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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.
Claudia Pérez Ortega; Chris Leininger; Jennifer Barry; Brad Poland; Nasser Yalpani; Dan Altier; Mark E. Nelson; Albert L. Lu. Coordinated binding of a two-component insecticidal protein from Alcaligenes faecalis to western corn rootworm midgut tissue. Journal of Invertebrate Pathology 2021, 183, 107597 .
AMA StyleClaudia Pérez Ortega, Chris Leininger, Jennifer Barry, Brad Poland, Nasser Yalpani, Dan Altier, Mark E. Nelson, Albert L. Lu. Coordinated binding of a two-component insecticidal protein from Alcaligenes faecalis to western corn rootworm midgut tissue. Journal of Invertebrate Pathology. 2021; 183 ():107597.
Chicago/Turabian StyleClaudia Pérez Ortega; Chris Leininger; Jennifer Barry; Brad Poland; Nasser Yalpani; Dan Altier; Mark E. Nelson; Albert L. Lu. 2021. "Coordinated binding of a two-component insecticidal protein from Alcaligenes faecalis to western corn rootworm midgut tissue." Journal of Invertebrate Pathology 183, no. : 107597.
Western corn rootworm (WCR), Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae), is a serious insect pest in the major corn growing areas of North America and in parts of Europe. WCR populations with resistance to Bacillus thuringiensis (Bt) toxins utilized in commercial transgenic traits have been reported, raising concerns over their continued efficacy in WCR management. Understanding the modes of action of Bt toxins is important for WCR control and resistance management. Although different classes of proteins have been identified as Bt receptors for lepidopteran insects, identification of receptors in WCR has been limited with no reports of functional validation. Our results demonstrate that heterologous expression of DvABCB1 in Sf9 and HEK293 cells conferred sensitivity to the cytotoxic effects of Cry3A toxins. The result was further validated using knockdown of DvABCB1 by RNAi which rendered WCR larvae insensitive to a Cry3A toxin. However, silencing of DvABCB2 which is highly homologous to DvABCB1 at the amino acid level, did not reduce the sensitivity of WCR larvae to a Cry3A toxin. Furthermore, our functional studies corroborate different mode-of-actions for other insecticidal proteins including Cry34Ab1/35Ab1, Cry6Aa1, and IPD072Aa against WCR. Finally, reduced expression and alternatively spliced transcripts of DvABCB1 were identified in a mCry3A-resistant strain of WCR. Our results provide the first clear demonstration of a functional receptor in the molecular mechanism of Cry3A toxicity in WCR and confirmed its role in the mechanism of resistance in a mCry3A resistant strain of WCR.
Xiping Niu; Adane Kassa; James Hasler; Samantha Griffin; Claudia Perez-Ortega; Lisa Procyk; Jun Zhang; Deirdre M. Kapka-Kitzman; Mark E. Nelson; Albert Lu. Functional validation of DvABCB1 as a receptor of Cry3 toxins in western corn rootworm, Diabrotica virgifera virgifera. Scientific Reports 2020, 10, 1 -13.
AMA StyleXiping Niu, Adane Kassa, James Hasler, Samantha Griffin, Claudia Perez-Ortega, Lisa Procyk, Jun Zhang, Deirdre M. Kapka-Kitzman, Mark E. Nelson, Albert Lu. Functional validation of DvABCB1 as a receptor of Cry3 toxins in western corn rootworm, Diabrotica virgifera virgifera. Scientific Reports. 2020; 10 (1):1-13.
Chicago/Turabian StyleXiping Niu; Adane Kassa; James Hasler; Samantha Griffin; Claudia Perez-Ortega; Lisa Procyk; Jun Zhang; Deirdre M. Kapka-Kitzman; Mark E. Nelson; Albert Lu. 2020. "Functional validation of DvABCB1 as a receptor of Cry3 toxins in western corn rootworm, Diabrotica virgifera virgifera." Scientific Reports 10, no. 1: 1-13.
Various lepidopteran insects are responsible for major crop losses worldwide. Although crop plant varieties developed to express Bacillus thuringiensis (Bt) proteins are effective at controlling damage from key lepidopteran pests, some insect populations have evolved to be insensitive to certain Bt proteins. Here, we report the discovery of a family of homologous proteins, two of which we have designated IPD083Aa and IPD083Cb, which are from Adiantum spp. Both proteins share no known peptide domains, sequence motifs, or signatures with other proteins. Transgenic soybean or corn plants expressing either IPD083Aa or IPD083Cb, respectively, show protection from feeding damage by several key pests under field conditions. The results from comparative studies with major Bt proteins currently deployed in transgenic crops indicate that the IPD083 proteins function by binding to different target sites. These results indicate that IPD083Aa and IPD083Cb can serve as alternatives to traditional Bt-based insect control traits with potential to counter insect resistance to Bt proteins.
Lu Liu; Eric Schepers; Amy Lum; Janet Rice; Nasser Yalpani; Ryan Gerber; Nuria Jiménez-Juárez; Fikru Haile; Alejandra Pascual; Jennifer Barry; Xiuli Qi; Adane Kassa; Matthew J. Heckert; Weiping Xie; Changkui Ding; Jarred Oral; Minh Nguyen; James Le; Lisa Procyk; Scott H. Diehn; Virginia C. Crane; Howard Damude; Carol Pilcher; Russ Booth; Genhai Zhu; Timothy M. Nowatzki; Mark E. Nelson; Albert L. Lu; Gusui Wu. Identification and Evaluations of Novel Insecticidal Proteins from Plants of the Class Polypodiopsida for Crop Protection against Key Lepidopteran Pests. Toxins 2019, 11, 383 .
AMA StyleLu Liu, Eric Schepers, Amy Lum, Janet Rice, Nasser Yalpani, Ryan Gerber, Nuria Jiménez-Juárez, Fikru Haile, Alejandra Pascual, Jennifer Barry, Xiuli Qi, Adane Kassa, Matthew J. Heckert, Weiping Xie, Changkui Ding, Jarred Oral, Minh Nguyen, James Le, Lisa Procyk, Scott H. Diehn, Virginia C. Crane, Howard Damude, Carol Pilcher, Russ Booth, Genhai Zhu, Timothy M. Nowatzki, Mark E. Nelson, Albert L. Lu, Gusui Wu. Identification and Evaluations of Novel Insecticidal Proteins from Plants of the Class Polypodiopsida for Crop Protection against Key Lepidopteran Pests. Toxins. 2019; 11 (7):383.
Chicago/Turabian StyleLu Liu; Eric Schepers; Amy Lum; Janet Rice; Nasser Yalpani; Ryan Gerber; Nuria Jiménez-Juárez; Fikru Haile; Alejandra Pascual; Jennifer Barry; Xiuli Qi; Adane Kassa; Matthew J. Heckert; Weiping Xie; Changkui Ding; Jarred Oral; Minh Nguyen; James Le; Lisa Procyk; Scott H. Diehn; Virginia C. Crane; Howard Damude; Carol Pilcher; Russ Booth; Genhai Zhu; Timothy M. Nowatzki; Mark E. Nelson; Albert L. Lu; Gusui Wu. 2019. "Identification and Evaluations of Novel Insecticidal Proteins from Plants of the Class Polypodiopsida for Crop Protection against Key Lepidopteran Pests." Toxins 11, no. 7: 383.
A novel Bacillus thuringiensis Cry protein, Cry8Hb, active against Diabrotica virgifera virgifera (Western corn rootworm, WCRW) was discovered. Unexpectedly, the anti-rootworm activity of the Cry8Hb toxin was enhanced significantly by fusing Escherichia coli maltose binding protein (MBP) to this Cry toxin. While the exact mechanism of the activity enhancement remains indefinite, it is probable that the enhancement is a result of increased solubility of the MBP-Cry8Hb fusion in the rootworm midgut. This hypothesis was examined using a synthetic Cry3 protein called IP3-1, which was not soluble at a neutral pH like Cry8Hb and marginally active to WCRW. When IP3-1 was fused to MBP, its anti-WCRW activity was enhanced 13-fold. To further test the hypothesis, DNA shuffling was performed on IP3-1 to increase the solubility without MBP. Screening of shuffled libraries found six new IP3 variants showing very high anti-WCRW activity without MBP. Sequence and 3D structure analysis of those highly active, shuffled IP3 variants revealed several charge-altering mutations such as Lys to Glu on the putative MBP-attaching side of the IP3 molecule. It is likely that those mutations make the protein acidic to substitute the functions of MBP including enhancing the solubility of IP3 at a neutral pH.
Jingtong Hou; Ruth Cong; Michi Izumi-Willcoxon; Hana Ali; Yi Zheng; Ericka Bermudez; Mark McDonald; Mark Nelson; Takashi Yamamoto. Engineering of Bacillus thuringiensis Cry Proteins to Enhance the Activity against Western Corn Rootworm. Toxins 2019, 11, 162 .
AMA StyleJingtong Hou, Ruth Cong, Michi Izumi-Willcoxon, Hana Ali, Yi Zheng, Ericka Bermudez, Mark McDonald, Mark Nelson, Takashi Yamamoto. Engineering of Bacillus thuringiensis Cry Proteins to Enhance the Activity against Western Corn Rootworm. Toxins. 2019; 11 (3):162.
Chicago/Turabian StyleJingtong Hou; Ruth Cong; Michi Izumi-Willcoxon; Hana Ali; Yi Zheng; Ericka Bermudez; Mark McDonald; Mark Nelson; Takashi Yamamoto. 2019. "Engineering of Bacillus thuringiensis Cry Proteins to Enhance the Activity against Western Corn Rootworm." Toxins 11, no. 3: 162.