Small RNA viruses of insects : expression in plants and RNA silencing

Interest in insect small RNA viruses (SRVs) has grown slowly but steadily. A number of new viruses have been analyzed at the sequence level, adding to our knowledge of their diversity at the level of both individual virus species and families. In particular, a number of possible new virus families have emerged. This research has largely been driven by interest in their potential for pest control, as well as in their importance as the causal agents of disease in beneficial arthropods. At the same time, research into known viruses has made valuable contributions to our understanding of an emerging new field of central importance to molecular biology-the existence of RNA-based gene silencing, developmental control, and adaptive immune systems in eukaryotes. Subject to RNA-based adaptive immune responses in their hosts, viruses have evolved a variety of genes encoding proteins capable of suppressing the immune response. Such genes were first identified in plant viruses, but the first examples known from animal viruses were identified in insect RNA viruses. This chapter will address the diversity of insect SRVs, and attempts to harness their simplicity in the engineering of transgenic plants expressing viruses for resistance to insect pests. We also describe RNA interference and antiviral pathways identified in plants and animals, how they have led viruses to evolve genes capable of suppressing such adaptive immunity, and the problems presented by these pathways for the strategy of expressing viruses in transgenic plants. Approaches for countering these problems are also discussed. © 2006 Elsevier Inc. All rights reserved.

Expression of Caenorhabditis elegans RNA-directed RNA polymerase in transgenic Drosophila melanogaster does not affect morphological development

Drosophila melanogaster, along with all insects and the vertebrates, lacks an RdRp gene. We created transgenic strains of Drosophila melanogaster in which the rrf-1 or ego-1 RdRp genes from C. elegans were placed under the control of the yeast GAL4 upstream activation sequence. Activation of the gene was performed by crossing these lines to flies carrying the GAL4 transgene under the control of various Drosophila enhancers. RT-PCR confirmed the successful expression of each RdRp gene. The resulting phenotypes indicated that introduction of the RdRp genes had no effect on D. melanogaster morphological development. © 2010 Springer Science+Business Media B.V.

The roles of plant dsRNA-binding proteins in RNAi-like pathways

Dicers are associated with double-stranded RNA-binding proteins (dsRBPs) in animals. In the plant, Arabidopsis, there are four dicer-like (DCL) proteins and five potential dsRBPs. These DCLs act redundantly and hierarchically. However, we show there is little or no redundancy or hierarchy amongst the DRBs in their DCL interactions. DCL1 operates exclusively with DRB1 to produce micro (mi)RNAs, DCL4 operates exclusively with DRB4 to produce trans-acting (ta) siRNAs and 21nt siRNAs from viral RNA. DCL2 and DCL3 produce viral siRNAs without requiring assistance from any dsRBP. DRB2, DRB3 and DRB5 appear unnecessary for mi-, tasi-, viral si-, or heterochromatinising siRNA production but act redundantly in a developmental pathway. © 2008 Federation of European Biochemical Societies.

Cloning and characterization of microRNAs from Brassica napus

A library containing approximately 40,000 small RNA sequences was constructed for Brassica napus. Analysis of 3025 sequences obtained from this library resulted in the identification of 11 conserved miRNA families, which were validated by secondary structure prediction using surrounding sequences in the Brassica genome. Two 21 nt small RNA sequences reside within the arm of a pre-miRNA like stem-loop structure, making them likely candidates for novel non-conserved miRNAs in B. napus. Most of the conserved miRNAs were expressed at similar levels in a F1 hybrid B. napus line and its four double haploid progeny that showed marked variations in phenotypes, but many were differentially expressed between B. napus and Arabidopsis. The miR169 family was expressed at high levels in young leaves and stems, but was undetectable in roots and mature leaves, suggesting that miR169 expression is developmentally regulated in B. napus. © 2007 Federation of European Biochemical Societies.

Posttranscriptional gene silencing is not compromised in the Arabidopsis CARPEL FACTORY (DICER-LIKE1) mutant, a homolog of dicer-1 from Drosophila

Posttranscriptional silencing (PTGS) in plants, nematodes, Drosophila, and perhaps all eukaryotes operates by sequence-specific degradation or translational inhibition of the target mRNA. These processes are mediated by duplexed RNA. In Drosophila and nematodes, double-stranded (ds)RNA or self-complementary RNA is processed into fragments of approximately 21 nt by Dicer-1 [1, 2]. These small interfering RNAs (siRNAs) serve as guides to target degradation of homologous single-stranded (ss)RNA [1, 3]. In some cases, the approximately 21 nt guide fragments derived from endogenous, imperfectly self-complementary RNAs cause translational inhibition of their target mRNAs, with which they have substantial, but not perfect sequence complementarity [4-6]. These small temporal RNAs (stRNAs) belong to a class of noncoding microRNAs (miRNAs), 20-24 nt in length, that are found in flies, plants, nematodes, and mammals [4, 6-12]. In nematodes, the Dicer-1 enzyme catalyzes the production of both siRNA and stRNA [2, 13-15]. Mutation of the Arabidopsis Dicer-1 homolog, CARPEL FACTORY (CAF), blocks miRNA production [1, 4, 16-18]. Here, we report that the same caf mutant does not block either PTGS or siRNA production induced by self-complementary hairpin RNA. This suggests either that this mutation only impairs miRNA formation or, more interestingly, that plants have two distinct dicer-like enzymes, one for miRNA and another for siRNAi production.

The genome organization and affinities of an Australian isolate of carrot mottle umbravirus

The genomic sequence of an Australian isolate of carrot mottle umbravirus (CMoV-A) was determined from cDNA generated from dsRNA. This provides the first data on the genome organization and phylogeny of an umbravirus. The 4201-nucleotide genome contains four major open reading frames (ORFs). Analysis suggests that ORF2 encodes an RNA-dependent RNA polymerase, that ORF4 encodes a movement protein, and that the virus has no coat protein gene. The functions of ORFs 1 and 3 remain unknown. ORF2 is probably translated following ribosomal frameshifting. ORFs 3 and 4 are probably translated from a subgenomic mRNA. Sequence comparisons showed CMoV-A to be closely related to pea enation mosaic RNA2 NA2), but also to have affinities with the Bromoviridae. These findings shed light on the relationships between the luteoviruses, PEMV, and the umbraviruses and on the relationships between the carmo-like viruses and the Bromoviridae.

Multilocus sequence analysis provides insights into molecular epidemiology of Chlamydia pecorum infections in Australian sheep, cattle and koalas

Chlamydia pecorum is a significant pathogen of domestic livestock and wildlife. We have developed a C. pecorum-specific multilocus sequence analysis (MLSA) scheme to examine the genetic diversity of and relationships between Australian sheep, cattle, and koala isolates. An MLSA of seven concatenated housekeeping gene fragments was performed using 35 isolates, including 18 livestock isolates (11 Australian sheep, one Australian cow, and six U.S. livestock isolates) and 17 Australian koala isolates. Phylogenetic analyses showed that the koala isolates formed a distinct clade, with limited clustering with C. pecorum isolates from Australian sheep. We identified 11 MLSA sequence types (STs) among Australian C. pecorum isolates, 10 of them novel, with koala and sheep sharing at least one identical ST (designated ST2013Aa). ST23, previously identified in global C. pecorum livestock isolates, was observed here in a subset of Australian bovine and sheep isolates. Most notably, ST23 was found in association with multiple disease states and hosts, providing insights into the transmission of this pathogen between livestock hosts. The complexity of the epidemiology of this disease was further highlighted by the observation that at least two examples of sheep were infected with different C. pecorum STs in the eyes and gastrointestinal tract. We have demonstrated the feasibility of our MLSA scheme for understanding the host relationship that exists between Australian C. pecorum strains and provide the first molecular epidemiological data on infections in Australian livestock hosts.

Epidemiological and molecular features of dengue virus type-1 in New Caledonia, South Pacific, 2001–2013

Background The epidemiology of dengue in the South Pacific has been characterized by transmission of a single dominant serotype for 3–5 years, with subsequent replacement by another serotype. From 2001 to 2008 only DENV-1 was reported in the Pacific. In 2008, DENV-4 emerged and quickly displaced DENV-1 in the Pacific, except in New Caledonia (NC) where DENV-1 and DENV-4 co-circulated in 2008–2009. During 2012–2013, another DENV-1 outbreak occurred in NC, the third DENV-1 outbreak in a decade. Given that dengue is a serotype-specific immunizing infection, the recurrent outbreaks of a single serotype within a 10-year period was unexpected. Findings This study aimed to inform this phenomenon by examining the phylogenetic characteristics of the DENV-1 viruses in NC and other Pacific islands between 2001 and 2013. As a result, we have demonstrated that NC experienced introductions of viruses from both the Pacific (genotype IV) and South-east Asia (genotype I). Moreover, whereas genotype IV and I were co-circulating at the beginning of 2012, we observed that from the second half of 2012, i.e. during the major DENV-1 outbreak, all analyzed viruses were genotype I suggesting that a genotype switch occurred. Conclusions Repeated outbreaks of the same dengue serotype, as observed in NC, is uncommon in the Pacific islands. Why the earlier DENV-1 outbreaks did not induce sufficient herd immunity is unclear, and likely multifactorial, but the robust vector control program may have played a role by limiting transmission and thus maintaining a large susceptible pool in the population. Keywords: Dengue; Phylogeny; Genotype; Epidemics; New Caledonia

UNIFOLIATA regulates leaf and flower morphogenesis in pea

Background The vegetative phenotype of the pea mutant unifoliata (uni) is a simplification of the wild-type compound leaf to a single leaflet. Mutant uni plants are also self-sterile and the flowers resemble known floral meristem and organ identity mutants. In Antirrhinum and Arabidopsis, mutations in the floral meristem identity gene FLORICAULA/LEAFY (FLO/LFY) affect flower development alone, whereas the tobacco FLO/LFY homologue, NFL, is expressed in vegetative tissues, suggesting that NFL specifies determinacy in the progenitor cells for both flowers and leaves. In this paper, we characterised the pea homologue of FLO/LFY. Results The pea cDNA homologue of FLO/LFY, PEAFLO, mapped to the uni locus in recombinant-inbred mapping populations and markers based on PEAFLO cosegregated with uni in segregating sibling populations. The characterisation of two spontaneous uni mutant alleles, one containing a deletion and the other a point mutation in the PEAFLO coding sequences, predicted that PEAFLO corresponds to UNI and that the mutant vegetative phenotype was conferred by the defective PEAFLO gene. Conclusions The uni mutant demonstrates that there are shared regulatory processes in the morphogenesis of leaves and flowers and that floral meristem identity genes have an extended role in plant development. Pleiotropic regulatory genes such as UNI support the hypothesis that leaves and flowers derive from a common ancestral sporophyll-like structure. The regulation of indeterminacy during leaf and flower morphogenesis by UNI may reflect a primitive function for the gene in the pre-angiosperm era.

Turtle ghrelin

In a recent paper, Wang and colleagues described the genomes of two turtles, the Chinese soft-shell turtle (Pelodiscus sinensis) and the green sea turtle (Chelonia mydas)1. A salient finding was an apparent absence of GHRL, the gene encoding the only known circulating orexigen, the peptide hormone ghrelin. The highly conserved GHRL encodes at least two bioactive peptide hormones, ghrelin2 and obestatin3, which are recognized to have a diverse range of functions in a number of cell types and physiological systems4, 5. Wang and colleagues hypothesized that the absence of ghrelin was associated with the low metabolic rate observed in these turtle species1.