Novel reassortant H7N9 viruses were associated with severe and fatal respiratory disease in three patients. (Funded by the National Basic Research Program of China and others.).

Citrus is a large genus that includes several major cultivated species, including C. sinensis (sweet orange), Citrus reticulata (tangerine and mandarin), Citrus limon (lemon), Citrus grandis (pummelo) and Citrus paradisi (grapefruit). In 2009, the global citrus acreage was 9 million hectares and citrus production was 122.3 million tons (FAO statistics, see URLs), which is the top ranked among all the fruit crops. Among the 10.9 million tons (valued at $9.3 billion) of citrus products traded in 2009, sweet orange accounted for approximately 60% of citrus production for both fresh fruit and processed juice consumption (FAO statistics, see URLs). Moreover, citrus fruits and juice are the prime human source of vitamin C, an important component of human nutrition.Citrus fruits also have some unique botanical features, such as nucellar embryony (nucellus cells can develop into apomictic embryos that are genetically identical to mother plant). Consequently, somatic embryos grow much more vigorously than the zygotic embryos in seeds such that seedlings are essentially clones of the maternal parent. Such citrus-unique characteristics have hindered the study of citrus genetics and breeding improvement 1,2 . Complete genome sequences would provide valuable genetic resources for improving citrus crops.Citrus is believed to be native to southeast Asia 3-5 , and cultivation of fruit crops occurred at least 4,000 years ago 3,6 . The genetic origin of the sweet orange is not clear, although there are some speculations that sweet orange might be derived from interspecific hybridization of some primitive citrus species 7,8 . Citrus is also in the order Sapindales, a sister order to the Brassicales in the Malvidae, making it valuable for comparative genomics studies with the model plant Arabidopsis.We aimed to sequence the genome of Valencia sweet orange (C. sinensis cv. Valencia), one of the most important sweet orange varieties cultivated worldwide and grown primarily for orange juice production. Normal sweet oranges are diploids, with nine pairs of chromosomes and an estimated genome size of ~367 Mb 9 . To reduce the complexity of the sequenced genome, we obtained a doublehaploid (dihaploid) line derived from the anther culture of Valencia sweet orange 10 . We first generated whole-genome shotgun pairedend-tag sequence reads from the dihaploid genomic DNA and built a de novo assembly as the citrus reference genome; we then produced shotgun sequencing reads from the parental diploid DNA and mapped the sequences to the haploid reference genome to obtain the complete genome information for Valencia sweet orange. In addition, we conducted comprehensive transcriptome sequencing analyses for four representative tissues using shotgun RNA sequencing (RNA-Seq) to capture all transcribed sequences and paired-end-tag RNA sequencing (RNA-PET) to demarcate the 5′ and 3′ ends of all transcripts. On the basis of the DNA and RNA sequencing data, we characterized the orange genome for its gene content, heterozygosity and evolutionary features. ...

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Precise and efficient genome editing is very important for gene functional characterization. In recent years, sequence-specific DNA nucleases have been developed to increase the efficiency of gene targeting or genome editing in animals and plants. Among them, Zinc-Finger Nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs) are two most commonly used sequencespecific chimeric proteins (Gaj et al., 2013). Recently, a breakthrough gene-targeting tool based on RNA-guided Cas9 nuclease from type II prokaryotic Cluster Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated system has been developed (Jinek et al., 2012). CRISPR/Cas system is an adaptive defense system in prokaryotes to fight against alien nucleic acids (Horvath and Barrangou, 2010). The CRISPR loci are variable short spacers separated by short repeats, which are transcribed into synthetic single-guide RNA (sgRNA). The sgRNA forms a functional complex with CRISPR-associated nuclease (Cas9) and guide the nuclease to genomic loci matching a 20-bp complementary invading DNA, cleaving it immediately upstream of a required 5'-NGG Protospacer Adjacent Motif (PAM). A chimeric sgRNA that mimics the natural synthetic sgRNA can be used to target Cas9 for genome editing in eukaryotic cells.Until now, the CRISPR/Cas system has been successfully applied to efficient genome editing in bacteria, animals and plants (Jiang et al., 2013; Mali et al., 2013; Xie and Yang, 2013). To inform the selection of target sites and avoid off-target effects, Hsu and colleagues characterized Cas9 targeting specificity in human cells by a volume of experiments, and built a computational tool for optimized sgRNA selection in human and animal genomes (Hsu et al., 2013). However, with the wide application of CRISPR-system in plant genome editing, tools for optimized sgRNA selection in plants are highly needed. In the current analysis, we present a web application tool-CRISPR-P, for CRISPR sgRNA design in more than 20 plant species. CRISPR-P allows users to search for highly specific Cas9 target sites within DNA sequences of interest, which also provides off-target loci prediction for further analysis and marks restriction enzyme cutting sites for convenience. CRISPR-P is freely available at http://cbi.hzau.edu.cn/crispr/.Genomic data and annotation of the analyzed plant genomes are obtained from public databases. The genome and annotation of Arabidopsis lyrata (v.1.0), Arabidopsis thaliana (TAIR10), Brachypodium distachyon (v1.0), Brassica

Human infection associated with a novel reassortant avian influenza H7N9 virus has recently been identified in China. A total of 132 confirmed cases and 39 deaths have been reported. Most patients presented with severe pneumonia and acute respiratory distress syndrome. Although the first epidemic has subsided, the presence of a natural reservoir and the disease severity highlight the need to evaluate its risk on human public health and to understand the possible pathogenesis mechanism. Here we show that the emerging H7N9 avian influenza virus poses a potentially high risk to humans. We discover that the H7N9 virus can bind to both avian-type (α2,3-linked sialic acid) and human-type (α2,6-linked sialic acid) receptors. It can invade epithelial cells in the human lower respiratory tract and type II pneumonocytes in alveoli, and replicated efficiently in ex vivo lung and trachea explant culture and several mammalian cell lines. In acute serum samples of H7N9-infected patients, increased levels of the chemokines and cytokines IP-10, MIG, MIP-1β, MCP-1, IL-6, IL-8 and IFN-α were detected. We note that the human population is naive to the H7N9 virus, and current seasonal vaccination could not provide protection.

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Cytochrome P450 enzymes (CYPs) play major roles in generating highly functionalized terpenoids, but identifying the exact biotransformation step(s) catalyzed by plant CYP in terpenoid biosynthesis is extremely challenging. Tanshinones are abietane-type norditerpenoid naphthoquinones that are the main lipophilic bioactive components of the Chinese medicinal herb danshen (Salvia miltiorrhiza). Whereas the diterpene synthases responsible for the conversion of (E,E,E)-geranylgeranyl diphosphate into the abietane miltiradiene, a potential precursor to tanshinones, have been recently described, molecular characterization of further transformation of miltiradiene remains unavailable. Here we report stableisotope labeling results that demonstrate the intermediacy of miltiradiene in tanshinone biosynthesis. We further use a next-generation sequencing approach to identify six candidate CYP genes being coregulated with the diterpene synthase genes in both the rhizome and danshen hairy roots, and demonstrate that one of these, CYP76AH1, catalyzes a unique four-electron oxidation cascade on miltiradiene to produce ferruginol both in vitro and in vivo. We then build upon the previous establishment of miltiradiene production in Saccharomyces cerevisiae, with incorporation of CYP76AH1 and phyto-CYP reductase genes leading to heterologous production of ferruginol at 10.5 mg/L. As ferruginol has been found in many plants including danshen, the results and the approaches that were described here provide a solid foundation to further elucidate the biosynthesis of tanshinones and related diterpenoids. Moreover, these results should facilitate the construction of microbial cell factories for the production of phytoterpenoids.phytoterpenoids biosynthesis | gene discovery | synthetic pathway | metabolic engineering

Gossypium hirsutum contributes the most production of cotton fibre, but G. barbadense is valued for its better comprehensive resistance and superior fibre properties. However, the allotetraploid genome of G. barbadense has not been comprehensively analysed. Here we present a high-quality assembly of the 2.57 gigabase genome of G. barbadense, including 80,876 protein-coding genes. The double-sized genome of the A (or At) (1.50 Gb) against D (or Dt) (853 Mb) primarily resulted from the expansion of Gypsy elements, including Peabody and Retrosat2 subclades in the Del clade, and the Athila subclade in the Athila/Tat clade. Substantial gene expansion and contraction were observed and rich homoeologous gene pairs with biased expression patterns were identified, suggesting abundant gene sub-functionalization occurred by allopolyploidization. More specifically, the CesA gene family has adapted differentially temporal expression patterns, suggesting an integrated regulatory mechanism of CesA genes from At and Dt subgenomes for the primary and secondary cellulose biosynthesis of cotton fibre in a “relay race”-like fashion. We anticipate that the G. barbadense genome sequence will advance our understanding the mechanism of genome polyploidization and underpin genome-wide comparison research in this genus.