Quantitative Real-Time Polymerase Chain Reaction, better known as qPCR, is the most sensitive and specific technique we have for the detection of nucleic acids. Even though it has been around for more than 30 years and is preferred in research applications, it has yet to win broad acceptance in routine practice. This requires a means to unambiguously assess the performance of specific qPCR analyses. Here we present methods to determine the limit of detection (LoD) and the limit of quantification (LoQ) as applicable to qPCR. These are based on standard statistical methods as recommended by regulatory bodies adapted to qPCR and complemented with a novel approach to estimate the precision of LoD.

Although transgenic crops expressing Bacillus thuringiensis (Bt) toxins have been used successfully for management of lepidopteran and coleopteran pest species, the sap-sucking insects (Hemiptera) are not particularly susceptible to Bt toxins. To overcome this limitation, we demonstrate that addition of a short peptide sequence selected for binding to the gut of the targeted pest species serves to increase toxicity against said pest. Insertion of a 12-aa pea aphid gut-binding peptide by adding to or replacing amino acids in one of three loops of the Bt cytolytic toxin, Cyt2Aa, resulted in enhanced binding and toxicity against both the pea aphid, Acyrthosiphon pisum, and the green peach aphid, Myzus persicae. This strategy may allow for transgenic plant-mediated suppression of other hemipteran pests, which include some of the most important pests of global agriculture.aphid management | biotechnology | insect resistance

Insect-borne plant viruses cause significant agricultural losses and jeopardize sustainable global food production. Although blocking plant virus transmission would allow for crop protection, virus receptors in insect vectors are unknown. Here we identify membrane alanyl aminopeptidase N (APN) as a receptor for pea enation mosaic virus (PEMV) coat protein (CP) in the gut of the pea aphid, Acyrthosiphon pisum, using a far-Western blot method. Pulldown and immunofluorescence binding assays and surface plasmon resonance were used to confirm and characterize CP-APN interaction. PEMV virions and a peptide comprised of PEMV CP fused to a proline-rich hinge (-P-) and green fluorescent protein (CP-P-GFP) specifically bound to APN. Recombinant APN expressed in Sf9 cells resulted in internalization of CP-P-GFP, which was visualized by confocal microscopy; such internalization is an expected hallmark of a functional gut receptor. Viruses that infect crop plants restrict our ability to consistently produce high yields from agricultural crops. Many of these viruses are transmitted to plants by pestiferous insects, with aphids transmitting nearly half of the 600 insect-borne plant viruses. Aphids therefore represent a significant threat to global agriculture (1). Viruses in the family Luteoviridae are phloem-restricted RNA viruses transmitted exclusively by aphids and cause disease in multiple food crops (2). Luteovirids are transmitted in a circulative and persistent manner which involves specific molecular interactions between the virus and the aphid (3). For this type of transmission, ingested virions cross the aphid gut and salivary gland epithelial barriers for transmission to additional plant hosts.Luteovirus-aphid interactions are mediated by the viral capsid proteins consisting of a major coat protein (CP; 22 kDa) and one minor coat protein readthrough domain (CP-RTD; 35 to 55 kDa) (3). The RTD is not required for virus particle assembly or for uptake of virus from the gut into the aphid hemocoel, but both CP and RTD are essential for aphid transmission and are the sole determinants of vector specificity (3). The virus binds to a receptor in either the midgut, hindgut, or both for transcytosis across the aphid gut epithelium and release into the hemocoel (4). A second receptor-mediated transcytosis event occurs at the accessory salivary glands (ASG) from which virus particles are secreted with saliva to inoculate the plant phloem during subsequent feedings (5). Only a fraction of the virions present in the aphid hemolymph cross into the ASG, and a threshold amount of virus in the hemocoel is required before transmission via the ASG can occur (3). Hence, impeding the binding of a plant virus to the aphid gut receptor could reduce the amount of virus present in the hemocoel to less-than-threshold levels, thereby reducing plant virus transmission. Citation Linz LB, Liu S, Chougule NP, Bonning BC. 2015. In vitro evidence supports membrane alanyl aminopeptidase N as a receptor for a plant virus in the pea aphid ve...

Pea enation mosaic virus (PEMV)—a plant RNA virus transmitted exclusively by aphids—causes disease in multiple food crops. However, the aphid-virus interactions required for disease transmission are poorly understood. For virus transmission, PEMV binds to a heavily glycosylated receptor aminopeptidase N in the pea aphid gut and is transcytosed across the gut epithelium into the aphid body cavity prior to release in saliva as the aphid feeds. To investigate the role of glycans in PEMV–aphid interactions and explore the possibility of viral control through blocking a glycan interaction, we synthesized insect N-glycan terminal trimannosides by automated solution-phase synthesis. The route features a mannose building block with C-5 ester enforcing a β-linkage, which also provides a site for subsequent chain extension. The resulting insect N-glycan terminal trimannosides with fluorous tags were used in a fluorous microarray to analyze binding with fluorescein isothiocyanate-labeled PEMV; however, no specific binding between the insect glycan and PEMV was detected. To confirm these microarray results, we removed the fluorous tag from the trimannosides for isothermal titration calorimetry studies with unlabeled PEMV. The ITC studies confirmed the microarray results and suggested that this particular glycan–PEMV interaction is not involved in virus uptake and transport through the aphid.

Insect transmission of plant viruses results in tremendous economic loss within the agricultural sector worldwide. Aphids account for nearly half of insect-borne plant virus transmission. Viruses in the family Luteoviridae are transmitted by aphids in a persistentcirculative manner that requires specific molecular interactions between the aphid and virus. Ingested virions cross the aphid gut and salivary gland epithelial barriers using receptors that have not been identified. We assessed the binding of a model luteovirid, Pea enation mosaic virus (PEMV), to brush border membrane vesicles (BBMV) of the pea aphid, Acyrthosiphon pisum using a two-dimensional far-western blot method. Pea aphid membrane alanyl aminopeptidase N (APN) was identified by mass spectrometry following specific binding to PEMV virions and to a PEMV coat protein-eGFP fusion peptide (CP-P-eGFP). The binding of PEMV to APN was confirmed by multiple methods including a pull-down assay, surface plasmon resonance (SPR) analysis, and by increased binding of CP-P-eGFP to baculovirusexpressed pea aphid APN in Sf9 cells. We also show that a peptide (GBP3.1) that was previously shown to impede uptake of PEMV into the pea aphid also binds to APN. Based on these results, we conclude that APN is a putative gut receptor for PEMV in the pea aphid and if confirmed would be the first insect receptor identified for a plant virus. Interestingly, PEMV appears to bind to a different, as yet unidentified, receptor in a second vector, Myzus persicae, suggesting that different gut receptors may be used by luteoviruses in different vector species. Luteoviruses are acquired when aphids ingest the phloem sap of an infected plant. Phloem proteins have been shown to associate with luteovirus particles and facilitate aphid transmission in in vitro feeding assays. We showed an increase of virus in the hemocoel of v aphids fed on artificial diet containing purified PEMV with bovine serum albumin (BSA) compared to aphids fed on virus in the absence of BSA. Interestingly, BSA reduced the amount of a mutant virus lacking the minor structural protein readthrough domain (RTD) detected in the aphid hemocoel. We also demonstrated that the PEMV RTD binds to multiple aphid proteins. SPR analysis indicated that the CP and RTD both bind to BSA. Based on these data, models are presented to account for the role of the RTD and mechanism by which BSA and plant proteins facilitate virus entry into the aphid hemocoel. Little is known about the role of glycans in mediating luteovirus-aphid interactions. We used the lectins Concanavalin A (ConA) and Galanthus nivalis agglutinin (GNA) for lectin blot analysis of BBMV and confirmed that pea aphid proteins are glycosylated with mannose and glucose moieties. APN, the PEMV gut receptor, is glycosylated with mannose residues. However, we did not detect any binding of PEMV to a synthesized tri-mannose glycan that is common in insects using both isothermal titration calorimetry or a carbohydrate microarray. These results suggest that mannose by itself...