Population-based nanopore sequencing of the HIV-1 pangenome to identify drug resistance mutations.

HIV-1 drug resistance genotypic tests have primarily been performed by Sanger sequencing of gene segments encoding different drug target proteins. Since the number of targets has increased with the addition of a new class of antiretroviral drugs, a simple high-throughput system for assessing nucleotide sequences throughout the HIV-1 genome is required. Here, we developed a new solution using nanopore sequencing of viral pangenomes amplified by PCR. Benchmark tests using HIV-1 molecular clones demonstrated an accuracy of up to 99.9%. In addition, validation tests of our protocol in 106 clinical samples demonstrated high concordance of drug resistance and tropism genotypes (92.5% and 98.1%, respectively) between the nanopore sequencing-based results and archived clinical determinations made based on Sanger sequencing data. These results suggest that our new approach will be a powerful solution for the comprehensive survey of HIV-1 drug resistance mutations in clinical settings.

Evaluation of the Geenius HIV 1/2 confirmatory assay for HIV-2 samples isolated in Japan.

BACKGROUND: Although the number of HIV-2-infected individuals is quite low in Japan, at least three groups of HIV-2 (A, B and CRF01_AB) have been detected thus far. In particular, CRF01_AB HIV-2 cases have been found only in limited areas, Cote d'Ivoire and Japan. Here, we demonstrate that Geenius HIV 1/2 Confirmatory Assay (Geenius, Bio-Rad Laboratories) is able to detect HIV-2 samples, including groups A, B and CRF01_AB, isolated in Japan. STUDY DESIGN: A total of 57 plasma samples, including three panels (Ⅰ: HIV-2-positive samples [n=9], Ⅱ: HIV-1 infection with HIV-2 antibody cross-reactivity samples [n=37], and Ⅲ: HIV negative with biological false-positive HIV-2 samples [n=11]) were tested by Geenius. RESULTS: Geenius determined Panel I to be "HIV-2 positive with/without HIV-1 cross-reactivity (n=4, respectively)", including HIV-2 group A and CRF01_AB. In the case with HIV-2 group B, all bands were detected, resulting in a Geenius interpretation of "HIV positive untypable". Geenius classified Panels II and III as "HIV-1 positive (n=37)" or "HIV negative (n=9)", "HIV indeterminate (n=1)" and "HIV-2 indeterminate (n=1)", suggesting 95.8% HIV-2 differentiation by Geenius. CONCLUSIONS: With Geenius, there were fewer false-positives for HIV-1/-2 negativity and fewer cross-reactions with HIV-2 among HIV-1-positive samples. Additionally, the assay could detect HIV-2 genetic group CRF01_AB. Geenius can be expected to be a useful diagnostic tool that is an alternative to conventional Western blotting.

Functional Characterization of Three Diterpene Synthases Responsible for Tetracyclic Diterpene Biosynthesis in Scoparia dulcis.

Scoparia dulcis produces unique biologically active diterpenoids such as scopadulcic acid B (SDB). They are biosynthesized from geranylgeranyl diphosphate (GGPP) via syn-copalyl diphosphate (syn-CPP) and scopadulanol as an important key intermediate. In this paper, we functionally characterized three diterpene synthases, SdCPS2, SdKSL1 and SdKSL2, from S. dulcis. The SdCPS2 catalyzed a cyclization reaction from GGPP to syn-CPP, and SdKSL1 did from syn-CPP to scopadulan-13α-ol. On the other hand, SdKSL2 was found to incorporate a non-sense mutation at 682. Therefore, we mutated the nucleotide residue from A to G in SdKSL2 to produce SdKSL2mut, and it was able to recover the catalytic function from syn-CPP to syn-aphidicol-16-ene, the precursor to scopadulin. From our results, SdCPS2 and SdKSL1 might be important key players for SDB biosynthesis in S. dulcis.

Divergent Evolution of the Diterpene Biosynthesis Pathway in Tea Plants (Camellia sinensis) Caused by Single Amino Acid Variation of ent-Kaurene Synthase.

Most plant terpenoids are classified as secondary metabolites. A small portion of them are products of primary metabolism biosynthesized by relatively conserved pathways. Gibberellins (GAs), which are essential for plant growth and development, are diterpenoid phytohormones. (E,E,E)-Geranylgeranyl diphosphate (GGPP) is the precursor for both GAs and other diterpenoids of secondary metabolism. ent-Kaurene biosynthesis from GGPP is a key step of GA formation, which is catalyzed by two sequential and dedicated diterpene synthases (diTPSs): ent-copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS) of the terpene synthase gene family. Sharing a common evolutionary origin, CPS and KS belong to different TPS subfamilies. Tea plant (Camellia sinensis), the subject of this study, is a leaf-based economic crop. Budbreak mainly manipulated by GAs is a primary factor for targeted tea breeding. The key genes for gibberellin biosynthesis are known; however, they have not yet been characterized in tea plants. Here, we identified and functionally characterized three diterpene biosynthesis-related genes, including one CPS and two highly similar KSs in tea plants. These genes were initially identified through transcriptome sequencing. The functional characterization determined by enzymatic activity assay indicated that CsCPS could catalyze GGPP to form ent-copalyl diphosphate (ent-CPP), which was further used as the substrate by CsKS1 to produce ent-kaurene or by CsKS2 to produce 16α-hydroxy-ent-kaurane with ent-kaurene as a minor product, respectively. We demonstrated that the divergent evolution of diterpene biosynthesis in tea plants resulted from gene duplication of KSs, followed by functional divergence caused by single amino acid variation. This study would provide an insight into the diterpenoid metabolism and GA biosynthesis in tea plants to further understand leaf bud development or insect resistance and to provide a genetic basis for tea plant breeding.

Functional characterization of NADPH-cytochrome P450 reductase and cinnamic acid 4-hydroxylase encoding genes from Scoparia dulcis L.

BACKGROUND: Most plant cytochrome P450 (P450) proteins need to be supplied with electrons from a redox partner, e.g. an NADPH-cytochrome P450 reductase (CPR), for the activation of oxygen molecules via heme. CPR is a flavoprotein with an N-terminal transmembrane domain, which transfers electrons from NADPH to the P450 via coenzymes flavin adenine dinucleotide and flavin mononucleotide. RESULTS: In this study, a novel CPR (SdCPR) was isolated from a tropical medicinal plant Scoparia dulcis L. The deduced amino acid of SdCPR showed high homology of > 76% with CPR from higher plants and belonged to the class II CPRs of dicots. Recombinant SdCPR protein reduced cytochrome c, ferricyanide (K3Fe(CN)6), and dichlorophenolindophenol in an NADPH-dependent manner. To elucidate the P450 monooxygenase activity of SdCPR, we isolated a cinnamic acid 4-hydroxylase (SdC4H, CYP73A111) gene from S. dulcis. Biochemical characterization of SdCPR/SdC4H demonstrated that SdCPR supports the oxidation step of SdC4H. Real-time qPCR results showed that expression levels of SdCPR and SdC4H were inducible by mechanical wounding treatment and phytohormone elicitation (methyl jasmonate, salicylic acid), which were consistent with the results of promotor analyses. CONCLUSIONS: Our results showed that the SdCPR and SdC4H are related to defense reactions, including the biosynthesis of secondary metabolites.

Characterization of ent-kaurene synthase and kaurene oxidase involved in gibberellin biosynthesis from Scoparia dulcis.

Gibberellins (GAs) are ubiquitous diterpenoids in higher plants, whereas some higher plants produce unique species-specific diterpenoids. In GA biosynthesis, ent-kaurene synthase (KS) and ent-kaurene oxidase (KO) are key players which catalyze early step(s) of the cyclization and oxidation reactions. We have studied the functional characterization of gene products of a KS (SdKS) and two KOs (SdKO1 and SdKO2) involved in GA biosynthesis in Scoparia dulcis. Using an in vivo heterologous expression system of Escherichia coli, we found that SdKS catalyzed a cyclization reaction from ent-CPP to ent-kaurene and that the SdKOs oxidized ent-kaurene to ent-kaurenoic acid after modification of the N-terminal region for adaptation to the E. coli expression system. The real-time PCR results showed that the SdKS, SdKO1 and SdKO2 genes were mainly expressed in the root and lateral root systems, which are elongating tissues. Based on these results, we suggest that these three genes may be responsible for the metabolism of GAs in S. dulcis.

Elucidation of terpenoid metabolism in Scoparia dulcis by RNA-seq analysis.

Scoparia dulcis biosynthesize bioactive diterpenes, such as scopadulcic acid B (SDB), which are known for their unique molecular skeleton. Although the biosynthesis of bioactive diterpenes is catalyzed by a sequence of class II and class I diterpene synthases (diTPSs), the mechanisms underlying this process are yet to be fully identified. To elucidate these biosynthetic machinery, we performed a high-throughput RNA-seq analysis, and de novo assembly of clean reads revealed 46,332 unique transcripts and 40,503 two unigenes. We found diTPSs genes including a putative syn-copalyl diphosphate synthase (SdCPS2) and two kaurene synthase-like (SdKSLs) genes. Besides them, total 79 full-length of cytochrome P450 (CYP450) genes were also discovered. The expression analyses showed selected CYP450s associated with their expression pattern of SdCPS2 and SdKSL1, suggesting that CYP450 candidates involved diterpene modification. SdCPS2 represents the first predicted gene to produce syn-copalyl diphosphate in dicots. In addition, SdKSL1 potentially contributes to the SDB biosynthetic pathway. Therefore, these identified genes associated with diterpene biosynthesis lead to the development of genetic engineering focus on diterpene metabolism in S. dulcis.

Induction, cloning and functional expression of a sesquiterpene biosynthetic enzyme, δ-guaiene synthase, of Aquilaria microcarpa cell cultures.

A homology-based cloning strategy yielded a cDNA clone presumably encoding δ-guaiene synthase, a sesquiterpene cyclase, from tissue cultures of Aquilaria microcarpa, which were treated with methyl jasmonate. Incubation of cell cultures of the plant with yeast extract also induced transcriptional activation of the sesquiterpene synthase gene. The translated protein of the gene obtained by heterologous expression in Escherichia coli catalyzed the cyclization of farnesyl diphosphate to liberate δ-guaiene with δ-guaiene and germacrene A as the minor products. The results obtained in the present study, together with the previously reported results, suggest that two classes of δ-guaiene synthase occur in Aquilaria; the enzyme proteins from A. microcarpa and A. sinensis liberate germacrene A as a minor product, while the protein from A. crassna generates α-humulene instead of germacrene A.

Enhanced accumulation of atropine in Atropa belladonna transformed by Rac GTPase gene isolated from Scoparia dulcis.

Leaf tissues of Atropa belladonna were transformed by Sdrac2, a Rac GTPase gene, that is isolated from Scoparia dulcis, and the change in atropine concentration of the transformants was examined. Re-differentiated A. belladonna overexpressing Sdrac2 accumulated considerable concentration of atropine in the leaf tissues, whereas the leaves of plants transformed by an empty vector accumulated only a very low concentration of the compound. A. belladonna transformed by CASdrac2, a modified Sdrac2 of which translate was expected to bind guanosine triphosphate (GTP) permanently, accumulated very high concentrations of atropine (approximately 2.4-fold excess to those found in the wild-type plant in its natural habitat). In sharp contrast, the atropine concentration in transformed A. belladonna prepared with negatively modified Sdrac2, DNSdrac2, expected to bind guanosine diphosphate instead of GTP, was very low. These results suggested that Rac GTPases play an important role in the regulation of secondary metabolism in plant cells and that overexpression of the gene(s) may be capable of enhancing the production of natural products accumulated in higher plant cells.

Modification and translocation of Rac/Rop guanosine 5'-triphosphate-binding proteins of Scoparia dulcis in response to stimulation with methyl jasmonate.

Translocation of two Rac/Rop guanosine 5'-triphosphate-binding proteins from Scoparia dulcis, Sdrac-1 and Sdrac-2, was examined employing transformed belladonna which overproduces these proteins as glutathione-S-transferase-tagged forms. The transferase activities of the fused proteins in microsomal fraction of belladonna markedly increased by the incubation with methyl jasmonate either in Sdrac-1 or Sdrac-2 transformant, while low and constant activities were observed in the untreated control. Recombinant Sdrac-2 protein was found to bind to prenyl chain in the presence of cell extracts prepared from methyl jasmonate-treated S. dulcis, however, Sdrac-1 was palmitoylated by the addition of the cell extracts. These results suggest that both Sdrac-1 and Sdrac-2 translocate to plant membranes by the stimulation with methyl jasmonate, however, targeting of these proteins is triggered by the independent modification mechanisms, palmitoylation for Sdrac-1 and prenylation for Sdrac-2.

Methyl jasmonate-induced enhancement of expression activity of Am-FaPS-1, a putative farnesyl diphosphate synthase gene from Aquilaria microcarpa.

A cDNA clone, designated Am-FaPS-1 (1310 bp), was isolated from callus culture derived from the leaf tissues of Aquilaria microcarpa. This gene contains an open reading frame encoding the protein of 342 amino acid residues with high homology to farnesyl diphosphate synthase from various plant sources. An appreciable increase in the transcriptional level of Am-FaPS-1 was reproducibly observed by the exposure of the cell culture to methyl jasmonate. The expression activity of the gene was also elevated when the cells were treated with yeast extract and Ca(2+)-ionophore A23187. These results suggest that Am-FaPS-1 and its translate play roles in methyl jasmonate- and yeast extract-induced responses of A. microcarpa, and Ca(2+) functions as an important messenger molecule in these processes. This set of the results would support our hypothesis that the activation of Ca(2+)-cascade evoked by the elevation of cytoplasmic Ca(2+) concentration is an essential early event in methyl jasmonate-induced responses of higher plant cells.

Transcriptional activation of putative calmodulin genes am-cam-1 and am-cam-2 from Aquilaria microcarpa, in response to external stimuli.

A homology-based cloning strategy yielded two cDNA clones designated Am-cam-1 and Am-cam-2, presumably encoding calmodulin protein from a callus culture derived from the leaf tissues of Aquilaria microcarpa. An appreciable increase in the transcriptional activity of Am-cam-1 was reproducibly observed by exposure of the cell culture to methyl jasmonate, as analyzed by a reverse-transcription polymerase chain reaction. The expression level of the gene also increased when the cells were treated with yeast extract. The transcription of Am-cam-2 was similarly stimulated by the treatment with methyl jasmonate and yeast extract, however, the intensities of the enhanced expression appeared to be lower as compared with that of Am-cam-1. In contrast, Ca(2+)-ionophore A23187 did not show inducing activity for the expression of these two calmodulin genes. These results suggest that Am-cam-1 and Am-cam-2 and their products play important roles in signal transduction processes in methyl jasmonate- and yeast extract-treated cells of A. microcarpa, accompanying the change in the transcriptional activities.

FLNA p.V528M substitution is neither associated with bilateral periventricular nodular heterotopia nor with macrothrombocytopenia.

Filamin A is encoded by the FLNA gene on chromosome Xq28 and functions in cross-linking actin filaments into orthogonal networks in the cortical cytoplasm. FLNA p.V528M was initially detected in a female autopsy case of X-linked bilateral periventricular nodular heterotopia (BPNH), a neuronal migration disorder characterized by subependymal nodules of gray matter. During our mutation analysis of FLNA in a boy with apparent X-linked thrombocytopenia, we detected the p.V528M variant. The patient, mother and sister, who were heterozygous for the substitution, did not have BPNH. We observed an allele frequency of 4.8% in healthy control Japanese, but did not observe the variant in Caucasian subjects. Hemizygous controls had a normal platelet count and size. We suggest that p.V528M is neither associated with BPNH nor with thrombocytopenia and giant platelets, and represents a functional polymorphism.

Cloning and characterization of a gene encoding Rac/Rop-like monomeric guanosine 5'-triphosphate-binding protein from Scoparia dulcis.

A cDNA clone, designated Sd-racrop (969 bp), was isolated from seedlings of Scoparia dulcis. This gene contains an open reading frame encoding the protein of 197 amino acid residues with high homology to Rac/Rop small guanosine 5'-triphosphate-binding proteins from various plant sources. In Southern hybridization analysis, the restriction digests prepared from genomic DNA of S. dulcis showed a main signal together with a few weakly hybridized bands. The transcriptional level of Sd-racrop showed a transient decrease by exposure of the leaf tissues of S. dulcis to the ethylene-generating reagent 2-chloroethylphosphonic acid. However, an appreciable increase in gene expression was reproducibly observed upon treatment of the plant with methyl jasmonate. These results suggest that the Sd-racrop product plays roles in ethylene- and methyl jasmonate-induced responses of S. dulcis accompanying the change in the transcriptional level, however, the cellular events mediated by this protein toward these external stimuli would be regulated by various mechanisms.

Cloning and characterization of Sdga gene encoding alpha-subunit of heterotrimeric guanosine 5'-triphosphate-binding protein complex in Scoparia dulcis.

A homology-based cloning strategy yielded Sdga, a cDNA clone presumably encoding alpha-subunit of heterotrimeric guanosine 5'-triphosphate-binding protein complex, from leaf tissues of Scoparia dulcis. Phylogenetic tree analysis of G-protein alpha-subunits from various biological sources suggested that, unlike in animal cells, classification of Galpha-proteins into specific subfamilies could not be applicable to the proteins from higher plants. Restriction digests of genomic DNA of S. dulcis showed a single hybridized signal in Southern blot analysis, suggesting that Sdga is a sole gene encoding Galpha-subunit in this plant. The expression level of Sdga appeared to be maintained at almost constant level after exposure of the leaves to methyl jasmonate as analyzed by reverse-transcription polymerase chain reaction. These results suggest that Sdga plays roles in methyl jasmonate-induced responses of S. dulcis without a notable change in the transcriptional level.

Enhanced secretory activity of Atropa belladonna hairy root culture over-expressing ADP-ribosylation factor gene.

Agrobacterium-mediated transformation of leaf tissues of Atropa belladonna with an adenosine 5'-diphosphate (ADP)-ribosylation factor gene of carrot, arf-001, was performed employing pBCR82 as an expression vector. This vector co-expresses rol gene cluster together with arf-001, and thus, the transformed host cells were obtained as hairy roots. Two cell lines of the transformed belladonna were established as the liquid cultures of hairy root tissues, and expression of arf-001 and accumulation of its product in the cells were confirmed by RT-PCR and Western blot analyses, respectively. A marked increase in extracellular protein concentrations was observed in the transformed belladonna root cultures as compared with the controls transformed with an empty vector. However, the secretion of the proteins of the transformants was markedly reduced in the presence of a physiological concentration of monensin. These results suggest that over-expression of arf-001 in belladonna results in the enhancement of secretory activity in the transformed cells.

The coiled-coil protein-binding motif in Fusarium verticillioides Fsr1 is essential for maize stalk rot virulence.

Fusarium verticillioides (Sacc.) Nirenberg (teleomorph Gibberella moniliformis Wineland) is one of the key pathogens of maize stalk rot disease. However, a clear understanding of stalk rot pathogenesis is still lacking. Previously, we identified the F. verticillioides FSR1 gene, which plays a key role in fungal virulence and sexual mating. The predicted Fsr1 protein contains multiple protein-binding domains, namely a caveolin-binding domain, a coiled-coil structure, and a calmodulin-binding motif at the N terminus and a WD40 repeat domain at the C terminus. Fsr1 shares significant similarity to a family of striatin proteins that play a critical role in cellular mechanisms that regulate a variety of developmental processes. Significantly, FSR1 function is conserved in Fusarium graminearum, where it also plays a direct role in pathogenesis. In this study, our goal was to determine the motif(s) in Fsr1 that are directly associated with fungal virulence. We complemented the FSR1 knockout (Deltafsr1) strain with mutated versions of the FSR1 gene, and determined that the Fsr1 C-terminal WD40 repeat domain is dispensable for vegetative growth and maize stalk rot virulence. We also examined the potential link between FSR1-mediated virulence and cell wall-degrading enzyme (alpha-amylase, pectinase and cellulase) activities. Further characterization of the N-terminal region revealed that the coiled-coil structure is essential for virulence in F. verticillioides. The coiled-coil domain is involved in a variety of protein-protein interactions in eukaryotic systems, and thus we hypothesize that the interaction between Fsr1 and the putative Fsr1-binding protein triggers downstream gene signalling that is associated with F. verticillioides virulence.

A translational enhancer element on the 3'-proximal end of the Panicum mosaic virus genome.

Panicum mosaic virus (PMV) is a single-stranded positive-sense RNA virus in the family Tombusviridae. PMV genomic RNA (gRNA) and subgenomic RNA (sgRNA) are not capped or polyadenylated. We have determined that PMV uses a cap-independent mechanism of translation. A 116-nucleotide translational enhancer (TE) region on the 3'-untranslated region of both the gRNA and sgRNA has been identified. The TE is required for efficient translation of viral proteins in vitro. For mutants with a compromised TE, addition of cap analog, or transposition of the cis-active TE to another location, both restored translational competence of the 5'-proximal sgRNA genes in vitro.

Tomato bushy stunt virus: a resilient model system to study virus-plant interactions.

UNLABELLED: SUMMARY Taxonomy: Tomato bushy stunt virus (TBSV) (Fig. 1) is the type species of the plant-infecting Tombusvirus genus in the family Tombusviridae. Physical properties: TBSV virions are non-enveloped icosahedral T = 3 particles assembled from 180 coat protein subunits (42 kDa) whose arrangement causes a granular appearance on the surface structure. The particles are approximately 33 nm in diameter and composed of 17% ribonucleic acid and 83% protein. Encapsidated within the virion is the TBSV genome that consists of a positive-sense single-stranded RNA of approximately 4.8 kb, which lacks the 5'-cap or 3'-poly(A) tail typical for eukaryotic mRNAs. HOST RANGE: In nature, TBSV has a fairly restricted host range, mostly encompassing a few dicotyledonous species in separate families, and affected agricultural crops comprise primarily vegetables. The experimental host range is broad, with over 120 plant species in more than 20 different families reported to be susceptible although in most plants the infection often remains localized around the site of entry. The differences between hosts with regards to requirements for cell-to-cell and long-distance movement have led to the development of TBSV as an attractive model system to obtain general insights into RNA transport through plants. SYMPTOMS: SYMPTOMS induced by TBSV are largely dependent on the host genotype; they can vary from necrotic and chlorotic lesions, to a systemic mild or severe mosaic, or they may culminate in a lethal necrosis. The original TBSV isolates from tomato plants caused a mottle, crinkle and downward curling of leaves with the youngest leaves exhibiting tip necrosis upon systemic infection. Tomato fruit yield can be greatly reduced by virus infection. Plants may be stunted and a proliferation of lateral shoots leads to a bushy appearance of the infected tomato plants, hence the nomenclature of the pathogen. Useful sites: http://image.fs.uidaho.edu/vide/descr825.htm; http://www.ictvdb.rothamsted.ac.uk/ICTVdB/74010001.htm (general information); http://mmtsb.scripps.edu/viper/info_page.php?vipPDB=2tbv (structural information).

Molecular cloning and characterization of a cDNA encoding a novel apoplastic protein preferentially expressed in a shikonin-producing callus strain of Lithospermum erythrorhizon.

A cDNA (LEPS-2) encoding a novel cell wall protein was cloned from shikonin-producing callus tissues of Lithospermum erythrorhizon by differential display between a shikonin-producing culture strain and a non-producing strain. The LEPS-2 cDNA encoded a polypeptide of 184 amino acids. The deduced amino acid sequence exhibited no significant homology with known proteins. Expression of LEPS-2 gene as well as accumulation of LEPS-2 protein was highly correlated with shikonin production in L. erythrorhizon cells in culture. In the intact plant, expression of LEPS-2 was detected only in the roots where shikonin pigments accumulated. Cell fractionation experiments and immunocytochemical analysis showed that the protein was localized in the apoplast fraction of the cell walls. The shikonin pigments were also stored on the cell walls as oil droplets. These results indicate that expression of the LEPS-2 is closely linked with shikonin biosynthesis and the LEPS-2 protein may be involved in the intra-cell wall trapping of shikonin pigments.