High-resolution methylome analysis uncovers stress-responsive genomic hotspots and drought-sensitive transposable element superfamilies in the clonal Lombardy poplar.

DNA methylation is environment-sensitive and can mediate stress responses. In trees, changes in the environment might cumulatively shape the methylome landscape over time. However, because high-resolution methylome studies usually focus on single environmental cues, the stress-specificity and long-term stability of methylation responses remain unclear. Here, we studied the methylome plasticity of a Populus nigra cv. 'Italica' clone widely distributed across Europe. Adult trees from different geographic locations were clonally propagated in a common garden experiment and exposed to cold, heat, drought, herbivory, rust infection, and salicylic acid treatments. Whole-genome bisulfite sequencing revealed stress-induced and naturally occurring DNA methylation variants. In CG/CHG contexts, the same genomic regions were often affected by multiple stresses, suggesting a generic methylome response. Moreover, these variants showed striking overlap with naturally occurring methylation variants between trees from different locations. Drought treatment triggered CHH hypermethylation of transposable elements, affecting entire superfamilies near drought-responsive genes. Thus, we revealed genomic hotspots of methylation change that are not stress-specific and that contribute to natural DNA methylation variation, and identified stress-specific hypermethylation of entire transposon superfamilies with possible functional consequences. Our results underscore the importance of studying multiple stressors in a single experiment for recognizing general versus stress-specific methylome responses.

Stress responses and epigenomic instability mark the loss of somatic embryogenesis competence in grapevine.

Somatic embryogenesis (SE) represents the most appropriate tool for next-generation breeding methods in woody plants such as grapevine (Vitis vinifera L.). However, in this species, the SE competence is strongly genotype-dependent and the molecular basis of this phenomenon is poorly understood. We explored the genetic and epigenetic basis of SE in grapevine by profiling the transcriptome, epigenome, and small RNAome of undifferentiated, embryogenic, and non-embryogenic callus tissues derived from two genotypes differing in competence for SE, Sangiovese and Cabernet Sauvignon. During the successful formation of embryonic callus, we observed the upregulation of epigenetic-related transcripts and short interfering RNAs in association with DNA hypermethylation at transposable elements in both varieties. Nevertheless, the switch to nonembryonic development matched the incomplete reinforcement of transposon silencing, and the evidence of such effect was more apparent in the recalcitrant Cabernet Sauvignon. Transcriptomic differences between the two genotypes were maximized already at early stage of culture where the recalcitrant variety expressed a broad panel of genes related to stress responses and secondary metabolism. Our data provide a different angle on the SE molecular dynamics that can be exploited to leverage SE as a biotechnological tool for fruit crop breeding.

Open chromatin in grapevine marks candidate CREs and with other chromatin features correlates with gene expression.

Vitis vinifera is an economically important crop and a useful model in which to study chromatin dynamics. In contrast to the small and relatively simple genome of Arabidopsis thaliana, grapevine contains a complex genome of 487 Mb that exhibits extensive colonization by transposable elements. We used Hi-C, ChIP-seq and ATAC-seq to measure how chromatin features correlate to the expression of 31 845 grapevine genes. ATAC-seq revealed the presence of more than 16 000 open chromatin regions, of which we characterize nearly 5000 as possible distal enhancer candidates that occur in intergenic space > 2 kb from the nearest transcription start site (TSS). A motif search identified more than 480 transcription factor (TF) binding sites in these regions, with those for TCP family proteins in greatest abundance. These open chromatin regions are typically within 15 kb from their nearest promoter, and a gene ontology analysis indicated that their nearest genes are significantly enriched for TF activity. The presence of a candidate cis-regulatory element (cCRE) > 2 kb upstream of the TSS, location in the active nuclear compartment as determined by Hi-C, and the enrichment of H3K4me3, H3K4me1 and H3K27ac at the gene are correlated with gene expression. Taken together, these results suggest that regions of intergenic open chromatin identified by ATAC-seq can be considered potential candidates for cis-regulatory regions in V. vinifera. Our findings enhance the characterization of a valuable agricultural crop, and help to clarify the understanding of unique plant biology.

The genome of the seagrass Zostera marina reveals angiosperm adaptation to the sea.

Seagrasses colonized the sea on at least three independent occasions to form the basis of one of the most productive and widespread coastal ecosystems on the planet. Here we report the genome of Zostera marina (L.), the first, to our knowledge, marine angiosperm to be fully sequenced. This reveals unique insights into the genomic losses and gains involved in achieving the structural and physiological adaptations required for its marine lifestyle, arguably the most severe habitat shift ever accomplished by flowering plants. Key angiosperm innovations that were lost include the entire repertoire of stomatal genes, genes involved in the synthesis of terpenoids and ethylene signalling, and genes for ultraviolet protection and phytochromes for far-red sensing. Seagrasses have also regained functions enabling them to adjust to full salinity. Their cell walls contain all of the polysaccharides typical of land plants, but also contain polyanionic, low-methylated pectins and sulfated galactans, a feature shared with the cell walls of all macroalgae and that is important for ion homoeostasis, nutrient uptake and O2/CO2 exchange through leaf epidermal cells. The Z. marina genome resource will markedly advance a wide range of functional ecological studies from adaptation of marine ecosystems under climate warming, to unravelling the mechanisms of osmoregulation under high salinities that may further inform our understanding of the evolution of salt tolerance in crop plants.

Genetic, epigenetic and genomic effects on variation of gene expression among grape varieties.

The transcriptional regulatory structure of plant genomes is still relatively unexplored, and little is known about factors that influence expression variation in plants. We used a genetic system consisting of 10 heterozygous grape varieties with high consanguinity and high haplotypic diversity to: (i) identify regions of haplotype sharing through whole-genome resequencing and single-nucleotide polymorphism (SNP) genotyping; (ii) analyse gene expression through RNA-seq in four stages of berry development; and (iii) associate gene expression variation with genetic and epigenetic properties. We found that haplotype sharing in and around genes was positively correlated with similarity in expression and was negatively correlated with the fraction of differentially expressed genes. Genetic and epigenetic properties of the gene and the surrounding region showed significant effects on the extent of expression variation, with negative associations for the level of gene body methylation and mean expression level, and with positive associations for nucleotide diversity, structural diversity and ratio of non-synonymous to synonymous nucleotide diversity. We also observed a spatial dependency of covariation of gene expression among varieties. These results highlight relevant roles for cis-acting factors, selective constraints and epigenetic features of the gene, and the regional context in which the gene is located, in the determination of expression variation. OPEN RESEARCH BADGES: This article has earned an Open Data Badge for making publicly available the digitally-shareable data necessary to reproduce the reported results. The data is available at https://www.ncbi.nlm.nih.gov/bioproject/PRJNA385116; https://www.ncbi.nlm.nih.gov/bioproject/PRJNA392287; https://www.ncbi.nlm.nih.gov/bioproject/PRJNA373967 (released upon publication); https://www.ncbi.nlm.nih.gov/bioproject/PRJNA490160 (released upon publication); https://www.ncbi.nlm.nih.gov/bioproject/PRJNA265039; https://www.ncbi.nlm.nih.gov/bioproject/PRJNA265040.

Grapevine field experiments reveal the contribution of genotype, the influence of environment and the effect of their interaction (G×E) on the berry transcriptome.

Changes in the performance of genotypes in different environments are defined as genotype × environment (G×E) interactions. In grapevine (Vitis vinifera), complex interactions between different genotypes and climate, soil and farming practices yield unique berry qualities. However, the molecular basis of this phenomenon remains unclear. To dissect the basis of grapevine G×E interactions we characterized berry transcriptome plasticity, the genome methylation landscape and within-genotype allelic diversity in two genotypes cultivated in three different environments over two vintages. We identified, through a novel data-mining pipeline, genes with expression profiles that were: unaffected by genotype or environment, genotype-dependent but unaffected by the environment, environmentally-dependent regardless of genotype, and G×E-related. The G×E-related genes showed different degrees of within-cultivar allelic diversity in the two genotypes and were enriched for stress responses, signal transduction and secondary metabolism categories. Our study unraveled the mutual relationships between genotypic and environmental variables during G×E interaction in a woody perennial species, providing a reference model to explore how cultivated fruit crops respond to diverse environments. Also, the pivotal role of vineyard location in determining the performance of different varieties, by enhancing berry quality traits, was unraveled.

Haemolymph removal by Varroa mite destabilizes the dynamical interaction between immune effectors and virus in bees, as predicted by Volterra's model.

The association between the deformed wing virus and the parasitic mite Varroa destructor has been identified as a major cause of worldwide honeybee colony losses. The mite acts as a vector of the viral pathogen and can trigger its replication in infected bees. However, the mechanistic details underlying this tripartite interaction are still poorly defined, and, particularly, the causes of viral proliferation in mite-infested bees. Here, we develop and test a novel hypothesis that mite feeding destabilizes viral immune control through the removal of both virus and immune effectors, triggering uncontrolled viral replication. Our hypothesis is grounded on the predator-prey theory developed by Volterra, which predicts prey proliferation when both predators and preys are constantly removed from the system. Consistent with this hypothesis, we show that the experimental removal of increasing volumes of haemolymph from individual bees results in increasing viral densities. By contrast, we do not find consistent support for alternative proposed mechanisms of viral expansion via mite immune suppression or within-host viral evolution. Our results suggest that haemolymph removal plays an important role in the enhanced pathogen virulence observed in the presence of feeding Varroa mites. Overall, these results provide a new model for the mechanisms driving pathogen-parasite interactions in bees, which ultimately underpin honeybee health decline and colony losses.

MicroRNAs as master regulators of the plant NB-LRR defense gene family via the production of phased, trans-acting siRNAs.

Legumes and many nonleguminous plants enter symbiotic interactions with microbes, and it is poorly understood how host plants respond to promote beneficial, symbiotic microbial interactions while suppressing those that are deleterious or pathogenic. Trans-acting siRNAs (tasiRNAs) negatively regulate target transcripts and are characterized by siRNAs spaced in 21-nucleotide (nt) "phased" intervals, a pattern formed by DICER-LIKE 4 (DCL4) processing. A search for phased siRNAs (phasiRNAs) found at least 114 Medicago loci, the majority of which were defense-related NB-LRR-encoding genes. We identified three highly abundant 22-nt microRNA (miRNA) families that target conserved domains in these NB-LRRs and trigger the production of trans-acting siRNAs. High levels of small RNAs were matched to >60% of all ∼540 encoded Medicago NB-LRRs; in the potato, a model for mycorrhizal interactions, phasiRNAs were also produced from NB-LRRs. DCL2 and SGS3 transcripts were also cleaved by these 22-nt miRNAs, generating phasiRNAs, suggesting synchronization between silencing and pathogen defense pathways. In addition, a new example of apparent "two-hit" phasiRNA processing was identified. Our data reveal complex tasiRNA-based regulation of NB-LRRs that potentially evolved to facilitate symbiotic interactions and demonstrate miRNAs as master regulators of a large gene family via the targeting of highly conserved, protein-coding motifs, a new paradigm for miRNA function.

Transgenerational Response to Nitrogen Deprivation in Arabidopsis thaliana.

Nitrogen (N) deficiency is one of the major stresses that crops are exposed to. It is plausible to suppose that a stress condition can induce a memory in plants that might prime the following generations. Here, an experimental setup that considered four successive generations of N-sufficient and N-limited Arabidopsis was used to evaluate the existence of a transgenerational memory. The results demonstrated that the ability to take up high amounts of nitrate is induced more quickly as a result of multigenerational stress exposure. This behavior was paralleled by changes in the expression of nitrate responsive genes. RNAseq analyses revealed the enduring modulation of genes in downstream generations, despite the lack of stress stimulus in these plants. The modulation of signaling and transcription factors, such as NIGTs, NFYA and CIPK23 might indicate that there is a complex network operating to maintain the expression of N-responsive genes, such as NRT2.1, NIA1 and NIR. This behavior indicates a rapid acclimation of plants to changes in N availability. Indeed, when fourth generation plants were exposed to N limitation, they showed a rapid induction of N-deficiency responses. This suggests the possible involvement of a transgenerational memory in Arabidopsis that allows plants to adapt efficiently to the environment and this gives an edge to the next generation that presumably will grow in similar stressful conditions.

Massive analysis of rice small RNAs: mechanistic implications of regulated microRNAs and variants for differential target RNA cleavage.

Small RNAs have a variety of important roles in plant development, stress responses, and other processes. They exert their influence by guiding mRNA cleavage, translational repression, and chromatin modification. To identify previously unknown rice (Oryza sativa) microRNAs (miRNAs) and those regulated by environmental stress, 62 small RNA libraries were constructed from rice plants and used for deep sequencing with Illumina technology. The libraries represent several tissues from control plants and plants subjected to different environmental stress treatments. More than 94 million genome-matched reads were obtained, resulting in more than 16 million distinct small RNA sequences. This allowed an evaluation of ~400 annotated miRNAs with current criteria and the finding that among these, ~150 had small interfering RNA-like characteristics. Seventy-six new miRNAs were found, and miRNAs regulated in response to water stress, nutrient stress, or temperature stress were identified. Among the new examples of miRNA regulation were members of the same miRNA family that were differentially regulated in different organs and had distinct sequences Some of these distinct family members result in differential target cleavage and provide new insight about how an agriculturally important rice phenotype could be regulated in the panicle. This high-resolution analysis of rice miRNAs should be relevant to plant miRNAs in general, particularly in the Poaceae.

Draft genome sequences from 127 Legionella spp. strains isolated in water systems linked to legionellosis outbreaks.

Legionnaires' disease is a severe form of pneumonia caused by Legionella spp. bacteria. According to the European Centre for Disease Prevention and Control, problems related to this pathogen showed a significant surge in recent years, making its monitoring critical.

Revitalizing agriculture: next-generation genotyping and -omics technologies enabling molecular prediction of resilient traits in the Solanaceae family.

This review highlights -omics research in Solanaceae family, with a particular focus on resilient traits. Extensive research has enriched our understanding of Solanaceae genomics and genetics, with historical varietal development mainly focusing on disease resistance and cultivar improvement but shifting the emphasis towards unveiling resilience mechanisms in genebank-preserved germplasm is nowadays crucial. Collecting such information, might help researchers and breeders developing new experimental design, providing an overview of the state of the art of the most advanced approaches for the identification of the genetic elements laying behind resilience. Building this starting point, we aim at providing a useful tool for tackling the global agricultural resilience goals in these crops.

Self-DNA Early Exposure in Cultivated and Weedy Setaria Triggers ROS Degradation Signaling Pathways and Root Growth Inhibition.

The accumulation of fragmented extracellular DNA reduces conspecific seed germination and plantlet growth in a concentration-dependent manner. This self-DNA inhibition was repeatedly reported, but the underlying mechanisms are not fully clarified. We investigated the species-specificity of self-DNA inhibition in cultivated vs. weed congeneric species (respectively, Setaria italica and S. pumila) and carried out a targeted real-time qPCR analysis under the hypothesis that self-DNA elicits molecular pathways that are responsive to abiotic stressors. The results of a cross-factorial experiment on root elongation of seedlings exposed to self-DNA, congeneric DNA, and heterospecific DNA from Brassica napus and Salmon salar confirmed a significantly higher inhibition by self-DNA as compared to non-self-treatments, with the latter showing a magnitude of the effect consistent with the phylogenetic distance between the DNA source and the target species. Targeted gene expression analysis highlighted an early activation of genes involved in ROS degradation and management (FSD2, ALDH22A1, CSD3, MPK17), as well as deactivation of scaffolding molecules acting as negative regulators of stress signaling pathways (WD40-155). While being the first exploration of early response to self-DNA inhibition at molecular level on C4 model plants, our study highlights the need for further investigation of the relationships between DNA exposure and stress signaling pathways by discussing potential applications for species-specific weed control in agriculture.

Boosting grapevine breeding for climate-smart viticulture: from genetic resources to predictive genomics.

The multifaceted nature of climate change is increasing the urgency to select resilient grapevine varieties, or generate new, fitter cultivars, to withstand a multitude of new challenging conditions. The attainment of this goal is hindered by the limiting pace of traditional breeding approaches, which require decades to result in new selections. On the other hand, marker-assisted breeding has proved useful when it comes to traits governed by one or few genes with great effects on the phenotype, but its efficacy is still restricted for complex traits controlled by many loci. On these premises, innovative strategies are emerging which could help guide selection, taking advantage of the genetic diversity within the Vitis genus in its entirety. Multiple germplasm collections are also available as a source of genetic material for the introgression of alleles of interest via adapted and pioneering transformation protocols, which present themselves as promising tools for future applications on a notably recalcitrant species such as grapevine. Genome editing intersects both these strategies, not only by being an alternative to obtain focused changes in a relatively rapid way, but also by supporting a fine-tuning of new genotypes developed with other methods. A review on the state of the art concerning the available genetic resources and the possibilities of use of innovative techniques in aid of selection is presented here to support the production of climate-smart grapevine genotypes.

Detailed analysis of a contiguous 22-Mb region of the maize genome.

Most of our understanding of plant genome structure and evolution has come from the careful annotation of small (e.g., 100 kb) sequenced genomic regions or from automated annotation of complete genome sequences. Here, we sequenced and carefully annotated a contiguous 22 Mb region of maize chromosome 4 using an improved pseudomolecule for annotation. The sequence segment was comprehensively ordered, oriented, and confirmed using the maize optical map. Nearly 84% of the sequence is composed of transposable elements (TEs) that are mostly nested within each other, of which most families are low-copy. We identified 544 gene models using multiple levels of evidence, as well as five miRNA genes. Gene fragments, many captured by TEs, are prevalent within this region. Elimination of gene redundancy from a tetraploid maize ancestor that originated a few million years ago is responsible in this region for most disruptions of synteny with sorghum and rice. Consistent with other sub-genomic analyses in maize, small RNA mapping showed that many small RNAs match TEs and that most TEs match small RNAs. These results, performed on approximately 1% of the maize genome, demonstrate the feasibility of refining the B73 RefGen_v1 genome assembly by incorporating optical map, high-resolution genetic map, and comparative genomic data sets. Such improvements, along with those of gene and repeat annotation, will serve to promote future functional genomic and phylogenomic research in maize and other grasses.

Draft Genome Sequences of Eight Bacilli Isolated from an Ancient Roman Amphora.

Paleomicrobiology, the study of ancient microbiological material, allows us to understand different evolutionary phenomena in bacteria. In this study, eight bacilli isolated from an ancient Roman amphora, which dates to the IV to V sec. AD, were sequenced and functionally annotated.

Distinct extremely abundant siRNAs associated with cosuppression in petunia.

Cosuppression is a classical form of eukaryotic post-transcriptional gene silencing. It was first reported in transgenic petunia, where a sense transgene meant to overexpress the host Chalcone Synthase-A (CHS-A) gene caused the degradation of the homologous transcripts and the loss of flower pigmentation. In this work, we used deep sequencing technology to characterize in detail the small RNA population generated from the CHS-A sequence in cosuppressed transgenic petunia. Unexpectedly, two distinct small interfering RNAs (siRNAs) were found to vastly predominate. Our demonstration that they guide prominent cleavage events in CHS-A mRNA provides compelling and previously lacking evidence of a causative association between induction of individual siRNAs and an example of cosuppression. The preferential accumulation of these siRNAs provides new insights about sense cosuppression that may apply to other natural and engineered RNA silencing events.

Distinct size distribution of endogeneous siRNAs in maize: Evidence from deep sequencing in the mop1-1 mutant.

Small RNAs from plants are known to be highly complex and abundant, with this complexity proportional to genome size. Most endogenous siRNAs in Arabidopsis are dependent on RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) for their biogenesis. Recent work has demonstrated that the maize MEDIATOR OF PARAMUTATION1 (mop1) gene is a predicted ortholog of RDR2. The mop1 gene is required for establishment of paramutation and maintenance of transcriptional silencing of transposons and transgenes, suggesting the potential involvement of small RNAs. We analyzed small RNAs in wild-type maize and in the isogenic mop1-1 loss-of-function mutant by using Illumina's sequencing-by-synthesis (SBS) technology, which allowed us to characterize the complement of maize small RNAs to considerable depth. Similar to rdr2 in Arabidopsis, in mop1-1, the 24-nucleotide (nt) endogenous heterochromatic short-interfering siRNAs were dramatically reduced, resulting in an enrichment of miRNAs and transacting siRNAs. In contrast to the Arabidopsis rdr2 mutant, the mop1-1 plants retained a highly abundant heterochromatic approximately 22-nt class of small RNAs, suggesting a second mechanism for heterochromatic siRNA production. The enrichment of miRNAs and loss of 24-nt heterochromatic siRNAs in mop1-1 should be advantageous for miRNA discovery as the maize genome becomes more fully sequenced.

Targeted Mutagenesis of the Female-Suppressor SyGI Gene in Tetraploid Kiwifruit by CRISPR/CAS9.

Kiwifruit belong to the genus Actinidia with 54 species apparently all functionally dioecious. The sex-determinants of the type XX/XY, with male heterogametic, operate independently of the ploidy level. Recently, the SyGI protein has been described as the suppressor of female development. In the present study, we exploited the CRISPR/Cas9 technology by targeting two different sites in the SyGI gene in order to induce a stable gene knock-out in two tetraploid male accessions of Actinidia chinensis var. chinensis. The two genotypes showed a regenerative efficiency of 58% and 73%, respectively. Despite not yet being able to verify the phenotypic effects on the flower structure, due to the long time required by tissue-cultured kiwifruit plants to flower, we obtained two regenerated lines showing near fixation of a unique modification in their genome, resulting in both cases in the onset of a premature stop codon, which induces the putative gene knock-out. Evaluation of gRNA1 locus for both regenerated plantlets resulted in co-amplification of a minor variant differing from the target region for a single nucleotide. A genomic duplication of the region in proximity of the Y genomic region could be postulated.

Transposable elements and the plant pan-genomes.

The comparative sequencing of several grass genomes has revealed that transposable elements are largely responsible for extensive variation in both intergenic and local genic content, not only between closely related species but also among individuals within a species. These observations indicate that a single genome sequence might not reflect the entire genomic complement of a species, and prompted us to introduce the concept of the plant pan-genome, which includes core genomic features that are common to all individuals and a dispensable genome composed of partially shared and/or non-shared DNA sequence elements. Uncovering the intriguing nature of the dispensable genome, namely its composition, origin and function, represents a step forward towards an understanding of the processes that generate genetic diversity and phenotypic variation. The developing view of transcriptional regulation as a complex and modular system, in which long-range interactions and the involvement of transposable elements are frequently observed, lends support to the possibility of an important functional role for the dispensable genome and could make it less dispensable than previously thought.