Identification of active target genes in bioassay screening is the first important step for application of RNA interference (RNAi) for pest control. Here, we describe the methodology for performing ...high-throughput RNAi target screening against important agriculture pest, Western corn rootworm in 96-well microplate. Two approaches are presented to identify active targets from random-cDNA library or testing a certain group of specific targets via in silico sequence analysis. Methods of PCR primer design, DNA template preparation, and dsRNA production described here can be applied for other pests.
Abstract
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.
Laser ablation tomography is a novel method for rapid quantitative and qualitative analysis of root anatomy, providing new opportunities to investigate interactions between roots and edaphic organisms.
As agricultural biotechnology continues to develop solutions for addressing crop pests through newly expressed proteins from novel source organisms, with different modes or sites of action and/or ...different spectra of activity, the safety of these proteins will be assessed. The results of hazard-identification and characterization studies for the insecticidal protein IPD079Ea, which is derived from a fern (Ophioglossum pendulum) and active against the maize pest western corn rootworm (Diabrotica virgifera virgifera, Coleoptera: Chrysomelidae) are provided. Collectively these results indicate that IPD079Ea is unlikely to present a hazard to human or animal health and support the safety of genetically modified maize expressing IPD079Ea.
•IPD079Ea is a novel insecticidal protein derived from Ophioglossum pendulum.•IPD079Ea offers a new site of action to protect against WCR in GM maize.•IPD079Ea protein safety was assessed through a weight-of-evidence approach.•No IPD079Ea protein toxin or allergen hazard was identified.•IPD079Ea is unlikely to be a hazard to human or animal health.
The aim of this study was to determine the role of Diabrotica virgifera virgifera (western corn rootworm, WCR) as a potential insect vector of the pathogenic bacterium Pantoea ananatis. The ...experiment included a greenhouse test to investigate if WCR was able to transfer the pathogenic bacteria from infected to healthy maize plants. Adult WCR specimens collected from maize fields near Rzeszów were used in the experiment. The plant materials were sweetcorn plants of the Waza variety. Pure cultures of previously verified P. ananatis strain M241 were the source of the inoculum. Insects caught under natural conditions were incubated in an isolator containing pathogen-negative plants. Randomly selected insects were then examined for the presence of bacterial pathogens of maize in their digestive tract. Pathogen-negative insects were used in the next stage of the experiment still carried out in isolators, in which the insects foraged on maize seedlings previously infected with P. ananatis. The control group consisted of healthy, uninfected insects and plants. After the incubation period, the presence of bacterial pathogens in the gastrointestinal tract of the WCR specimens was confirmed. Subsequent insects that acquired P. ananatis were bred on pathogen-negative maize plants. After incubation, the presence of pathogenic bacteria in the body of the examined WCR beetles was confirmed, and the presence of symptoms of the bacterial disease was noted on maize plants, fulfilling Koch's postulates and indicating that the WCR is a vector of P. ananatis on maize plants. This is the first report on the vectorization of P. ananatis by D. virgifera globally.
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•WCR transmits bacterial pathogen (Pantoea ananatis) to maize (Zea mays) plants.•This is the first report of D. virgifera transmitting P. ananatis.•The insects seem to be the key transmitters of P. ananatis.•Results are important for global programs of maize protection.
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.
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•AfIP-1A/1B act as a binary insecticidal protein against western corn rootworm.•AfIP-1A, but not AfIP-1B exhibits specific binding directly to WCR midgut tissue.•AfIP-1A is essential ...to enable specific binding of AfIP-1B.•AfIP-1A/1B exhibits pore formation in artificial lipid bilayers.•Binding of AfIP-1A/1B was reduced in midgut tissue from Cry34Ab1/Cry35Ab1-resistant WCR.
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.
Reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays are a highly accurate and precise method for measuring transcript expression levels. A major drawback of RT-qPCR is the ...extensive optimization and validation necessary to produce high-quality assays, as described in the guidelines "Minimum Information for Publication of Quantitative Real-Time PCR Experiments." This chapter describes use of designed and optimized RT-qPCR assays that accurately detect expression of eight genes predicted to be centrally involved in the RNA interference (RNAi) pathways of western corn rootworm (WCR), and appropriate accompanying parameters. Assay gene targets include drosha, dicer-1, dicer-2, pasha, loquacious, r2d2, argonaute 1, and argonaute 2, and detection has been validated at nine different points in the WCR life cycle. These assays can be used with this procedure to assess expression of any one of these core RNAi pathway genes in up to 96 samples per 384-well qPCR plate.
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•The O. nubilalis orthologs of Ago2, Dcr2, and R2D2 were sequenced and characterized.•Transcripts of all three genes were detected in all stages and tissues.•Comparison of ...evolutionary distances for domains revealed significant variations.•Injection of dsGFP had no effect on Ago2, Dcr2, or R2D2 expression.•Ingestion of dsGFP could induce up/down-regulation of some transcripts.
RNA interference (RNAi) is commonly used in the laboratory to analyze gene function, and RNAi-based pest management strategies are now being employed. Unfortunately, RNAi is hindered by inefficient and highly-variable results when different insects are targeted, especially lepidopterans, such as the European corn borer (ECB), Ostrinia nubilalis (Lepidoptera: Crambidae). Previous efforts to achieve RNAi-mediated gene suppression in ECB revealed low RNAi efficiency with both double-stranded RNA (dsRNA) injection and ingestion. One mechanism that can affect RNAi efficiency in insects is the expression and function of core RNAi pathway genes, such as those encoding Argonaut 2 (Ago2), Dicer 2 (Dcr2), and a dsRNA binding protein (R2D2). To determine if deficiencies in these core RNAi pathway genes contribute to low RNAi efficiency in ECB, full-length complementary DNAs encoding OnAgo2, OnDcr2, and OnR2D2 were cloned, sequenced, and characterized. A comparison of domain architecture suggested that all three predicted proteins contained the necessary domains to function. However, a comparison of evolutionary distances revealed potentially important variations in the first RNase III domain of OnDcr2, the double-stranded RNA binding domains of OnR2D2, and both the PAZ and PIWI domains of OnAgo2, which may indicate functional differences in enzymatic activity between species. Expression analysis indicated that transcripts for all three genes were expressed in all developmental stages and tissues investigated. Interestingly, the introduction of non-target dsRNA into ECB second-instar larvae via microinjection did not affect OnAgo2, OnDcr2, or OnR2D2 expression. In contrast, ingestion of the same dsRNAs resulted in upregulation of OnDcr2 but downregulation of OnR2D2. The unexpected transcriptional responses of the core machinery and the divergence in amino-acid sequence between specific domains in each core RNAi protein may possibly contribute to low RNAi efficiency in ECB. Understanding the contributions of different RNAi pathway components is critical to adapting this technology for use in controlling lepidopteran pests that exhibit low RNAi efficiency.
Western corn rootworm, Diabrotica virgifera virgifera (LeConte) (Coleoptera: Chrysomelidae), is the most serious economic pest of maize, Zea mays (L.) (Poales: Poaceae), in the U.S. Corn Belt and ...also threatens production in Europe. Traditional management options have repeatedly failed over time as western corn rootworm rapidly develops resistance to insecticides, transgenic maize and even crop rotation. Traits that improve host plant resistance and tolerance are highly sought after by plant breeders for crop protection and pest management. However, maize resistance to western corn rootworm appears to be highly complex and despite over 75 yr of breeding efforts, there are no naturally resistant hybrids available commercially. Using phenotypic data from field and greenhouse experiments on a highly diverse collection of 282 inbred lines, we screened and genetically mapped western corn rootworm-related traits to identify genetic loci which may be useful for future breeding or genetic engineering efforts. Our results confirmed that western corn rootworm resistance is complex with relatively low heritability due in part to strong genotype by environment impacts and the inherent difficulties of phenotyping below ground root traits. The results of the Genome Wide Associated Study identified 29 loci that are potentially associated with resistance to western corn rootworm. Of these loci, 16 overlap with those found in previous transcription or mapping studies indicating a higher likelihood they are truly involved in maize western corn rootworm resistance. Taken together with previous studies, these results indicate that breeding for natural western corn rootworm resistance will likely require the stacking of multiple small effect loci.
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.
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•DsRNA was highly unstable when incubated in gut contents of Ostrinia nubilalis larvae.•DsRNA was degraded under physiologically relevant pH conditions.•Degradation in gut contents and hemolymph was due to enzymatic activity.•Degradation of dsRNA was not size or sequence-dependent.
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