Hetero-oligomerization of Bacillus thuringiensis Cry1A proteins enhance binding to the ABCC2 transporter of Spodoptera exigua.

The ATP binding cassette (ABC) transporters are membrane proteins that can act as putative receptors for Cry proteins from Bacillus thuringiensis (Bt) in the midgut of different insects. For the beet armyworm, Spodoptera exigua, ABCC2 and ABCC3 have been found to interact with Cry1A proteins, the main insecticidal proteins used in Bt crops, as well as Bt-based pesticides. The ABCC2 has shown to have specific binding towards Cry1Ac and is involved in the toxic process of Cry1A proteins, but the role of this transporter and how it relates with the Cry1A proteins is still unknown. Here, we have characterized the interactions between the SeABCC2 and the main proteins that bind to the receptor. By labeling the Cry1Aa protein, we have found that virtually all of the binding is in an oligomeric state, a conformation that allowed higher levels of specific binding that could not be achieved by the monomeric protein on its own. Furthermore, we have observed that Cry1A proteins can hetero-oligomerize in the presence of the transporter, which is reflected in an increase in binding and toxicity to SeABCC2-expressing cells. This synergism can be one of the reasons why B. thuringiensis co-expresses different Cry1 proteins that can apparently have similar binding preferences. The results from in vitro competition and ex vivo competition showed that Cry1Aa, Cry1Ab and Cry1Ac share functional binding sites. By using Cry1Ab-Cry1Ac chimeras, the presence of domain I from Cry1A proteins was revealed to be critical for oligomer formation.

New Paralogs of the Heliothis virescens ABCC2 Transporter as Potential Receptors for Bt Cry1A Proteins.

The ATP-binding cassette (ABC) transporters are a superfamily of membrane proteins. These active transporters are involved in the export of different substances such as xenobiotics. ABC transporters from subfamily C (ABCC) have also been described as functional receptors for different insecticidal proteins from Bacillus thuringiensis (Bt) in several lepidopteran species. Numerous studies have characterized the relationship between the ABCC2 transporter and Bt Cry1 proteins. Although other ABCC transporters sharing structural and functional similarities have been described, little is known of their role in the mode of action of Bt proteins. For Heliothis virescens, only the ABCC2 transporter and its interaction with Cry1A proteins have been studied to date. Here, we have searched for paralogs to the ABCC2 gene in H. virescens, and identified two new ABC transporter genes: HvABCC3 and HvABCC4. Furthermore, we have characterized their gene expression in the midgut and their protein topology, and compared them with that of ABCC2. Finally, we discuss their possible interaction with Bt proteins by performing protein docking analysis.

Evaluation of a Tube Voltage-Based Contrast Media Adaptation in Coronary Computed Tomography Angiography Using Personalized Triphasic Injection Protocols: A Matched Case-Control Study.

RATIONALE AND OBJECTIVES: Coronary CT angiography (CCTA) has recently been established as a first-line test in patients with suspected coronary artery disease (CAD). Due to the increased use of CCTA, strategies to reduce radiation and contrast medium (CM) exposure are of high importance. The aim of this study was to evaluate the performance of automated tube voltage selection (ATVS)-adapted CM injection protocol for CCTA compared to a clinically established triphasic injection protocol in terms of image quality, radiation exposure, and CM administration MATERIAL AND METHODS: Patients undergoing clinically indicated CCTA were prospectively enrolled from July 2021 to July 2023. Patients underwent CCTA using a modified triphasic CM injection protocol tailored to the tube voltage by the ATVS algorithm, in a range of 70 to 130 kV with a 10 kV interval. The injection protocol consisted of two phases of mixed CM and saline boluses with different proportions to assure a voltage-specific iodine delivery rate, followed by a third phase of saline flush. This cohort was compared to a control group identified retrospectively and scanned on the same CT system but with a standard triphasic CM protocol. Radiation and contrast dose, subjective and objective image quality (contrast-to-noise-ratio [CNR] and signal-to-noise-ratio [SNR]) were compared between the two groups. RESULTS: The final population consisted of 120 prospective patients matched with 120 retrospective controls, with 20 patients in each kV group. The 120 kV group was excluded from the statistical analysis due to insufficient sample size. A significant CM reduction was achieved in the prospective group overall (46.0 [IQR 37.0-52.0] vs. 51.3 [IQR 40.1-73.0] mL, p < 0.001) and at all kV levels too (all pairwise p < 0.001). There were no significant differences in radiation dose (6.13 ± 4.88 vs. 5.97 ± 5.51 mSv, p = 0.81), subjective image quality (median score of 4 [3-5] vs. 4 [3-5], p = 0.40), CNR, and SNR in the aorta and the left anterior descending coronary artery (all p > 0.05). CONCLUSION: ATVS-adapted CM injection protocol allows for diagnostic quality CCTA with reduced CM volume while maintaining similar radiation exposure, subjective and objective image quality.

Accuracy of ultra-high resolution and virtual non-calcium reconstruction algorithm for stenosis evaluation with photon-counting CT: results from a dynamic phantom study.

BACKGROUND: We compared ultra-high resolution (UHR), standard resolution (SR), and virtual non-calcium (VNCa) reconstruction for coronary artery stenosis evaluation using photon-counting computed tomography (PC-CT). METHODS: One vessel phantom (4-mm diameter) containing solid calcified lesions with 25% and 50% stenoses inside a thorax phantom with motion simulation underwent PC-CT using UHR (0.2-mm slice thickness) and SR (0.6-mm slice thickness) at heart rates of 60 beats per minute (bpm), 80 bpm, and 100 bpm. A paired t-test or Wilcoxon test with Bonferroni correction was used. RESULTS: For 50% stenosis, differences in percent mean diameter stenosis between UHR and SR at 60 bpm (51.0 vs 60.3), 80 bpm (51.7 vs 59.6), and 100 bpm (53.7 vs 59.0) (p ≤ 0.011), as well as between VNCa and SR at 60 bpm (50.6 vs 60.3), 80 bpm (51.5 vs 59.6), and 100 bpm (53.7 vs 59.0) were significant (p ≤ 0.011), while differences between UHR and VNCa at all heart rates (p ≥ 0.327) were not significant. For 25% stenosis, differences between UHR and SR at 60 bpm (28.0 vs 33.7), 80 bpm (28.4 vs 34.3), and VNCa vs SR at 60 bpm (29.1 vs 33.7) were significant (p ≤ 0.015), while differences for UHR vs SR at 100 bpm (29.9 vs 34.0), as well as for VNCa vs SR at 80 bpm (30.7 vs 34.3) and 100 bpm (33.1 vs 34.0) were not significant (p ≥ 0.028). CONCLUSION: Stenosis quantification accuracy with PC-CT improved using either UHR acquisition or VNCa reconstruction. RELEVANCE STATEMENT: PC-CT offers to scan with UHR mode and the reconstruction of VNCa images both of them could provide improved coronary stenosis quantification at increased heart rates, allowing a more accurate stenosis grading at low and high heart rates compared to SR. KEY POINTS: Evaluation of coronary stenosis with conventional CT is challenging at high heart rates. PC-CT allows for scanning with ECG-gated UHR and SR modes. UHR and VNCa images were compared in a dynamic phantom. UHR improves stenosis quantification up to 100 bpm. VNCa reconstruction improves stenosis evaluation up to 80 bpm.

Improved Detection of Small and Low-Density Plaques in Virtual Noncontrast Imaging-based Calcium Scoring at Photon-Counting Detector CT.

Purpose To investigate the impact of plaque size and density on virtual noncontrast (VNC)-based coronary artery calcium scoring (CACS) using photon-counting detector CT and to provide safety net reconstructions for improved detection of subtle plaques in patients whose VNC-based CACS would otherwise be erroneously zero when compared with true noncontrast (TNC)-based CACS. Materials and Methods In this prospective study, CACS was evaluated in a phantom containing calcifications with different diameters (5, 3, and 1 mm) and densities (800, 400, and 200 mg/cm3) and in participants who underwent TNC and contrast-enhanced cardiac photon-counting detector CT (July 2021-March 2022). VNC images were reconstructed at different virtual monoenergetic imaging (55-80 keV) and quantum iterative reconstruction (QIR) levels (QIR,1-4). TNC scans at 70 keV with QIR off served as the reference standard. In vitro CACS was analyzed using standard settings (3.0-mm sections, kernel Qr36, 130-HU threshold). Calcification detectability and CACS of small and low-density plaques were also evaluated using 1.0-mm sections, kernel Qr44, and 120- or 110-HU thresholds. Safety net reconstructions were defined based on background Agatston scores and evaluated in vivo in TNC plaques initially nondetectable using standard VNC reconstructions. Results The in vivo cohort included 63 participants (57.8 years ± 15.5 [SD]; 37 [59%] male, 26 [41%] female). Correlation and agreement between standard CACSVNC and CACSTNC were higher in large- and medium-sized and high- and medium-density than in low-density plaques (in vitro: intraclass correlation coefficient [ICC] ≥ 0.90; r > 0.9 vs ICC = 0.20-0.48; r = 0.5-0.6). Small plaques were not detectable using standard VNC reconstructions. Calcification detectability was highest using 1.0-mm sections, kernel Qr44, 120- and 110-HU thresholds, and QIR level of 2 or less VNC reconstructions. Compared with standard VNC, using safety net reconstructions (55 keV, QIR 2, 110-HU threshold) for in vivo subtle plaque detection led to higher detection (increased by 89% [50 of 56]) and improved correlation and agreement of CACSVNC with CACSTNC (in vivo: ICC = 0.51-0.61; r = 0.6). Conclusion Compared with TNC-based calcium scoring, VNC-based calcium scoring was limited for small and low-density plaques but improved using safety net reconstructions, which may be particularly useful in patients with low calcium scores who would otherwise be treated based on potentially false-negative results. Keywords: Coronary Artery Calcium CT, Photon-Counting Detector CT, Virtual Noncontrast, Plaque Size, Plaque Density Supplemental material is available for this article. © RSNA, 2024.

Artificial Intelligence Provides Accurate Quantification of Thoracic Aortic Enlargement and Dissection in Chest CT.

This study evaluated a deep neural network (DNN) algorithm for automated aortic diameter quantification and aortic dissection detection in chest computed tomography (CT). A total of 100 patients (median age: 67.0 [interquartile range 55.3/73.0] years; 60.0% male) with aortic aneurysm who underwent non-enhanced and contrast-enhanced electrocardiogram-gated chest CT were evaluated. All the DNN measurements were compared to manual assessment, overall and between the following subgroups: (1) ascending (AA) vs. descending aorta (DA); (2) non-obese vs. obese; (3) without vs. with aortic repair; (4) without vs. with aortic dissection. Furthermore, the presence of aortic dissection was determined (yes/no decision). The automated and manual diameters differed significantly (p < 0.05) but showed excellent correlation and agreement (r = 0.89; ICC = 0.94). The automated and manual values were similar in the AA group but significantly different in the DA group (p < 0.05), similar in obese but significantly different in non-obese patients (p < 0.05) and similar in patients without aortic repair or dissection but significantly different in cases with such pathological conditions (p < 0.05). However, in all the subgroups, the automated diameters showed strong correlation and agreement with the manual values (r > 0.84; ICC > 0.9). The accuracy, sensitivity and specificity of DNN-based aortic dissection detection were 92.1%, 88.1% and 95.7%, respectively. This DNN-based algorithm enabled accurate quantification of the largest aortic diameter and detection of aortic dissection in a heterogenous patient population with various aortic pathologies. This has the potential to enhance radiologists' efficiency in clinical practice.

Impact of reconstruction parameters on the accuracy of myocardial extracellular volume quantification on a first-generation, photon-counting detector CT.

BACKGROUND: The potential role of cardiac computed tomography (CT) has increasingly been demonstrated for the assessment of diffuse myocardial fibrosis through the quantification of extracellular volume (ECV). Photon-counting detector (PCD)-CT technology may deliver more accurate ECV quantification compared to energy-integrating detector CT. We evaluated the impact of reconstruction settings on the accuracy of ECV quantification using PCD-CT, with magnetic resonance imaging (MRI)-based ECV as reference. METHODS: In this post hoc analysis, 27 patients (aged 53.1 ± 17.2 years (mean ± standard deviation); 14 women) underwent same-day cardiac PCD-CT and MRI. Late iodine CT scans were reconstructed with different quantum iterative reconstruction levels (QIR 1-4), slice thicknesses (0.4-8 mm), and virtual monoenergetic imaging levels (VMI, 40-90 keV); ECV was quantified for each reconstruction setting. Repeated measures ANOVA and t-test for pairwise comparisons, Bland-Altman plots, and Lin's concordance correlation coefficient (CCC) were used. RESULTS: ECV values did not differ significantly among QIR levels (p = 1.000). A significant difference was observed throughout different slice thicknesses, with 0.4 mm yielding the highest agreement with MRI-based ECV (CCC = 0.944); 45-keV VMI reconstructions showed the lowest mean bias (0.6, 95% confidence interval 0.1-1.4) compared to MRI. Using the most optimal reconstruction settings (QIR4. slice thickness 0.4 mm, VMI 45 keV), a 63% reduction in mean bias and a 6% increase in concordance with MRI-based ECV were achieved compared to standard settings (QIR3, slice thickness 1.5 mm; VMI 65 keV). CONCLUSIONS: The selection of appropriate reconstruction parameters improved the agreement between PCD-CT and MRI-based ECV. RELEVANCE STATEMENT: Tailoring PCD-CT reconstruction parameters optimizes ECV quantification compared to MRI, potentially improving its clinical utility. KEY POINTS: • CT is increasingly promising for myocardial tissue characterization, assessing focal and diffuse fibrosis via late iodine enhancement and ECV quantification, respectively. • PCD-CT offers superior performance over conventional CT, potentially improving ECV quantification and its agreement with MRI-based ECV. • Tailoring PCD-CT reconstruction parameters optimizes ECV quantification compared to MRI, potentially improving its clinical utility.

Intra-individual comparison of coronary CT angiography-based FFR between energy-integrating and photon-counting detector CT systems.

BACKGROUND: Coronary computed tomography angiography (CCTA)-based fractional flow reserve (CT-FFR) allows for noninvasive determination of the functional severity of anatomic lesions in patients with coronary artery disease. The aim of this study was to intra-individually compare CT-FFR between photon-counting detector (PCD) and conventional energy-integrating detector (EID) CT systems. METHODS: In this single-center prospective study, subjects who underwent clinically indicated CCTA on an EID-CT system were recruited for a research CCTA on PCD-CT within 30 days. Image reconstruction settings were matched as closely as possible between EID-CT (Bv36 kernel, iterative reconstruction strength level 3, slice thickness 0.5 mm) and PCD-CT (Bv36 kernel, quantum iterative reconstruction level 3, virtual monoenergetic level 55 keV, slice thickness 0.6 mm). CT-FFR was measured semi-automatically using a prototype on-site machine learning algorithm by two readers. CT-FFR analysis was performed per-patient and per-vessel, and a CT-FFR ≤ 0.75 was considered hemodynamically significant. RESULTS: A total of 22 patients (63.3 ± 9.2 years; 7 women) were included. Median time between EID-CT and PCD-CT was 5.5 days. Comparison of CT-FFR values showed no significant difference and strong agreement between EID-CT and PCD-CT in the per-vessel analysis (0.88 [0.74-0.94] vs. 0.87 [0.76-0.93], P = 0.096, mean bias 0.02, limits of agreement [LoA] -0.14/0.19, r = 0.83, ICC = 0.92), and in the per-patient analysis (0.81 [0.60-0.86] vs. 0.76 [0.64-0.86], P = 0.768, mean bias 0.02, LoA -0.15/0.19, r = 0.90, ICC = 0.93). All included patients were classified into the same category (CT-FFR > 0.75 vs ≤0.75) with both CT systems. CONCLUSIONS: CT-FFR evaluation is feasible with PCD-CT and it shows a strong agreement with EID-CT-based evaluation when images are similarly reconstructed.

Intra-individual comparison of image quality of the coronary arteries between photon-counting detector and energy-integrating detector CT systems.

PURPOSE: To intra-individually compare the objective and subjective image quality of coronary computed tomography angiography (CCTA) between photon-counting detector CT (PCD-CT) and energy-integrating detector CT (EID-CT). METHOD: Consecutive patients undergoing clinically indicated CCTA on an EID-CT system were prospectively enrolled for a research CCTA performed on a PCD-CT system within 30 days. Polychromatic images were reconstructed for both EID- and PCD-CT, while virtual monoenergetic images (VMI) were generated at 40, 45, 50, 55, 60 and 70 keV for PCD-CT. Two blinded readers calculated contrast-to-noise ratio (CNR) for each major coronary artery and rated image noise, vessel attenuation, vessel sharpness, and overall quality on a 1-5 Likert scale. Patients were then stratified by body mass index (BMI) [high (>30 kg/m2) vs low (<30 kg/m2)] for subgroup analysis. RESULTS: A total of 20 patients (67.5 ± 9.0 years, 75% male) were included in the study. Compared with EID-CT, coronary artery CNR values from PCD-CT monoenergetic and polychromatic reconstructions were all significantly higher than CNR values from EID-CT, with incrementally greater differences in obese subjects (all p < 0.008). Subjective image noise and sharpness were also significantly higher for all VMI reconstructions compared to EID-CT (all p < 0.008). All subjective scores were significantly higher for 55, 60, and 70 keV PCD-CT than EID-CT values (all p < 0.05). CONCLUSIONS: The improved objective and subjective image quality of PCD-CT compared to EID-CT may provide better visualization of the coronary arteries for a wide array of patients, especially those with a high BMI.

Impact of Cardiac Motion on coronary artery calcium scoring using a virtual non-iodine algorithm on photon-counting detector CT: a dynamic phantom study.

This study assessed the impact of cardiac motion and in-vessel attenuation on coronary artery calcium (CAC) scoring using virtual non-iodine (VNI) against virtual non-contrast (VNC) reconstructions on photon-counting detector CT. Two artificial vessels containing calcifications and different in-vessel attenuations (500, 800HU) were scanned without (static) and with cardiac motion (60, 80, 100 beats per minute [bpm]). Images were post-processed using a VNC and VNI algorithm at 70 keV and quantum iterative reconstruction (QIR) strength 2. Calcium mass, Agatston scores, cardiac motion susceptibility (CMS)-indices were compared to physical mass, static scores as well as between reconstructions, heart rates and in-vessel attenuations. VNI scores decreased with rising heart rate (p < 0.01) and showed less underestimation than VNC scores (p < 0.001). Only VNI scores were similar to the physical mass at static measurements, and to static scores at 60 bpm. Agatston scores using VNI were similar to static scores at 60 and 80 bpm. Standard deviation of CMS-indices was lower for VNI-based than for VNC-based CAC scoring. VNI scores were higher at 500 than 800HU (p < 0.001) and higher than VNC scores (p < 0.001) with VNI scores at 500 HU showing the lowest deviation from the physical reference. VNI-based CAC quantification is influenced by cardiac motion and in-vessel attenuation, but least when measuring Agatston scores, where it outperforms VNC-based CAC scoring.

Radiation Dose Reduction for Coronary Artery Calcium Scoring Using a Virtual Noniodine Algorithm on Photon-Counting Detector Computed-Tomography Phantom Data.

Background: On the basis of the hypothesis that virtual noniodine (VNI)-based coronary artery calcium scoring (CACS) is feasible at reduced radiation doses, this study assesses the impact of radiation dose reduction on the accuracy of this VNI algorithm on a photon-counting detector (PCD)-CT. Methods: In a systematic in vitro setting, a phantom for CACS simulating three chest sizes was scanned on a clinical PCD-CT. The standard radiation dose was chosen at volumetric CT dose indices (CTDIVol) of 1.5, 3.3, 7.0 mGy for small, medium-sized, and large phantoms, and was gradually reduced by adjusting the tube current resulting in 100, 75, 50, and 25%, respectively. VNI images were reconstructed at 55 keV, quantum iterative reconstruction (QIR)1, and at 60 keV/QIR4, and evaluated regarding image quality (image noise (IN), contrast-to-noise ratio (CNR)), and CACS. All VNI results were compared to true noncontrast (TNC)-based CACS at 70 keV and standard radiation dose (reference). Results: INTNC was significantly higher than INVNI, and INVNI at 55 keV/QIR1 higher than at 60 keV/QIR4 (100% dose: 16.7 ± 1.9 vs. 12.8 ± 1.7 vs. 7.7 ± 0.9; p < 0.001 for every radiation dose). CNRTNC was higher than CNRVNI, but it was better to use 60 keV/QIR4 (p < 0.001). CACSVNI showed strong correlation and agreement at every radiation dose (p < 0.001, r > 0.9, intraclass correlation coefficient > 0.9). The coefficients of the variation in root-mean squared error were less than 10% and thus clinically nonrelevant for the CACSVNI of every radiation dose. Conclusion: This phantom study suggests that CACSVNI is feasible on PCD-CT, even at reduced radiation dose while maintaining image quality and CACS accuracy.

Alteration of a Cry1A Shared Binding Site in a Cry1Ab-Selected Colony of Ostrinia furnacalis.

The Asian corn borer, Ostrinia furnacalis (Guenée, 1854), is a highly damaging pest in Asia and the Pacific islands, and larvae feed mainly from corn crops. To determine the suitability of Bt-corn technology for the future control of this pest, understanding the potential to develop resistance to Cry1Ab and the basis of cross-resistance to other Cry1 proteins is of great interest. Here, we have explored the binding of Cry1A proteins to brush border membrane vesicles from two O. furnacalis colonies, one susceptible (ACB-BtS) and one laboratory-selected with Cry1Ab (ACB-AbR). The insects developed resistance to Cry1Ab and showed cross-resistance to Cry1Aa, Cry1Ac, and Cry1F. Binding assays with radiolabeled Cry1Ab and brush border membrane vesicles from susceptible insects showed that Cry1A proteins shared binding sites, though the results were not conclusive for Cry1F. The results were confirmed using radiolabeled Cry1Aa. The resistant insects showed a reduction of the specific binding of both Cry1Ab and Cry1Aa, suggesting that part of the binding sites were lost or altered. Competition binding assays showed full competition between Cry1Ab and Cry1Aa proteins in the susceptible colony but only partial competition in resistant insects, confirming the alteration of some, but not all, binding sites for these two proteins. The binding site model for Cry1A proteins in O. furnacalis is in agreement with the occurrence of multiple membrane receptors for these proteins.

Response Mechanisms of Invertebrates to Bacillus thuringiensis and Its Pesticidal Proteins.

Extensive use of chemical insecticides adversely affects both environment and human health. One of the most popular biological pest control alternatives is bioinsecticides based on Bacillus thuringiensis This entomopathogenic bacterium produces different protein types which are toxic to several insect, mite, and nematode species. Currently, insecticidal proteins belonging to the Cry and Vip3 groups are widely used to control insect pests both in formulated sprays and in transgenic crops. However, the benefits of B. thuringiensis-based products are threatened by insect resistance evolution. Numerous studies have highlighted that mutations in genes coding for surrogate receptors are responsible for conferring resistance to B. thuringiensis Nevertheless, other mechanisms may also contribute to the reduction of the effectiveness of B. thuringiensis-based products for managing insect pests and even to the acquisition of resistance. Here, we review the relevant literature reporting how invertebrates (mainly insects and Caenorhabditis elegans) respond to exposure to B. thuringiensis as either whole bacteria, spores, and/or its pesticidal proteins.

Reduced Membrane-Bound Alkaline Phosphatase Does Not Affect Binding of Vip3Aa in a Heliothis virescens Resistant Colony.

The Vip3Aa insecticidal protein from Bacillus thuringiensis (Bt) is produced by specific transgenic corn and cotton varieties for efficient control of target lepidopteran pests. The main threat to this technology is the evolution of resistance in targeted insect pests and understanding the mechanistic basis of resistance is crucial to deploy the most appropriate strategies for resistance management. In this work, we tested whether alteration of membrane receptors in the insect midgut might explain the >2000-fold Vip3Aa resistance phenotype in a laboratory-selected colony of Heliothis virescens (Vip-Sel). Binding of 125I-labeled Vip3Aa to brush border membrane vesicles (BBMV) from 3rd instar larvae from Vip-Sel was not significantly different from binding in the reference susceptible colony. Interestingly, BBMV from Vip-Sel larvae showed dramatically reduced levels of membrane-bound alkaline phosphatase (mALP) activity, which was further confirmed by a strong downregulation of the membrane-bound alkaline phosphatase 1 (HvmALP1) gene. However, the involvement of HvmALP1 as a receptor for the Vip3Aa protein was not supported by results from ligand blotting and viability assays with insect cells expressing HvmALP1.

The Spodoptera exigua ABCC2 Acts as a Cry1A Receptor Independently of its Nucleotide Binding Domain II.

ABC proteins are primary-active transporters that require the binding and hydrolysis of ATP to transport substrates across the membrane. Since the first report of an ABCC2 transporter as receptor of Cry1A toxins, the number of ABC transporters known to be involved in the mode of action of Cry toxins has increased. In Spodoptera exigua, a mutation in the SeABCC2 gene is described as genetically linked to resistance to the Bt-product XentariTM. This mutation affects an intracellular domain involved in ATP binding, but not the extracellular loops. We analyzed whether this mutation affects the role of the SeABCC2 as a functional receptor to Cry1A toxins. The results show that Sf21 cells expressing the truncated form of the transporter were susceptible to Cry1A toxins. Moreover, specific Cry1Ac binding was observed in those cells expressing the truncated SeABCC2. Additionally, no differences in the irreversible Cry1Ac binding component (associated with the toxin insertion into the membrane) were observed when tested in Sf21 cells expressing either the full-length or the truncated form of the SeABCC2 transporter. Therefore, our results point out that the partial lack of the nucleotide binding domain II in the truncated transporter does not affect its functionality as a Cry1A receptor.

Role of Bacillus thuringiensis Cry1A toxins domains in the binding to the ABCC2 receptor from Spodoptera exigua.

Cry proteins from Bacillus thuringiensis (Bt) have been used to control insect pests either as formulated sprays or as in Bt-crops. However, field-evolved resistance to Bt proteins is threatening the long-term use of Bt products. The SeABCC2 locus has been genetically linked to resistance to a Bt bioinsecticide (Xentari™) in Spodoptera exigua (a mutation producing a truncated form of the transporter lacking an ATP binding domain was found in the resistant insects). Here, we investigated the role of SeABCC2 in the mode of action of Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ca, and two Cry1A-1Ca hybrids by expressing the receptor in Sf21 and HEK293T cell lines. Cell toxicity assays showed that Sf21 cells expressing SeABCC2 become susceptible to Cry1A proteins. HEK293T cells expressing the transporter were found susceptible to Cry1A proteins but not to Cry1Ca. The results with the Cry1A-1Ca hybrids suggest that domain II from Cry1Ab/c is crucial for the toxicity to Sf21 cells, whereas domain III from Cry1Aa/b is crucial for the toxicity to HEK293T cells. Binding assays showed that the Cry1Ac binding is of high affinity and specific to cells expressing the SeABCC2 transporter. Heterologous competition experiments support a model in which domain II of Cry1Ab/c has a common binding site in the SeABCC2 protein, whereas domain III of Cry1Aa/b binds to a different binding site in the SeABCC2 protein.