Dissecting the effect of soil on plant phenology and berry transcriptional plasticity in two Italian grapevine varieties (Vitis vinifera L.).

Grapevine embodies a fascinating species as regards phenotypic plasticity and genotype-per-environment interactions. The terroir, namely the set of agri-environmental factors to which a variety is subjected, can influence the phenotype at the physiological, molecular, and biochemical level, representing an important phenomenon connected to the typicality of productions. We investigated the determinants of plasticity by conducting a field-experiment where all terroir variables, except soil, were kept as constant as possible. We isolated the effect of soils collected from different areas, on phenology, physiology, and transcriptional responses of skin and flesh of a red and a white variety of great economic value: Corvina and Glera. Molecular results, together with physio-phenological parameters, suggest a specific effect of soil on grapevine plastic response, highlighting a higher transcriptional plasticity of Glera in respect to Corvina and a marked response of skin compared to flesh. Using a novel statistical approach, we identified clusters of plastic genes subjected to the specific influence of soil. These findings could represent an issue of applicative value, posing the basis for targeted agricultural practices to enhance the desired characteristics for any soil/cultivar combination, to improve vineyards management for a better resource usage and to valorize vineyards uniqueness maximizing the terroir-effect.

Apyrase-mediated amplification of secretory IgA promotes intestinal homeostasis.

Secretory immunoglobulin A (SIgA) interaction with commensal bacteria conditions microbiota composition and function. However, mechanisms regulating reciprocal control of microbiota and SIgA are not defined. Bacteria-derived adenosine triphosphate (ATP) limits T follicular helper (Tfh) cells in the Peyer's patches (PPs) via P2X7 receptor (P2X7R) and thereby SIgA generation. Here we show that hydrolysis of extracellular ATP (eATP) by apyrase results in amplification of the SIgA repertoire. The enhanced breadth of SIgA in mice colonized with apyrase-releasing Escherichia coli influences topographical distribution of bacteria and expression of genes involved in metabolic versus immune functions in the intestinal epithelium. SIgA-mediated conditioning of bacteria and enterocyte function is reflected by differences in nutrient absorption in mice colonized with apyrase-expressing bacteria. Apyrase-induced SIgA improves intestinal homeostasis and attenuates barrier impairment and susceptibility to infection by enteric pathogens in antibiotic-induced dysbiosis. Therefore, amplification of SIgA by apyrase can be leveraged to restore intestinal fitness in dysbiotic conditions.

A COMPASS for VESPUCCI: A FAIR Way to Explore the Grapevine Transcriptomic Landscape.

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/.

Transcriptome Profiles of Strawberry (Fragaria vesca) Fruit Interacting With Botrytis cinerea at Different Ripening Stages.

Gray mold caused by Botrytis cinerea is a major cause of economic losses in strawberry fruit production, limiting fruit shelf life and commercialization. When the fungus infects Fragaria × ananassa strawberry at flowering or unripe fruit stages, symptoms develop after an extended latent phase on ripe fruits before or after harvesting. To elucidate the growth kinetics of B. cinerea on flower/fruit and the molecular responses associated with low susceptibility of unripe fruit stages, woodland strawberry Fragaria vesca flowers and fruits, at unripe white and ripe red stages, were inoculated with B. cinerea. Quantification of fungal genomic DNA within 72 h postinoculation (hpi) showed limited fungal growth on open flower and white fruit, while on red fruit, the growth was exponential starting from 24 hpi and sporulation was observed within 48 hpi. RNA sequencing applied to white and red fruit at 24 hpi showed that a total of 2,141 genes (12.5% of the total expressed genes) were differentially expressed due to B. cinerea infection. A broad transcriptional reprogramming was observed in both unripe and ripe fruits, involving in particular receptor and signaling, secondary metabolites, and defense response pathways. Membrane-localized receptor-like kinases and nucleotide-binding site leucine-rich repeat genes were predominant in the surveillance system of the fruits, most of them being downregulated in white fruits and upregulated in red fruits. In general, unripe fruits exhibited a stronger defense response than red fruits. Genes encoding for pathogenesis-related proteins and flavonoid polyphenols as well as genes involved in cell-wall strengthening were upregulated, while cell-softening genes appeared to be switched off. As a result, B. cinerea remained quiescent in white fruits, while it was able to colonize ripe red fruits.

First step toward gene expression data integration: transcriptomic data acquisition with COMMAND>_.

BACKGROUND: Exploring cellular responses to stimuli using extensive gene expression profiles has become a routine procedure performed on a daily basis. Raw and processed data from these studies are available on public databases but the opportunity to fully exploit such rich datasets is limited due to the large heterogeneity of data formats. In recent years, several approaches have been proposed to effectively integrate gene expression data for analysis and exploration at a broader level. Despite the different goals and approaches towards gene expression data integration, the first step is common to any proposed method: data acquisition. Although it is seemingly straightforward to extract valuable information from a set of downloaded files, things can rapidly get complicated, especially as the number of experiments grows. Transcriptomic datasets are deposited in public databases with little regard to data format and thus retrieving raw data might become a challenging task. While for RNA-seq experiments such problem is partially mitigated by the fact that raw reads are generally available on databases such as the NCBI SRA, for microarray experiments standards are not equally well established, or enforced during submission, and thus a multitude of data formats has emerged. RESULTS: COMMAND>_ is a specialized tool meant to simplify gene expression data acquisition. It is a flexible multi-user web-application that allows users to search and download gene expression experiments, extract only the relevant information from experiment files, re-annotate microarray platforms, and present data in a simple and coherent data model for subsequent analysis. CONCLUSIONS: COMMAND>_ facilitates the creation of local datasets of gene expression data coming from both microarray and RNA-seq experiments and may be a more efficient tool to build integrated gene expression compendia. COMMAND>_ is free and open-source software, including publicly available tutorials and documentation.

Dual Transcriptome and Metabolic Analysis of Vitis vinifera cv. Pinot Noir Berry and Botrytis cinerea During Quiescence and Egressed Infection.

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.

Discovering Causal Relationships in Grapevine Expression Data to Expand Gene Networks. A Case Study: Four Networks Related to Climate Change.

In recent years the scientific community has been heavily engaged in studying the grapevine response to climate change. Final goal is the identification of key genetic traits to be used in grapevine breeding and the setting of agronomic practices to improve climatic resilience. The increasing availability of transcriptomic studies, describing gene expression in many tissues and developmental, or treatment conditions, have allowed the implementation of gene expression compendia, which enclose a huge amount of information. The mining of transcriptomic data represents an effective approach to expand a known local gene network (LGN) by finding new related genes. We recently published a pipeline based on the iterative application of the PC-algorithm, named NES2RA, to expand gene networks in Escherichia coli and Arabidopsis thaliana. Here, we propose the application of this method to the grapevine transcriptomic compendium Vespucci, in order to expand four LGNs related to the grapevine response to climate change. Two networks are related to the secondary metabolic pathways for anthocyanin and stilbenoid synthesis, involved in the response to solar radiation, whereas the other two are signaling networks, related to the hormones abscisic acid and ethylene, possibly involved in the regulation of cell water balance and cuticle transpiration. The expansion networks produced by NES2RA algorithm have been evaluated by comparison with experimental data and biological knowledge on the identified genes showing fairly good consistency of the results. In addition, the algorithm was effective in retaining only the most significant interactions among the genes providing a useful framework for experimental validation. The application of the NES2RA to Vitis vinifera expression data by means of the BOINC-based implementation is available upon request (valter.cavecchia@cnr.it).

The genome sequence and transcriptome of Potentilla micrantha and their comparison to Fragaria vesca (the woodland strawberry).

Background: The genus Potentilla is closely related to that of Fragaria, the economically important strawberry genus. Potentilla micrantha is a species that does not develop berries but shares numerous morphological and ecological characteristics with Fragaria vesca. These similarities make P. micrantha an attractive choice for comparative genomics studies with F. vesca. Findings: In this study, the P. micrantha genome was sequenced and annotated, and RNA-Seq data from the different developmental stages of flowering and fruiting were used to develop a set of gene predictions. A 327 Mbp sequence and annotation of the genome of P. micrantha, spanning 2674 sequence contigs, with an N50 size of 335,712, estimated to cover 80% of the total genome size of the species was developed. The genus Potentilla has a characteristically larger genome size than Fragaria, but the recovered sequence scaffolds were remarkably collinear at the micro-syntenic level with the genome of F. vesca, its closest sequenced relative. A total of 33,602 genes were predicted, and 95.1% of bench-marking universal single-copy orthologous genes were complete within the presented sequence. Thus, we argue that the majority of the gene-rich regions of the genome have been sequenced. Conclusions: Comparisons of RNA-Seq data from the stages of floral and fruit development revealed genes differentially expressed between P. micrantha and F. vesca.The data presented are a valuable resource for future studies of berry development in Fragaria and the Rosaceae and they also shed light on the evolution of genome size and organization in this family.

Abscisic Acid Is a Major Regulator of Grape Berry Ripening Onset: New Insights into ABA Signaling Network.

Grapevine is a world-wide cultivated economically relevant crop. The process of berry ripening is non-climacteric and does not rely on the sole ethylene signal. Abscisic acid (ABA) is recognized as an important hormone of ripening inception and color development in ripening berries. In order to elucidate the effect of this signal at the molecular level, pre-véraison berries were treated ex vivo for 20 h with 0.2 mM ABA and berry skin transcriptional modulation was studied by RNA-seq after the treatment and 24 h later, in the absence of exogenous ABA. This study highlighted that a small amount of ABA triggered its own biosynthesis and had a transcriptome-wide effect (1893 modulated genes) characterized by the amplification of the transcriptional response over time. By comparing this dataset with the many studies on ripening collected within the grapevine transcriptomic compendium Vespucci, an extended overlap between ABA- and ripening modulated gene sets was observed (71% of the genes), underpinning the role of this hormone in the regulation of berry ripening. The signaling network of ABA, encompassing ABA metabolism, transport and signaling cascade, has been analyzed in detail and expanded based on knowledge from other species in order to provide an integrated molecular description of this pathway at berry ripening onset. Expression data analysis was combined with in silico promoter analysis to identify candidate target genes of ABA responsive element binding protein 2 (VvABF2), a key upstream transcription factor of the ABA signaling cascade which is up-regulated at véraison and also by ABA treatments. Two transcription factors, VvMYB143 and VvNAC17, and two genes involved in protein degradation, Armadillo-like and Xerico-like genes, were selected for in vivo validation by VvABF2-mediated promoter trans-activation in tobacco. VvNAC17 and Armadillo-like promoters were induced by ABA via VvABF2, while VvMYB143 responded to ABA in a VvABF2-independent manner. This knowledge of the ABA cascade in berry skin contributes not only to the understanding of berry ripening regulation but might be useful to other areas of viticultural interest, such as bud dormancy regulation and drought stress tolerance.

Dual RNA-Seq of Lysobacter capsici AZ78 - Phytophthora infestans interaction shows the implementation of attack strategies by the bacterium and unsuccessful oomycete defense responses.

Biological interactions in the microbial communities of the rhizosphere continuously shape the gene expression patterns of each individual microorganism. A dual RNA-Seq approach was applied to obtain a comprehensive overview of the molecular mechanisms activated during the interaction between the biocontrol rhizobacterium Lysobacter capsici AZ78 and the soilborne phytopathogenic oomycete Phytophthora infestans. The RNA-Seq transcriptional profile of L. capsici AZ78 was characterized by up-regulation of genes concerned in the biogenesis of type 4 pilus and lytic enzymes, involved, respectively, in host colonization and subsequent attack of the P. infestans cell wall. The activation of detoxification processes allowed L. capsici AZ78 to overcome the attempted defense processes of P. infestans. Moreover, the genes involved in antibiotic biosynthesis were up-regulated in L. capsici AZ78 and caused cell death in P. infestans, with the activation of putative apoptotic processes. The consequences of P. infestans cell death resulted in the down-regulation of primary metabolic pathways, such as carbohydrates, nucleic acids and protein metabolisms. Overall, the mechanism of action of L. capsici AZ78 was related to parasitism and predatory activities that cause the death of P. infestans.

Molecular analysis of the early interaction between the grapevine flower and Botrytis cinerea reveals that prompt activation of specific host pathways leads to fungus quiescence.

Grape quality and yield can be impaired by bunch rot, caused by the necrotrophic fungus Botrytis cinerea. Infection often occurs at flowering, and the pathogen stays quiescent until fruit maturity. Here, we report a molecular analysis of the early interaction between B. cinerea and Vitis vinifera flowers, using a controlled infection system, confocal microscopy and integrated transcriptomic and metabolic analysis of the host and the pathogen. Flowers from fruiting cuttings of the cultivar Pinot Noir were infected with green fluorescent protein (GFP)-labelled B. cinerea and studied at 24 and 96 hours post-inoculation (h.p.i.). We observed that penetration of the epidermis by B. cinerea coincided with increased expression of genes encoding cell-wall-degrading enzymes, phytotoxins and proteases. Grapevine responded with a rapid defence reaction involving 1193 genes associated with the accumulation of antimicrobial proteins, polyphenols, reactive oxygen species and cell wall reinforcement. At 96 h.p.i., the reaction appears largely diminished both in the host and in the pathogen. Our data indicate that the defence responses of the grapevine flower collectively are able to restrict invasive fungal growth into the underlying tissues, thereby forcing the fungus to enter quiescence until the conditions become more favourable to resume pathogenic development.

Insights into the Role of the Berry-Specific Ethylene Responsive Factor VviERF045.

During grape ripening, numerous transcriptional and metabolic changes are required in order to obtain colored, sweet, and flavored berries. There is evidence that ethylene, together with other signals, plays an important role in triggering the onset of ripening. Here, we report the functional characterization of a berry-specific Ethylene Responsive Factor (ERF), VviERF045, which is induced just before véraison and peaks at ripening. Phylogenetic analysis revealed it is close to the SHINE clade of ERFs, factors involved in the regulation of wax biosynthesis and cuticle morphology. Transgenic grapevines lines overexpressing VviERF045 were obtained, in vitro propagated, phenotypically characterized, and analyzed for the content of specific classes of metabolites. The effect of VviERF045 was correlated with the level of transgene expression, with high-expressing lines showing stunted growth, discolored and smaller leaves, and a lower level of chlorophylls and carotenoids. One line with intermediate expression, L15, was characterized at the transcriptomic level and showed 573 differentially expressed genes compared to wild type plants. Microscopy and gene expression analyses point toward a major role of VviERF045 in epidermis patterning by acting on waxes and cuticle. They also indicate that VviERF045 affects phenolic secondary metabolism and induces a reaction resembling a plant immune response with modulation of receptor like-kinases and pathogen related genes. These results suggest also a possible role of this transcription factor in berry ripening, likely related to changes in epidermis and cuticle of the berry, cell expansion, a decrease in photosynthetic capacity, and the activation of several defense related genes as well as from the phenylpropanoid metabolism. All these processes occur in the berry during ripening.

Complete genome sequence of Bacillus amyloliquefaciens subsp. plantarum S499, a rhizobacterium that triggers plant defences and inhibits fungal phytopathogens.

Bacillus amyloliquefaciens subsp. plantarum S499 is a plant beneficial rhizobacterium with a good antagonistic potential against phytopathogens through the release of active secondary metabolites. Moreover, it can induce systemic resistance in plants by producing considerable amounts of surfactins. The complete genome sequence of B. amyloliquefaciens subsp. plantarum S499 includes a circular chromosome of 3,927,922bp and a plasmid of 8,008bp. A remarkable abundance in genomic regions of putative horizontal origin emerged from the analysis. Furthermore, we highlighted the presence of genes involved in the establishment of interactions with the host plants at the root level and in the competition with other soil-borne microorganisms. More specifically, genes related to the synthesis of amylolysin, amylocyclicin, and butirosin were identified. These antimicrobials were not known before to be part of the antibiotic arsenal of the strain. The information embedded in the genome will support the upcoming studies regarding the application of B. amyloliquefaciens isolates as plant-growth promoters and biocontrol agents.

VESPUCCI: Exploring Patterns of Gene Expression in Grapevine.

Large-scale transcriptional studies aim to decipher the dynamic cellular responses to a stimulus, like different environmental conditions. In the era of high-throughput omics biology, the most used technologies for these purposes are microarray and RNA-Seq, whose data are usually required to be deposited in public repositories upon publication. Such repositories have the enormous potential to provide a comprehensive view of how different experimental conditions lead to expression changes, by comparing gene expression across all possible measured conditions. Unfortunately, this task is greatly impaired by differences among experimental platforms that make direct comparisons difficult. In this paper, we present the Vitis Expression Studies Platform Using COLOMBOS Compendia Instances (VESPUCCI), a gene expression compendium for grapevine which was built by adapting an approach originally developed for bacteria, and show how it can be used to investigate complex gene expression patterns. We integrated nearly all publicly available microarray and RNA-Seq expression data: 1608 gene expression samples from 10 different technological platforms. Each sample has been manually annotated using a controlled vocabulary developed ad hoc to ensure both human readability and computational tractability. Expression data in the compendium can be visually explored using several tools provided by the web interface or can be programmatically accessed using the REST interface. VESPUCCI is freely accessible at http://vespucci.colombos.fmach.it.

The Lysobacter capsici AZ78 Genome Has a Gene Pool Enabling it to Interact Successfully with Phytopathogenic Microorganisms and Environmental Factors.

Lysobacter capsici AZ78 has considerable potential for biocontrol of phytopathogenic microorganisms. However, lack of information about genetic cues regarding its biological characteristics may slow down its exploitation as a biofungicide. In order to obtain a comprehensive overview of genetic features, the L. capsici AZ78 genome was sequenced, annotated and compared with the phylogenetically related pathogens Stenotrophomonas malthophilia K729a and Xanthomonas campestris pv. campestris ATCC 33913. Whole genome comparison, supported by functional analysis, indicated that L. capsici AZ78 has a larger number of genes responsible for interaction with phytopathogens and environmental stress than S. malthophilia K729a and X. c. pv. campestris ATCC 33913. Genes involved in the production of antibiotics, lytic enzymes and siderophores were specific for L. capsici AZ78, as well as genes involved in resistance to antibiotics, environmental stressors, fungicides and heavy metals. The L. capsici AZ78 genome did not encompass genes involved in infection of humans and plants included in the S. malthophilia K729a and X. c. pv. campestris ATCC 33913 genomes, respectively. The L. capsici AZ78 genome provides a genetic framework for detailed analysis of other L. capsici members and the development of novel biofungicides based on this bacterial strain.

COLOMBOS v3.0: leveraging gene expression compendia for cross-species analyses.

COLOMBOS is a database that integrates publicly available transcriptomics data for several prokaryotic model organisms. Compared to the previous version it has more than doubled in size, both in terms of species and data available. The manually curated condition annotation has been overhauled as well, giving more complete information about samples' experimental conditions and their differences. Functionality-wise cross-species analyses now enable users to analyse expression data for all species simultaneously, and identify candidate genes with evolutionary conserved expression behaviour. All the expression-based query tools have undergone a substantial improvement, overcoming the limit of enforced co-expression data retrieval and instead enabling the return of more complex patterns of expression behaviour. COLOMBOS is freely available through a web application at http://colombos.net/. The complete database is also accessible via REST API or downloadable as tab-delimited text files.

Whole-genome comparative analysis of virulence genes unveils similarities and differences between endophytes and other symbiotic bacteria.

Plant pathogens and endophytes co-exist and often interact with the host plant and within its microbial community. The outcome of these interactions may lead to healthy plants through beneficial interactions, or to disease through the inducible production of molecules known as virulence factors. Unravelling the role of virulence in endophytes may crucially improve our understanding of host-associated microbial communities and their correlation with host health. Virulence is the outcome of a complex network of interactions, and drawing the line between pathogens and endophytes has proven to be conflictive, as strain-level differences in niche overlapping, ecological interactions, state of the host's immune system and environmental factors are seldom taken into account. Defining genomic differences between endophytes and plant pathogens is decisive for understanding the boundaries between these two groups. Here we describe the major differences at the genomic level between seven grapevine endophytic test bacteria, and 12 reference strains. We describe the virulence factors detected in the genomes of the test group, as compared to endophytic and non-endophytic references, to better understand the distribution of these traits in endophytic genomes. To do this, we adopted a comparative whole-genome approach, encompassing BLAST-based searches through the GUI-based tools Mauve and BRIG as well as calculating the core and accessory genomes of three genera of enterobacteria. We outline divergences in metabolic pathways of these endophytes and reference strains, with the aid of the online platform RAST. We present a summary of the major differences that help in the drawing of the boundaries between harmless and harmful bacteria, in the spirit of contributing to a microbiological definition of endophyte.

Comparative analysis of gene expression: uncovering expression conservation and divergence between Salmonella enterica serovar Typhimurium strains LT2 and 14028S.

Different strains of the same organism can share a large amount of their genetic material, the so called core pangenome. Nevertheless, these species can display different lifestyles and it is still not well known to what extent the core pangenome plays a role in the divergence of lifestyles between the two organisms. Here, we present a procedure for uncovering the conservation and divergence of gene expression by using large expression compendia. We will use data from two Salmonella enterica serovar Typhimurium strains as an example here, strain LT2 and strain 14028S, to assess if there are orthologous gene pairs with different expression domains related in both strains.

Draft Genome Sequence of Lysobacter capsici AZ78, a Bacterium Antagonistic to Plant-Pathogenic Oomycetes.

Lysobacter capsici AZ78, isolated from tobacco rhizosphere, effectively controls Phytophthora infestans and Plasmopara viticola on tomato and grapevine plants, respectively. We report the first draft genome sequence of the L. capsici species.

COLOMBOS v2.0: an ever expanding collection of bacterial expression compendia.

The COLOMBOS database (http://www.colombos.net) features comprehensive organism-specific cross-platform gene expression compendia of several bacterial model organisms and is supported by a fully interactive web portal and an extensive web API. COLOMBOS was originally published in PLoS One, and COLOMBOS v2.0 includes both an update of the expression data, by expanding the previously available compendia and by adding compendia for several new species, and an update of the surrounding functionality, with improved search and visualization options and novel tools for programmatic access to the database. The scope of the database has also been extended to incorporate RNA-seq data in our compendia by a dedicated analysis pipeline. We demonstrate the validity and robustness of this approach by comparing the same RNA samples measured in parallel using both microarrays and RNA-seq. As far as we know, COLOMBOS currently hosts the largest homogenized gene expression compendia available for seven bacterial model organisms.