The analysis of the first plant genomes provided unexpected evidence for genome duplication events in species that had previously been considered as true diploids on the basis of their genetics [1][2][3] . These polyploidization events may have had important consequences in plant evolution, in particular for species radiation and adaptation and for the modulation of functional capacities 4-10 . Here we report a high-quality draft of the genome sequence of grapevine (Vitis vinifera) obtained from a highly homozygous genotype. The draft sequence of the grapevine genome is the fourth one produced so far for flowering plants, the second for a woody species and the first for a fruit crop (cultivated for both fruit and beverage). Grapevine was selected because of its important place in the cultural heritage of humanity beginning during the Neolithic period 11 . Several large expansions of gene families with roles in aromatic features are observed. The grapevine genome has not undergone recent genome duplication, thus enabling the discovery of ancestral traits and features of the genetic organization of flowering plants. This analysis reveals the contribution of three ancestral genomes to the grapevine haploid content. This ancestral arrangement is common to many dicotyledonous plants but is absent from the genome of rice, which is a monocotyledon. Furthermore, we explain the chronology of previously described whole-genome duplication events in the evolution of flowering plants.All grapevine varieties are highly heterozygous; preliminary data showed that there was as much as 13% sequence divergence between alleles, which would hinder reliable contig assembly when a wholegenome shotgun strategy was used for sequencing. Our consortium therefore selected the grapevine PN40024 genotype for sequencing. This line, originally derived from Pinot Noir, has been bred close to full homozygosity (estimated at about 93%) by successive selfings, permitting a high-quality whole-genome shotgun assembly.A total of 6.2 million end-reads were produced by our consortium, representing an 8.4-fold coverage of the genome. Within the assembly, performed with Arachne 12 , 316 supercontigs represent putative allelic haplotypes that constitute 11.6 million bases (Mb). These values are in good fit with the 7% residual heterozygosity of PN40024 assessed by using genetic markers. When considering only one of the haplotypes in each heterozygous region, the assembly (Table 1a) consists of 19,577 contigs (N 50 5 65.9 kilobases (kb), where N 50 corresponds to the size of the shorter supercontig or contig in a subset representing half of the assembly size) and 3,514 supercontigs (N 50 5 2.07 Mb) totalling 487 Mb. This value is close to the 475 Mb previously reported for the grapevine genome size 13 .Using a set of 409 molecular markers from the reference grapevine map 14 , 69% of the assembled 487 Mb, arranged into 45 ultracontigs

While genome assembly projects have been successful in a number of haploid or inbred species, one of the main current challenges is assembling non-inbred or rearranged heterozygous genomes. To address this critical need, we introduce the open-source FALCON and FALCON-Unzip algorithms (https://github.com/PacificBiosciences/FALCON/) to assemble Single Molecule Real-Time (SMRT®) Sequencing data into highly accurate, contiguous, and correctly phased diploid genomes. We demonstrate the quality of this approach by assembling new reference sequences for three heterozygous samples, including an F1 hybrid of the model species Arabidopsis thaliana, the widely cultivated Vitis vinifera cv. Cabernet Sauvignon, and the coral fungus Clavicorona pyxidata that have challenged short-read assembly approaches. The FALCON-based assemblies were substantially more contiguous and complete than alternate short or long-read approaches. The phased diploid assembly enabled the study of haplotype structures and heterozygosities between the homologous chromosomes, including identifying widespread heterozygous structural variations within the coding sequences.

Recognition of an avirulent pathogen triggers the rapid production of the reactive oxygen intermediates superoxide (O2-) and hydrogen peroxide (H2O2). This oxidative burst drives crosslinking of the cell wall, induces several plant genes involved in cellular protection and defence, and is necessary for the initiation of host cell death in the hypersensitive disease-resistance response. However, this burst is not enough to support a strong disease-resistance response. Here we show that nitric oxide, which acts as a signal in the immune, nervous and vascular systems, potentiates the induction of hypersensitive cell death in soybean cells by reactive oxygen intermediates and functions independently of such intermediates to induce genes for the synthesis of protective natural products. Moreover, inhibitors of nitric oxide synthesis compromise the hypersensitive disease-resistance response of Arabidopsis leaves to Pseudomonas syringae, promoting disease and bacterial growth. We conclude that nitric oxide plays a key role in disease resistance in plants.

Nitric oxide (NO) and reactive oxygen intermediates (ROIs) play key roles in the activation of disease resistance mechanisms both in animals and plants. In animals NO cooperates with ROIs to kill tumor cells and for macrophage killing of bacteria. Such cytotoxic events occur because unregulated NO levels drive a diffusionlimited reaction with O 2 ؊ to generate peroxynitrite (ONOO ؊ ), a mediator of cellular injury in many biological systems. Here we show that in soybean cells unregulated NO production at the onset of a pathogen-induced hypersensitive response (HR) is not sufficient to activate hypersensitive cell death. The HR is triggered only by balanced production of NO and ROIs. Moreover, hypersensitive cell death is activated after interaction of NO not with O 2 ؊ but with H 2O2 generated from O2 ؊ by superoxide dismutase. Increasing the level of O 2 ؊ reduces NO-mediated toxicity, and ONOO ؊ is not a mediator of hypersensitive cell death. During the HR, superoxide dismutase accelerates O 2 ؊ dismutation to H2O2 to minimize the loss of NO by reaction with O 2 ؊ and to trigger hypersensitive cell death through NO͞H 2O2 cooperation. However, O2 ؊ rather than H 2O2 is the primary ROI signal for pathogen induction of glutathione S-transferase, and the rates of production and dismutation of O 2 ؊ generated during the oxidative burst play a crucial role in the modulation and integration of NO͞H 2O2 signaling in the HR. Thus although plants and animals use a similar repertoire of signals in disease resistance, ROIs and NO are deployed in strikingly different ways to trigger host cell death.A ttempted infection of plants by an avirulent pathogen elicits a battery of defenses often accompanied by the collapse of challenged host cells. This hypersensitive cell death results in a restricted lesion delimited from surrounding healthy tissue and is thought to contribute to pathogen restriction. An early event in this hypersensitive response (HR) is the generation of superoxide (O 2 Ϫ ) and accumulation of hydrogen peroxide (H 2 O 2 ) in an oxidative burst reminiscent of that producing such reactive oxygen intermediates (ROIs) in activated macrophages (1).Activation of the oxidative burst in the plant HR is part of a highly amplified and integrated signal system that also involves salicylic acid and perturbations of cytosolic Ca 2ϩ to trigger defense mechanisms (2) and to mediate the establishment of systemic immunity (3). The oxidative burst is necessary but not sufficient to trigger host cell death, and recent data indicate that nitric oxide (NO) cooperates with ROIs in the activation of hypersensitive cell death (4).NO and ROIs also interact in the mammalian native immune system where macrophage killing of pathogens and tumor cells involves the diffusion-limited reaction of NO and O 2 to generate ONOO Ϫ , a long lived and highly reactive oxidant species that freely crosses membranes (5), which may modulate NO signal functions (6). ONOO Ϫ induces apoptosis in some human tumor cells (7), and it is also directly cytotoxic...

We developed a genome-wide transcriptomic atlas of grapevine (Vitis vinifera) based on 54 samples representing green and woody tissues and organs at different developmental stages as well as specialized tissues such as pollen and senescent leaves. Together, these samples expressed ;91% of the predicted grapevine genes. Pollen and senescent leaves had unique transcriptomes reflecting their specialized functions and physiological status. However, microarray and RNA-seq analysis grouped all the other samples into two major classes based on maturity rather than organ identity, namely, the vegetative/ green and mature/woody categories. This division represents a fundamental transcriptomic reprogramming during the maturation process and was highlighted by three statistical approaches identifying the transcriptional relationships among samples (correlation analysis), putative biomarkers (O2PLS-DA approach), and sets of strongly and consistently expressed genes that define groups (topics) of similar samples (biclustering analysis). Gene coexpression analysis indicated that the mature/woody developmental program results from the reiterative coactivation of pathways that are largely inactive in vegetative/green tissues, often involving the coregulation of clusters of neighboring genes and global regulation based on codon preference. This global transcriptomic reprogramming during maturation has not been observed in herbaceous annual species and may be a defining characteristic of perennial woody plants.