Search

BackgroundAlternative splicing (AS) produces transcript variants playing potential roles in proteome diversification and gene expression regulation. AS modulation is thus essential to respond to developmental and environmental stimuli. In grapevine, a better understanding of berry development is crucial for implementing breeding and viticultural strategies allowing adaptation to climate changes. Although profound changes in gene transcription have been shown to occur in the course of berry ripening, no detailed study on splicing modifications during this period has been published so far. We report here on the regulation of gene AS in developing berries of two grapevine (Vitis vinifera L.) varieties, Gewurztraminer (Gw) and Riesling (Ri), showing distinctive phenotypic characteristics. Using the software rMATS, the transcriptomes of berries at four developmental steps, from the green stage to mid-ripening, were analysed in pairwise comparisons between stages and varieties.ResultsA total of 305 differential AS (DAS) events, affecting 258 genes, were identified. Interestingly, 22% of these AS events had not been reported before. Among the 80 genes that underwent the most significant variations during ripening, 22 showed a similar splicing profile in Gw and Ri, which suggests their involvement in berry development. Conversely, 23 genes were subjected to splicing regulation in only one variety. In addition, the ratios of alternative isoforms were different in Gw and Ri for 35 other genes, without any change during ripening. This last result indicates substantial AS differences between the two varieties. Remarkably, 8 AS events were specific to one variety, due to the lack of a splice site in the other variety. Furthermore, the transcription rates of the genes affected by stage-dependent splicing regulation were mostly unchanged, identifying AS modulation as an independent way of shaping the transcriptome.ConclusionsThe analysis of AS profiles in grapevine varieties with contrasting phenotypes revealed some similarity in the regulation of several genes with developmental functions, suggesting their involvement in berry ripening. Additionally, many splicing differences were discovered between the two varieties, that could be linked to phenotypic specificities and distinct adaptive capacities. Together, these findings open perspectives for a better understanding of berry development and for the selection of grapevine genotypes adapted to climate change.

The genome sequence assembly of the diploid and highly homozygousV. viniferagenotype PN40024 serves as the reference for many grapevine studies. Despite several improvements of the PN40024 genome assembly, its current version PN12X.v2 is quite fragmented and only represents the haploid state of the genome with mixed haplotypes. In fact, despite the PN40024 genome is nearly homozygous, it still contains various heterozygous regions. Taking the opportunity of the improvements that long-read sequencing technologies offer to fully discriminate haplotype sequences and considering that severalVitissp. genomes have recently been assembled with these approaches, an improved version of the reference, called PN40024.v4, was generated.Through incorporating long genomic sequencing reads to the assembly, the continuity of the 12X.v2 scaffolds was highly increased. The number of scaffolds decreased from 2,059 to 640 and the number of N bases was reduced by 88%. Additionally, the full alternative haplotype sequence was built for the first time, the chromosome anchoring was improved and the amount of unplaced scaffolds were reduced by half. To obtain a high-quality gene annotation that outperforms previous versions, a liftover approach was complemented with an optimized annotation workflow forVitis. Integration of the gene reference catalogue and its manual curation have also assisted in improving the annotation, while defining the most reliable estimation to date of 35,230 genes. Finally, we demonstrate that PN40024 resulted from selfings of cv. ‘Helfensteiner’ (cross of cv. ‘Pinot noir’ and ‘Schiava grossa’) instead of a single ‘Pinot noir’. These advances will help maintaining the PN40024 genome as a gold-standard reference also contributing in the eventual elaboration of the grapevine pangenome.

BackgroundNumerous studies have documented modifications in vector orientation behavior, settling and feeding behavior, and/or fecundity and survival due to virus infection in host plants. These alterations are often expected to enhance virus transmission, which has led to the hypothesis that such effects are vector manipulations by the virus. However, until now, the gene expression changes correlating with these effects and indicative of modified vector pathways and mechanisms are mostly unknown.ResultsTranscriptome profiling ofMyzus persicaeaphids feeding on turnip yellows virus (TuYV) and cauliflower mosaic virus (CaMV) infectedArabidopsis thalianaandCamelina sativarevealed a substantial proportion of commonly deregulated genes, amongst them many with general functions in plant-virus-aphid interactions. We identified also aphid genes specifically deregulated by CaMV or TuYV infection, which might be related to the viral transmission mode. Furthermore, we observed strong host-specific differences in the gene expression patterns with plant virus infection causing more deregulations of aphid genes onA. thalianathan onC. sativa, likely related to the differences in susceptibility of the plant hosts to these viruses. Finally, stress-related aphid genes were downregulated inM. persicaeon both infected plants, regardless of the virus.ConclusionsTuYV, relying on the circulative persistent mode of transmission, tended to affect developmental genes. This could increase the proportion of alate aphids, but also affect their locomotion, neuronal activity, and lifespan. CaMV, using the non-circulative non-persistent mode of transmission, had a strong impact on feeding-related genes and in particular those related to salivary proteins. In general, these transcriptome alterations targeted pathways that seem to be particularly adapted to the transmission mode of the corresponding virus and could be evidence of vector manipulation by the virus.

BackgroundEvidence accumulates that plant viruses alter host-plant traits in ways that modify their insect vectors’ behavior. These alterations often enhance virus transmission, which has led to the hypothesis that these effects are manipulations caused by viral adaptation. However, the genetic basis of these indirect, plant-mediated effects on vectors and their dependence on the plant host and the mode of virus transmission is hardly known.ResultsTranscriptome profiling of Arabidopsis thaliana and Camelina sativa plants infected with turnip yellows virus (TuYV) or cauliflower mosaic virus (CaMV) and infested with the common aphid vector Myzus persicae revealed strong virus- and host-specific differences in the gene expression patterns. CaMV infection caused more severe effects on the phenotype of both plant hosts than did TuYV infection, and the severity of symptoms correlated strongly with the proportion of differentially expressed genes, especially photosynthesis genes. Accordingly, CaMV infection modified aphid behavior and fecundity stronger than did infection with TuYV.ConclusionsOverall, infection with CaMV – relying on the non-circulative transmission mode – tends to have effects on metabolic pathways with strong potential implications for insect-vector / plant-host interactions (e.g. photosynthesis, jasmonic acid, ethylene and glucosinolate biosynthetic processes), while TuYV – using the circulative transmission mode – alters these pathways only weakly. These virus-induced deregulations of genes that are related to plant physiology and defense responses might impact aphid probing and feeding behavior on both infected host plants, with potentially distinct effects on virus transmission.

Virus infection of plants can result in various degrees of detrimental impacts and disparate symptom types and severities. Although great strides have been made in our understanding of the virus–host interactions in herbaceous model plants, the mechanisms underlying symptom development are poorly understood in perennial fruit crops. Grapevine fanleaf virus (GFLV) causes variable symptoms in most vineyards worldwide. To better understand GFLV-grapevine interactions in relation to symptom development, field and greenhouse trials were conducted with a grapevine genotype that exhibits distinct symptoms in response to a severe and a mild strain of GFLV. After validation of the infection status of the experimental vines by high-throughput sequencing, the transcriptomic and metabolomic profiles in plants infected with the two viral strains were tested and compared by RNA-Seq and LC-MS, respectively, in the differentiating grapevine genotype. In vines infected with the severe GFLV strain, 1023 genes, among which some are implicated in the regulation of the hypersensitive-type response, were specifically deregulated, and a higher accumulation of resveratrol and phytohormones was observed. Interestingly, some experimental vines restricted the virus to the rootstock and remained symptomless. Our results suggest that GFLV induces a strain- and cultivar-specific defense reaction similar to a hypersensitive reaction. This type of defense leads to a severe stunting phenotype in some grapevines, whereas others are resistant. This work is the first evidence of a hypersensitive-like reaction in grapevine during virus infection.

Carotenoid levels in plant tissues depend on the relative rates of synthesis and degradation of the molecules in the pathway. While plant carotenoid biosynthesis has been extensively characterized, research on carotenoid degradation and catabolism into apocarotenoids is a relatively novel field. To identify apocarotenoid metabolic processes, we characterized the transcriptome of transgenic Arabidopsis (Arabidopsis thaliana) roots accumulating high levels of β-carotene and, consequently, β-apocarotenoids. Transcriptome analysis revealed feedback regulation on carotenogenic gene transcripts suitable for reducing β-carotene levels, suggesting involvement of specific apocarotenoid signaling molecules originating directly from β-carotene degradation or after secondary enzymatic derivatizations. Enzymes implicated in apocarotenoid modification reactions overlapped with detoxification enzymes of xenobiotics and reactive carbonyl species (RCS), while metabolite analysis excluded lipid stress response, a potential secondary effect of carotenoid accumulation. In agreement with structural similarities between RCS and β-apocarotenoids, RCS detoxification enzymes also converted apocarotenoids derived from β-carotene and from xanthophylls into apocarotenols and apocarotenoic acids in vitro. Moreover, glycosylation and glutathionylation-related processes and translocators were induced. In view of similarities to mechanisms found in crocin biosynthesis and cellular deposition in saffron (Crocus sativus), our data suggest apocarotenoid metabolization, derivatization and compartmentalization as key processes in (apo)carotenoid metabolism in plants.

Background A key step in domestication of the grapevine was the transition from separate sexes (dioecy) in wild Vitis vinifera ssp. sylvestris (V. sylvestris) to hermaphroditism in cultivated Vitis vinifera ssp. sativa (V. vinifera). It is known that V. sylvestris has an XY system and V. vinifera a modified Y haplotype (Yh) and that the sex locus is small, but it has not previously been precisely characterized. Results We generate a high-quality de novo reference genome for V. sylvestris, onto which we map whole-genome re-sequencing data of a cross to locate the sex locus. Assembly of the full X, Y, and Yh haplotypes of V. sylvestris and V. vinifera sex locus and examining their gene content and expression profiles during flower development in wild and cultivated accessions show that truncation and deletion of tapetum and pollen development genes on the X haplotype likely causes male sterility, while the upregulation of a Y allele of a cytokinin regulator (APRT3) may cause female sterility. The downregulation of this cytokinin regulator in the Yh haplotype may be sufficient to trigger reversal to hermaphroditism. Molecular dating of X and Y haplotypes is consistent with the sex locus being as old as the Vitis genus, but the mechanism by which recombination was suppressed remains undetermined. Conclusions We describe the genomic and evolutionary characterization of the sex locus of cultivated and wild grapevine, providing a coherent model of sex determination in the latter and for transition from dioecy to hermaphroditism during domestication.

In a grapevine segregating population, genomic regions governing berry pH were identified, paving the way for breeding new grapevine varieties best adapted to a warming climate. As a consequence of global warming, grapevine berry acidity is expected to dramatically decrease. Adapting grapevine (Vitis vinifera L.) varieties to the climatic conditions of the future requires a better understanding of the genetic architecture of acidity-related traits. For this purpose, we studied during five growing seasons 120 individuals from a grapevine biparental cross. Each offspring was genotyped by simple sequence repeats markers and by hybridization on a 20-K Grapevine Illumina® SNP chip. Quantitative trait loci (QTLs) for pH colocalized with QTLs for the ratio between potassium and tartaric acid concentrations, on chromosomes 10, 11 and 13. Strong QTLs for malic acid concentration or for the malic acid-to-tartaric acid ratio, on chromosomes 6 and 8, were not associated with variations of pH but can be useful for controlling pH stability under high temperatures. Our study highlights the interdependency between acidity parameters and consequently the constraints and degrees of freedom for designing grapevine genotypes better adapted to the expected warmer climatic conditions. In particular, it is possible to create grapevine genotypes with a high berry acidity as the result of both high tartaric acid concentrations and low K+ accumulation capacities.

Inducible plant defences against pathogens are stimulated by infections and comprise several classes of pathogenesis-related (PR) proteins. Endo-β-1,3-glucanases (EGases) belong to the PR-2 class and their expression is induced by many pathogenic fungi and oomycetes, suggesting that EGases play a role in the hydrolysis of pathogen cell walls. However, reports of a direct effect of EGases on cell walls of plant pathogens are scarce. Here, we characterized three EGases from Vitis vinifera whose expression is induced during infection by Plasmopara viticola, the causal agent of downy mildew. Recombinant proteins were expressed in Escherichia coli. The enzymatic characteristics of these three enzymes were measured in vitro and in planta. A functional assay performed in vitro on germinated P. viticola spores revealed a strong anti-P. viticola activity for EGase3, which strikingly was that with the lowest in vitro catalytic efficiency. To our knowledge, this work shows, for the first time, the direct effect against downy mildew of EGases of the PR-2 family from Vitis.

International audience; Key messageIn grapevine interspecific hybrids, meiotic recombination is suppressed in homeologous regions and enhanced in homologous regions of recombined chromosomes, whereas crossover rate remains unchanged when chromosome pairs are entirely homeologous.AbstractVitis rotundifolia, an American species related to the cultivated European grapevine Vitis vinifera, has a high level of resistance to several grapevine major diseases and is consequently a valuable resource for grape breeding. However, crosses between both species most often lead to very few poorly fertile hybrids. In this context, identifying genetic and genomic features that make cross-breeding between both species difficult is essential. To this end, three mapping populations were generated by pseudo-backcrosses using V. rotundifolia as the donor parent and several V. vinifera cultivars as the recurrent parents. Genotyping-by-sequencing was used to establish high-density genetic linkage maps and to determine the genetic composition of the chromosomes of each individual. A good collinearity of the SNP positions was observed between parental maps, confirming the synteny between both species, except on lower arm of chromosome 7. Interestingly, recombination rate in V. rotundifoliaxV. vinifera interspecific hybrids depends on the length of the introgressed region. It is similar to grapevine for chromosome pairs entirely homeologous. Conversely, for chromosome pairs partly homeologous, recombination is suppressed in the homeologous regions, whereas it is enhanced in the homologous ones. This balance leads to the conservation of the total genetic length of each chromosome between V. vinifera and hybrid maps, whatever the backcross level and the proportion of homeologous region. Altogether, these results provide new insight to optimize the use of V. rotundifolia in grape breeding and, more generally, to improve the introgression of gene of interest from wild species related to crops.

International audience; Cytochromes P450 are enzymes that participate in a wide range of functions in plants, from hormonal signaling and biosynthesis of structural polymers, to defense or communication with other organisms. They represent one of the largest gene/protein families in the plant kingdom. The manual annotation of cytochrome P450 genes in the genome of Vitis vinifera PN40024 revealed 579 P450 sequences, including 279 complete genes. Most of the P450 sequences in grapevine genome are organized in physical clusters, resulting from tandem or segmental duplications. Although most of these clusters are small (2 to 35, median = 3), some P450 families, such as CYP76 and CYP82, underwent multiple duplications and form large clusters of homologous sequences. Analysis of gene expression revealed highly specific expression patterns, which are often the same within the genes in large physical clusters. Some of these genes are induced upon biotic stress, which points to their role in plant defense, whereas others are specifically activated during grape berry ripening and might be responsible for the production of berry-specific metabolites, such as aroma compounds. Our work provides an exhaustive and robust annotation including clear identification, structural organization, evolutionary dynamics and expression patterns for the grapevine cytochrome P450 families, paving the way to efficient functional characterization of genes involved in grapevine defense pathways and aroma biosynthesis.

The Oomycete Plasmopara viticola is responsible for downy mildew, which is one of the most damaging grapevine diseases. Due to the strictly biotrophic way of life of P. viticola, its metabolome is relatively poorly characterized. In this work, we have used a mass spectrometry-based non-targeted metabolomic approach to identify potential Plasmopara-specific metabolites. This has led to the characterization and structural elucidation of compounds belonging to three families of atypical lipids, which are not detected in healthy grapevine tissues. These lipids include ceramides and derivatives of arachidonic and eicosapentaenoic acid, most of which had not been previously described in Oomycetes. Furthermore, we show that these lipids can be detected in Plasmopara-infected tissues at very early stages of the infection process, long before the appearance the first visible symptoms of the disease. Therefore, the potential use of these specific lipids as markers to monitor the development of P. viticola is discussed.

Inducible plant defences against pathogens are stimulated by infections and comprise several classes of pathogenesis-related (PR) proteins. Endo-beta-1,3-glucanases (EGases) belong to the PR2 class and their expression is induced by many pathogenic fungi and oomycetes, suggesting that EGases play a role in the hydrolysis of pathogen cell walls. However, reports of a direct effect of EGases on cell walls of plant pathogens are scarce. Here, we characterized three EGases from Vitis vinifera whose expression is induced during infection by Plasmopara viticola, the causal agent of downy mildew. Recombinant proteins were expressed in Escherichia coli. The enzymatic characteristics of these three enzymes were measured in vitro and in planta. A functional assay performed in vitro on germinated P. viticola spores revealed a strong anti-P. viticola activity for EGase3, which strikingly was that with the lowest in vitro catalytic efficiency. To our knowledge, this work shows, for the first time, the direct effect against downy mildew of EGases of the PR-2 family from Vitis.

International audience

Background Wheat grains are an important source of food, stock feed and raw materials for industry, but current production levels cannot meet world needs. Elucidation of the molecular mechanisms underlying wheat grain development will contribute valuable information to improving wheat cultivation. One of the most important mechanisms implicated in plant developmental processes is the ubiquitin-proteasome system (UPS). Among the different roles of the UPS, it is clear that it is essential to hormone signaling. In particular, E3 ubiquitin ligases of the UPS have been shown to play critical roles in hormone perception and signal transduction. Results A NimbleGen microarray containing 39,179 UniGenes was used to study the kinetics of gene expression during wheat grain development from the early stages of cell division to the mid-grain filling stage. By comparing 11 consecutive time-points, 9284 differentially expressed genes were identified and annotated during this study. A comparison of the temporal profiles of these genes revealed dynamic transcript accumulation profiles with major reprogramming events that occurred during the time intervals of 80-120 and 220-240°Cdays. The list of the genes expressed differentially during these transitions were identified and annotated. Emphasis was placed on E3 ligase and hormone-related genes. In total, 173 E3 ligase coding genes and 126 hormone-related genes were differentially expressed during the cell division and grain filling stages, with each family displaying a different expression profile. Conclusions The differential expression of genes involved in the UPS and plant hormone pathways suggests that phytohormones and UPS crosstalk might play a critical role in the wheat grain developmental process. Some E3 ligase and hormone-related genes seem to be up- or down-regulated during the early and late stages of the grain development.

To improve our understanding of the organization and regulation of the wheat (Triticum aestivum) gene space, we established a transcription map of a wheat chromosome (3B) by hybridizing a newly developed wheat expression microarray with bacterial artificial chromosome pools from a new version of the 3B physical map as well as with cDNA probes derived from 15 RNA samples. Mapping data for almost 3,000 genes showed that the gene space spans the whole chromosome 3B with a 2-fold increase of gene density toward the telomeres due to an increase in the number of genes in islands. Comparative analyses with rice (Oryza sativa) and Brachypodium distachyon revealed that these gene islands are composed mainly of genes likely originating from interchromosomal gene duplications. Gene Ontology and expression profile analyses for the 3,000 genes located along the chromosome revealed that the gene islands are enriched significantly in genes sharing the same function or expression profile, thereby suggesting that genes in islands acquired shared regulation during evolution. Only a small fraction of these clusters of cofunctional and coexpressed genes was conserved with rice and B. distachyon, indicating a recent origin. Finally, genes with the same expression profiles in remote islands (coregulation islands) were identified suggesting long-distance regulation of gene expression along the chromosomes in wheat.

Background Because of its size, allohexaploid nature and high repeat content, the wheat genome has always been perceived as too complex for efficient molecular studies. We recently constructed the first physical map of a wheat chromosome (3B). However gene mapping is still laborious in wheat because of high redundancy between the three homoeologous genomes. In contrast, in the closely related diploid species, barley, numerous gene-based markers have been developed. This study aims at combining the unique genomic resources developed in wheat and barley to decipher the organisation of gene space on wheat chromosome 3B. Results Three dimensional pools of the minimal tiling path of wheat chromosome 3B physical map were hybridised to a barley Agilent 15K expression microarray. This led to the fine mapping of 738 barley orthologous genes on wheat chromosome 3B. In addition, comparative analyses revealed that 68% of the genes identified were syntenic between the wheat chromosome 3B and barley chromosome 3 H and 59% between wheat chromosome 3B and rice chromosome 1, together with some wheat-specific rearrangements. Finally, it indicated an increasing gradient of gene density from the centromere to the telomeres positively correlated with the number of genes clustered in islands on wheat chromosome 3B. Conclusion Our study shows that novel structural genomics resources now available in wheat and barley can be combined efficiently to overcome specific problems of genetic anchoring of physical contigs in wheat and to perform high-resolution comparative analyses with rice for deciphering the organisation of the wheat gene space.

L'article original est publié par The American Society of Plant Biologists; To improve our understanding of the organization and evolution of the wheat (Triticum aestivum) genome, we sequenced and annotated 13-Mb contigs (18.2 Mb) originating from different regions of its largest chromosome, 3B (1 Gb), and produced a 2x chromosome survey by shotgun Illumina/Solexa sequencing. All regions carried genes irrespective of their chromosomal location. However, gene distribution was not random, with 75% of them clustered into small islands containing three genes on average. A twofold increase of gene density was observed toward the telomeres likely due to high tandem and interchromosomal duplication events. A total of 3222 transposable elements were identified, including 800 new families. Most of them are complete but showed a highly nested structure spread over distances as large as 200 kb. A succession of amplification waves involving different transposable element families led to contrasted sequence compositions between the proximal and distal regions. Finally, with an estimate of 50,000 genes per diploid genome, our data suggest that wheat may have a higher gene number than other cereals. Indeed, comparisons with rice (Oryza sativa) and Brachypodium revealed that a high number of additional noncollinear genes are interspersed within a highly conserved ancestral grass gene backbone, supporting the idea of an accelerated evolution in the Triticeae lineages.