in various biological processes and stress responses, but the gene function and expression patterns of its members in the common bean remain unclear. Here, we systematically identified 63 U-box genes, including 8 tandem genes and 55 non-tandem genes, in the common bean. These PvPUB genes were unevenly distributed across 11 chromosomes, with chromosome 2 holding the most members of the PUB family, containing 10 PUB genes. The analysis of the phylogenetic tree classified the 63 PUB genes into three groups. Moreover, transcriptome analysis based on cold-tolerant and cold-sensitive varieties identified 4 differentially expressed PvPUB genes, suggesting their roles in cold tolerance. Taken together, this study serves as a valuable resource for exploring the functional aspects of the common bean U-box gene family and offers crucial theoretical support for the development of new cold-tolerant common bean varieties. Full article

Ubiquitination plays a crucial role in regulating signal pathways during the post-translation stage of protein synthesis in response to various environmental stresses. E3 ubiquitin ligase has been discovered to ultimately control various intracellular activities by imparting specificity to proteins to be degraded. This study was conducted to confirm biological and genetic functions of the U-box type E3 ubiquitin ligase (PUB) gene against biotic stress in rice (Oryza sativa L.). OsPUB9 gene-specific sgRNA were designed and transformants were developed through Agrobacterium-mediated transformation. Deep sequencing using callus was performed to confirm the mutation type of T₀ plants, and a total of three steps were performed to select null individuals without T-DNA insertion. In the case of the OsPUB9 gene-edited line, a one bp insertion was generated by gene editing, and it was confirmed that early stop codon and multiple open reading frame (ORF) sites were created by inserting thymine. It is presumed that ubiquitination function also changed according to the change in protein structure of U-box E3 ubiquitin ligase. The OsPUB9 gene-edited null lines were inoculated with bacterial leaf blight, and finally confirmed to have a resistance phenotype similar to Jinbaek, a bacterial blight-resistant cultivar. Therefore, it is assumed that the amino acid sequence derived from the OsPUB9 gene is greatly changed, resulting in a loss of the original protein functions related to biological mechanisms. Comprehensively, it was confirmed that resistance to bacterial leaf blight stress was enhanced when a mutation occurred at a specific site of the OsPUB9 gene. Full article

The U-box protein family of ubiquitin ligases is important in the biological processes of plant growth, development, and biotic and abiotic stress responses. Plants in the genus Zoysia are recognized as excellent warm-season turfgrass species with drought, wear and salt tolerance. In this study, we conducted the genome-wide identification of plant U-box (PUB) genes in Zoysia japonica based on U-box domain searching. In total, 71 ZjPUB genes were identified, and a protein tree was constructed of AtPUBs, OsPUBs, and ZjPUBs, clustered into five groups. The gene structures, characteristics, cis-elements and protein interaction prediction network were analyzed. There were mainly ABRE, ERE, MYB and MYC cis-elements distributed in the promoter regions of ZjPUBs. ZjPUBs were predicted to interact with PDR1 and EXO70B1, related to the abscisic acid signaling pathway. To better understand the roles of ZjPUBs under salt stress, the expression levels of 18 ZjPUBs under salt stress were detected using transcriptome data and qRT-PCR analysis, revealing that 16 ZjPUBs were upregulated in the roots under salt treatment. This indicates that ZjPUBs might participate in the Z. japonica salt stress response. This research provides insight into the Z. japonica PUB gene family and may support the genetic improvement in the molecular breeding of salt-tolerant zoysiagrass varieties. Full article

Kiwifruit bacterial canker caused by Pseudomonas syringae pv. actinidiae (Psa) is the most serious disease threatening kiwifruit production. Our previous study found genes encoding the U-box containing proteins were significantly regulated by Psa infection. Here, we report a U-box type E3 ubiquitin ligase PUB23 in kiwifruit which acts as a negative regulator of immune responses against Psa. PUB23 was found to physically interact with GT1, a trihelix transcription factor, in vitro and in vivo. The expression of GT1 was up-regulated in PUB23-silenced plants, indicating that interacting with PUB23 may directly or indirectly suppress GT1 expression. The silencing of PUB23 led to enhanced immune responses of PAMP-triggered immunity (PTI), including a higher expression level of defense marker genes PR1 and RIN4, and increased accumulation of hydrogen peroxide and superoxide anion. Our results reveal a negative role PUB23 plays in kiwifruit immune responses against Psa and may regulate gene expression by interacting with GT1. Full article

Members of the WRKY transcription factor (TF) family are unique to plants and serve as important regulators of diverse physiological processes, including the ability of plants to manage biotic and abiotic stressors. However, the functions of specific WRKY family members in the context of maize responses to fungal pathogens remain poorly understood, particularly in response to Ustilago maydis (DC.) Corda (U. maydis), which is responsible for the devastating disease known as corn smut. A systematic bioinformatic approach was herein employed for the characterization of the maize WRKY TF family, leading to the identification of 120 ZmWRKY genes encoded on 10 chromosomes. Further structural and phylogenetic analyses of these TFs enabled their classification into seven different subgroups. Segmental duplication was established as a major driver of ZmWRKY family expansion in gene duplication analyses, while the Ka/Ks ratio suggested that these ZmWRKY genes had experienced strong purifying selection. When the transcriptional responses of these genes to pathogen inoculation were evaluated, seven U. maydis-inducible ZmWRKY genes were identified, as validated using a quantitative real-time PCR approach. All seven of these WKRY proteins were subsequently tested using a yeast one-hybrid assay approach, which revealed their ability to directly bind the ZmSWEET4b W-box element, thereby controlling the U. maydis-inducible upregulation of ZmSWEET4b. These results suggest that these WRKY TFs can control sugar transport in the context of fungal infection. Overall, these data offer novel insight into the evolution, transcriptional regulation, and functional characteristics of the maize WRKY family, providing a basis for future research aimed at exploring the mechanisms through which these TFs control host plant responses to common smut and other fungal pathogens. Full article