Volatile-mediated oviposition preference for healthy over root-infested plants by the European corn borer.

The selection of oviposition sites by female moths is crucial in shaping their progeny performance and survival, and consequently in determining insect fitness. Selecting suitable plants that promote the performance of the progeny is referred to as the Preference-Performance hypothesis (or 'mother-knows-best'). While root infestation generally reduces the performance of leaf herbivores, little is known about its impact on female oviposition. We investigated whether maize root infestation by the Western corn rootworm (WCR) affects the oviposition preference and larval performance of the European corn borer (ECB). ECB females used leaf volatiles to select healthy plants over WCR-infested plants. Undecane, a compound absent from the volatile bouquet of healthy plants, was the sole compound to be upregulated upon root infestation and acted as a repellent for first oviposition. ECB larvae yet performed better on plants infested below-ground than on healthy plants, suggesting an example of 'bad motherhood'. The increased ECB performance on WCR-infested plants was mirrored by an increased leaf consumption, and no changes in the plant primary or secondary metabolism were detected. Understanding plant-mediated interactions between above- and below-ground herbivores may help to predict oviposition decisions, and ultimately, to manage pest outbreaks in the field.

IPD072Aa from Pseudomonas chlororaphis Targets Midgut Epithelial Cells in Killing Western Corn Rootworm (Diabrotica virgifera virgifera).

IPD072Aa from Pseudomonas chlororaphis is a new insecticidal protein that has been shown to have high activity against western corn rootworm (WCR). IPD072 has no sequence signatures or predicted structural motifs with any known protein revealing little insight into its mode of action using bioinformatic tools. As many bacterially derived insecticidal proteins are known to act through mechanisms that lead to death of midgut cells, we evaluated whether IPD072Aa also acts by targeting the cells of WCR midgut. IPD072Aa exhibits specific binding to brush border membrane vesicles (BBMVs) prepared from WCR guts. The binding was found to occur at binding sites that are different than those recognized by Cry3A or Cry34Ab1/Cry35Ab1, proteins expressed by current maize traits that target WCR. Using fluorescence confocal microscopy, immuno-detection of IPD072Aa in longitudinal sections from whole WCR larvae that were fed IPD072Aa revealed the association of the protein with the cells that line the gut. High-resolution scanning electron microscopy of similar whole larval sections revealed the disruption of the gut lining resulting from cell death caused by IPD072Aa exposure. These data show that the insecticidal activity of IPD072Aa results from specific targeting and killing of rootworm midgut cells. IMPORTANCE Transgenic traits targeting WCR based on insecticidal proteins from Bacillus thuringiensis have proven effective in protecting maize yield in North America. High adoption has led to WCR populations that are resistant to the trait proteins. Four proteins have been developed into commercial traits, but they represent only two modes of action due to cross-resistance among three. New proteins suited for trait development are needed. IPD072Aa, identified from the bacterium Pseudomonas chlororaphis, was shown to be effective in protecting transgenic maize against WCR. To be useful, IPD072Aa must work through binding to different receptors than those utilized by current traits to reduce risk of cross-resistance and understanding its mechanism of toxicity could aid in countering resistance development. Our results show that IPD072Aa binds to receptors in WCR gut that are different than those utilized by current commercial traits and its targeted killing of midgut cells results in larval death.

Early Identification of Root Damages Caused by Western Corn Rootworms Using a Minimally Invasive Root Phenotyping Robot-MISIRoot.

Western corn rootworm (WCR) is one of the most devastating corn rootworm species in North America because of its ability to cause severe production loss and grain quality damage. To control the loss, it is important to identify the infection of WCR at an early stage. Because the root system is the earliest feeding source of the WCR at the larvae stage, assessing the direct damage in the root system is crucial to achieving early detection. Most of the current methods still necessitate uprooting the entire plant, which could cause permanent destruction and a loss of the original root's structural information. To measure the root damages caused by WCR non-destructively, this study utilized MISIRoot, a minimally invasive and in situ automatic plant root phenotyping robot to collect not only high-resolution images but also 3D positions of the roots without uprooting. To identify roots in the images and to study how the damages were distributed in different types of roots, a deep convolution neural network model was trained to differentiate the relatively thick and thin roots. In addition, a color camera was used to capture the above-ground morphological features, such as the leaf color, plant height, and side-view leaf area. To check if the plant shoot had any visible symptoms in the inoculated group compared to the control group, several vegetation indices were calculated based on the RGB color. Additionally, the shoot morphological features were fed into a PLS-DA model to differentiate the two groups. Results showed that none of the above-ground features or models output a statistically significant difference between the two groups at the 95% confidence level. On the contrary, many of the root structural features measured using MISIRoot could successfully differentiate the two groups with the smallest t-test p-value of 1.5791 × 10-6. The promising outcomes were solid proof of the effectiveness of MISIRoot as a potential solution for identifying WCR infestations before the plant shoot showed significant symptoms.

Root volatile profiles and herbivore preference are mediated by maize domestication, geographic spread, and modern breeding.

MAIN CONCLUSION: Domestication affected the abundances and diversity of maize root volatiles more than northward spread and modern breeding, and herbivore preference for roots was correlated with volatile diversity and herbivore resistance. Studies show that herbivore defenses in crops are mediated by domestication, spread, and breeding, among other human-driven processes. They also show that those processes affected chemical communication between crop plants and herbivores. We hypothesized that (i) preference of the herbivore (Diabrotica virgifera virgifera) larvae for embryonic roots of maize (Zea mays mays) would increase and (ii) root volatile diversity would decrease with the crop's domestication, northward spread to present-day USA, and modern breeding. We used Balsas teosinte (Zea mays parviglumis), Mexican and USA landrace maizes, and US inbred maize lines to test these hypotheses. We found that herbivore preference and volatile diversity increased with maize domestication and northward spread but decreased with modern breeding. Additionally, we found that the abundances of single volatiles did not consistently increase or decrease with maize domestication, spread, and breeding; rather, volatiles grouped per their abundances were differentially affected by those processes, and domestication had the greatest effects. Altogether, our results suggested that: the herbivore's preference for maize roots is correlated with volatile diversity and herbivore resistance; changes in abundances of individual volatiles are evident at the level of volatile groups; and maize domestication, but not spread and breeding, affected the abundances of some green leaf volatiles and sesquiterpenes/sesquiterpenoids. In part, we discussed our results in the context of herbivore defense evolution when resources for plant growth and defense vary across environments. We suggested that variability in relative abundance of volatiles may be associated with their local, functional relevance across wild and agricultural environments.

Host resistance to Bacillus thuringiensis is linked to altered bacterial community within a specialist insect herbivore.

Evolution of resistance to transgenic crops producing toxins from Bacillus thuringiensis (Bt) threatens the sustainability of the technology. Examination of resistance mechanisms has largely focused on characterization of mutations in proteins serving as Bt toxin binding sites. However, insect microbial communities have the potential to provide host resistance to pesticides in a myriad of ways. Previous findings suggest the killing mechanism of Bt relies on enteric bacteria becoming pathogenic in the disrupted gut environment of the insect following Bt intoxication. Thus, here we hypothesized that resistance to Bt would alter the microbiome composition of the insect. Previous studies have manipulated the microbiome of susceptible insects and monitored their response to Bt. In our study, we characterized the associated bacterial communities of Bt-resistant and -susceptible western corn rootworms, a widespread pest of maize in the United States. We found resistant insects harbor a bacterial community that is less rich and distinct from susceptible insects. After feeding on Bt-expressing maize, susceptible insects exhibited dysbiosis of the associated bacterial community, whereas the community within resistant insects remained relatively unchanged. These results suggest resistance to Bt produces alterations in the microbiome of the western corn rootworm that may contribute to resistance. We further demonstrated that by itself, feeding on Bt toxin-expressing seedlings caused a shift in the microbiota. This work provides a broader picture of the effect stressors have on microbiome composition, and the potential heritable changes induced as a result of intense selection.

Detection of alternative splicing in western corn rootworm (Diabrotica virgifera virgifera LeConte) in association with eCry3.1Ab resistance using RNA-seq and PacBio Iso-Seq.

Alternative splicing is a common feature in eukaryotes that not only increases the transcript diversity, but also has functional consequences. In insects, alternative splicing has been found associated with resistance to pesticides and Bt toxins. Up to date, the alternative splicing in western corn rootworm (Diabrotica virgifera virgifera LeConte) has not been studied. To investigate its alternative splicing pattern and relation to Bt resistance, we carried out single-molecule real-time (SMRT) transcript sequencing and Iso-seq analysis on resistant, eCry3.1Ab-selected and susceptible, unselected, western corn rootworm neonate midguts which fed on seedling maize with and without eCry3.1Ab for 12 and 24 h. We present transcriptome-wide alternative splicing patterns of western corn rootworm midgut in response to feeding on eCry3.1Ab-expressing corn using a comprehensive approach that combines both RNA-seq and SMRT transcript sequencing techniques. The results showed genes in western corn rootworm are highly alternatively spliced, which happens on 67.73% of multi-exon genes. One of the alternative splicing events we identified was a novel peritrophic matrix protein with two alternative splicing isoforms. Analysis of differential exon usage between resistant and susceptible colonies showed that in eCry3.1Ab-resistant western corn rootworm, expression of one isoform was significantly higher than in the susceptible colony, while no significant differences between colonies were observed with the other isoform. Our results provide the first survey of alternative splicing in western corn rootworm and suggest that the observed alternatively spliced isoforms of peritrophic matrix protein may be associated with eCry3.1Ab resistance in western corn rootworm.

Linking land use patterns and pest outbreaks in Bt maize.

Western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), is a major pest of maize in the United States and is an invasive pest in Europe. Maize is the only agricultural crop on which western corn rootworm larvae can survive and this insect requires two consecutive years of maize cultivation to complete its life cycle. Transgenic maize producing insecticidal proteins derived from the bacterium Bacillus thuringiensis (Bt) is often used to manage rootworm populations. The first Bt trait, Cry3Bb1, was introduced in 2003, but larval resistance to this toxin appeared in northeastern Iowa in 2009. Rootworm management occurs on a field-by-field basis, but adult rootworm may disperse among fields. It is known that growing consecutive years of Cry3Bb1 maize within a field can lead to resistance, but the relationship of the surrounding landscape to the development of resistance is unknown. Using geospatial tools and publicly available land-use data, we examined circular areas (buffers) surrounding fields that had previously experienced high levels of rootworm injury to Cry3Bb1 maize and rootworm resistance to Cry3Bb1 maize (problem fields). We calculated the proportion of area inside each buffer planted to maize continuously for 1-9 yr, and compared these values to those for randomly selected control points throughout the state. We also calculated the proportion of the state planted to maize for at least three consecutive years for 2003 through 2018, and its relationship with the annual value of maize. We found that areas surrounding problem fields had significantly more continuous maize compared to controls, with the most continuous maize within 1.6 km of problem fields. We also found that the cultivation of continuous maize in Iowa increased significantly between 2003 and 2018, and this was correlated with average annual price of maize. We hypothesize a scenario in which continuous cultivation of Cry3Bb1 maize in local landscapes, driven in part by the increased value of maize, facilitated selection for Cry3Bb1 resistance. These results suggest that land use in areas surrounding problem fields affect the rate of resistance evolution and approaches for resistance management can be enhanced by taking a landscape-level perspective.

Molecular characterization and RNA interference responses of the lethal giant larvae gene in Diabrotica virgifera virgifera adults.

High specificity for silencing target genes and single-copy target genes that yield clear phenotypes are two important factors for the success of RNA interference (RNAi). The lethal giant larvae (Lgl) gene appears to be an ideal gene for RNAi because RNAi can effectively suppress its expression and results in molting defects and mortality in Tribolium castaneum. To investigate the suitability of this gene for RNAi in other insects, we identified and characterized DvLgl from the western corn rootworm, Diabrotica virgifera virgifera, a species exhibiting high RNAi efficiency. DvLgl was expressed in all developmental stages and tissues investigated. The deduced DvLgl protein showed high amino-acid sequence identities and similar domain architecture to Lgls from other insect species. Despite many similarities among insect Lgls, RNAi-mediated suppression of DvLgl failed to produce a phenotype in D. v. virgifera adults. The difference in developing phenotypes could be attributed greatly to the level of gene suppression and the insect developmental stages for RNAi. These results highlight the variability in RNAi response among insects and showcase the importance of screening multiple target genes when conducting RNAi studies. Our findings are expected to help the design of future RNAi studies and future investigations of Lgl in insects.

Coordinated binding of a two-component insecticidal protein from Alcaligenes faecalis to western corn rootworm midgut tissue.

AfIP-1A/1B is a two-component insecticidal protein identified from the soil bacterium Alcaligenes faecalis that has high activity against western corn rootworm (WCR; Diabrotica virgifera virgifera LeConte). Previous results revealed that AfIP-1A/1B is cross-resistant to the binary protein from Bacillus thuringiensis (Bt), Cry34Ab1/Cry35Ab1 (also known as Gpp34Ab1/Tpp35Ab1; Crickmore et al., 2020), which was attributed to shared binding sites in WCR gut tissue (Yalpani et al., 2017). To better understand the interaction of AfIP-1A/1B with its receptor, we have systematically evaluated the binding of these proteins with WCR brush border membrane vesicles (BBMVs). Our findings show that AfIP-1A binds directly to BBMVs, while AfIP-1B does not; AfIP-1B binding only occurred in the presence of AfIP-1A which was accompanied by the presence of stable, high molecular weight oligomers of AfIP-1B observed on denaturing protein gels. Additionally, we show that AfIP-1A/1B forms pores in artificial lipid membranes. Finally, binding of AfIP-1A/1B was found to be reduced in BBMVs from Cry34Ab1/Cry35Ab1-resistant WCR where Cry34Ab1/Cry35Ab1 binding was also reduced. The reduced binding of both proteins is consistent with recognition of a shared receptor that has been altered in the resistant strain. The coordination of AfIP-1B binding by AfIP-1A, the similar structures between AfIP-1A and Cry34Ab1, along with their shared binding sites and cross-resistance, suggest a similar role for AfIP1A and Cry34Ab1 in receptor recognition and docking site for their cognate partners, AfIP-1B and Cry35Ab1, respectively.

Aegerolysins from the fungal genus Pleurotus - Bioinsecticidal proteins with multiple potential applications.

The aegerolysin proteins ostreolysin A6, pleurotolysin A2 and erylysin A are produced by mushrooms of the genus Pleurotus. These aegerolysins can interact specifically with sphingolipid-enriched membranes. In particular, they strongly bind insect cells and to artificial lipid membranes that contain physiologically relevant concentrations of the main invertebrate-specific sphingolipid, ceramide phosphoethanolamine. Moreover, the aegerolysins permeabilise these membranes when combined with their protein partner pleurotolysin B, which contains a membrane-attack-complex/perforin domain. These aegerolysin/ pleurotolysin B complexes show strong and selective toxicity towards western corn rootworm larvae and adults and Colorado potato beetle larvae. Their insecticidal activities arise through aegerolysin binding to ceramide phosphoethanolamine in the insect midgut. This mode of membrane binding is different from those described for similar aegerolysin-based complexes of bacterial origin (e.g., Cry34Ab1/Cry35Ab1), or other Bacillus thuringiensis proteinaceous crystal toxins, which associate with protein receptors. The ability of Pleurotus aegerolysins to specifically interact with sphingolipid-enriched domains in mammalian cells can be further exploited to visualize lipid rafts in living cells, and to treat certain types of tumours and metabolic disorders. Finally, these proteins can strongly enhance fruiting initiation of P. ostreatus even when applied externally. In this review, we summarise the current knowledge of the potential biotechnological and biomedical applications of the Pleurotus aegerolysins, either alone or when complexed with pleurotolysin B, with special emphasis on their bioinsecticidal effects.

Meeting technical challenges for protein characterization and surrogate equivalence studies that resulted from insecticidal protein co-expression in maize event MZIR098.

Safety assessment of genetically modified plants includes protein characterization to confirm the intended trait protein expression. In addition, to conduct safety tests, the large amount of purified protein needed is usually met through the use of a surrogate, microbially produced protein source. Characterization of the eCry3.1Ab and mCry3A proteins as derived from Event MZIR098 maize was challenging because of the difficulty in purifying/isolating these proteins that are of similar molecular weight and have considerable shared sequence and immunogenicity. This also applies to establishing the biochemical equivalence to the microbially produced surrogate proteins, as highly-purified plant protein is required. While use of crude plant extracts facilitated functional equivalence testing with the surrogate proteins, a separate technical challenge had to be met. The eCry3.1Ab and mCry3A proteins display differentiated modes of action toward CRW pests, however, with the same overall target pest spectrum, no differential test organism existed to allow equivalence testing for one insecticidal protein in the presence of the other. To establish that the microbially produced proteins are suitable surrogates for the plant-produced proteins, the challenges in the protein purification and bioactivity testing had to be addressed. This article describes technical solutions to assess and characterize the insecticidal proteins in this new event and thereby confirm equivalence/suitability of the microbially produced protein surrogates.

Enhanced metabolism and selection of pyrethroid-resistant western corn rootworms (Diabrotica virgifera virgifera LeConte).

Western corn rootworm (WCR) pyrethroid resistance has been previously reported in the United States (US) western Corn Belt, and cross-resistance and synergism studies suggested that both target site insensitivity and enhanced metabolism may be conferring WCR resistance to pyrethroids. The present study aimed to investigate the potential mechanisms of WCR pyrethroid resistance and to estimate the heritability of the resistance trait. Biochemical assays using model substrates and spectrophotometry revealed 2-4-fold higher activity of P450s and esterases in pyrethroid-resistant WCR populations, whereas the biological activity of glutathione S-transferase was similar between populations tested. No mutation in the voltage-gated sodium channel was detected in pyrethroid-resistant WCR individuals by sequencing PCR products containing the para-homologous L1014, T929, and M918 amino acid positions that are commonly associated with target site mutations in other pyrethroid-resistant insects. A pilot estimation of pyrethroid resistance heritability obtained during laboratory selection of a WCR population suggested a major genetic component of the resistance trait and predicted a 10-fold increase in WCR bifenthrin resistance within ~7 generations of insecticide lethal exposure. Results support earlier indirect evidence that enhanced metabolism may be contributing to WCR resistance to pyrethroids and illustrates the potential of WCR pyrethroid resistance evolution.

Stability of double-stranded RNA in gut contents and hemolymph of Ostrinia nubilalis larvae.

RNA interference (RNAi) is a revolutionary technique for silencing gene expression, but the success of this technique is dependent upon the stability of double-stranded RNA (dsRNA) molecules. In many insects, especially lepidopteran species, RNAi efficiency is limited by high instability of dsRNA in the gut and/or hemolymph, preventing the development of RNAi-based strategies for many serious pests. Previous attempts to perform RNAi on Ostrinia nubilalis (ECB, Lepidoptera: Crambidae) indicate low RNAi efficiency with both dsRNA injection and feeding. To investigate the contribution of dsRNA instability to low RNAi efficiency in ECB, a serious of ex vivo incubation experiments were performed where dsRNA integrity was assessed following incubation in larval gut continents and hemolymph using gel electrophoresis or RT-qPCR. DsRNA was less stable in the gut contents from ECB than in gut contents from Diabrotica virgifera virgifera, a coleopteran exhibiting high RNAi efficiency. Furthermore, characterization of dsRNA stability in ECB gut contents and hemolymph revealed that dsRNA was rapidly degraded under physiologically relevant conditions as a result of enzymatic activity that was neither size- nor sequence-dependent. These findings suggest that instability of dsRNA in ECB tissues is a contributing factor to the poor efficiency of RNAi in this pest. This work advances our understanding of mechanisms impacting RNAi efficiency in ECB and related lepidopteran insects for which novel pest management strategies are needed, and may facilitate the development of strategies for enhancing dsRNA stability in ECB tissues.

A knowledge-based approach to designing control strategies for agricultural pests.

Chemical control of insect pests remains vital to agricultural productivity, but limited mechanistic understanding of the interactions between crop, pest and chemical control agent have restricted our capacity to respond to challenges such as the emergence of resistance and demands for tighter environmental regulation. Formulating effective control strategies that integrate chemical and non-chemical management for soil-dwelling pests is particularly problematic owing to the complexity of the soil-root-pest system and the variability that occurs between sites and between seasons. Here, we present a new concept, termed COMPASS, that integrates ecological knowledge on pest development and behaviour together with crop physiology and mechanistic understanding of chemical distribution and toxic action within the rhizosphere. The concept is tested using a two-dimensional systems model (COMPASS-Rootworm) that simulates root damage in maize from the corn rootworm Diabrotica spp. We evaluate COMPASS-Rootworm using 119 field trials that investigated the efficacy of insecticidal products and placement strategies at four sites in the USA over a period of ten years. Simulated root damage is consistent with measurements for 109 field trials. Moreover, we disentangle factors influencing root damage and pest control, including pest pressure, weather, insecticide distribution, and temporality between the emergence of crop roots and pests. The model can inform integrated pest management, optimize pest control strategies to reduce environmental burdens from pesticides, and improve the efficiency of insecticide development.

Structural and functional insights into the Diabrotica virgifera virgifera ATP-binding cassette transporter gene family.

BACKGROUND: The western corn rootworm, Diabrotica virgifera virgifera, is a pervasive pest of maize in North America and Europe, which has adapted to current pest management strategies. In advance of an assembled and annotated D. v. virgifera genome, we developed transcriptomic resources to use in identifying candidate genes likely to be involved in the evolution of resistance, starting with members of the ATP-binding cassette (ABC) transporter family. RESULTS: In this study, 65 putative D. v. virgifera ABC (DvvABC) transporters were identified within a combined transcriptome assembly generated from embryonic, larval, adult male, and adult female RNA-sequence libraries. Phylogenetic analysis placed the deduced amino-acid sequences of the DvvABC transporters into eight subfamilies (A to H). To supplement our sequence data with functional analysis, we identified orthologs of Tribolium castaneum ABC genes which had previously been shown to exhibit overt RNA interference (RNAi) phenotypes. We identified eight such D. v. virgifera genes, and found that they were functionally similar to their T. castaneum counterparts. Interestingly, depletion of DvvABCB_39715 and DvvABCG_3712 transcripts in adult females produced detrimental reproductive and developmental phenotypes, demonstrating the potential of these genes as targets for RNAi-mediated insect control tactics. CONCLUSIONS: By combining sequence data from four libraries covering three distinct life stages, we have produced a relatively comprehensive de novo transcriptome assembly for D. v. virgifera. Moreover, we have identified 65 members of the ABC transporter family and provided the first insights into the developmental and physiological roles of ABC transporters in this pest species.

Laser ablation tomography for visualization of root colonization by edaphic organisms.

Soil biota have important effects on crop productivity, but can be difficult to study in situ. Laser ablation tomography (LAT) is a novel method that allows for rapid, three-dimensional quantitative and qualitative analysis of root anatomy, providing new opportunities to investigate interactions between roots and edaphic organisms. LAT was used for analysis of maize roots colonized by arbuscular mycorrhizal fungi, maize roots herbivorized by western corn rootworm, barley roots parasitized by cereal cyst nematode, and common bean roots damaged by Fusarium. UV excitation of root tissues affected by edaphic organisms resulted in differential autofluorescence emission, facilitating the classification of tissues and anatomical features. Samples were spatially resolved in three dimensions, enabling quantification of the volume and distribution of fungal colonization, western corn rootworm damage, nematode feeding sites, tissue compromised by Fusarium, and as well as root anatomical phenotypes. Owing to its capability for high-throughput sample imaging, LAT serves as an excellent tool to conduct large, quantitative screens to characterize genetic control of root anatomy and interactions with edaphic organisms. Additionally, this technology improves interpretation of root-organism interactions in relatively large, opaque root segments, providing opportunities for novel research investigating the effects of root anatomical phenes on associations with edaphic organisms.

RNase If -treated quantitative PCR for dsRNA quantitation of RNAi trait in genetically modified crops.

BACKGROUND: RNA interference (RNAi) technology has been widely used to knockdown target genes via post-transcriptional silencing. In plants, RNAi is used as an effective tool with diverse applications being developed such as resistance against insects, fungi, viruses, and metabolism manipulation. To develop genetically modified (GM) RNAi traits for insect control, a transgene is created and composed of an inversely-repeated sequence of the target gene with a spacer region inserted between the repeats. The transgene design is subject to form a self-complementary hairpin RNA (hpRNA) and the active molecules are > 60 bp doubled-stranded RNA (dsRNA) derived from the hpRNA. However, in some cases, an undesirable intermediate such as single-stranded RNA (ssRNA) may be formed, which is not an active molecule. The aforementioned characteristics of RNAi traits lead to increase the challenges for RNAi-derived dsRNA quantitation. RESULTS: To quantify the dsRNA and distinguish it from the ssRNA in transgenic maize, an analytical tool is required to be able to effectively quantify dsRNA which contains a strong secondary structure. Herein, we develop a modified qRT-PCR method (abbreviated as RNase If -qPCR) coupled with a ssRNA preferred endonuclease (i.e., RNase If). This method enables the precise measurement of the active molecules (i.e., dsRNA) derived from RNAi traits of GM crops and separately quantifies the dsRNA from ssRNA. Notably, we also demonstrate that the RNase If -qPCR is comparable to a hybridization-based method (Quantigene Plex 2.0). CONCLUSIONS: To our best knowledge, this is the first report of a method combining RNase If with modified qRT-PCR protocol. The method represents a reliable analytical tool to quantify dsRNA for GM RNAi crops. It provides a cost-effective and feasible analytical tool for general molecular laboratory without using additional equipment for other methods. The RNase If -qPCR method demonstrates high sensitivity (to 0.001 pg/ µL of dsRNA), precision and accuracy. In this report, we demonstrated the deployment of this method to characterize the RNAi events carrying v-ATPase C in maize during trait development process. The method can be utilized in any application which requires the dsRNA quantification such as double-stranded RNA virus or sprayable dsRNA as herbicide.

A selective insecticidal protein from Pseudomonas mosselii for corn rootworm control.

The coleopteran insect western corn rootworm (WCR, Diabrotica virgifera virgifera) is an economically important pest in North America and Europe. Transgenic corn plants producing Bacillus thuringiensis (Bt) insecticidal proteins have been useful against this devastating pest, but evolution of resistance has reduced their efficacy. Here, we report the discovery of a novel insecticidal protein, PIP-47Aa, from an isolate of Pseudomonas mosselii. PIP-47Aa sequence shows no shared motifs, domains or signatures with other known proteins. Recombinant PIP-47Aa kills WCR, two other corn rootworm pests (Diabrotica barberi and Diabrotica undecimpunctata howardi) and two other beetle species (Diabrotica speciosa and Phyllotreta cruciferae), but it was not toxic to the spotted lady beetle (Coleomegilla maculata) or seven species of Lepidoptera and Hemiptera. Transgenic corn plants expressing PIP-47Aa show significant protection from root damage by WCR. PIP-47Aa kills a WCR strain resistant to mCry3A and does not share rootworm midgut binding sites with mCry3A or AfIP-1A/1B from Alcaligenes that acts like Cry34Ab1/Cry35Ab1. Our results indicate that PIP-47Aa is a novel insecticidal protein for controlling the corn rootworm pests.

Knockdown of RNA interference pathway genes in western corn rootworm, Diabrotica virgifera virgifera, identifies no fitness costs associated with Argonaute 2 or Dicer-2.

The use of transgenic crops that induce silencing of essential genes using double-stranded RNA (dsRNA) through RNA interference (RNAi) in western corn rootworm, Diabrotica virgifera virgifera, is likely to be an important component of new technologies for the control of this important corn pest. Previous studies have demonstrated that the dsRNA response in D. v. virgifera depends on the presence of RNAi pathway genes including Dicer-2 and Argonaute 2, and that downregulation of these genes limits the lethality of environmental dsRNA. A potential resistance mechanism to lethal dsRNA may involve loss of function of RNAi pathway genes. Howver, the potential for resistance to evolve may depend on whether these pathway genes have essential functions such that the loss of function of core proteins in the RNAi pathway will have fitness costs in D. v. virgifera. Fitness costs associated with potential resistance mechanisms have a central role in determining how resistance can evolve to RNAi technologies in western corn rootworm. We evaluated the effect of dsRNA and microRNA pathway gene knockdown on the development of D. v. virgifera larvae through short-term and long-term exposures to dsRNA for Dicer and Argonaute genes. Downregulation of Argonaute 2, Dicer-2, Dicer-1 did not significantly affect larval survivorship or development through short and long-term exposure to dsRNA. However, downregulation of Argonaute 1 reduced larval survivorship and delayed development. The implications of these results as they relate to D. v. virgifera resistance to lethal dsRNA are discussed.

Asymmetric Total Synthesis of Four Stereoisomers of the Sex Pheromone of the Western Corn Rootworm.

A convergent synthesis of four stereoisomers of the sex pheromone of the western corn rootworm (8-methyldecan-2-yl propionate, 1) from commercially available chiral starting materials is reported. The key step was Julia-Kocienski olefination between chiral BT-sulfone and chiral aldehyde. This synthetic route provided the four stereoisomers of 1 in 24-29% total yield via a six-step sequence. The simple scale-up strategy provides a new way to achieve the asymmetric synthesis of the sex pheromone.