Characterization of Anopheles stephensi Odorant Receptor 8, an Abundant Component of the Mouthpart Chemosensory Transcriptome

Several mosquito species within the genus Anopheles are vectors for human malaria, and the spread of this disease is driven by the propensity of certain species to feed preferentially on humans. The study of olfaction in mosquitoes is important to understand dynamics of host-seeking and host-selection; however, the majority of these studies focus on Anopheles gambiae or An. coluzzii, both vectors of malaria in Sub-Saharan Africa. Other malaria vectors may recognize different chemical cues from potential hosts; therefore, in this study, we investigated An. stephensi, the south Asian malaria mosquito. We specifically focused on the mouthparts (primarily the maxillary palp and labella) that have been much less investigated compared to the antennae but are also important for host-seeking. To provide a broad view of chemoreceptor expression, RNAseq was used to examine the transcriptomes from the mouthparts of host-seeking females, blood-fed females, and males. Notably, AsOr8 had a high transcript abundance in all transcriptomes and was, therefore, cloned and expressed in the Drosophila empty neuron system. This permitted characterization with a panel of odorants that were selected, in part, for their presence in the human odor profile. The responsiveness of AsOr8 to odorants was highly similar to An. gambiae Or8 (AgOr8), except for sulcatone, which was detected by AsOr8 but not AgOr8. Subtle differences in the receptor sensitivity to specific odorants may provide clues to species- or strain-specific approaches to host-seeking and host selection. Further exploration of the profile of An. stephensi chemosensory proteins may yield a better understanding of how different malaria vectors navigate host-finding and host-choice.

Identification of Novel Genomic Associations and Gene Candidates for Grain Starch Content in Sorghum

Starch accumulated in the endosperm of cereal grains as reserve energy for germination serves as a staple in human and animal nutrition. Unraveling genetic control for starch metabolism is important for breeding grains with high starch content. In this study, we used a sorghum association panel with 389 individuals and 141,557 single nucleotide polymorphisms (SNPs) to fit linear mixed models (LMM) for identifying genomic regions and potential candidate genes associated with starch content. Three associated genomic regions, one in chromosome (chr) 1 and two novel associations in chr-8, were identified using combination of LMM and Bayesian sparse LMM. All significant SNPs were located within protein coding genes, with SNPs ∼ 52 Mb of chr-8 encoding a Casperian strip membrane protein (CASP)-like protein (Sobic.008G111500) and a heat shock protein (HSP) 90 (Sobic.008G111600) that were highly expressed in reproductive tissues including within the embryo and endosperm. The HSP90 is a potential hub gene with gene network of 75 high-confidence first interactors that is enriched for five biochemical pathways including protein processing. The first interactors of HSP90 also showed high transcript abundance in reproductive tissues. The candidates of this study are likely involved in intricate metabolic pathways and represent candidate gene targets for source-sink activities and drought and heat stress tolerance during grain filling.

Glycosylation of bioactive C13-apocarotenols in Nicotiana benthamiana and Mentha × piperita

SummaryC13-apocarotenoids (norisoprenoids) are carotenoid-derived oxidation products, which perform important physiological functions in plants. Although their biosynthetic pathways have been extensively studied, their metabolism including glycosylation remains elusive. Candidate uridine-diphosphate glycosyltransferase genes (UGTs) were selected for their high transcript abundance in comparison with other UGTs in vegetative tissues of Nicotiana benthamiana and Mentha × piperita, as these tissues are rich sources of apocarotenoid glucosides. Hydroxylated C13-apocarotenol substrates were produced by P450-catalyzed biotransformation and microbial/plant enzyme systems were established for the synthesis of glycosides. Natural substrates were identified by physiological aglycone libraries prepared from isolated plant glycosides. In total, we identified six UGTs that catalyze the unprecedented glucosylation of C13-apocarotenols, where glucose is bound either to the cyclohexene ring or butane side chain. MpUGT86C10 is a superior novel enzyme that catalyzes the glucosylation of allelopathic 3-hydroxy-α-damascone, 3-oxo-α-ionol, 3-oxo-7,8-dihydro-α-ionol (Blumenol C) and 3-hydroxy-7,8-dihydro-β-ionol, while a germination test demonstrated the higher phytotoxic potential of a norisoprenoid glucoside in comparison to its aglycone. Glycosylation of C13-apocarotenoids has several functions in plants, including increased allelopathic activity of the aglycone, facilitating exudation by roots and allowing symbiosis with arbuscular mycorrhizal fungi. The results enable in-depth analyses of the roles of glycosylated norisoprenoid allelochemicals, the physiological functions of apocarotenoids during arbuscular mycorrhizal colonization and the associated maintenance of carotenoid homeostasis.One-sentence summaryWe identified six transferases in Nicotiana benthamiana and Mentha x piperita, two rich sources of glycosylated apocarotenoids that catalyze the unprecedented glycosylation of a range of hydroxylated α- and β-ionone/ionol derivatives and were able to modify bioactivity by glucosylation.

An RNA-Seq analysis of the peach transcriptome with a focus on genes associated with skin colour

Red pigmentation of the skin is an important consumer trait in peach (Prunus persica). The pigment consists largely of anthocyanin. Here, a transcriptomic contrast, based on RNA-Seq technology, was drawn between a white-skinned (Feitao) and a red-skinned (Qiuxue) peach cultivar. The analysis identified 2407 genes as differentially transcribed in the fruit skin of the two cultivars. Among these were a number of genes known to contribute to anthocyanin synthesis. A quantitative real-time PCR assay was used to validate the RNA-Seq-based estimates of transcript abundance for 14 differentially transcribed genes. Anthocyanin synthesis was observed in the skin of Qiuxue fruit during the late ripening stage, matching the high transcript abundance of the gene encoding UDP glucose: flavonoid 3-O-glucosyltransferase, the final step in the synthesis of anthocyanin.

Hammondia hammondi Harbors Functional Orthologs of the Host-Modulating Effectors GRA15 and ROP16 but Is Distinguished from Toxoplasma gondii by a Unique Transcriptional Profile

ABSTRACTToxoplasma gondiiand its nearest extant relative,Hammondia hammondi, are phenotypically distinct despite their remarkable similarity in gene content, synteny, and functionality. To begin to identify genetic differences that might drive distinct infection phenotypes ofT. gondiiandH. hammondi, in the present study we (i) determined whether two known host-interacting proteins, dense granule protein 15 (GRA15) and rhoptry protein 16 (ROP16), were functionally conserved inH. hammondiand (ii) performed the first comparative transcriptional analysis ofH. hammondiandT. gondiisporulated oocysts. We found that GRA15 and ROP16 fromH. hammondi(HhGRA15 and HhROP16) modulate the host NF-κB and STAT6 pathways, respectively, when expressed heterologously inT. gondii. We also found the transcriptomes ofH. hammondiandT. gondiito be highly distinct. Consistent with the spontaneous conversion ofH. hammonditachyzoites into bradyzoites bothin vitroandin vivo,H. hammondihigh-abundance transcripts are enriched for genes that are of greater abundance inT. gondiibradyzoites. We also identified genes that are of high transcript abundance inH. hammondibut are poorly expressed in multipleT. gondiilife stages, suggesting that these genes are uniquely expressed inH. hammondi. Taken together, these data confirm the functional conservation of knownT. gondiivirulence effectors inH. hammondiand point to transcriptional differences as a potential source of the phenotypic differences between these species.

Characterization of the polydnaviral ‘T. rostrale virus’ (TrV) gene family: TrV1 expression inhibits in vitro cell proliferation

Tranosema rostrale ichnovirus (TrIV) is a polydnavirus (PDV) transmitted by the endoparasitic wasp T. rostrale to its host Choristoneura fumiferana during oviposition. PDV genes are expressed in infected caterpillars, causing physiological disturbances that promote the survival of the developing endoparasite. The previously sequenced genome of TrIV contains ~86 genes organized in multigene families and distributed on multiple segments of circular dsDNA. Among these, the ‘T. rostrale virus’ (TrV) family comprises seven genes that are absent in other PDV genomes examined to date and whose function(s) remain(s) unknown. Here, we initiated a functional analysis of the TrV family using qPCR, transfection and RNAi approaches. TrV family genes were weakly expressed in wasp ovaries, but some displayed high transcript abundance in parasitized caterpillars. Whilst TrV1 was the most highly transcribed TrV gene in infected caterpillars, transcript levels for TrV5 and TrV6 were nearly undetectable, indicating that they may be pseudogenes. Temporal and tissue-specific patterns of transcript abundance were similar for all expressed TrV family genes, indicative of an apparent lack of difference in function or tissue specificity. Infection of Cf-203 and Sf-21 insect cells with TrIV led to a dose-dependent inhibition of cell proliferation with no sign of apoptosis. Whilst similar inhibition was observed following transfection of cells with a cloned genome segment carrying the TrV1 gene, RNA interference targeting TrV1 largely restored cell growth in TrIV-infected cells, indicating that TrV1 expression was responsible for the observed inhibition. We suggest that TrV genes may contribute to host developmental disruption by interfering with host-cell proliferation during parasitism.

Quantitative characterization of prostaglandins in the uterus of early pregnant cattle

Prostaglandins (PGs) are important regulators of reproductive processes including early embryonic development. We analyzed the most relevant PG in bovine uteri at different preimplantation pregnancy stages when compared with non-pregnant controls. Additionally, endometrium and trophoblast tissues were examined regarding specific enzymes and receptors involved in PG generation and function. Simmental heifers were artificially inseminated or received seminal plasma only. At days 12, 15, or 18, post-estrus uteri were flushed for PG determination by liquid chromatography–tandem mass spectrometry. Endometrium and trophoblast tissues were sampled for RNA extraction and quantitative real-time PCR analysis. At all days and points of time examined, the concentration of 6-keto PGF1α (stable metabolite of PGI2) was predominant followed by PGF2α>PGE2>PGD2≈TXB2 (stable metabolite of TXA2). At days 15 and 18, PG increased from overall low levels at day 12, with a much more pronounced increase during pregnancy. The PGF2α/PGE2 ratio was not influenced by status. The highest PG concentration was measured at day 15 with 6-keto PGF1α (6.4 ng/ml) followed by PGF2α (1.1 ng/ml) and PGE2 (0.3 ng/ml). Minor changes in endometrial PG biosynthesis enzymes occurred due to pregnancy. Trophoblasts revealed high transcript abundance of general and specific PG synthases contributing to uterine PG. As PGI2 and PGF2α receptors were abundantly expressed by the trophoblast, abundant amounts of PGI2 and PGF2α in the uterine lumen point towards an essential role of PG for the developing embryo. High amounts of PG other than PGE2 in the preimplantation uterus may be essential rather than detrimental for successful reproduction.