Motivation Genome annotation files (GFF3/GTF) are the standard for storing genomic feature data, yet their flexibility often results in formatting inconsistencies that create bottlenecks for downstream bioinformatics analyses. A robust, unified framework is required to parse, standardise, and validate these files to ensure interoperability and facilitate complex comparative genomic tasks.Results We present AEGIS (Annotation Extraction and Genomic Integration Suite), a comprehensive toolkit designed to parse, correct, and standardise genome annotations. Beyond quality control, AEGIS provides advanced modules for flexible feature extraction (e.g., coding sequences, promoters) and comparative genomic analysis. Uniquely, it integrates multiple lines of evidence, including sequence homology, synteny, and coordinate-based lift-overs, to assess gene model correspondence and infer orthology. We demonstrate the utility of AEGIS by quantifying complex structural changes between Arabidopsis annotation versions and identifying high-confidence orthologues across diverse plant genomes.Availability AEGIS is implemented in Python. Source code and documentation are freely available under the GPL-3 license at https://github.com/Tomsbiolab/aegis and as a Docker container at https://hub.docker.com/r/tomsbiolab/aegis. The package is also available on PyPI (pip install aegis-bio).

Terpenes contribute to the characteristic flavor and aroma of grapes and their derived products while serving protective roles in plants against radiation, oxidative stress, and biotic challenges. The phytohormone methyl jasmonate (MeJA) mediates many of these processes and enhances terpene content in grape berries, but its underlying regulatory mechanism remains unclear. To address this, we treated Vitis vinifera cv. Gamay Fréaux berry cell suspensions with MeJA (100 µM) and cyclodextrins (50 mM), generating transcriptomic data. Upregulated genes (URGs) were enriched in jasmonic acid biosynthesis and signaling pathways, and transcription factors (TF) that represent novel candidates modulating MeJA responses. Inspection of these TFs in terms of their co-expressed genes allowed us to focus on MYC2 , the sole bHLH IIIe subgroup member in grapevine. Using DAP-seq, we identified MYC2-bound genes and integrated them with MeJA-URGs and MYC2 co-expressed genes (CEGs) to produce a high-confidence target list. These targets were bound via a conserved G-box motif and included jasmonate-related genes (e.g., LOX and JAZ ) and TFs such as MYB24, previously found to interact with MYC2 to activate terpenoid biosynthesis genes. Consistently, MeJA treatment induced 15 terpene synthase genes ( TPS ), eleven of which were bound by MYB24, MYC2, or both. Terpenoid compounds associated with these induced TPSs accumulated both intra- and extracellularly following treatment with MeJA and cyclodextrins, but not with abscisic acid (ABA). Our findings suggest that MYC2 regulates the jasmonate pathway and cooperates with MYB24 to mediate MeJA-induced terpene biosynthesis, shedding light on mechanisms that may extend to flower and fruit development, where this MYB-bHLH complex is also expressed. ### Competing Interest Statement The authors have declared no competing interest.

The cell wall (CW) is the dynamic structure of a plant cell, acting as a barrier against biotic and abiotic stresses. In grape berries, the modifications of pulp and skin CW during softening ensure flexibility during cell expansion and determine the final berry texture. In addition, the CW of grape berry skin is of fundamental importance for winemaking, controlling secondary metabolite extractability. Grapevine varieties with contrasting CW characteristics generally respond differently to biotic and abiotic stresses. In the context of climate change, it is important to investigate the CW dynamics occurring upon different stresses, to define new adaptation strategies. This review summarizes the molecular mechanisms underlying CW modifications during grapevine berry fruit ripening, plant-pathogen interaction, or in response to environmental stresses, also considering the most recently published transcriptomic data. Furthermore, perspectives of new biotechnological approaches aiming at modifying the CW properties based on other crops’ examples are also presented. we provide a comprehensive overview of the mechanisms behind cell wall modeling in grapevine during berry development and environmental stresses, with a focus on biotech applications in fleshy fruit trees.

Terpenes are responsible for flavors and aromas of grapes, however, they also protect from radiation, participate in biotic stress and antioxidant mechanisms. The phytohormone methyl jasmonate (MeJA) mediates many of these stress responses and has been associated with increased terpene content in berries. Here, we generated transcriptomic data of Vitis vinifera cv. 'Gamay' cells treated with MeJA (100 μM) and cyclodextrins (50 μM) to understand these responses. Ontology analysis revealed that up-regulated genes (URGs) were enriched in jasmonic acid biosynthesis and signaling terms, as expected. Inspection of transcription factors (TFs) among URGs allowed us to study uncharacterized TFs. MapMan enrichment analysis on their TOP420 co-expressed genes (CEGs) allowed us to delimit some TFs highly enriched in jasmonate-related terms. This was the case of VviMYC2, the only grape member of the bHLH IIIe subgroup, and the best candidate for studying the regulation of jasmonate signaling. We confirmed the binding potential of MYC2 by DAP-seq, and combining it to the list of MeJA-URGs and MYC2-CEGs, we generated a list of high-confidence targets that included jasmonate-related genes and TFs such as MYB24, previously found to interact with MYC2 and required for the activation of terpenoid genes. In concordance, our MeJA data showed 13 significantly induced TPS genes, 9 of which are bound by MYB24, MYC2 or both. A few terpenoid compounds associated with the induced TPSs were significantly accumulated by MeJA. Our data suggests MYC2 regulates the jasmonate pathway and mediates terpene production cooperating with MYB24 in response to MeJA.

Addressing the opportunities and challenges of genomics methods in grapevine (Vitis vinifera L.) requires the development of a comprehensive and accurate reference genome and annotation. We aimed to create a new gene annotation for the PN40024 grapevine reference genome by integrating the highly accurate and complete T2T assembly and the manually curated PN40024.v4 annotation. Here, we present a novel workflow to enhance the annotation of the T2T genome by incorporating past community input found in PN40024.v4. The pipeline's containerization will improve the workflow's reproducibility and flexibility, facilitating its inclusion as a shared workflow on the Grapedia portal, the grapevine genomics encyclopedia. The pipeline includes both structural and functional annotation of genes, including lncRNAs and miRNAs. Moreover, we provide conversions between different annotation versions, allowing the comparison and integration of various grapevine datasets. To ensure transparency and traceability, we have modified the structure of the gene IDs, retaining the Vitvi prefix but also referencing the genome version and annotation. This will allow us to handle any gene model issues between different annotation versions and to easily distinguish the version from the annotation in publications. The annotation workflow will soon be available on the Grapedia portal (https://grapedia.org/), where it can potentially be applied to other cultivars. This annotation version will also serve as the basis for the new grape gene reference catalogue, which will provide a comprehensive and updated resource for grapevine genomics.

Overarching surveys of QTL (Quantitative Trait Loci) studies in both model plants and staple crops have facilitated the access to information and boosted the impact of existing data on plant improvement activities. Today, the grapevine community is ready to take up the challenge of making the wealth of QTL information F.A.I.R.. To ensure that all valuable published data can be used more effectively, the myriad of identified QTLs have to be captured, standardised and stored in a dedicated public database. As an outcome of the GRAPEDIA initiative, QTL-dedicated experts from around the world have gathered to compile the grapevine QTLome: the complete information (e.g., map positions, associated phenotypes) describing all experimentally supported QTLs for a specific trait. This has led to the collection of more than 150 published QTL papers and to the FAIRification of the fields relevant to the grapevine QTL database. A grapevine-QTL frontend application for uploading data has been developed to support QTL curators.For each specific trait, the QTLome will be anchored firstly to the grapevine reference PN40024.T2T(v5) genome/annotation and secondly to the published diverse genome assemblies. The generated "Grapevine QTL browser" will (i) enhance the understanding of the genetic architecture of diverse phenotypes, (ii) reveal consistent QTLs across studies (consensus genomic intervals), which are particularly valuable for marker-assisted breeding, (iii) assist the identification of candidate genes (relevant alleles) and their integration into biological/biotechnological applications. The potential of this resource will be demonstrated by a case study.

Drug resistance observed with many anti-infectives clearly highlights the need for new broad-spectrum agents to treat especially neglected tropical diseases (NTDs) caused by eukaryotic parasitic pathogens, including fungal infections. Herein, we show that the simple modification of one of the most well-known antifungal drugs, fluconazole, with organometallic moieties not only improves the activity of the parent drug but also broadens the scope of application of the new derivatives. These compounds were highly effective in vivo against pathogenic fungal infections and potent against parasitic worms such as Brugia, which causes lymphatic filariasis and Trichuris, one of the soil-transmitted helminths that infects millions of people globally. Notably, the identified molecular targets indicate a mechanism of action that differs greatly from that of the parental antifungal drug, including targets involved in biosynthetic pathways that are absent in humans, offering great potential to expand our armamentarium against drug-resistant fungal infections and neglected tropical diseases (NTDs) targeted for elimination by 2030.

The taxonomic identification of organisms based on the amplification of specific genetic markers (metabarcoding) implicitly requires adequate discriminatory information and taxonomic coverage of environmental DNA sequences in taxonomic databases. These requirements were quantitatively examined by comparing the determination of cyanobacteria and microalgae obtained by metabarcoding and light microscopy. We used planktic and biofilm samples collected in 37 lakes and 22 rivers across the Alpine region. We focused on two of the most used and best represented genetic markers in the reference databases, namely the 16S rRNA and 18S rRNA genes. A sequence gap analysis using blastn showed that, in the identity range of 99-100%, approximately 30% (plankton) and 60% (biofilm) of the sequences did not find any close counterpart in the reference databases (NCBI GenBank). Similarly, a taxonomic gap analysis showed that approximately 50% of the cyanobacterial and eukaryotic microalgal species identified by light microscopy were not represented in the reference databases. In both cases, the magnitude of the gaps differed between the major taxonomic groups. Even considering the species determined under the microscope and represented in the reference databases, 22% and 26% were still not included in the results obtained by the blastn at percentage levels of identity ≥95% and ≥97%, respectively. The main causes were the absence of matching sequences due to amplification and/or sequencing failure and potential misidentification in the microscopy step. Our results quantitatively demonstrated that in metabarcoding the main obstacles in the classification of 16S rRNA and 18S rRNA sequences and interpretation of high-throughput sequencing biomonitoring data were due to the existence of important gaps in the taxonomic completeness of the reference databases and the short length of reads. The study focused on the Alpine region, but the extent of the gaps could be much greater in other less investigated geographic areas.

Successfully integrating transcriptomic experiments is a challenging task with the ultimate goal of analyzing gene expression data in the broader context of all available measurements, all from a single point of access. In its second major release VESPUCCI, the integrated database of gene expression data for grapevine, has been updated to be FAIR-compliant, employing standards and created with open-source technologies. It includes all public grapevine gene expression experiments from both microarray and RNA-seq platforms. Transcriptomic data can be accessed in multiple ways through the newly developed COMPASS GraphQL interface, while the expression values are normalized using different methodologies to flexibly satisfy different analysis requirements. Sample annotations are manually curated and use standard formats and ontologies. The updated version of VESPUCCI provides easy querying and analyzing of integrated grapevine gene expression (meta)data and can be seamlessly embedded in any analysis workflow or tools. VESPUCCI is freely accessible and offers several ways of interaction, depending on the specific goals and purposes and/or user expertise; an overview can be found at https://vespucci.readthedocs.io/.

Grapevine (Vitis vinifera) is a valuable crop in Europe for both economical and cultural reasons, but highly susceptible to Downy mildew (DM). The generation of resistant vines is of critical importance for a sustainable viticulture and can be achieved either by introgression of resistance genes in susceptible varieties or by mutation of Susceptibility (S) genes, e.g., by gene editing. This second approach offers several advantages: it maintains the genetic identity of cultivars otherwise disrupted by crossing and generally results in a broad-spectrum and durable resistance, but it is hindered by the poor knowledge about S genes in grapevines. Candidate S genes are Downy mildew Resistance 6 (DMR6) and DMR6-Like Oxygenases (DLOs), whose mutations confer resistance to DM in Arabidopsis. In this work, we show that grapevine VviDMR6-1 complements the Arabidopsis dmr6-1 resistant mutant. We studied the expression of grapevine VviDMR6 and VviDLO genes in different organs and in response to the DM causative agent Plasmopara viticola. Through an automated evaluation of causal relationships among genes, we show that VviDMR6-1, VviDMR6-2, and VviDLO1 group into different co-regulatory networks, suggesting distinct functions, and that mostly VviDMR6-1 is connected with pathogenesis-responsive genes. Therefore, VviDMR6-1 represents a good candidate to produce resistant cultivars with a gene-editing approach.

Fruit firmness and in particular the individual components of texture and moisture loss, are considered the key quality traits when describing blueberry fruit quality, and whilst these traits are genetically regulated, the mechanisms governing their control are not clearly understood. In this investigation, RNAseq was performed on fruits of two blueberry cultivars with very different storage properties, ‘Bluecrop’ and ‘Legacy’, at harvest, three weeks storage in a non-modified environment at 4 °C and after three weeks storage at 4 °C followed by three days at 21 °C, with the aim of understanding the transcriptional changes that occur during storage in cultivars with very different post-harvest fruit quality. De novo assemblies of the transcriptomes of the two cultivars were performed separately and a total of 39,335 and 41,896 unigenes for ‘Bluecrop’ and ‘Legacy’ respectively were resolved. Differential gene expression analyses were grouped into four cluster profiles based on changes in transcript abundance between harvest and 24 days post-harvest. A total of 290 unigenes were up-regulated in ‘Legacy’ only, 685 were up-regulated in ‘Bluecrop’, 252 were up-regulated in both cultivars and 948 were down-regulated in both cultivars between harvest and 24 days post-harvest. Unigenes showing significant differential expression between harvest and following post-harvest cold-storage were grouped into classes of biological processes including stress responses, cell wall metabolism, wax metabolism, calcium metabolism, cellular components, and biological processes. In total 21 differentially expressed unigenes with a putative role in regulating the response to post-harvest cold-storage in the two cultivars were identified from the de novo transcriptome assemblies performed. The results presented provide a stable foundation from which to perform further analyses with which to functionally validate the candidate genes identified, and to begin to understand the genetic mechanisms controlling changes in firmness in blueberry fruits post-harvest.

The ripening of climacteric fruits, such as apple, is represented by a series of genetically programmed events orchestrated by the action of several hormones. In this study, we investigated the existence of a hormonal crosstalk between ethylene and auxin during the post-harvest ripening of three internationally known apple cultivars: 'Golden Delicious', 'Granny Smith' and 'Fuji'. The normal climacteric ripening was impaired by the exogenous application of 1-methylcyclopropene (1-MCP) that affected the production of ethylene and the physiological behaviour of specific ethylene-related quality traits, such as fruit texture and the production of volatile organic compounds. The application of 1-MCP induced, moreover, a de-novo accumulation of auxin. The RNA-Seq wide-transcriptome analysis evidenced as the competition at the level of the ethylene receptors induced a cultivar-dependent transcriptional re-programming. The DEGs annotation carried out through the KEGG database identified as most genes were assigned to the plant hormone signaling transduction category, and specifically related to auxin and ethylene. The interplay between these two hormones was further assessed through a candidate gene analysis that highlighted a specific activation of GH3 and ILL genes, encoding key steps in the process of the auxin homeostasis mechanism. Our results showed that a compromised ethylene metabolism at the onset of the climacteric ripening in apple can stimulate, in a cultivar-dependent fashion, an initial de-novo synthesis and de-conjugation of auxin as a tentative to restore a normal ripening progression.

Lemon (Citrus limon (L.) Burm. f.) is an evergreen tree belonging to the genus Citrus. The fruits are particularly prized for the organoleptic and nutraceutical properties of the juice and for the quality of the essential oils in the peel. Herein, we report, for the first time, the release of a high-quality reference genome of the two haplotypes of lemon. The sequencing has been carried out coupling Illumina short reads and Oxford Nanopore data leading to the definition of a primary and an alternative assembly characterized by a genome size of 312.8 Mb and 324.74 Mb respectively, which agree well with an estimated genome size of 312 Mb. The analysis of the transposable element (TE) allowed the identification of 2878 regions on the primary and 2897 on the alternative assembly distributed across the nine chromosomes. Furthermore, an in silico analysis of the microRNA genes was carried out using 246 mature miRNA and the respective pre-miRNA hairpin sequences of Citrus sinensis. Such analysis highlighted a high conservation between the two species with 233 mature miRNAs and 51 pre-miRNA stem-loops aligning with perfect match on the lemon genome. In parallel, total RNA was extracted from fruit, flower, leaf, and root enabling the detection of 35,020 and 34,577 predicted transcripts on primary and alternative assemblies respectively. To further characterize the annotated transcripts based on their function, a gene ontology and a gene orthology analysis with other Citrus and Citrus-related species were carried out. The availability of a reference genome is an important prerequisite both for the setup of high-throughput genotyping analysis and for functional genomic approaches toward the characterization of the genetic determinism of traits of agronomic interest.

Fungal infections represent a global problem and there is an urgent need for new drugs. In this work, we prepared four novel organometallic derivatives of the frontline antifungal drug fluconazole with very promising in vivo activity.

Tagatose is a rare sugar with no negative impacts on human health and selective inhibitory effects on plant-associated microorganisms. Tagatose inhibited mycelial growth and negatively affected mitochondrial processes in Phytophthora infestans, but not in Phytophthora cinnamomi. The aim of this study was to elucidate metabolic changes and transcriptional reprogramming activated by P. infestans and P. cinnamomi in response to tagatose, in order to clarify the differential inhibitory mechanisms of tagatose and the species-specific reactions to this rare sugar. Phytophthora infestans and P. cinnamomi activated distinct metabolic and transcriptional changes in response to the rare sugar. Tagatose negatively affected mycelial growth, sugar content and amino acid content in P. infestans with a severe transcriptional reprogramming that included the downregulation of genes involved in transport, sugar metabolism, signal transduction, and growth-related process. Conversely, tagatose incubation upregulated genes related to transport, energy metabolism, sugar metabolism and oxidative stress in P. cinnamomi with no negative effects on mycelial growth, sugar content and amino acid content. Differential inhibitory effects of tagatose on Phytophthora spp. were associated with an attempted reaction of P. infestans, which was not sufficient to attenuate the negative impacts of the rare sugar and with an efficient response of P. cinnamomi with the reprogramming of multiple metabolic processes, such as genes related to glucose transport, pentose metabolism, tricarboxylic acid cycle, reactive oxygen species detoxification, mitochondrial and alternative respiration processes. Knowledge on the differential response of Phytophthora spp. to tagatose represent a step forward in the understanding functional roles of rare sugars.

Fungal infections are an alarming global problem, most importantly for immunocompromised patients in a hospital environment. The appearance of multidrug resistance in several fungal species is a strong indication that alternative treatments are required. Azoles represent the mainstay of antifungal drugs, and their mode of action involves the binding mode of these molecules to the fungal lanosterol 14α-demethylase target enzyme. In this work, by rational design, we have prepared and characterized four novel organometallic derivatives of the frontline antifungal drug fluconazole (1a-4a). All compounds showed excellent in vitro activity against the yeast C. robusta, clearly surpassing the progenitor organic drug fluconazole. As anticipated, due to the presence of the ferrocenyl moiety in 1a-4a, a modest increase in ROS generation was observed on C. robusta upon treatment. Very importantly, enzyme inhibition and chemogenetic profiling demonstrated that lanosterol 14α-demethylase was the main target of the most active compound of the series, (N-(ferrocenylmethyl)-2-(2,4-difluorophenyl)-2-hydroxy-N-methyl-3-(1H-1,2,4-triazol-1-yl)propan-1-aminium chloride, 2a). Transmission electron microscopy (TEM) studies suggested that 2a induced a loss in wall integrity as well as intracellular features ascribable to late apoptosis or necrosis. The impressive activity of 2a was further confirmed on clinical isolates, where antimycotic potency up to 400 times higher than fluconazole was observed. Also, 2a showed activity towards azole-resistant strains. This finding is very interesting since the target of 2a is primarily the same as that of fluconazole, emphasizing the role played by the organometallic moiety. In vivo experiments conducted with 2a at a dose of 10 mg/Kg in mice model of Candida infections, while not decreasing fungal burden in the kidney, reduced distal distribution to liver and brain and greatly improved the inflammatory pathology in the kidney and colon, compared to untreated mice.

Botrytis cinerea is an important necrotroph in vineyards. Primary infections are mostly initiated by airborne conidia from overwintered sources around bloom, then the fungus remains quiescent from bloom till maturity and egresses at ripeness. We previously described in detail the process of flower infection and quiescence initiation. Here, we complete the characterization studying the cross-talk between the plant and the fungus during pathogen quiescence and egression by an integrated transcriptomic and metabolic analysis of the host and the pathogen. Flowers from fruiting cuttings of the cv. Pinot Noir were inoculated with a GFP-labeled strain of B. cinerea at full cap-off stage, and molecular analyses were carried out at 4 weeks post inoculation (wpi, fungal quiescent state) and at 12 wpi (fungal pre-egression and egression states). The expressed fungal transcriptome highlighted that the fungus remodels its cell wall to evade plant chitinases besides undergoing basal metabolic activities. Berries responded by differentially regulating genes encoding for different PR proteins and genes involved in monolignol, flavonoid, and stilbenoid biosynthesis pathways. At 12 wpi, the transcriptome of B. cinerea in the pre-egressed samples showed that virulence-related genes were expressed, suggesting infection process was initiated. The egressed B. cinerea expressed almost all virulence and growth related genes that enabled the pathogen to colonize the berries. In response to egression, ripe berries reprogrammed different defense responses, though futile. Examples are activation of membrane localized kinases, stilbene synthases, and other PR proteins related to SA and JA-mediated responses. Our results indicated that hard-green berries defense program was capable to hamper B. cinerea growth. However, ripening associated fruit cell wall self-disassembly together with high humidity created the opportunity for the fungus to egress and cause bunch rot.