Transformation and gene silencing technologies to control Helicoverpa armigera

Stable transformation is an essential tool for molecular biologists working on non-model organisms. The ability to introduce and express genes of choice in an organism provides a means to investigate important molecular questions such as gene function, biochemical pathway analysis, reporter gene studies and developmental processes. My PhD studies have focused on the transformation of the pest Helicoverpa armigera with the reporter gene EGFP (enhanced green fluorescent protein). There are essentially two parts to transformation, 1) DNA delivery and 2) target gene integration. Biolistics is a technique for DNA delivery that involves coating microscopic gold particles with the DNA of choice and accelerating them at high velocity into cells. Biolistics has been widely used to transform many kinds of plant tissue, and has had mixed success transforming Drosophila embryos. Extensive attempts to adapt biolistics to transform H. armigera embryos proved fruitless, with too many technical hurdles to overcome. These difficulties led me to use microinjection delivery of DNA into embryos. Compared to biolistics, microinjection is a lower-throughput technique delivering DNA to individual embryos, however, this method is well established, with none of the technological hurdles raised by biolistics. Results for microinjection were encouraging, with a high frequency of transient EGFP expression and the generation of two putative EGFP stably transformed H. armigera lines. Following DNA delivery, integration of target genes into insect genomes is commonly mediated by transposon-based gene movement. I used the class II transposon piggyBac to facilitate the movement of the EGFP reporter gene into the genome of H. armigera embryos as a visual proof of integration. The development of an effective microinjection technique also allowed exploration of the role of RNA interference (RNAi) in H. armigera. This highly specific silencing technique was used with a view to knocking down the expression of genes essential for the growth and development of this insect. This in turn will form the basis for the development of a targeted genetic control mechanism. By co-injecting an EGFP construct and either siRNA or dsRNA against EGFP into embryos, I observed a significant reduction in the frequency and level of EGFP fluorescence in embryos. Quantitative real time PGR validated these observations, showing a reduction in EGFP transcript upon co-injection with dsRNA or siRNA. These results suggest that the RNAi pathway is conserved in H. armigera and provide a basis for testing phenotypic effects of silencing specific genes in this insect. For RNAi to be developed as an effective pest control mechanism, the parameters of RNAi in specific pests must be thoroughly understood. In particular, is systemic RNAi functional in H. armigera? For RNAi to be most effective, the silencing signal must be able to spread throughout all cells in the organism. One gene identified in C. elegans, known as SID-1, plays a role in mediating systemic spread of the RNAi signal, which may involve the cell-cell movement of siRNAs. Not all organisms contain a SID-1 gene. For example, no SID-1 homologue has been identified in Drosophila, and as a result systemic silencing is absent. I identified two different SID-1-like genes in H. armigera, strongly suggesting the possibility of systemic RNAi in this organism and supporting further studies into the use of RNAi as a pest control mechanism

RNA interference in Lepidoptera: an overview of successful and unsuccessful studies and implications for experimental design

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A Novel Proteinase, SNOWY COTYLEDON4, Is Required for Photosynthetic Acclimation to Higher Light Intensities in Arabidopsis.

International audience: Excess light can have a negative impact on photosynthesis; thus, plants have evolved many different ways to adapt to different light conditions to both optimize energy use and avoid damage caused by excess light. Analysis of the Arabidopsis (Arabidopsis thaliana) mutant snowy cotyledon4 (sco4) revealed a mutation in a chloroplast-targeted protein that shares limited homology with CaaX-type endopeptidases. The SCO4 protein possesses an important function in photosynthesis and development, with point mutations rendering the seedlings and adult plants susceptible to photooxidative stress. The sco4 mutation impairs the acclimation of chloroplasts and their photosystems to excess light, evidenced in a reduction in photosystem I function, decreased linear electron transfer, yet increased nonphotochemical quenching. SCO4 is localized to the chloroplasts, which suggests the existence of an unreported type of protein modification within this organelle. Phylogenetic and yeast complementation analyses of SCO4-like proteins reveal that SCO4 is a member of an unknown group of higher plant-specific proteinases quite distinct from the well-described CaaX-type endopeptidases RAS Converting Enzyme1 (RCE1) and zinc metallopeptidase STE24 and lacks canonical CaaX activity. Therefore, we hypothesize that SCO4 is a novel endopeptidase required for critical protein modifications within chloroplasts, influencing the function of proteins involved in photosynthesis required for tolerance to excess light

RNA interference in Lepidoptera: An overview of successful and unsuccessful studies and implications for experimental design

Gene silencing through RNA interference (RNAi) has revolutionized the study of gene function, particularly in non-model insects. However, in Lepidoptera (moths and butterflies) RNAi has many times proven to be difficult to achieve. Most of the negative results have been anecdotal and the positive experiments have not been collected in such a way that they are possible to analyze. In this review, we have collected detailed data from more than 150 experiments including all to date published and many unpublished experiments. Despite a large variation in the data, trends that are found are that RNAi is particularly successful in the family Saturniidae and in genes involved in immunity. On the contrary, gene expression in epidermal tissues seems to be most difficult to silence. In addition, gene silencing by feeding dsRNA requires high concentrations for success. Possible causes for the variability of success in RNAi experiments in Lepidoptera are discussed. The review also points to a need to further investigate the mechanism of RNAi in lepidopteran insects and its possible connection to the innate immune response. Our general understanding of RNAi in Lepidoptera will be further aided in the future as our public database at http://insectacentral.org/RNAi will continue to gather information on RNAi experiments

A novel proteinase, SNOWY COTYLEDON4, is required for photosynthetic acclimation to higher light intensities in Arabidopsis

Excess light can have a negative impact on photosynthesis; thus, plants have evolved many different ways to adapt to different light conditions to both optimize energy use and avoid damage caused by excess light. Analysis of the Arabidopsis (Arabidopsis

Evidence for a SAL1-PAP Chloroplast Retrograde Pathway That Functions in Drought and High Light Signaling in Arabidopsis

Compartmentation of the eukaryotic cell requires a complex set of subcellular messages, including multiple retrograde signals from the chloroplast and mitochondria to the nucleus, to regulate gene expression. Here, we propose that one such signal is a phosphonucleotide (3'-phosphoadenosine 5'-phosphate [PAP]), which accumulates in Arabidopsis thaliana in response to drought and high light (HL) stress and that the enzyme SAL1 regulates its levels by dephosphorylating PAP to AMP. SAL1 accumulates in chloroplasts and mitochondria but not in the cytosol. sal1 mutants accumulate 20-fold more PAP without a marked change in inositol phosphate levels, demonstrating that PAP is a primary in vivo substrate. Significantly, transgenic targeting of SAL1 to either the nucleus or chloroplast of sal1 mutants lowers the total PAP levels and expression of the HL-inducible ASCORBATE PEROXIDASE2 gene. This indicates that PAP must be able to move between cellular compartments. The mode of action for PAP could be inhibition of 5' to 3' exoribonucleases (XRNs), as SAL1 and the nuclear XRNs modulate the expression of a similar subset of HL and drought-inducible genes, sal1 mutants accumulate XRN substrates, and PAP can inhibit yeast (Saccharomyces cerevisiae) XRNs. We propose a SAL1-PAP retrograde pathway that can alter nuclear gene expression during HL and drought stress

Evidence for a SAL1-PAP Chloroplast Retrograde Pathway That Functions in Drought and High Light Signaling in Arabidopsis

Compartmentation of the eukaryotic cell requires a complex set of subcellular messages, including multiple retrograde signals from the chloroplast and mitochondria to the nucleus, to regulate gene expression. Here, we propose that one such signal is a phosphonucleotide (3'-phosphoadenosine 5'-phosphate [PAP]), which accumulates in Arabidopsis thaliana in response to drought and high light (HL) stress and that the enzyme SAL1 regulates its levels by dephosphorylating PAP to AMP. SAL1 accumulates in chloroplasts and mitochondria but not in the cytosol. sal1 mutants accumulate 20-fold more PAP without a marked change in inositol phosphate levels, demonstrating that PAP is a primary in vivo substrate. Significantly, transgenic targeting of SAL1 to either the nucleus or chloroplast of sal1 mutants lowers the total PAP levels and expression of the HL-inducible ASCORBATE PEROXIDASE2 gene. This indicates that PAP must be able to move between cellular compartments. The mode of action for PAP could be inhibition of 5' to 3' exoribonucleases (XRNs), as SAL1 and the nuclear XRNs modulate the expression of a similar subset of HL and drought-inducible genes, sal1 mutants accumulate XRN substrates, and PAP can inhibit yeast (Saccharomyces cerevisiae) XRNs. We propose a SAL1-PAP retrograde pathway that can alter nuclear gene expression during HL and drought stress