Nuclear Factor Y (NF-Y) represents a group of transcription factors commonly present in higher eukaryotes, typically consisting of three subunits: NF-YA, NF-YB, and NF-YC. They play crucial roles in the embryonic development, photosynthesis, flowering, abiotic stress responses, and other essential processes in plants. To better understand the genome-wide NF-Y domain-containing proteins, the protein physicochemical properties, chromosomal localization, synteny, phylogenetic relationships, genomic structure, promoter cis-elements, and protein interaction network of NtNF-Ys in tobacco (Nicotiana tabacum L.) were systematically analyzed. In this study, we identified 58 NtNF-Ys in tobacco, respectively, and divided into three subfamilies corresponding to their phylogenetic relationships. Their tissue specificity and expression pattern analyses for leaf development, drought and saline-alkali stress, and ABA response were carried out using RNA-seq or qRT-PCR. These findings illuminate the role of NtNF-Ys in regulating plant leaf development, drought and saline-alkali stress tolerance, and ABA response. This study offers new insights to enhance our understanding of the roles of NtNF-Ys and identify potential genes involved in leaf development, as well as drought and saline-alkali stress tolerance of plants.

The impact of microplastic pollution has emerged as a significant global ecological concern. Various organisms have exhibited alterations in behavior or metabolic activities following exposure to microplastics (MPs). Mosquitoes, as crucial disease vectors, are particularly susceptible MPs exposure in the environment. Recent studies have demonstrated that MPs ingested by mosquitoes can be detected in vivo, potentially being transmitted during their different life cycles. However, it remains unclear whether MPs in vivo could affect mosquito infection with arboviruses. In this study, we identified that the physical adsorption effect of MPs is also effective against arboviruses, enabling the adsorption of Zika virus particles onto their surfaces. We established an exposure model by feeding adult Aedes albopictus (Skuse, 1895) (Diptera: Culicidae) with 1 μm MPs at concentrations of 5 and 50 μg/mL in 8 % sucrose solution. The transmission rate of ZIKV and population transmission rate in the laboratorial Ae. albopictus exposure model began to decrease from day 7, showing statistically significant differences compared to the control group on days 10 and 14 (**, p < 0.01), significantly affecting their vector efficiency. This phenomenon is not solely dependent on the physical adsorption of MPs to arboviruses. Transcriptome analysis indicated that exposure to MPs influenced the expression levels of genes associated with mosquito virus infection, altering the function of relevant pathways, which consequently reduces their capability to transmit arbovirus. These findings suggest that exposure to MPs significantly affects the vector efficiency of mosquitoes, providing novel perspectives for the ecological risk assessment of MPs.

The white button mushroom (Agaricus bisporus), widely cultivated worldwide as an edible mushroom, is susceptible to browning, which significantly impacts its nutritional and commercial value. Extensive research has enhanced our understanding of the mechanisms underlying this browning process. Although the role of DNA methylation in regulating gene expression has been studied in many fungi, information specifically concerning DNA methylation during the browning in A. bisporus is still limited. In this study, we initially evaluated the impact of temperatures (4 ℃ and room temperature) on discoloration in A. bisporus, and samples with similar discoloration under different temperatures were collected for transcriptome and DNA methylation sequencing. The results revealed that DNA methylation was positively correlated with browning, suggesting its involvement during the browning in A. bisporus. Further analysis showed the heightened methylation levels were primarily attributed to increased methylation at CHG and CHH sites. By joint analysis of transcriptome and DNA methylome, 342 genes with significant expression changes were identified to be affected by DNA methylation, and finally 13 genes were considered as important browning genes under different signaling pathways, such as ABA/ET pathway. Notably, four DNA methyltransferases were identified and validated to play important role during browning in A. bisporus. Altogether, this study provides theoretical insights into the functions of DNMTs in A. bisporus, and offers new perspectives on the role of DNA methylation in edible mushrooms.

Fungal secondary metabolites are considered as important resources for drug discovery. Despite various methods being employed to facilitate the discovery of new fungal secondary metabolites, the trend of identifying novel secondary metabolites from fungi is inevitably slowing down. Under laboratory conditions, the majority of biosynthetic gene clusters, which store information for secondary metabolites, remain inactive. Therefore, establishing the link between biosynthetic gene clusters and secondary metabolites would contribute to understanding the genetic logic underlying secondary metabolite biosynthesis and alleviating the current challenges in discovering novel natural products. Bioinformatics methods have garnered significant attention due to their powerful capabilities in data mining and analysis, playing a crucial role in various aspects. Thus, we have summarized successful cases since 2016 in which bioinformatics methods were utilized to establish the link between fungal biosynthetic gene clusters and secondary metabolites, focusing on their biosynthetic gene clusters and associated secondary metabolites, with the goal of aiding the field of natural product discovery.

Phages have garnered increasing attention due to their potential roles in biogeochemical cycling. However, their impacts on nitrogen cycling have primarily been inferred from the presence of putative auxiliary metabolic genes (AMGs) and the virus-host linkage, despite of very limited direct experimental evidence. In this study, a series of microcosms were established with the inoculation of either native or non-native phages to simulate coastal wetlands with different phage sources and different levels of copper (Cu) contamination. Metagenomics and metatranscriptomics were combined to reveal phages' regulation on microbially-driven nitrogen cycling and to explore how the effects were mediated by Cu stress. Phages significantly impacted denitrification-related genes, with their effects depending on Cu level. Phages inhibited nirK-type denitrification under Cu stress but led to up-regulation of nirS gene in the treatments without Cu addition. Non-native phages also promoted the transcription of genes related to nitrogen assimilation and organic nitrogen transformation. Detection of viral AMGs involved in glutamate synthesis suggested that horizontal gene transfer may be a crucial pathway for phages to facilitate microbial nitrogen uptake. Overall, these findings enhance the understanding of phages' impact on biogeochemical metabolism in coastal wetland, offering novel insights into the links of phages' regulation on microbial nitrogen cycling with Cu stress.

Cell elongation is an important process during cotton fiber development, ultimately determining the length of mature fibers. Myo-inositol oxygenase (MIOX) pathway provides pivotal precursors for the synthesis of non-cellulosic polysaccharides in plant cell walls. However, the role of MIOX gene in cotton fiber development has not been reported. Here, we hypothesized that Gossypium barbadense MIOX gene GbMIOX8 (GbM_D05G1480.1) could regulate fiber length by modulating cell wall composition. To test this hypothesis, we characterized the functional properties of GbMIOX8. GbMIOX8 preferentially expressed during fiber initiation and elongation in cotton and encodes non-secretory protein targeted to the cytoplasm. Overexpression of GbMIOX8 afforded transgenic A. thaliana significantly longer leaf trichomes, as well as longer hypocotyl cells compared to the wild type, with increases of at least 11 % and up to 23 %. We further overexpressed GbMIOX8 in cotton and found that transgenic cotton displayed fiber length that was increased by an average of 1.61 mm in the T1 generation and 1.93 mm in the T2 generation, respectively. Similar to Arabidopsis, transgenic cotton exhibited at least a threefold increase in myo-inositol oxygenase activity and content, boosting glucuronic acid production and reducing inositol. Furthermore, pectin and cellulose contents rose in transgenic cottons, with average rises of 19 % and 38 % respectively, indicating enhanced biosynthesis of these two cell wall components. These results revealed that GbMIOX8 played an important role in the elongation of plant cells by altering cell wall components and could be valuable for cotton fiber quality improvement.

The characterization of gene sets is a recurring task in computational biology. Identifying specific properties of a hit set compared to a reference set can reveal biological roles and mechanisms, and can lead to the prediction of new hits. However, collecting the features to evaluate can be time consuming, and implementing an informative but compact graphical representation of the multiple comparisons can be challenging, particularly for bench scientists. Here, I present Qarles (quick characterization of large sets of genes), a web server that annotates Saccharomyces cerevisiae gene sets by querying a database of 31 features widely used by the yeast community and that identifies their specific properties, providing publication-ready figures and reliable statistics. Qarles has a deliberately simple user interface with all the functionality in a single web page and a fast response time to facilitate adoption by the scientific community. Qarles provides a rich and compact graphical output, including up to five gene set comparisons across 31 features in a single dotplot, and interactive boxplots to enable the identification of outliers. Qarles can also predict new hit genes by using a random forest trained on the selected features. The web server is freely available at https://qarles.org.

Despite considerable scrutiny of mammalian arterivirus genomes, their genomic architecture remains incomplete, with several unannotated non-structural proteins (NSPs) and the enigmatic absence of methyltransferase (MTase) domains. Additionally, the host range of arteriviruses has expanded to include seven newly sequenced genomes from non-mammalian hosts, which remain largely unannotated and await detailed comparisons alongside mammalian isolates. Utilizing comparative genomics approaches and comprehensive sequence-structure analysis, we provide enhanced genomic architecture and annotations for arterivirus genomes. We identified the previously unannotated C-terminal domain of NSP3 as a winged helix-turn-helix domain and classified NSP7 as a new small β-barrel domain, both likely involved in interactions with viral RNA. NSP12 is identified as a derived variant of the N7-MTase-like Rossmann fold domain that retains core structural alignment with N7-MTases in Nidovirales but likely lacks enzymatic functionality due to the erosion of catalytic residues, indicating a unique role specific to mammalian arteriviruses. In contrast, non-mammalian arteriviruses sporadically retain a 2'-O-MTase and an exonuclease (ExoN) domain, which are typically absent in mammalian arteriviruses, highlighting contrasting evolutionary trends and variations in their molecular toolkit. Similar lineage-specific patterns are observed in the diversification of papain-like proteases and structural proteins. Overall, the study extends our knowledge of arterivirus genomic diversity and evolution.

Long noncoding RNAs (lncRNAs) can regulate gene expression by "sponging" microRNAs (miRNAs), reducing their inhibitory effects on mRNAs. However, this mechanism has been minimally investigated in preimplantation embryo development. In this study, we revisited existing RNA sequencing and small RNA sequencing data to investigate the role of lncRNAs in in vitro-produced bovine preimplantation embryos. Our findings revealed that although lncRNAs exhibit expression patterns similar to mRNAs, maternal lncRNAs degrade earlier than mRNAs during embryonic genome activation (EGA). Weighted gene co-expression network analysis identified 27 modules of mRNA and lncRNA, with enrichment analysis showing a significant negative correlation between the polycomb repressive complex pathway and blastocyst formation (R2 = -0.98). Additionally, bioinformatics analysis was used to predict and construct lncRNA-miRNA-mRNA networks, highlighting that lncRNAs bind more to miRNAs compared with mRNAs. Moreover, lncRNA-induced lncRNA-miRNA-mRNA axes participated in mRNA degradation and biogenesis around the EGA stage. These interactions became stronger after EGA, especially after the 16-cell stage. Overall, our study provides new insights into lncRNA-mediated regulatory networks during bovine preimplantation development.

Identifying structural relationships between proteins is crucial for understanding their functions and evolutionary histories. We present ISS_ProtSci, a Python package designed for structural similarity searches within the AlphaFold Database v2 (AFDB2). ISS_ProtSci incorporates DaliLite to identify geometrically similar structures and uses a transitive closure algorithm to iteratively explore neighboring shells of proteins. The precomputed all-against-all comparisons generated by Foldseek, chosen for its speed, are validated by DaliLite for precision. Search results are annotated with metadata from UniProtKB and Pfam protein family classifications, using hmmsearch to identify protein domains. Outputs, including Dali pairwise alignment data, are provided in TSV format for easy filtering and analysis. Our method offers a significant improvement in recall over existing tools like Foldseek, especially in detecting more distantly related proteins. This is particularly valuable in structurally diverse protein families where traditional sequence-based or fast structural methods struggle. ISS_ProtSci delivers practical runtimes and flexibility, allowing users to input a PDB file, define the minimum size of the common core, and evaluate results using Pfam clans. In evaluating our method across 12 test cases based on Pfam clans, we achieved over 99% recall of relevant proteins, even in challenging cases where Foldseek's recall dropped below 50%. ISS_ProtSci not only identifies closely related proteins but also uncovers previously unrecognized structural relationships, contributing to more accurate protein family classifications. The software can be downloaded from http://ekhidna2.biocenter.helsinki.fi/ISS_ProtSci/.

Fruits undergo a similar ripening process, yet they exhibit a range of differences in color, taste, and shape, both across different species and within the same species. How does this diversity arise? We uncovered a conserved fruit ripening process in lychee fruit in which a NAC transcription factor, LcNAC1, acts as a master regulator. LcNAC1 regulates the expression of two terpene synthase genes, LcTPSa1 and LcTPSa2, which belong to a gene cluster consisting of four TPS genes. LcTPSa1-LcTPSa3 are responsible for catalyzing the production of farnesol, which in turn dictates the aromatic diversity in fruit of different lychee varieties. Through comparative, transcriptomic, and genomic analyses across various lychee varieties, we found these four TPS genes exhibit distinct expression levels due to natural genetic variation. These include copy number variations, presence/absence variations, insertions and deletions, and single nucleotide polymorphisms, many of which affect the binding affinity of LcNAC1. A single nucleotide mutation in LcTPSa1 caused a premature translational termination, resulting in a truncated version of the TPS protein, which surprisingly remains functional. All these genomic changes in the LcNAC1-regulated TPS genes are likely to contribute to the great aromatic diversity observed in lychee fruit. This diversification of fruit aroma in lychee varieties offers a compelling example of how species- or variety-specific traits evolve - the phenotypic diversity is primarily derived from natural genetic variation accumulated in downstream structural genes within an evolutionarily conserved regulatory circuit.

Crop protection is undergoing significant evolution, transitioning towards sustainable approaches that minimize impacts on the environment and human health. Exogenous application of double-stranded RNA (dsRNA) that silences pest or pathogen genes via RNA interference (RNAi) has promise as a safe and effective next-generation crop protection platform without the need for genetic modification. However, exogenous dsRNA application at scale presents challenges. Specifically, a single dsRNA sequence needs to balance targeting the standing variation in a target pest or pathogen group against the potential for adverse impacts in a vast array of non-target and beneficial organisms at the application site and broader environment. To address these competing demands, we present dsRNAmax (https://github.com/sfletc/dsRNAmax), a software package that employs k-mer-based assembly of chimeric dsRNA sequences to target multiple related RNA sequences, to broaden the target spectrum. The package ensures that designed dsRNAs have no defined contiguous sequence homology with any off-target sequences, which can range from single transcriptomes through to metagenome sequence data and beyond. The efficacy of this package is demonstrated by a dsRNAmax-designed dsRNA that inhibits multiple root-knot nematode species but not a non-target nematode species, despite its susceptibility to environmental RNAi and high homology of the target gene.

Since the discovery of spliceosomal introns in eukaryotic genomes, the proximate molecular and evolutionary processes that generate new introns have remained a critical mystery. Specialized transposable elements (TEs), introners, are thought to be one of the major drivers of intron gain in diverse eukaryotes. However, the molecular mechanism(s) and evolutionary processes driving introner propagation within and between lineages remain elusive. Here, we analyze 8,716 genomes, revealing 1,093 introner families in 201 species spanning 1.7 billion years of evolution. Introners are derived from functionally diverse TEs including families of terminal-inverted-repeat DNA TEs, retrotransposons, cryptons, and helitrons as well as mobile elements with unknown molecular mechanisms. We identify eight cases where introners recently transferred between divergent host species and show that giant viruses that integrate into genomes may facilitate introner transfer across lineages. We propose that ongoing intron gain is primarily a consequence of TE activity in eukaryotes, thereby resolving a key mystery of genome structure evolution.

Apicomplexan parasites are single-celled obligate intracellular eukaryotic organisms that cause significant animal and human disease and pose a substantial health and socioeconomic burden worldwide. Eimeria acervulina is one such parasite of chickens, representative of several Eimeria species causing coccidiosis disease. A complete assembly of the E. acervulina genome may help identify markers of drug-resistance and design recombinant vaccines. We sequenced E. acervulina APU1 strain using Oxford Nanopore Sequencing and Illumina technology in combination with a Hi-C (Omni-C) proximity linkage library and achieved a chromosomal scale assembly using the MaSuRCA assembler. The final assembly was 52 Mb. with 15 chromosomes and 99% BUSCO completeness. A total of 7,621 genes were predicted using a pipeline of BRAKER3, GeneMark-ETP and AUGUSTUS, of which 4,647 (60.97%) have a predicted Pfam function and 1,962 (25.74%) have Gene Ontology (GO) terms matching molecular, biological, and functional classes. Stage-specific transcriptome analysis revealed 9,761 transcripts. This genome assembly and transcriptome analysis provides the foundation for identifying biologically important candidates for anticoccidial drug and vaccine development.

Limonene is a chiral, high-demand monoterpene that has wide applications in therapeutics, cosmetics, biofuels, agri-food, biomaterials, and solvent industries. However, its biosynthesis by microbial cell factories is often limited by the poor activity of limonene synthase (LS). Optimization of the rate-limiting enzyme is thus crucial for boosting limonene production. Here, we report the identification of ten LS homologues from sequence data mining and their testing in cells accumulating geranyl pyrophosphate (GPP) or neryl pyrophosphate (NPP) for limonene production. The selectivity of these enzymes toward GPP or NPP was investigated, leading to the identification of a limonene synthase from Agastache rugosa that displays a clear substrate preference for NPP over GPP in vivo. This enzyme was selected as a template for engineering. Using in silico analyses and mutagenesis, several mutants were engineered that revealed differences in substrate specificity. Among them, a combination of mutations (S8K/I265V/E276P/P277R/A281K/N282T/I285Q/I286L) improved limonene production by 4.8- and 1.9-fold with the GPP and NPP pathways, respectively. The mutant predominantly produced (+)-limonene from GPP and a mixture of limonene from NPP, with ∼85-90% of (+)-limonene. This decreased the selectivity for NPP by 2.4-fold. This supports the improved biological production of limonene enantiomers from renewable carbon sources.

Rhynchosporium commune is a fungal pathogen responsible for causing scald disease in barley, leading to significant yield losses and reduced grain quality in susceptible cultivars. Effector proteins secreted by R. commune play crucial roles in manipulating host defenses and facilitating infection. Hence, this study aimed to identify and characterize effector candidates (ECs) in R. commune using a comprehensive bioinformatics approach combined with experimental validation. Initially, a dataset of 12,211 genes from the R. commune strain UK7 genome was analyzed to identify potential ECs, resulting in the selection of 48 candidate proteins. These candidates were further validated using RNA-Seq analysis, which confirmed significant expression of 27 ECs during infection. Our analysis re-identified key effectors, including CZT06923 and CZT13833, with 100% identity to NIP3 and NIP2, respectively, in R. commune. Novel ECs, such as CZT07600, CZT13755, and CZT13375, were identified with lower identity to NIP2, suggesting potential variants. Additionally, structural analysis revealed that CZT07873 EC indicates significant structural similarity to known fungal effector. qRT-PCR validation confirmed the differential expression of CZS93219 and CZT13755, with peak expression at 9 and 12 dpi, respectively. This comprehensive approach enhances our understanding of R. commune's pathogenic mechanisms and provides insights into potential targets for developing disease management strategies in barley cultivation.

Nuclear receptors (NRs) constitute a superfamily of transcription factors that regulate diverse biological processes. In insects, NRs not only govern essential physiological functions including metabolism, development, and reproduction, but also play pivotal roles in regulating caste differentiation and division of labor within social insect colonies. Pseudoregma bambucicola is a species of social aphid in which adults exhibit a specialized reproductive division of labor. This unique system produces first-instar nymphs and soldiers, which share an identical genetic background yet exhibit distinct morphological and behavioral traits. Although NRs exhibit pleiotropic regulatory capacities, their roles in the unique developmental patterns of P. bambucicola remain unclear.

This study identified 21 NR genes based on the genomic data of P. bambucicola and analyzed the duplication and loss events of these genes through phylogenetic analysis. Additionally, differential expression of NR genes was analyzed using transcriptomic data. The TLL exhibited significant differential expression in adults with distinct reproductive behaviors, suggesting its involvement in the regulation of reproductive division of labor. E75 and HNF4 were found to be important for the post-embryonic development of soldiers. Furthermore, quantitative real-time PCR confirmed caste-specific expression patterns of HR4 and HR39, indicating their potential involvement in morphological differentiation and developmental regulation among castes.

This study conducted bioinformatic identification of NR genes in the social aphid P. bambucicola, and investigated their potential roles in morphological differentiation and behavioral division through analysis of differential gene expression. The findings provide preliminary evidence for the functional significance of NR genes in social aphids, while offering novel insights for subsequent research exploration.

Whereas alignment has been fundamental to sequence-based assessments of protein homology, it is ineffective for intrinsically disordered regions (IDRs) due to their lowered sequence conservation and unique sequence properties. Here, we present a web server implementation of SHARK (bio-shark.org), an alignment-free algorithm for homology classification that compares the overall amino acid composition and short regions (k-mers) shared between sequences (SHARK-scores). The output of such k-mer-based comparisons is used by SHARK-dive, a machine learning classifier to detect homology between unalignable, disordered sequences. SHARK-web provides sequence-versus-database assessment of protein sequence homology akin to conventional tools such as BLAST and HMMER. Additionally, we provide precomputed sets of IDR sequences from 16 model organism proteomes facilitating searches against species-specific IDR-omes. SHARK-dive offers superior overall homology detection performance to BLAST and HMMER, driven by a large increase in sensitivity to low sequence identity homologs, and can be used to facilitate the study of sequence-function relationships in disordered, difficult-to-align regions.

Lipoxygenases (LOXs) are key enzymes in plant lipid metabolism and stress responses, yet their genomic organization and functional dynamics in Luffa aegyptiaca-a species of culinary, medicinal, and ornamental importance-remain unexplored. Here, we present the first genome-wide identification and characterization of the LOX gene family in L. aegyptiaca, revealing 29 LOX genes, including 14 members of 13S-lipoxygenases (13-LOX) and 15 members of 9S-lipoxygenases (9-LOX), respectively. Notably, tandem duplication events shaped the expansion of LOX genes, with 24 genes clustered in two loci, suggesting functional diversification to enhance environmental adaptability. Phylogenetic analysis demonstrated evolutionary conservation of LOX genes across Cucurbitaceae species, while collinearity analysis highlighted conserved genomic organization. Promoter cis-element profiling identified stress- and hormone-responsive motifs, implicating LOX genes in developmental and stress regulatory networks. Tissue-specific expression patterns revealed 18 LOX genes predominantly expressed in tendril, fruit, root, and male flower, linking them to organ-specific physiological roles. Crucially, under heat stress, 9 out of 11 expressed LOX genes were significantly downregulated, indicating their potential role in thermal stress adaptation through metabolic reconfiguration. This study provides foundational insights into the LOX family's contribution to L. aegyptiaca's resilience and offers genetic targets for breeding strategies to improve stress tolerance in cucurbit crops.

Caspases are cysteine-dependent aspartate-directed proteases which have critical functions in programmed cell death and inflammation. Their catalytic activity depends on a catalytic dyad of cysteine and histidine within a characteristic protein fold, the so-called caspase domain. Here, we investigated the evolution of caspase-16 (CASP16), an enigmatic member of the caspase family, for which only a partial human gene had been reported previously. The presence of CASP16 orthologs in placental mammals, marsupials and monotremes suggests that caspase-16 originated prior to the divergence of the main phylogenetic clades of mammals. Caspase-16 proteins of various species contain a carboxy-terminal caspase domain and an amino-terminal prodomain predicted to fold into a caspase domain-like structure, which is a unique feature among caspases known so far. Comparative sequence analysis indicates that the prodomain of caspase-16 has evolved by the duplication of exons encoding the caspase domain, whereby the catalytic site was lost in the amino-terminal domain and conserved in the carboxy-terminal domain of caspase-16. The murine and human orthologs of CASP16 contain frameshift mutations and therefore represent pseudogenes (CASP16P). CASP16 of the chimpanzee displays more than 98% nucleotide sequence identity with the human CASP16P gene but, like CASP16 genes of other primates, has an intact protein coding sequence. We conclude that caspase-16 structurally differs from other mammalian caspases, and the pseudogenization of CASP16 distinguishes humans from their phylogenetically closest relatives.

Elsholtzia splendens, a perennial herb native to East Asia, is valued for its ornamental and medicinal uses, particularly in treating inflammatory and febrile conditions. Recent studies have highlighted its antibacterial, anti-inflammatory, antidepressant, antithrombotic, and lipid-lowering properties of its compounds. Additionally, E. splendens shows potential for phytoremediation owing to its ability to hyperaccumulate copper (Cu), lead (Pb), zinc (Zn), and cadmium (Cd). However, its role in remediation conflicts with its medicinal use because of the risk of heavy metal accumulation. Genome sequencing will be key to boosting beneficial compound production and reducing heavy metal risks. In this study, we generated a high-resolution, haplotype-resolved, chromosome-scale genome sequence of E. splendens using PacBio Revio long-read, Illumina short-read, and Hi-C sequencing technologies. The haplotype genome assemblies, spanned 275.4 and 265.0 Mbp with a scaffold N50 of 33.9 and 33.8 Mbp for haplotype 1 and 2, respectively. This assembly provides valuable insights into medicinal compound biosynthesis and supports genetic conservation efforts, facilitating future genetic and biotechnological applications of E. splendens for medicinal and ecological uses.

Syntrophic cocultures (hitherto assumed to be commensalistic) of Clostridium acetobutylicum and Clostridium ljungdahlii, whereby CO2 and H2 produced by the former feed the latter, result in interspecies cell fusion involving large-scale exchange of protein, RNA, and DNA between the two organisms. Although mammalian cell fusion is mechanistically dissected, the mechanism for such microbial-cell fusions is unknown. To start exploring this mechanism, we used RNA sequencing to identify genes differentially expressed in this coculture using two types of comparisons. One type compared coculture to the two monocultures, capturing the combined impact of interactions through soluble signals in the medium and through direct cell-to-cell interactions. The second type compared membrane-separated versus -unseparated cocultures, isolating the impact of interspecies physical contact. While we could not firmly identify specific genes that might drive cell fusion, consistent with our hypothesized model for this interspecies microbial cell fusion, we observed differential regulation of genes involved in C. ljungdahlii's autotrophic Wood-Ljungdahl pathway metabolism and genes of the motility machinery. Unexpectedly, we also identified differential regulation of biosynthetic genes of several amino acids, and notably of arginine and histidine. We verified that they are produced by C. acetobutylicum and are metabolized by C. ljungdahlii to its growth advantage. These and other findings, and notably upregulation of C. acetobutylicum ribosomal-protein genes, paint a more complex syntrophic picture and suggest a mutualistic relationship, whereby beyond CO2 and H2, C. acetobutylicum feeds C. ljungdahlii with growth-boosting amino acids, while benefiting from the H2 utilization by C. ljungdahlii.IMPORTANCEThe construction and study of synthetic microbial cocultures is a growing research area due to the untapped potential of defined multi-species industrial bioprocesses and the utility of defined cocultures for generating insight into complex, undefined, natural microbial consortia. Our previous work showed that coculturing C. acetobutylicum and C. ljungdahlii leads to a unique metabolic phenotype (production of isopropanol) and heterologous cell fusion events. Here, we used RNAseq to explore genes involved in and impacted by these fusions. First, we compared gene expression in coculture to each monoculture. Second, we utilized a transwell system to compare gene expression in mixed cocultures to cocultures with both species physically separated by a permeable membrane, isolating the impact of interspecies "touching" on the transcriptome. This study deepens our mechanistic understanding of the C. acetobutylicum-C. ljungdahlii coculture phenotype, laying the groundwork for reverse genetic studies of heterologous cell fusion in Clostridium cocultures.

Members of the heat shock protein 90 family (HSP90s) are evolutionarily conserved and play crucial roles in protein transport, immune regulation and antigen presentation. In this study, five hsp90s were identified from the genome of large yellow croaker (Larimichthys crocea) and analyzed using bioinformatics. All five identified hsp90s encode proteins with HATPase_c and HSP90 domains, and are mainly localized in the cytoplasm, mitochondria and endoplasmic reticulum. Chromosomal mapping revealed their distribution across three distinct chromosomes. Quantitative real-time PCR (qPCR) analysis showed differential expression patterns of the five hsp90s in 11 tissues. Additionally, their expression dynamics in the liver, spleen, head kidney, gill and blood were analyzed at 3 h, 12 h, 24 h and 48 h post thermal stress, Vibrio parahaemolyticus infection or under a combination of these two stressors. Results showed that the L. crocea hsp90s exhibited distinct expression patterns in response to the above three stimuli in different immune tissues. Notably, hsp90s in the spleen were most responsive. This study systematically clarified for the first time the gene structure characteristics, tissue expression patterns, and environmental stress response mechanisms of the HSP90 family in L. crocea. It confirmed that hsp90s show significant functional differentiation and synergy in response to biotic (pathogen infection) and abiotic (thermal stress) stresses, and provides important clues for a deeper understanding of the genetic basis of environmental adaptation in L. crocea.

Ethylene response factors (ERFs), belonging to the AP2/ERF superfamily, play vital roles in plant growth, development, and stress responses. The evolutionary and expression features of the members of the ERF gene family have not yet been extensively analyzed through comprehensive comparative genomics across various diploid, tetraploid, and hexaploid wheat genomes. In this study, we identified a total of 2,967 ERF genes across one diploid, two tetraploid, and five hexaploid wheat genomes using the characteristics of conserved domains of ERF proteins. Phylogenetic analysis revealed that ERF genes clustered into two main groups. Analyses of expansion of the ERF gene family indicated that the members of IIIc and IX (sub)groups were observed to show the expansion in tetraploid and hexaploid wheat compared to diploid wheat. Tandem duplication was identified as a key mechanism for ERF gene family expansion, with varying proportions across different wheat genomes. Ancient evolutionary evidence was traced using Amborella trichopoda as a reference, revealing the retention of gene copies in both tetraploid and hexaploid wheat. Then, we analyzed the expression of ERF genes under salt stress in Triticum aestivum, identifying 86 consistently up-regulated and 14 down-regulated ERF genes, and reported the stress tolerant and disease resistant ERF genes in hexaploid wheat. These findings provide valuable insights into the evolutionary dynamics and functional features of ERF genes in wheat, paving the way for genetic breeding and molecular improvement of wheat species.

Gypsophila vaccaria Sm., a member of the Caryophyllaceae family, is known for its dry mature seeds, which are widely used in traditional Chinese medicine as "Wang Bu Liu Xing". This study presents a high-quality, chromosome-scale genome assembly of G. vaccaria, integrating Hi-C technology with PacBio and Illumina sequencing data. The final assembled genome measures 1.09 Gb in total length, with a contig N50 of 9.73 Mb and a scaffold N50 of 73.3 Mb, and complete benchmarking universal single-copy orthologs (BUSCO) for the genome and protein modes were 95.9% and 94.9%. Notably, 99.93% of the sequences are anchored to 15 pseudo-chromosomes. A total of 21,795 protein-coding genes were predicted, and repetitive elements were found to constitute 80.43% of the assembled genome. This chromosome-level genome assembly serves as an invaluable resource for future research, including functional genomics and molecular breeding of G. vaccaria.

Proposal for a new fourth PEBP gene group (SFT-like) in a genomic context different from 21 the other three. FT/TFL groups evolved from MFT, but then became sub-, neo-functionalized. The phosphatidylethanolamine-binding protein (PEBP) gene family plays crucial roles in plant development, principally involved in flowering time regulation and seed development. Traditionally, PEBP genes are classified into three clades: MOTHER OF FT AND TFL1 (MFT), FLOWERING LOCUS T (FT), and TERMINAL FLOWER 1 (TFL). We used phylogenomic and microsynteny network analyses to explore the PEBP family across 275 plant genomes from different lineages. The phylogenetic tree of the identified 3707 PEBP proteins allows us to visualize a fourth clade within the PEBP family. This new clade, named SFT (Sibling of FT/TFL), is closely related to the MFT clade but sister to the branch point of FT/TFL subfamilies, suggesting a long-standing evolutionary divergence. In addition, the SFT subfamily is in a different genomic context, whereas FT and TFL share a common origin with MFT. Motif analyzes show differences between this new clade and those already reported, suggesting functions other than flowering or seed development. The Ka/Ks analysis also suggests that SFT clade had fewer duplication events, so these genes could have an important function for the plant that had not yet been elucidated. These findings offer new insights into the evolutionary history and functional diversification of PEBP genes in plants. This study provides an update on the classification of the PEBP family. By understanding the syntenic relationships and evolutionary dynamics within the PEBP family, this research sets the stage for future functional studies on PEBP genes in plant biology, particularly the recently identified SFT clade.

One of the major challenges in precision oncology is the identification of pathogenic, actionable variants and the selection of personalized treatments. We present Onkopus, a variant interpretation framework based on a modular architecture, for interpreting and prioritizing genetic alterations in cancer patients. A multitude of tools and databases are integrated into Onkopus to provide a comprehensive overview about the consequences of a variant, each with its own semantic, including pathogenicity predictions, allele frequency, biochemical and protein features, and therapeutic options. We present the characteristics of variants and personalized therapies in a clear and concise form, supported by interactive plots. To support the interpretation of variants of unknown significance (VUS), we present a protein analysis based on protein structures, which allows variants to be analyzed within the context of the entire protein, thereby serving as a starting point for understanding the underlying causes of variant pathogenicity. Onkopus has the potential to significantly enhance variant interpretation and the selection of actionable variants for identifying new targets, drug screens, drug testing using organoids, or personalized treatments in molecular tumor boards. We provide a free public instance of Onkopus at https://mtb.bioinf.med.uni-goettingen.de/onkopus.

A wide variety of human diseases are associated with loss of microbial diversity in the human gut, inspiring a great interest in the diagnostic or therapeutic potential of the microbiota. However, the ecological forces that drive diversity reduction in disease states remain unclear, rendering it difficult to ascertain the role of the microbiota in disease emergence or severity. One hypothesis to explain this phenomenon is that microbial diversity is diminished as disease states select for microbial populations that are more fit to survive environmental stress caused by inflammation or other host factors. Here, we tested this hypothesis on a large scale, by developing a software framework to quantify the enrichment of microbial metabolisms in complex metagenomes as a function of microbial diversity. We applied this framework to over 400 gut metagenomes from individuals who are healthy or diagnosed with inflammatory bowel disease (IBD). We found that high metabolic independence (HMI) is a distinguishing characteristic of microbial communities associated with individuals diagnosed with IBD. A classifier we trained using the normalized copy numbers of 33 HMI-associated metabolic modules not only distinguished states of health vs IBD, but also tracked the recovery of the gut microbiome following antibiotic treatment, suggesting that HMI is a hallmark of microbial communities in stressed gut environments.

A virulent bacteriophage, P12L, infecting hypermucoviscous Klebsiella pneumoniae of capsule-type K2 was characterized. The phage was found to have podovirus-like morphology, with an icosahedral head and a short tail. It exhibited efficient adsorption with a burst size of 183 PFU/cell. The viral genome is a linear dsDNA molecule that is 42,343 bp in length and contains 62 putative open reading frames (ORFs). It lacks genes associated with drug resistance or virulence factors and encodes two predicted domains associated with depolymerase activity. Because depolymerase can degrade polysaccharide capsules and promote efficient phage-host interactions, phage P12L shows potential as a biocontrol agent.

Anther is a key male reproductive organ that is essential for the plant life cycle, from the sporophyte to the gametophyte generation. To explore isoform-level transcriptional landscape of developing anthers in maize (Zea mays L.), we analyzed Iso-Seq data from anthers collected at 10 developmental stages, together with strand-specific RNA-seq, CAGE-seq, and PAS-seq data. Of the 152,026 high-confidence full-length isoforms identified, 68.8% have not been described; these include 22,365 isoforms that originate from previously unannotated loci and 82,167 novel isoforms that originate from annotated protein-coding genes. Using our newly developed strategy to detect dynamic expression patterns of isoforms, we identified 13,899 differentially variable regions (DVRs); surprisingly, 1,275 genes contain more than two DVRs, revealing highly efficient utilization of limited genic regions. We identified 7,876 long non-coding RNAs (lncRNAs) from 4,098 loci, most of which were preferentially expressed during cell differentiation and meiosis. We also detected 371 long-range interactions involving intergenic lncRNAs (lincRNAs); interestingly, 243 were lincRNA-gene ones, and the interacting genes were highly expressed in anthers, suggesting that many potential lncRNA regulators of key genes are required for anther development. This study provides valuable resources and fundamental information for studying the essential transcripts of key genes during anther development.

Methylesterases (MESs) are a class of enzymes responsible for the demethylation of methylated compounds in plants, play a vital role in plant growth and development. However, studies on MES enzymes in tomato (Solanum lycopersicum) are limited.

This study systematically identified MES genes in tomatoes for the first time and studied their physicochemical properties, evolutionary relationships, and expression patterns. Sixteen Solanum lycopersicum methylesterase (SlMES) genes were identified through comprehensive bioinformatics analysis and were categorized into three subfamilies. Members of the same subfamily exhibited similar gene structures, structural domains, and conserved motifs. Chromosomal analysis revealed an uneven distribution of SlMESs across the five chromosomes, with evidence of gene duplication. Cis-acting element analyses suggested that the SlMES family may have important regulatory functions in tomato growth, development, and stress responses. Among them, Solyc02g065260 was further examined for its role in tomato fruit ripening and stress responses. Its tissue-specific expression patterns, dynamic expression during fruit ripening, and responses to pathogens, low temperatures, and hormones, such as methyl jasmonate (MeJA), methyl salicylate (MeSA), abscisic acid (ABA), and ethylene (ET), were analyzed. The results provided further evidence towards understanding the roles of the SlMES family in the tomatoes.

The results established a theoretical foundation for future investigations into the functional characterization of MES genes during tomato growth and development.

Cooperativity is a central feature to protein folding and is important for protein design. Repeat proteins are good systems for quantifying the thermodynamic basis of cooperativity. Analysis of repeat proteins composed of identical consensus repeats show that repeats strongly drive the folding of their neighbors through extensive tertiary contacts. Here, we use the consensus approach to quantify the cooperativity of folding of spectrin repeat arrays. Spectrin repeats are unique among tandem repeat proteins in that they share an elongated α-helix that spans neighboring repeats. We generate a consensus spectrin repeat sequence and show that this sequence is structured by CD and NMR spectroscopy, and is considerably more stable than extant spectrin repeats. By generating pairs of consensus spectrin repeats, we find tandem repeats to be further stabilized, demonstrating cooperative stabilization by neighboring repeats. Using an Ising model to analyze single- and tandem spectrin repeat unfolding, we find that the consensus stability increase results from intrinsic but not interfacial stabilization. By introducing mutations and insertions at the boundary between consensus repeats, we find that cooperativity is driven primarily by helical propagation; to a lesser extent, helix propagation also stabilizes partly folded states where one of two repeats is unfolded.

Nature constructs matter by employing protein folding motifs, many of which have been synthetically reconstituted to exploit function. A less understood motif whose structure-function relationships remain unexploited is formed by parallel β-strands arranged in a helical repetitive pattern, termed a β-helix. Herein we reconstitute a protein β-helix by design and endow it with biological function. Unlike β-helical proteins, which are contiguous covalent structures, this β-helix self-assembles from an elementary sequence of 18 amino acids. Using a combination of experimental and computational methods, we demonstrate that the resulting assemblies are discrete cylindrical structures exhibiting conserved dimensions at the nanoscale. We provide evidence for the structures to form a carpet-like three-dimensional scaffold promoting and inhibiting the growth of human and bacterial cells, respectively, while being able to mediate intracellular gene delivery. The study introduces a self-assembled β-helix as a self-contained bio- and multi-functional motif for exploring and exploiting mechanistic biology.

Pseudocercospora abelmoschi causes black mould on the leaves of okra. The disease is prevalent post-rainy season when high moisture and warm temperatures prevail. Severe defoliation is observed during favourable environments, leading to a significant loss in productivity. Based on the importance of the pathogen agriculturally, the P. abelmoschi isolate Cer 86 - 18 (MCC:9491) was selected for genome sequencing. The genome assembly of P. abelmoschi resulted in a genome of 31.90 Mb with an overall GC content of 54.26%. Quantitative genome assessment using BUSCO (Benchmarking Universal Single-Copy Orthologs) identified 1,664 (97.53%) complete BUSCOs, reflecting a high representation of conserved genes with minimal duplication and strong orthologous uniqueness. Gene prediction analysis identified 11,325 protein-coding genes, of which 3,857 were annotated using the KEGG database. As per analyses, 410 genes were predicted to encode carbohydrate-active enzymes, whereas 369 genes were predicted to encode peptidases. Eighteen gene clusters involved in secondary metabolite biosynthesis were also identified. A total of 143 proteins were predicted to be effectors using the in-silico pipeline. This is the first report on the genome organisation of P. abelmoschi. This study was designed to address this gap by enhancing our understanding of the genome organisation of P. abelmoschi and gene annotation, thereby paving the way for functional genomics studies, such as identifying virulence genes to aid in resistance breeding. Also, this genome could be another addition to the available genomic resources of the genus Pseudocercospora and can provide valuable insights into host-pathogen interactions and evolutionary relationships.

Acute pancreatitis (AP) is a common acute abdominal condition in clinical practice, associated with high morbidity and mortality rates. Forsythia constitutes a component of traditional Chinese medicinal decoctions used for clinical AP treatment; however, the efficacy of its active monomer in treating AP has yet to be completely substantiated. Here, we engineered an AP cell and mouse model by administering a combination of caerulein and LPS. In vitro experiments utilizing AR42J cells demonstrated that forsythoside B (FST·B) was the most effective monomer in mitigating cellular inflammation. Subsequently, a comprehensive evaluation of FST·B concentrations and efficacy was performed in animal models. Next Mass spectrometry analysis of pancreatic from AP mice treated with 50 mg/kg FST·B was conducted to elucidate its primary regulatory molecular signaling and key targets. FST·B effectively mitigated pathological damage in mice with acute pancreatitis, leading to a reduction in the expression of inflammatory cytokines in both pancreatic tissue and serum. Proteomics and phosphoproteomic profiles revealed that FST·B significantly enhanced the level of oxidative phosphorylation and spliceosome pathway in the AP mice. This research provides initial evidence of the regulatory molecular signals and targets of FST·B in AP, laying a potential foundation for its clinical use in treating AP. SIGNIFICANCE: Acute pancreatitis (AP) is a common acute abdominal condition in clinical practice, associated with high morbidity and mortality rates, and the global incidence of AP has increased by approximately 25 % over the past 15 years. Despite the complexity of AP's causes and the high susceptibility of proteins to degradation during lesions, systems biology studies, such as proteomics, have been limited in investigating the molecular mechanisms involved in its pharmacological treatment. Forsythoside B, a phenylethanol glycoside isolated from the air-dried fruit of forsythia, is a traditional oriental anti-inflammatory drug commonly used in clinical practice. We demonstrated in the AP mouse model that forsythoside B can alleviate pancreatic inflammatory damage in vivo. To elucidate the molecular mechanisms underlying the anti-inflammatory effect of forsythoside B, a comprehensive proteomic and phosphoproteomic analysis was conducted on AP mice models prior to and subsequent to forsythoside B intervention. Finally, 1640 significantly differentially expressed proteins, 1448 significantly differentially expressed phosphoproteins corresponding to 2496 significantly differentially expressed phosphosites were identified. Functional analysis revealed that forsythoside B significantly enhanced the level of oxidative phosphorylation in the AP mice in proteomic profiles, and the spliceosome pathway at the phosphorylation level was significantly affected by forsythoside B. This research provides initial evidence of the regulatory molecular signals and targets of forsythoside B in AP, laying a potential foundation for its clinical use in treating AP.

Antheraea assamensis Helfer (A. assamensis) or Muga silkworm is popularly known for producing golden silk and endemic to the region of Northeast India. The present work characterizes a cypovirus variant infecting A. assamensis larvae, exhibiting characteristic symptoms of flacherie disease. Scanning electron microscope and transmission electron microscope imaging revealed the presence of polyhedral occlusion bodies (OBs) and virion particles measuring 40-50 nm in size. The cypovirus strain comprised of 10 dsRNA genome segments, which were sequenced, assembled and annotated. The encoded viral proteins from different genomic fragments were studied. The phylogenetic analysis of the RNA-dependent RNA polymerase and polyhedrin revealed a close relationship with the previously classified Antheraea mylitta cypovirus 4. The strain was characterized as Antheraea assamensis cypovirus 4 (AaCPV4) with substantial genomic and proteomic evidence that was previously unexplored. The peptide fingerprints of the polyhedrin protein were analysed in the diseased and healthy silkworm lysate by using LC-MS/MS. The polyhedrin protein of AaCPV4 was modelled by different in silico methods and compared with the previously reported cypovirus strains. The multimeric models of polyhedrin were studied and demonstrated the mechanism of formation of OB geometry. Our study provides new insights into the complete genome of AaCPV4 and its viral proteins, which were previously unknown. The present work will help in understanding the differentiation of CPV4 variants infecting Antheraea species and different host adaptations.

Yellow lupin (Lupinus luteus) gives valuable high-quality protein and has good sustainability due to its ability in nitrogen fixation and exudation of organic acids, which reduces the need for chemical-based phosphate fertilization in acid soils. However, the crop needs further improvements to contribute in a major way to sustainable agriculture and food security.In this study, we present the first chromosome-level genome assembly of L. luteus. The results provide insights into its genomic organization, evolution, and functional attributes. Using integrated genomic approaches, we unveil the genetic bases governing its adaptive responses to environmental stress, delineating the intricate interplay among alkaloid biosynthesis, mechanisms of pathogen resistance, and secondary metabolite transporters. Our comparative genomic analysis of closely related species highlights recent speciation events within the Lupinus genus, exposing extensive synteny preservation alongside notable structural alterations, particularly chromosome translocations. Remarkable expansions of gene families implicated in terpene metabolism, stress responses, and conglutin proteins were identified, elucidating the genetic basis of L. luteus' superior nutritional profile and defensive capabilities. Additionally, a diverse array of disease resistance-related (R) genes was uncovered, alongside the characterization of pivotal enzymes governing quinolizidine alkaloid biosynthesis, thus shedding light on the molecular mechanisms underlying "bitterness" in lupin seeds.This comprehensive genomic analysis serves as a valuable resource to improve this species in terms of resilience, yield, and seed protein levels to contribute to food and feed to face the worldwide challenge of sustainable agriculture and food security.

Citrus, a globally vital economic crop, faces severe challenges due to extreme climatic conditions and diseases/pests attack. Poncirus trifoliata is closely related to citrus and shows unique cold tolerance, making it a crucial material for unraveling genes involved in cold tolerance. NAC (NAM, ATAF1/2, CUC2) transcription factors play important roles in plant growth, development, and stress responses. However, their evolution patterns and gene functions in citrus remain poorly studied. This study aims to elucidate the genomic characteristics and evolution of the NAC genes in P. trifoliata, and to analyze their expression patterns and conduct functional validation under cold stress.

Genome-wide analysis identified 135 PtrNAC genes in P. trifoliata with non-random chromosomal distribution, including 20 gene clusters. 57.78% of the NAC genes are located in the chromosomes 3, 4 and 5. Gene duplication analysis revealed that proximal and tandem duplications as primary expansion mechanisms, with tandem repeats specifically driving gene expansion in citrus lineages (subfamilies IV, V, and VII). Collinearity analysis showed that 24.44% of the PtrNAC genes were retained in homologous regions, and Ka/Ks ratio analysis further confirmed that purifying selection dominated their evolutionary process. Transcriptome landscapes revealed that Pt5g024390 (PtrNAC2) was induced to the greatest degree under the cold stress. Meanwhile, expression level of PtrNAC2 in tetraploid was more than two folds higher compared to diploid counterpart in the presence of cold stress. Virus-induced gene silencing of PtrNAC2 led to significantly enhanced cold tolerance, implying that it plays a negative role in regulation of cold tolerance.

This study systematically elucidated the global distribution and evolutionary patterns of NAC genes in P. trifoliata. In addition, the NAC gene exhibit adaptive expansion driven by tandem duplications. The identification of PtrNAC2, a negative regulator of cold tolerance in P. trifoliata, provides valuable insights into unravelling potential candidates for engineering cold tolerance in citrus.

In this study, we present an updated transcriptome assembly for the Iberian ribbed newt, Pleurodeles waltl (P. waltl), a widely used model organism in regeneration research. The existing publicly available transcriptome for this species is limited by the inclusion of only three libraries from the limb and two from the heart, tissues of particular interest for regeneration studies. Additionally, the previous annotation was limited, reducing the utility of the dataset for further in-depth research. To provide a more complete transcriptome with a more comprehensive annotation, we utilized 58 previously published and 9 newly sequenced libraries, expanding the available transcriptomic data for key tissues, especially limb and heart tissues. Our assessment demonstrates that the new assembly offers a more comprehensive representation of reads and proteins compared to previous versions. Furthermore, we significantly improved the functional annotation by using the Trinotate pipeline, which includes the identification of complete ORFs, Pfam motifs, gene names, GO terms, and KEGG Orthology, facilitating more robust transcriptomic analyses. We also examined various stages of limb regeneration and development, gaining insights into the key signaling pathways involved. This work provides a valuable resource for researchers investigating the molecular mechanisms underlying P. waltl's regenerative abilities, enabling more detailed gene expression studies and broader biological insights.

Velvet transcription factors are found throughout the fungal kingdom and share a common velvet domain with a fold similar to that of animal NF-κB. They act as homodimeric or heterodimeric master regulators of fungal secondary metabolism, development, and differentiation. A comparison of velvet domains from 4,999 fungal velvet proteins revealed a conserved overall architecture, including an N-terminal DNA-binding region of approximately 30 amino acids and a C-terminal dimerization region of about 100 amino acids. The dimerization region comprises α- and β-subunits separated by a linker region. A detailed analysis of the velvet domain in Aspergillus nidulans VelB identified glycine (glycine 240) and leucine (leucine 331) residues in the dimerization region as critical for interactions with velvet proteins. These core amino acid residues are conserved and also essential for the function of VeA or VosA, which corroborates their general importance in functional fungal velvet domains. Functional studies of VelB dimerization linkers suggest its tolerance for length shortening. These findings enhance our understanding of the working mechanisms of fungal velvet regulators.IMPORTANCEFungi, as relatives of animals within Opisthokonta, are closely connected to human life through interactions such as food, pathogenicity, and medicines. Similar to animals, fungi have developed NF-κB-like velvet family regulators to respond to various environmental and internal signals. Velvet regulators form homo- or heterodimers in implementing different functional roles. However, the molecular mechanism by which velvet proteins interact remains incompletely understood. In this study, we found a common architecture of fungal velvet domains and resolved the dimerization region using Aspergillus nidulans VelB as a paradigm. The growing understanding of the fungal velvet regulatory network may help to control fungi for pathogenic and industrial purposes and shed light on the general mechanisms shared with the animal NF-κB system in cellular responses to stimuli.