Electroporation-mediated RNA interference reveals a role of the multicopper oxidase 2 gene in dragonfly cuticular pigmentation

Dragonflies are colorful insects, and recent RNA sequencing studies have identified a number of candidate genes potentially involved in their color pattern formation and color vision. However, functional aspects of such genes have not been assessed due to the lack of molecular genetic tools applicable to dragonflies. We established an electroporation-mediated RNA interference (RNAi) procedure using the tiny dragonfly Nannophya pygmaea Rambur, 1842 (Odonata: Libellulidae) that targets the multicopper oxidase 2 gene (MCO2; also known as laccase2 gene) responsible for cuticular pigmentation in many insects. RNA sequencing of N. pygmaea and genomic survey of the dragonfly Ladona fulva identified four multicopper oxidase family genes: MCO1, MCO2, MCO3 and multicopper oxidase-related protein gene (MCORP). In N. pygmaea, MCO2 was specifically expressed around the cuticular pigmentation period, whereas MCO1 was constantly expressed. MCORP was expressed at adult stages, and MCO3 was scarcely expressed. When we applied in vivo electroporation, final instar larvae injected with MCO2 small interfering RNA became adults with patchy unpigmented regions. RNAi without in vivo electroporation did not affect cuticular pigmentation, suggesting that dragonflies do not show a systemic RNAi response. These results indicate that MCO2 is required for cuticular pigmentation across diverse insects, and highlight the usefulness of the electroporation-mediated RNAi method in dragonflies.     

Trojan Horse Strategy for Non-invasive Interference of <Emphasis Type="Italic">Clock</Emphasis> Gene in the Oyster <Emphasis Type="Italic">Crassostrea gigas</Emphasis>

RNA interference is a powerful method to inhibit specific gene expression. Recently, silencing target genes by feeding has been successfully carried out in nematodes, insects, and small aquatic organisms. A non-invasive feeding-based RNA interference is reported here for the first time in a mollusk bivalve, the pacific oyster Crassostrea gigas. In this Trojan horse strategy, the unicellular alga Heterocapsa triquetra is the food supply used as a vector to feed oysters with Escherichia coli strain HT115 engineered to express the double-stranded RNA targeting gene. To test the efficacy of the method, the Clock gene, a central gene of the circadian clock, was targeted for knockout. Results demonstrated specific and systemic efficiency of the Trojan horse strategy in reducing Clock mRNA abundance. Consequences of Clock disruption were observed in Clock-related genes (Bmal, Tim1, Per, Cry1, Cry2, Rev.-erb, and Ror) and triploid oysters were more sensitive than diploid to the interference. This non-invasive approach shows an involvement of the circadian clock in oyster bioaccumulation of toxins produced by the harmful alga Alexandrium minutum.     

The complete chloroplast genome sequence of <Emphasis Type="Italic">Pseudoroegneria libanotica</Emphasis>, genomic features,and phylogenetic relationship with <Emphasis Type="Italic">Triticeae</Emphasis> species

Pseudoroegneria libanotica is an important herbage diploid species possessing the St genome. The St genome participates in the formation of nine perennial genera in Triticeae (Poaceae). The whole chloroplast (cp) genome of P. libanotica is 135 026 bp in length. The typical quadripartite structure consists of one large single copy of 80 634 bp, one small single copy of 12 766 bp and a pair of inverted regions (20 813 bp each). The cp genome contains 76 coding genes, four ribosomal RNA and 30 transfer RNA genes. Comparative sequence analysis suggested that: 1) the 737 bp deletion in the cp of P. libanotica was specific in Triticeae species and might transfer into its nuclear genome; 2) hot-spot regions, indels in intergenic regions and protein coding sequences mainly led to the length variation in Triticeae; 3) highly divergence regions combined with negative selection in rpl2, rps12, ccsA, rps8, ndhH, petD, ndhK, psbM, rps3, rps18, and ndhA were identified as effective molecular markers and could be considered in future phylogenetic studies of Triticeae species; and 4) ycf3 gene with rich cpSSRs was suitable for phylogeny analysis or could be used for DNA barcoding at low taxonomic levels. The cpSSRs distribution in the coding regions of diploid Triticeae species was shown for the first time and provided a valuable source for developing primers to study specific simple sequence repeat loci.     

Contribution of <Emphasis Type="Italic">Eutrema salsugineum</Emphasis> Cold Shock Domain Structure to the Interaction with RNA

Plant cold shock domain proteins (CSDPs) are DNA/RNA-binding proteins. CSDPs contain the conserved cold shock domain (CSD) in the N-terminal part and a varying number of the CCHC-type zinc finger (ZnF) motifs alternating with glycine-rich regions in the C-terminus. CSDPs exhibit RNA chaperone and RNA-melting activities due to their non-specific interaction with RNA. At the same time, there are reasons to believe that CSDPs also interact with specific RNA targets. In the present study, we used three recombinant CSDPs from the saltwater cress plant (Eutrema salsugineum)-EsCSDP1, EsCSDP2, EsCSDP3 with 6, 2, and 7 ZnF motifs, respectively, and showed that their nonspecific interaction with RNA is determined by their C-terminal fragments. All three proteins exhibited high affinity to the single-stranded regions over four nucleotides long within RNA oligonucleotides. The presence of guanine in the single-or double-stranded regions was crucial for the interaction with CSDPs. Complementation test using E. coli BX04 cells lacking four cold shock protein genes (ΔcspA, ΔcspB, ΔcspE, ΔcspG) revealed that the specific binding of plant CSDPs with RNA is determined by CSD.     

Genetic and phenotypic characterizations of <Emphasis Type="Italic">Xenorhabdus</Emphasis> species (Enterobacteriales: Enterobacteriaceae) isolated from steinernematid nematodes (Rhabditida: Steinernematidae) in Japan

The bacterial species of the genus Xenorhabdus in the family Enterobacteriaceae have a mutualistic association with steinernematid entomopathogenic nematodes (EPNs), which have been used as biological control agents against soil insect pests. In this study we present the genetic and phenotypic characterizations of the Xenorhabdus species isolated from steinernematid nematodes in Japan. The 18 Japanese Xenorhabdus isolates were classified into five bacterial species based on 16S ribosomal RNA (16S rRNA) gene sequences: Xenorhabdus bovienii, Xenorhabdus hominickii, Xenorhabdus indica, Xenorhabdus ishibashii, and Xenorhabdus japonica. There was no genetic variation between the 16S RNA sequences among the three X. ishibashii isolates, 0–0.1% variation among the five X. hominickii isolates, and 0–0.5% among the eight X. bovienii isolates. Phenotypic characterization demonstrated that representative isolates of the five bacterial species shared common characteristics of the genus Xenorhabdus, and only X. hominickii isolates produced indole. Symbiotic association and co-speciation of Xenorhabdus bacteria with Steinernema nematodes from Japan are discussed.     

Polyphasic characterisation of three novel species of <Emphasis Type="Italic">Paraboeremia</Emphasis>

Paraboeremia was recently introduced for a distinct lineage in the family Didymellaceae. Currently, three species are included, i.e. P. adianticola, P. putaminum and P. selaginellae, all of which are plant pathogens. Paraboeremia is morphologically similar to Phoma but phylogenetically distinct. In this paper, three new species, i.e. Paraboeremia camelliae isolated from Camellia sp., P. litseae from Litsea sp., and P. oligotrophica from cave limestone, are described, illustrated and compared with closely related taxa. Phylogenetic analysis based on the multi-locus sequences of the internal transcribed spacer regions 1 and 2 and 5.8S nuclear ribosomal RNA gene (ITS), partial large subunit 28S nrDNA region (LSU), partial β-tubulin (TUB2) gene and RNA polymerase II (RPB2) gene regions confirmed the distinction of these species in Paraboeremia. These three new species were discovered from habitats and hosts that are previously unknown from this genus.     

Two new species of <Emphasis Type="Italic">Pseudopyricularia</Emphasis> from Iran

Two new species, Pseudopyricularia hyrcaniana and P. iraniana, are described, illustrated, and discussed in this paper. Phylogenetic analyses of the large subunit of the ribosomal RNA gene cluster and their internal transcribed spacer regions, and protein encoding gene introns and exons, including the largest subunit of the RNA polymerase II and calmodulin, confirmed their placement in Pseudopyricularia. Pseudopyricularia hyrcaniana was isolated as a pathogen from leaves of Cyperus alternifolius, while P. iraniana was from Juncus sp. Conidia of P. hyrcaniana are obclavate and 1-septate; those of P. iraniana are fusiform or cylindrical and 2-septate. Analyses of multigene sequences confirm the distinction between P. hyrcaniana and P. iraniana, and reveal their relationship with their allies in Pyriculariaceae.     

<Emphasis Type="Italic">Cortinarius majestaticus</Emphasis> comb. nov.: phylogenetic evidence for the transfer of <Emphasis Type="Italic">Descolea majestatica</Emphasis> to <Emphasis Type="Italic">Cortinarius</Emphasis>

Descolea majestatica is an agaric with features described as intermediate between the genera Descolea Singer and Rozites P. Karst. (≡ Cortinarius). Molecular phylogenetic analysis of nuclear ribosomal internal transcribed spacer (ITS), large ribosomal subunit (LSU) and RNA polymerase second largest subunit (RPB2) sequences indicates that D. majestatica is nested within the genus Cortinarius, with its closest relative a sequestrate Cortinarius species from Argentina. Taxonomic recombination is made to restore the monophyly of Cortinarius and Descolea.     

Heterologous expression of an RNA-binding protein affects flowering time as well as other developmental processes in <Emphasis Type="Italic">Solanaceae</Emphasis>

Flowering time in members of the Solanaceae plant family, such as pepper (Capsicum spp.) and tomato (Solanum lycopersicum), is an important agronomic trait for controlling shoot architecture and improving yield. To investigate the feasibility of flowering time regulation in tomato, an RNA-binding protein (RBP) encoding gene homologous to human Nucleolar protein interacting with the forkhead-associated (FHA) domain of pKI-67 (NIFK), CaRBP, was isolated from hot pepper. The function of CaRBP was determined in transgenic tomato. The deduced amino acid sequence includes an RNA recognition motif (RRM) and showed most similarity to the RRM present in a putative RBP encoded by human NIFK. CaRBP was highly expressed in the vegetative and reproductive tissues, such as leaves and fruits, respectively. Subcellular localization analysis indicated that CaRBP is a nucleolar protein. Heterologous expression of CaRBP under 35S promoter in tomato plants induced severe alteration of flowering with additional defects of vegetative organs. This floral retardation was associated with the alteration of SFT/SP3D and SlSOC1s as floral integrators. Furthermore, CaRBP reduces the expression levels of SlCOLs/TCOLs via changes in the expression of SlCDF3, SlFBHs, and SlFKF1s. This indicates a repressive effect of CaRBP on the regulation of flowering time in tomato. Overall, these results suggest that alteration in CaRBP expression levels may provide an effective means of controlling flowering time in day-neutral Solanaceae.     

Genome-Wide Identification of the Dicer-Like,Argonaute, and RNA-Dependent RNA Polymerase Gene Families in Cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.)

Dicer, Argonaute (AGO), and RNA-dependent RNA polymerase (RDR) comprise the core components of RNA-induced silencing complexes, which trigger RNA silencing. Here, we performed a complete analysis of the cucumber Dicer-like, AGO, and RDR gene families including the gene structure, genomic localization, and phylogenetic relationships among family members. We identified seven CsAGO genes, five CsDCL genes, and eight CsRDR genes in cucumber. Based on phylogenetic analysis, each of these genes families was categorized into three or four clades. The orthologs of CsAGOs, CsDCLs, and CsRDRs were identified in apple, peach, wild strawberry, foxtail millet, and maize, and the evolutionary relationships among the orthologous gene pairs were investigated. We also investigated the expression levels of CsAGOs, CsDCLs, and CsRDRs in various cucumber tissues. All CsAGOs were relatively higher upregulated in leaves and tendrils than in other organs, especially CsAGO1c, CsAGO1d, and CsAGO7. All CsDCL genes were relatively higher upregulated in tendrils, with almost no expression detected for CsDCL1, CsDCL4a, or CsDCL4b in other organs. In addition, CsRDR1a, CsRDR2, CsRDR3, and CsRDR6 had relatively higher upregulation in tendrils, whereas almost all CsRDRs were downregulation in other organs. The results of this study will facilitate further studies of gene silencing pathways in cucumber.     

Clavicipitaceous entomopathogens: new species in <Emphasis Type="Italic">Metarhizium</Emphasis> and a new genus <Emphasis Type="Italic">Nigelia</Emphasis>

In several surveys in the tropical forests in Thailand, specimens that looked morphologically similar to Metarhizium martiale and Cordyceps variegata, as well as other Metarhizium species were collected and cultured in vitro. A combined phylogeny of several genes including the small (18S) and large (28S) subunits of the ribosomal DNA, elongation factor 1-α (TEF), RNA polymerase II subunits 1 and 2 (RPB1, RPB2) genes has shown these to be new taxa in the Clavicipitaceae. Nigelia is described as a new genus closely related to Metarhizium, to the scale insect pathogens Aschersonia (Hypocrella), Samuelsia and Moelleriella, and to plant pathogens in Claviceps and Balansia, and other relatives. Nigelia comprises M. martiale and a new species Nigelia aurantiaca, which has been found infecting lepidopteran larvae and which produces pseudoimmersed, obliquely arranged, obpyriform perithecia with curved or bent ostioles and with whole (non-separating) cylindric ascospores. Metarhizium chaiyaphumense, M. kalasinense, M. prachinense, M. samlanense, and M. takense are described as new species of Metarhizium. Metarhizium martiale is transferred to Nigelia, and Paecilomyces reniformis is transferred to Metarhizium.     

On the Mechanism of Action of <Emphasis Type="Italic">lac</Emphasis> Repressor

Chen et al. have proved conclusively that lac repressor and RNA polymerase bind independently to wild type lac DNA in vitro. To explain the lacp ^s mutation, which causes competitive binding between repressor and polymerase, they suggest that a new promoter site has been created near the lac operator.     

Complete mitochondrial DNA sequence and phylogenetic analysis of Zhikong scallop <Emphasis Type="Italic">Chlamys farreri</Emphasis> (Bivalvia: Pectinidae)

The complete mitochondrial genome of Zhikong scallop Chlamys farreri is 21,695 bp in length and contains 12 protein-coding genes (the atp8 gene is absent, as in most bivalves), 2 ribosomal RNA genes, and 22 transfer RNA genes. The heavy strand has an overall A+T content of 58.7%. GC and AT skews for the mt genome of C. farreri are 0.337 and ?0.184, respectively, indicating the nucleotide bias against C and A. The mitochondrial gene order of C. farreri differs drastically from the scallops Argopecten irradians, Mimachlamys nobilis and Placopecten magellanicus, which belong to the same family Pectinidae. 6623 bp non-coding nucleotides exist intergenically in the mitogenome of C. farreri, with a large continuous sequence (4763 bp) between tRNA ^Val and tRNA ^Asn . Two repeat families are found in the large continuous sequence, which seems to be a common feature of scallops. Phylogenetic analysis based on 12 concatenated amino acid sequences of protein-coding genes supports the monophyly of Pectinidae and paraphyletic Pteriomorphia with respect to Heteroconchia.     

Identification of a novel <Emphasis Type="Italic">Drosophila</Emphasis> gene, <Emphasis Type="Italic">beltless</Emphasis>, using injectable embryonic and adult RNA interference (RNAi)

Background

RNA interference (RNAi) is a process triggered by a double-stranded RNA that leads to targeted down-regulation/silencing of gene expression and can be used for functional genomics; i.e. loss-of-function studies. Here we report on the use of RNAi in the identification of a developmentally important novel Drosophila (fruit fly) gene (corresponding to a putative gene CG5652/GM06434), that we named beltless based on an embryonic loss-of-function phenotype.

Results

Beltless mRNA is expressed in all developmental stages except in 0–6 h embryos. In situ RT-PCR localized beltless mRNA in the ventral cord and brain of late stage embryos and in the nervous system, ovaries, and the accessory glands of adult flies. RNAi was induced by injection of short (22 bp) beltless double-stranded RNAs into embryos or into adult flies. Embryonic RNAi altered cuticular phenotypes ranging from partially-formed to missing denticle belts (thus beltless) of the abdominal segments A2–A4. Embryonic beltless RNAi was lethal. Adult RNAi resulted in the shrinkage of the ovaries by half and reduced the number of eggs laid. We also examined Df(1)RK4 flies in which deletion removes 16 genes, including beltless. In some embryos, we observed cuticular abnormalities similar to our findings with beltless RNAi. After differentiating Df(1)RK4 embryos into those with visible denticle belts and those missing denticle belts, we assayed the presence of beltless mRNA; no beltless mRNA was detectable in embryos with missing denticle belts.

Conclusions

We have identified a developmentally important novel Drosophila gene, beltless, which has been characterized in loss-of-function studies using RNA interference. The putative beltless protein shares homologies with the C. elegans nose resistant to fluoxetine (NRF) NRF-6 gene, as well as with several uncharacterized C. elegans and Drosophila melanogaster genes, some with prominent acyltransferase domains. Future studies should elucidate the role and mechanism of action of beltless during Drosophila development and in adults, including in the adult nervous system.