Implementing the sterile insect technique with RNA interference – a review

RNA interference (RNAi) of insect pests is reviewed and its potential for implementing Sterile Insect Technique (SIT)-related control is considered. The molecular mechanisms that support RNAi in pest species are reviewed in detail, drawing on literature from a range of species including Drosophila melanogaster and Homo sapiens. The underlying genes that enable RNAi are generally conserved across taxa, although variance exists in both their form and function. RNAi represents a plausible, non-GM system for targeting populations of insects for control purposes, if RNA interference (RNAi) effector molecules can be delivered environmentally (eRNAi). We consider studies of eRNAi from across several insect orders and review to what extent taxonomy, genetics and differing methods of double stranded (ds)RNA synthesis and delivery can influence the efficiency of gene knockdown. Several factors, including the secondary structure of the target mRNA and the specific nucleotide sequence of dsRNA effector molecules, can affect the potency of eRNAi. However, taxonomic relationships between insects cannot be used to reliably forecast the efficiency of an eRNAi response. The mechanisms by which insects acquire dsRNA from their environment require further research, but the evidence to date suggests that endocytosis and transport channels both play key roles. Delivery of RNA molecules packaged in intermediary carriers such as bacteria or nanoparticles may facilitate their entry into and through the gut, and enable the evasion of host defense systems, such as toxic pH, that would otherwise attenuate the potential for RNAi

Adaptive sequence evolution is driven by biotic stress in a pair of orchid species (Dactylorhiza) with distinct ecological optima

This work was funded by an Austrian Science Fund (FWF) project (Y661-B16) awarded to OP and a Marie Curie IEF fellowship (PERG-GA-2011-299608-TRANSADAPTATION) to FB.The orchid family is the largest in the angiosperms, but little is known about the molecular basis of the significant variation they exhibit. We investigate here the transcriptomic divergence between two European terrestrial orchids, Dactylorhiza incarnata and D. fuchsii, and integrate these results in the context of their distinct ecologies that we also document. Clear signals of lineage-specific adaptive evolution of protein-coding sequences are identified, notably targeting elements of biotic defence, including both physical and chemical adaptations in the context of divergent pools of pathogens and herbivores. In turn, a substantial regulatory divergence between the two species appears linked to adaptation/acclimation to abiotic conditions. Several of the pathways affected by differential expression are also targeted by deviating post-transcriptional regulation via sRNAs. Finally, Dactylorhiza incarnata appears to suffer from insufficient sRNA control over the activity of RNA-dependent DNA polymerase, resulting in increased activity of class I transposable elements and, over time, in larger genome size than that of D. fuchsii. The extensive molecular divergence between the two species suggests significant genomic and transcriptomic shock in their hybrids and offers insights into the difficulty of coexistence at the homoploid level. Altogether, biological response to selection, accumulated during the history of these orchids, appears governed by their microenvironmental context, in which biotic and abiotic pressures act synergistically to shape transcriptome structure, expression and regulation.Publisher PDFPeer reviewe

Quantification of dsRNA using stable isotope labeling dilution liquid chromatography mass spectrometry

RATIONALE: Recent developments in RNA interference (RNAi) have created a need for cost-effective and large scale synthesis of double stranded RNA (dsRNA), in conjunction with high throughput analytical techniques to fully characterise and accurately quantify dsRNA prior to downstream RNAi applications. METHODS: Stable isotope labeled dsRNA was synthesised both in vivo (15N) and in vitro (13C,15N guanosine-containing dsRNA) prior to purification and quantification. The stable isotope labeled dsRNA standards were subsequently spiked into total RNA extracted from E. coli engineered to express dsRNA. RNase mass mapping approaches were subsequently performed using LC-ESI-MS for both the identification and absolute quantification of the dsRNA using the ratios of the light and heavy oligonucleotides pairs. RESULTS: Absolute quantification was performed based on the resulting light and heavy oligoribonicleotides identified using mass spectrometry. Using this approach we determined that 624.6 ng/μl and 466.5 ng/μl of dsRNA was present in 80 μl total RNA extracted from 108E. coli cells expressing 765 bp and 401 bp dsRNA respectively. CONCLUSIONS: Stable isotopic labelling of dsRNA in conjunction with mass spectrometry enabled the characterisation and quantification of dsRNA in complex total RNA mixtures

Regulation of Gene Expression in Plants through miRNA Inactivation

Eukaryotic organisms possess a complex RNA-directed gene expression regulatory network allowing the production of unique gene expression patterns. A recent addition to the repertoire of RNA-based gene regulation is miRNA target decoys, endogenous RNA that can negatively regulate miRNA activity. miRNA decoys have been shown to be a valuable tool for understanding the function of several miRNA families in plants and invertebrates. Engineering and precise manipulation of an endogenous RNA regulatory network through modification of miRNA activity also affords a significant opportunity to achieve a desired outcome of enhanced plant development or response to environmental stresses. Here we report that expression of miRNA decoys as single or heteromeric non-cleavable microRNA (miRNA) sites embedded in either non-protein-coding or within the 3′ untranslated region of protein-coding transcripts can regulate the expression of one or more miRNA targets. By altering the sequence of the miRNA decoy sites, we were able to attenuate miRNA inactivation, which allowed for fine regulation of native miRNA targets and the production of a desirable range of plant phenotypes. Thus, our results demonstrate miRNA decoys are a flexible and robust tool, not only for studying miRNA function, but also for targeted engineering of gene expression in plants. Computational analysis of the Arabidopsis transcriptome revealed a number of potential miRNA decoys, suggesting that endogenous decoys may have an important role in natural modulation of expression in plants

Silencing Nicotiana attenuata

The plant hormone jasmonic acid (JA) plays a pivotal role in plant-insect interactions. Herbivore attack usually elicits dramatic increases in JA concentrations, which in turn activate the accumulation of metabolites that function as defenses against herbivores. Although almost all enzymes involved in the biosynthesis pathway of JA have been identified and characterized, the mechanism by which plants regulate JA biosynthesis remains unclear. Calcium-dependent protein kinases (CDPKs) are plant-specific proteins that sense changes in [Ca(2+)] to activate downstream responses. We created transgenic Nicotiana attenuata plants, in which two CDPKs, NaCDPK4 and NaCDPK5, were simultaneously silenced (IRcdpk4/5 plants). IRcdpk4/5 plants were stunted and aborted most of their flower primordia. Importantly, after wounding or simulated herbivory, IRcdpk4/5 plants accumulated exceptionally high JA levels. When NaCDPK4 and NaCDPK5 were silenced individually, neither stunted growth nor high JA levels were observed, suggesting that NaCDPK4 and NaCDPK5 have redundant roles. Attack from Manduca sexta larvae on IRcdpk4/5 plants induced high levels of defense metabolites that slowed M. sexta growth. We found that NaCDPK4 and NaCDPK5 affect plant resistance against insects in a JA- and JA-signaling-dependent manner. Furthermore, IRcdpk4/5 plants showed overactivation of salicylic-acid-induced protein kinase, a mitogen-activated protein kinase involved in various stress responses, and genetic analysis indicated that the increased salicylic-acid-induced protein kinase activity in IRcdpk4/5 plants was a consequence of the exceptionally high JA levels and was dependent on CORONATINE INSENSITIVE1. This work reveals the critical roles of CDPKs in modulating JA homeostasis and highlights the complex duet between JA and mitogen-activated protein kinase signaling

CDPKs CPK6 and CPK3 Function in ABA Regulation of Guard Cell S-Type Anion- and Ca(2+)- Permeable Channels and Stomatal Closure

Abscisic acid (ABA) signal transduction has been proposed to utilize cytosolic Ca(2+) in guard cell ion channel regulation. However, genetic mutants in Ca(2+) sensors that impair guard cell or plant ion channel signaling responses have not been identified, and whether Ca(2+)-independent ABA signaling mechanisms suffice for a full response remains unclear. Calcium-dependent protein kinases (CDPKs) have been proposed to contribute to central signal transduction responses in plants. However, no Arabidopsis CDPK gene disruption mutant phenotype has been reported to date, likely due to overlapping redundancies in CDPKs. Two Arabidopsis guard cell–expressed CDPK genes, CPK3 and CPK6, showed gene disruption phenotypes. ABA and Ca(2+) activation of slow-type anion channels and, interestingly, ABA activation of plasma membrane Ca(2+)-permeable channels were impaired in independent alleles of single and double cpk3cpk6 mutant guard cells. Furthermore, ABA- and Ca(2+)-induced stomatal closing were partially impaired in these cpk3cpk6 mutant alleles. However, rapid-type anion channel current activity was not affected, consistent with the partial stomatal closing response in double mutants via a proposed branched signaling network. Imposed Ca(2+) oscillation experiments revealed that Ca(2+)-reactive stomatal closure was reduced in CDPK double mutant plants. However, long-lasting Ca(2+)-programmed stomatal closure was not impaired, providing genetic evidence for a functional separation of these two modes of Ca(2+)-induced stomatal closing. Our findings show important functions of the CPK6 and CPK3 CDPKs in guard cell ion channel regulation and provide genetic evidence for calcium sensors that transduce stomatal ABA signaling

Direct Identification of the Meloidogyne incognita Secretome Reveals Proteins with Host Cell Reprogramming Potential

The root knot nematode, Meloidogyne incognita, is an obligate parasite that causes significant damage to a broad range of host plants. Infection is associated with secretion of proteins surrounded by proliferating cells. Many parasites are known to secrete effectors that interfere with plant innate immunity, enabling infection to occur; they can also release pathogen-associated molecular patterns (PAMPs, e.g., flagellin) that trigger basal immunity through the nematode stylet into the plant cell. This leads to suppression of innate immunity and reprogramming of plant cells to form a feeding structure containing multinucleate giant cells. Effectors have generally been discovered using genetics or bioinformatics, but M. incognita is non-sexual and its genome sequence has not yet been reported. To partially overcome these limitations, we have used mass spectrometry to directly identify 486 proteins secreted by M. incognita. These proteins contain at least segmental sequence identity to those found in our 3 reference databases (published nematode proteins; unpublished M. incognita ESTs; published plant proteins). Several secreted proteins are homologous to plant proteins, which they may mimic, and they contain domains that suggest known effector functions (e.g., regulating the plant cell cycle or growth). Others have regulatory domains that could reprogram cells. Using in situ hybridization we observed that most secreted proteins were produced by the subventral glands, but we found that phasmids also secreted proteins. We annotated the functions of the secreted proteins and classified them according to roles they may play in the development of root knot disease. Our results show that parasite secretomes can be partially characterized without cognate genomic DNA sequence. We observed that the M. incognita secretome overlaps the reported secretome of mammalian parasitic nematodes (e.g., Brugia malayi), suggesting a common parasitic behavior and a possible conservation of function between metazoan parasites of plants and animals