Search

RNA alternative polyadenylation contributes to the complexity of information transfer from genome to phenome, thus amplifying gene function. Here, we report the first X. tropicalis resource with 127,914 alternative polyadenylation (APA) sites derived from embryos and adults. Overall, APA networks play central roles in coordinating the maternal-zygotic transition (MZT) in embryos, sexual dimorphism in adults and longitudinal growth from embryos to adults. APA sites coordinate reprogramming in embryos before the MZT, but developmental events after the MZT due to zygotic genome activation. The APA transcriptomes of young adults are more variable than growing adults and male frog APA transcriptomes are more divergent than females. The APA profiles of young females were similar to embryos before the MZT. Enriched pathways in developing embryos were distinct across the MZT and noticeably segregated from adults. Briefly, our results suggest that the minimal functional units in genomes are alternative transcripts as opposed to genes.

Construction of next-generation sequencing (NGS) libraries involves RNA manipulation, which often creates noisy, biased, and artifactual data that contribute to errors in transcriptome analysis. In this study, a total of 19 whole transcriptome termini site sequencing (WTTS-seq) and seven RNA sequencing (RNA-seq) libraries were prepared from Xenopus tropicalis adult and embryo samples to determine the most effective library preparation method to maximize transcriptomics investigation. We strongly suggest that appropriate primers/adaptors are designed to inhibit amplification detours and that PCR overamplification is minimized to maximize transcriptome coverage. Furthermore, genome annotation must be improved so that missing data can be recovered. In addition, a complete understanding of sequencing platforms is critical to limit the formation of false-positive results. Technically, the WTTS-seq method enriches both poly(A)+ RNA and complementary DNA, adds 5'- and 3'-adaptors in one step, pursues strand sequencing and mapping, and profiles both gene expression and alternative polyadenylation (APA). Although RNA-seq is cost prohibitive, tends to produce false-positive results, and fails to detect APA diversity and dynamics, its combination with WTTS-seq is necessary to validate transcriptome-wide APA.

Bactericera cockerelli (Šulc) (Hemiptera: Triozidae) is a vector of ' Candidatus Liberibacter solanacearum' (Lso), the pathogen that causes potato zebra chip. Zebra chip incidence varies regionally, perhaps because of geographic differences in species of noncrop hosts available to the vector and in susceptibility of those hosts to Lso. Native and introduced species of Lycium (Solanales: Solanaceae) are important noncrop hosts of B. cockerelli in some regions of North America. Susceptibility of native Lycium species to Lso is uncertain. We investigated the use of two native species of Lycium by B. cockerelli in South Texas and tested whether they are susceptible to Lso. Bactericera cockerelli adults and nymphs were collected frequently from L. berlandieri Dunal and L. carolinianum Walter. Greenhouse assays confirmed that B. cockerelli develops on both species and showed that Lso infects L. carolinianum. Molecular gut content analysis provided evidence that B. cockerelli adults disperse between potato and Lycium. These results demonstrate that L. berlandieri and L. carolinianum are likely noncrop sources of potato-colonizing B. cockerelli in South Texas and that L. carolinianum is a potential source of Lso-infected psyllids. We also routinely collected the congeneric psyllid, Bactericera dorsalis (Crawford), from both Lycium species. These records are the first for this psyllid in Texas. Bactericera dorsalis completed development on both native Lycium species, albeit with high rates of mortality on L. berlandieri. B. dorsalis acquired and transmitted Lso on L. carolinianum under greenhouse conditions but did not transmit Lso to potato. These results document a previously unknown vector of Lso. [ABSTRACT FROM AUTHOR]

Auchenorrhyncha (Hemiptera) includes several families of sap-feeding insects that tend to feed on a wide-range of host plants. Some species within Auchenorrhyncha are major agricultural pests that transmit plant pathogens or cause direct feeding damage. Nearly all pest Auchenorrhyncha are highly polyphagous, have mobile nymphs, and colonize crops from noncrop habitats. We examined whether methods for gut content analysis developed for more specialized Hemiptera identify dietary history of polyphagous Auchenorrhyncha. We used high-throughput sequencing of the plant genes trnF and ITS to examine the dietary history of Circulifer tenellus (Baker) (Cicadellidae), Colladonus geminatus (Van Duzee) (Cicadellidae) , Colladonus montanus reductus (Van Duzee) (Cicadellidae), and Lycorma delicatula (White) (Fulgoridae). C. tenellus is a vector of the vegetable pathogens Candidatus Phytoplasma trifolii and beat curly top virus. Both Colladonus species are vectors of Ca. Phytoplasma pruni, the pathogen associated with X-disease of stone fruits. Lycorma delicatula is an invasive pest of grape and ornamentals in the eastern United States. Results showed spring hosts for C. tenellus included Brassicaceae, especially Sisymbrium sp. (tumble mustard), and spring hosts for both Colladonus species included Taraxacum sp. (dandelion). Gut content analysis also detected a decrease in host-breadth by L. delicatula from early to late instars. Results demonstrate that directed sequencing of plant DNA identified the dietary history of leafhopper and planthopper pests. Expanded use of gut content analysis will help identify the noncrop sources of phytoplasma-infected C. tenellus and Colladonus, and to examine seasonal changes in host shifts by L. delicatula. [ABSTRACT FROM AUTHOR]

Insects harbor bacterial endosymbionts that provide their hosts with nutritional benefit or with protection against natural enemies, plant defenses, insecticides, or abiotic stresses. We used directed sequencing of 16S rDNA to identify and compare endosymbionts of Bactericera maculipennis (Crawford) and the western, central, and northwestern haplotypes of B. cockerelli (Šulc) (Hemiptera: Psylloidea: Triozidae). Both species are native to North America, are known to harbor the plant pathogen 'Candidatus Liberibacter solanacearum' and develop on shared host plants within the Convolvulaceae. The Old-World species Heterotrioza chenopodii (Reuter) (Psylloidea: Triozidae), now found in North America, was included as an outgroup. 16S sequencing confirmed that both Bactericera species harbor 'Candidatus Liberibacter solanacearum' and revealed that both species harbor unique strains of Wolbachia and Sodalis. However, the presence of Wolbachia and Sodalis varied among haplotypes of B. cockerelli. The central and western haplotypes harbored the same strains of Wolbachia, which was confirmed by Sanger sequencing of the wsp and ftsZ genes. Wolbachia was also detected in very low abundance from the northwestern haplotype by high-throughput sequencing of 16S but was not detected from this haplotype by PCR screening. The northwestern and central haplotypes also harbored Sodalis, which was not detected in the western haplotype. Heterotrioza chenopodii harbored an entirely different community of potential endosymbionts compared with the Bactericera spp. that included Rickettsia and an unidentified bacterium in the Enterobacteriaceae. Results of this study provide a foundation for further research on the interactions between psyllids and their bacterial endosymbionts. [ABSTRACT FROM AUTHOR]

Functional genomics approaches, which use combined computational and expression-based analyses of large amounts of sequence information, are emerging as powerful tools to accelerate the comprehensive understanding of cellular metabolism in specialized tissues and whole organisms. As part of an ongoing effort to identify genes of essential oil (monoterpene) biosynthesis, we have obtained sequence information from 1,316 randomly selected cDNA clones, or expressed sequence tags (ESTs), from a peppermint (Mentha x piperita) oil gland secretory cell cDNA library. After bioinformatic selection, candidate genes putatively involved in essential oil biosynthesis and secretion have been subcloned into suitable expression vectors for functional evaluation in Escherichia coli. On the basis of published and preliminary data on the functional properties of these clones, it is estimated that the ESTs involved in essential oil metabolism represent about 25% of the described sequences. An additional 7% of the recognized genes code for proteins involved in transport processes, and a subset of these is likely involved in the secretion of essential oil terpenes from the site of synthesis to the storage cavity of the oil glands. The integrated approaches reported here represent an essential step toward the development of a metabolic map of oil glands and provide a valuable resource for defining molecular targets for the genetic engineering of essential oil formation.

The genome sequences of the plastid and mitochondrion of white spruce (Picea glauca) were assembled from whole-genome shotgun sequencing data using ABySS. The sequencing data contained reads from both the nuclear and organellar genomes, and reads of the organellar genomes were abundant in the data as each cell harbors hundreds of mitochondria and plastids. Hence, assembly of the 123-kb plastid and 5.9-Mb mitochondrial genomes were accomplished by analyzing data sets primarily representing low coverage of the nuclear genome. The assembled organellar genomes were annotated for their coding genes, ribosomal RNA, and transfer RNA. Transcript abundances of the mitochondrial genes were quantified in three developmental tissues and five mature tissues using data from RNA-seq experiments. C-to-U RNA editing was observed in the majority of mitochondrial genes, and in four genes, editing events were noted to modify ACG codons to create cryptic AUG start codons. The informatics methodology presented in this study should prove useful to assemble organellar genomes of other plant species using whole-genome shotgun sequencing data. [ABSTRACT FROM AUTHOR]

Construction of next-generation sequencing (NGS) libraries involves RNA manipulation, which often creates noisy, biased, and artifactual data that contribute to errors in transcriptome analysis. In this study, a total of 19 whole transcriptome termini site sequencing (WTTS-seq) and seven RNA sequencing (RNA-seq) libraries were prepared from Xenopus tropicalis adult and embryo samples to determine the most effective library preparation method to maximize transcriptomics investigation. We strongly suggest that appropriate primers/adaptors are designed to inhibit amplification detours and that PCR overamplification is minimized to maximize transcriptome coverage. Furthermore, genome annotation must be improved so that missing data can be recovered. In addition, a complete understanding of sequencing platforms is critical to limit the formation of false-positive results. Technically, the WTTS-seq method enriches both poly(A)+ RNA and complementary DNA, adds 59- and 39-adaptors in one step, pursues strand sequencing and mapping, and profiles both gene expression and alternative polyadenylation (APA). Although RNA-seq is cost prohibitive, tends to produce false-positive results, and fails to detect APA diversity and dynamics, its combination with WTTS-seq is necessary to validate transcriptome-wide APA. [ABSTRACT FROM AUTHOR]

(-)-Menthol is the most familiar of the monoterpenes as both a pure natural product and as the principal and characteristic constituent of the essential oil of peppermint (Mentha x piperita). In this paper, we review the biosynthesis and molecular genetics of (-)-menthol production in peppermint. In Mentha species, essential oil biosynthesis and storage is restricted to the peltate glandular trichomes (oil glands) on the aerial surfaces of the plant. A mechanical method for the isolation of metabolically functional oil glands, has provided a system for precursor feeding studies to elucidate pathway steps, as well as a highly enriched source of the relevant biosynthetic enzymes and of their corresponding transcripts with which cDNA libraries have been constructed to permit cloning and characterization of key structural genes. The biosynthesis of (-)-menthol from primary metabolism requires eight enzymatic steps, and involves the formation and subsequent cyclization of the universal monoterpene precursor geranyl diphosphate to the parent olefin (-)-(4S)-limonene as the first committed reaction of the sequence. Following hydroxylation at C3, a series of four redox transformations and an isomerization occur in a general "allylic oxidation-conjugate reduction" scheme that installs three chiral centers on the substituted cyclohexanoid ring to yield (-)-(1R, 3R, 4S)-menthol. The properties of each enzyme and gene of menthol biosynthesis are described, as are their probable evolutionary origins in primary metabolism. The organization of menthol biosynthesis is complex in involving four subcellular compartments, and regulation of the pathway appears to reside largely at the level of gene expression. Genetic engineering to up-regulate a flux-limiting step and down-regulate a side route reaction has led to improvement in the composition and yield of peppermint oil.

The complex mixture of monoterpenes, sesquiterpenes, and diterpenes that comprises oleoresin provides the primary defense of conifers against bark beetles and their associated fungal pathogens. Monoterpene synthases produce the turpentine fraction of oleoresin, which allows mobilization of the diterpene resin acid component (rosin) and is also toxic toward invading insects; this is particularly the case for alpha-pinene, a prominent bicyclic monoterpene of pine turpentine. The stereochemistry of alpha-pinene is a critical determinant of host defense capability and has implications for host selection, insect pheromone biosynthesis, and tritrophic-level interactions. Pines produce both enantiomers of alpha-pinene, which appear to arise through antipodal reaction mechanisms by distinct enzymes. Using a cDNA library constructed with mRNA from flushing needles of loblolly pine (Pinus taeda), we employed a homology-based cloning strategy to isolate, and confirm by functional expression, the genes encoding (+)-(3R:5R)-alpha-pinene synthase, (-)-(3S:5S)-alpha-pinene synthase, and several other terpene synthases. The pinene synthases, which produce mirror-image products, share only 66% amino acid identity (72% similarity) but are similar in general properties to other monoterpene synthases of gymnosperms. The stereochemical control of monoterpene cyclization reactions, the evolution of "antipodal" enzymes, and the implications of turpentine composition in ecological interactions are discussed.