Hanneke Huvenne

Delivery of dsRNA for RNAi in insects: an overview and future directions

Abstract  RNA interference (RNAi) refers to the process of exogenous double‐stranded RNA (dsRNA) silencing the complementary endogenous messenger RNA. RNAi has been widely used in entomological research for functional genomics in a variety of insects and its potential for RNAi‐based pest control has been increasingly emphasized mainly because of its high specificity. This review focuses on the approaches of introducing dsRNA into insect cells or insect bodies to induce effective RNAi. The three most common delivery methods, namely, microinjection, ingestion, and soaking, are illustrated in details and their advantages and limitations are summarized for purpose of feasible RNAi research. In this review, we also briefly introduce the two possible dsRNA uptake machineries, other dsRNA delivery methods and the history of RNAi in entomology. Factors that influence the specificity and efficiency of RNAi such as transfection reagents, selection of dsRNA region, length, and stability of dsRNA in RNAi research are discussed for further studies.

Bombyx mori DNA/RNA non-specific nuclease: Expression of isoforms in insect culture cells, subcellular localization and functional assays

A DNA/RNA non-specific alkaline nuclease (BmdsRNase) was isolated from the digestive juice of Bombyx mori. While originally reported to be produced by the midgut only, in this project it was found that the mRNA of this enzyme was also expressed in the epidermis, fat body, gut, thoracic muscles, Malpighian tubules, brain, and silk glands of 5th instar larvae, indicating additional functions to its reported role in nucleic acid digestion in the midgut. In order to study the functional properties of BmdsRNase, three pEA-BmdsRNase expression constructs were generated, characterized by presence or absence of a signal peptide and a propeptide, and used for expression in lepidopteran Hi5 tissue culture cells. Western blot indicated that these different forms of BmdsRNase protein were not secreted into the growth medium, while they were detected in the pellets and supernatants of Hi5 cell extracts. Nucleic acids cleavage experiments indicated that full-length BmdsRNase could digest dsRNA and that the processed form (absence of signal peptide and propeptide) of BmdsRNase could degrade both DNA and dsRNA in Hi5 cell culture. Using a reporter assay targeted by transfected homologous dsRNA, it was shown that the digestive property of the processed form could interfere with the RNAi response. Immunostaining of processed BmdsRNase protein showed asymmetric localization in the cellular cytoplasm and co-localization with Flag-tagged Dicer-2 was also observed. In conclusion, our in vitro studies indicated that intracellular protein isoforms of BmdsRNase can be functional and involved in the regulation of nucleic acid metabolism in the cytoplasm. In particular, because of its propensity to degrade dsRNA, the enzyme might be involved in the innate immune response against invading nucleic acids such as RNA viruses.

Search for limiting factors in the RNAi pathway in silkmoth tissues and the Bm5 cell line: the RNA-binding proteins R2D2 and Translin.

RNA interference (RNAi), an RNA-dependent gene silencing process that is initiated by double-stranded RNA (dsRNA) molecules, has been applied with variable success in lepidopteran insects, in contrast to the high efficiency achieved in the coleopteran Tribolium castaneum. To gain insight into the factors that determine the efficiency of RNAi, a survey was carried out to check the expression of factors that constitute the machinery of the small interfering RNA (siRNA) and microRNA (miRNA) pathways in different tissues and stages of the silkmoth, Bombyx mori. It was found that the dsRNA-binding protein R2D2, an essential component in the siRNA pathway in Drosophila, was expressed at minimal levels in silkmoth tissues. The silkmoth-derived Bm5 cell line was also deficient in expression of mRNA encoding full-length BmTranslin, an RNA-binding factor that has been shown to stimulate the efficiency of RNAi. However, despite the lack of expression of the RNA-binding proteins, silencing of a luciferase reporter gene was observed by co-transfection of luc dsRNA using a lipophilic reagent. In contrast, gene silencing was not detected when the cells were soaked in culture medium supplemented with dsRNA. The introduction of an expression construct for Tribolium R2D2 (TcR2D2) did not influence the potency of luc dsRNA to silence the luciferase reporter. Immunostaining experiments further showed that both TcR2D2 and BmTranslin accumulated at defined locations within the cytoplasm of transfected cells. Our results offer a first evaluation of the expression of the RNAi machinery in silkmoth tissues and Bm5 cells and provide evidence for a functional RNAi response to intracellular dsRNA in the absence of R2D2 and Translin. The failure of TcR2D2 to stimulate the intracellular RNAi pathway in Bombyx cells is discussed.

Colorado potato beetle (Coleoptera) gut transcriptome analysis: expression of RNA interference-related genes.

In the search for new methods of pest control, the potential of RNA interference (RNAi) is being explored. Because the gut is the first barrier for the uptake of double‐stranded (ds)RNA, pyrosequencing of the gut transcriptome is a powerful tool for obtaining the necessary sequences for specific dsRNA‐mediated pest control. In the present study, a dataset representing the gut transcriptome of the Colorado potato beetle (CPB; Leptinotarsa decemlineata) was generated and analysed for the presence of RNAi‐related genes. Almost all selected genes that were implicated in silencing efficiency at different levels in the RNAi pathway (core machinery, associated intracellular factors, dsRNA uptake, antiviral RNAi, nucleases), which uses different types of small RNA (small interfering RNA, microRNA and piwi‐RNA), were expressed in the CPB gut. Although the database is of lower quality, the majority of the RNAi genes are also found to be present in the gut transcriptome of the tobacco hornworm [TH; Manduca sexta (19 out of 35 genes analysed)]. The high quality of the CPB transcriptome database will lay the foundation for future gene expression and functional studies regarding the gut and RNAi.

Brenneria salicis, the bacterium causing watermark disease in willow, resides as an endophyte in wood

Brenneria salicis has been studied in willow wood only in relation to watermark disease. In this pathogenic condition, the bacterium occurs at high concentrations. Pathogenicity of B. salicis is still uncontrollable and the disease unpredictable because the plant-bacteria interaction is not understood. Thanks to molecular techniques B. salicis can be detected at low concentrations, which are found in most non-pathogenic interactions. Brenneria salicis was identified and traced with a new specific three-primer polymerase chain reaction and its identity and relative concentration in biological samples confirmed through denaturing gradient gel electrophoresis profiling. Brenneria salicis was found in symptomless willows sampled randomly in Flanders agricultural areas, in young nursery willows, and also in poplar (Populus) and alder (Alnus). It harboured the nitrogenase reductase gene NifH and promoted growth and chlorophyll in willow. Inoculated luminescence-marked B. salicis circulated through the whole plant without inducing disease and exuded at the leaf margins. Other willow endophytes identified were Rahnella, Sphingomonas and Methylobacterium. In conclusion, because endophytic B. salicis is generally observed in willow, disease must not be dependent on infection. Leaf-to-leaf spread is proposed as an important mechanism for spread of B. salicis.

Nutritional imbalance caused by nitrogen excess is correlated with the occurrence of watermark disease in white willow

Willows in the Flemish region are threatened by the watermark disease, a wood disease caused by the bacterium Brenneria salicis (Hauben et al. Syst Appl Microbiol 21:384–397, 1998). Affected trees are observed to grow at specific sites which seem more sensitive to periodical watermark symptom development than other sites. In this study we aim at revealing abiotic site factors that could play a role in triggering the watermark disease. During three consecutive years we analyzed stands of young isogenic willows (Salix alba L. cv. Lichtenvoorde), which were planted on four different sites in an agricultural environment. These test sites were chosen, based on the occurrence/absence of watermark disease in the old willows that grow on these sites. Two sites had old willow pollards with clear watermark symptoms, and on the two other sites the old willows were unaffected. Differences in abiotic characteristics between the sites were analysed and their effects on the nutrient status in willow were measured in the isogenic willows that were newly planted. Brenneria salicis-specific multiplex PCR revealed the presence of this pathogenic bacterium in both the old and the freshly planted young willows. At each site, the physical and chemical characteristics of soil and groundwater were investigated; the young tree growth was monitored and nutritional status of the willow plantings was assessed by foliar analysis. Physical soil properties including soil type and profile water availability, and chemical soil analysis were unable to explain differences between healthy and affected sites. Diseased sites, however, had significantly higher N concentrations and traces of herbicides in the groundwater. In young trees, foliar levels of N and S were always greater at the affected sites, while P contents were lower. Dry mass based N concentration in the leaves of the newly planted trees increased systematically over the years, but more at affected than at healthy sites. Also, chlorophyll content was significantly greater at the affected sites. Growth rate of the young isogenic trees was twice as fast at the diseased sites. The presented data suggest that application of fertilisers in agriculture causes an imbalance in the nutritional status of Salix alba L. Excess nitrogen makes this species fast-growing, reflected by an average foliar N:P ratio >10, and more susceptible for watermark disease, indicated by an average foliar N:Ca ratio exceeding 1.5.

Real-time PCR mediated monitoring of Fusarium foetens in symptomatic and non-symptomatic hosts

Fusarium foetens is a recently described aggressive vascular pathogen of Begonia x hiemalis. Since 2004, it has caused severe losses for Begonia growers in Northern Europe and North America. F. foetens is likely to be of exotic origin. Little is known about the accumulation of the fungus in Begonia plants before and during symptom expression and about its host range. We have optimised a molecular detection method for F. foetens by only using the plant part containing the largest amount of the pathogen and by optimising the tissue maceration and DNA extraction techniques. This allowed a reliable detection limit of 2310 spore equivalents per plant and a theoretical detection limit of as low as 84 to 167 spore equivalents per plant. Using this method, we demonstrated exponential accumulation of F. foetens DNA in Begonia roots, resulting in symptoms at a threshold of approximately 107 spore equivalents and levelling off at 109 spore equivalents per plant. The observed rate of accumulation and the amount of pathogen DNA in non-symptomatic plants can be combined to determine whether the cuttings were infected after delivery at the Begonia nursery and to calculate the estimated timing of symptom development. To test the host range, we applied the optimised molecular detection technique. During these tests, only Begonia x hiemalis plants became symptomatic, but many other plant species supported growth of the pathogen. This information can be used to aid pathogen control and has implications for pest risk assessment.

Identification of quorum sensing signal molecules and oligolignols associated with watermark disease in willow (Salix sp.).

The bacterium Brenneria salicis is the causal agent of watermark disease in willow. This work shows the importance of in situ studies and high-resolution separation of biological samples with ultrahigh performance liquid chromatography combined with ion trap mass spectrometry to unambiguously identify molecular compounds associated with this disease. Approximately 40 oligolignols accumulated in wood sap of watermark diseased willow, and are indicative for degradation of the xylem cell wall, of which 15 were structurally assigned based on an earlier study. Many bacteria are known to produce and release quorum sensing signal molecules that switch on the expression of specific, sometimes pathogenic functions. Two quorum sensing signal molecules, N-(3-oxohexanoyl)-l-homoserine lactone and N-(hexanoyl)-l-homoserine lactone, were present in 4/1 ratios in diseased wood and in high-density in vitro cultures of B. salicis at 0.13-1.2 microM concentrations, and absent in healthy wood and in low-density in vitro cultures of B. salicis. Although it is not a proof, it can be an indication for involvement of quorum sensing in B. salicis pathogenesis. Cyclic dipeptides were present at high concentrations in high-density in vitro cultures of B. salicis, but not in situ, and were found not to be involved in quorum sensing signaling, therefore, the attribution of quorum signal properties to cyclic dipeptides isolated from in vitro cultures of pathogenic bacteria should be reconsidered.

Willow wood sap promotes the density‐dependent pathogenesis of Brenneria salicis

Brenneria salicis resides in symptomless willow (Salix spp.) and other tree species, but only willow trees develop watermark disease. To understand the conversion of B. salicis into a pathogen, its pathogenicity and differential growth in the various tree species are studied. Brenneria salicis was detected by plating and polymerase chain reaction-based techniques. Cell wall degradation and quorum sensing (QS) were assayed as possible pathogenicity mechanisms in wood. Differences in B. salicis growth capacities were tested in wood sap of the trees. Watermark diseased willow wood contained high concentrations of B. salicis with QS-induced cellulase activity. In the fall, wood sap of willow, and not of poplar and alder, promoted high density growth of B. salicis. In situ, B. salicis was the dominant bacterial type in willow wood during the fall and winter period. Willow sustains high densities of B. salicis at the time of leaf shedding. The cellulase in the immobilized wood sap has then a long-lasting contact with the xylem cell wall. Timing of dormancy and subsequent winter conditions might interfere with sap composition, B. salicis density, activity and survival, and be the reason, at least partly, for the variable occurrence of the disease.