Source: N Y AGRICULTURAL EXPT STATION submitted to NRP
UNDERSTANDING THE MOLECULAR GENETIC BASIS OF A NOVEL MECHANISM OF BT RESISTANCE IN INSECTS OFR RISK ASSESSMENT OF BT-CROPS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
1027098
Grant No.
2021-33522-35383
Cumulative Award Amt.
$500,000.00
Proposal No.
2021-04217
Multistate No.
(N/A)
Project Start Date
Sep 1, 2021
Project End Date
Aug 31, 2025
Grant Year
2021
Program Code
[HX]- Biotechnology Risk Assessment
Recipient Organization
N Y AGRICULTURAL EXPT STATION
(N/A)
GENEVA,NY 14456
Performing Department
Entomology
Non Technical Summary
The soil bacterium Bacillus thuringiensis (Bt) is an important microbial insect pathogen and has been used as a major biological insecticide for insect pest control. Bt genes coding for insecticidal toxins are the primary insecticidal genes used in insect-resistant transgenic crops (Bt-crops). Bt-crops have been widely adopted and the acreage of Bt-crops worldwide has reached to >100 million hectares with proven economic and environmental benefits. However, the widespread application of Bt toxins in agriculture imposes intense selection pressure on insects to evolve resistance to the Bt toxins, and cases of Bt resistance have been increasingly reported. The risk of resistance development in insects in response to the widespread planting of Bt-crops continuously requires urgent attention. Assessment of Bt resistance development is an important component of the regulatory framework for insect-resistant GE-crops.Assessment and management of the risk of resistance development to Bt-crops critically relies on better understanding of various molecular genetic mechanisms of insect resistance to Bt toxins. Discovery and understanding of new Bt resistance genes and resistance-conferring mutations in insects continue to be an urgent need for adequate assessment and management of the risk of development of insect resistance to Bt-crops. We have recently discovered that the high-level resistance to the Bt toxin Cry1Ac in the cabbage looper, Trichoplusia ni, which enables the insects to feed and survive on Bt-cotton plants, is conferred by multi-gene mutations with a novel resistance gene to be identified and understood. We have successfully isolated the new resistance traits and established a biological research system to study the novel resistance gene and the mechanism of resistance. This project is designed to discover the novel Bt resistance gene and to reveal the new molecular genetic mechanism of Bt resistance in insects, to provide fundamental knowledge required for assessment and management of the risk of insect resistance to Bt-crops and to generate insect field population information necessary for molecular monitoring of resistance development in insects in the field.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2113110104030%
2113110108060%
2113110115010%
Knowledge Area
211 - Insects, Mites, and Other Arthropods Affecting Plants;

Subject Of Investigation
3110 - Insects;

Field Of Science
1040 - Molecular biology; 1080 - Genetics; 1150 - Toxicology;
Goals / Objectives
Bacillus thuringiensis (Bt) is an environment-friendly microbial insecticide, and Bt genes coding for insecticidal toxins have been the primary transgenes in transgenic crops (Bt-crops) to confer insect resistance. Bt-crops have been widely adopted in the US since 1996, providing significant environmental and economic benefits. However, widespread applications of Bt toxins in agriculture impose intense selection pressure on insects to evolve resistance to the Bt toxins, and cases of Bt resistance have been increasingly reported. Therefore, the risk of Bt resistance development in insects in response to the widespread adoption of Bt-crops continuously requires urgent attention.For assessment and management of the risk of Bt-resistance evolved in insects, it is essential to understand the molecular genetic mechanisms conferring Bt resistance. Recent studies on Bt resistance have indicated that high-level Bt-resistance may require multi-gene mutations in insects, but new resistance genes and mutations have yet to be identified and understood. The cabbage looper, Trichoplusia ni, evolved Bt resistance in commercial greenhouses, and resistant T. ni strains can survive on the major commercial Bt-cotton varieties. We have recently identified that the high-level resistance to Bt toxin Cry1Ac in T. ni is conferred by multi-gene mutations involving a novel resistance gene to be identified and understood. This project is a designed "research concerning the introduction of genetically engineered (GE) organisms into the environment" and aims to generate "new information that will assist Federal regulatory agencies in making science-based decisions about the environmental effects of introducing genetically engineered (GE) organisms by recombinant nucleic acid techniques". We will use the unique T. ni strains to discover and understand the novel Bt-resistance gene and the resistance mechanism by reaching the specific objectives: 1) to map the genetic locus of the novel Cry1Ac resistance gene in T. ni genome, 2) to identify the novel gene and associated mutation(s) for the resistance, 3) to functionally verify the novel resistance gene and the mutation(s) conferring resistance to Cry1Ac, and 4) to detect and examine the Cry1Ac resistance-associated gene mutations in T. ni from field populations.
Project Methods
From the greenhouse derived Bt resistant T. ni populations, we have discovered a high-level Bt Cry1Ac resistance mechanism conferred by two gene mutations and have established the research system to identify the novel resistance gene in T. ni and to functionally study the resistance mechanism with two gene mutations. In this project, we aim to identify the novel Cry1Ac resistance gene Cry1Ac-R2 and functionally understand the Cry1Ac-R2 gene in Cry1Ac resistance in insects, and to examine the Cry1Ac resistance conferring gene mutation in T. ni from the field, by (1) mapping the genetic locus of the novel gene Cry1Ac-R2 in T. ni genome, using the genomics resources and tools we have developed for T. ni research, (2) identifying the Cry1Ac-R2 gene and associated mutation(s) in T. ni genome, using genomic and molecular approaches, (3) functionally verifying and understanding the gene mutation(s) conferring the novel resistance to Bt-crops, using gene editing, biological and OMICS-based analyses and (4) detecting and examining the novel resistant gene mutations in T. ni from field populations. To reach these research objectives, we will:1) Use biphasic genetic linkage mapping to (1) identify the linkage group (the specific chromosome) harboring the Cry1Ac-R2 gene, (2) localize Cry1AC-R2 in a region in the identified chromosome, and then (3) localize the resistance gene in a finely mapped resistance locus, to answer the question "where in the genome is the novel Cry1Ac-R2 gene localized?". Illumina genome sequencing and amplicon sequencing (Amp-seq) of T. ni segregating mapping populations will be conducted to determine the association of the resistance with the molecular markers in T. ni genome.2) Examine the mutations in the Cry1Ac-R2 locus by whole genome resequencing of the Cry1Ac resistant T. ni strain and determine the expression level of the genes in the resistance locus by midgut RNA-seq; and 2) identify the resistance associated mutations by cDNA sequencing of genes in the resistance locus and genomic DNA sequencing of the mutation sites, to answer the research questions "What is the novel resistance gene Cry1Ac-R2 and what mutation(s) in the gene is(are) associated with the resistance?".3) Use CRISPR/Cas9 gene mutagenesis to generate T. ni mutants to test and verify the Cry1Ac-R2 gene mutations that confers the resistance, to answer the question "Do the identified Cry1Ac-R2 gene and mutations confer the high-level Cry1Ac resistance with Cry1Ac-R1 in T. ni?".4) Detect and examine the Cry1Ac resistance-associated mutations and variations in the Cry1Ac resistance genes in field populations of T. ni, as an initial step to generate field population information necessary for molecular monitoring of resistance gene selection in the field.

Progress 09/01/23 to 08/31/24

Outputs
Target Audience:The target audience of this project are researchers and technologists from both academia and industry in the areas of agricultural biotechnology, insect pathology, insect pest control and management of insect resistance to Bt toxins. Audience of this project also include insect pest management professionals and crop growers, and students, postdocs and other trainees in relevant areas, relevant government agencies, and the general public. During this project period, target audience are reached through our publications in scientific journals and presentations of research our findings in scientific meetings, and in classroom lectures, laboratory training of graduate students, undergraduate research interns and other researchers, by presentations in academic workshops and in educational open house and other outreach events to the general public. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided training opportunities to 2 graduate students and 4 undergraduate students, and other trainees who obtained knowledge and skills for career development from the research. How have the results been disseminated to communities of interest?During this project period, results from the project have been delivered to the audience through our publications in scientific journals and presentations of research our findings in scientific meetings, and in classroom lectures, laboratory training of graduate students, undergraduate research interns and other researchers, and by presentations in academic workshops and educational open house events to the general public. What do you plan to do during the next reporting period to accomplish the goals?The project will continue as proposed to identify the resistance gene Cry1Ac-R2 in Trichoplusia ni and functionally understand the resistance conferring mutation in the insects from the field.

Impacts
What was accomplished under these goals? This project aims to identify a novel Cry1Ac resistance gene Cry1Ac-R2 in Trichoplusia ni, and functionally understand the Cry1Ac-R2 gene and its mutation in Cry1Ac resistance in insects. The project is designed and has been carried to map the genetic locus of the novel gene Cry1Ac-R2 in T. ni genome, identify the Cry1Ac-R2 gene and associated mutation(s), and functionally verify and understand the gene mutation(s) conferring the novel resistance to Bt toxins. We have genetically determined the Cry1Ac-R2 trait to be incompletely dominant in resistance to Cry1Ac. The linkage group of Cry1Ac-R2 has been identified in T. ni genome by genetic linkage analysis of the dominant resistance trait using bulked segregant analysis sequencing (BSA-seq) and amplified fragment length polymorphism (AFLP) markers. The resistance gene locus in the linkage group has been further localized using PCR-amplicon sequencing (AmpSeq). The genes in the resistance locus have been annotated and the genes expressed in the midgut were identified. In addition, the expression levels of those midgut genes in resistant T. ni have been comparatively analyzed with the susceptible T. ni, using data from RNA-seq of transcripts of the larval midgut from susceptible and resistant T. ni. To identify mutations in the candidate resistance genes in the Cry1Ac-R2 locus in T. ni genome, a genomic DNA contig of the Cry1Ac-R2 locus from the resistant T. ni has been assembled de novo, using whole genome sequencing data of the resistant T. ni. Midgut-expressing genes in the resistance locus have been identified and candidate resistance associated mutations have been identified. For the major genes identified to carry mutations in the resistant strain, the cDNAs from both susceptible and resistant T. ni larvae have been sequenced in full length to confirm the mutations in the CDS, and their expression levels have been determined by quantitative real-time RT-PCR. Furthermore, the gene mutations found in the de novo assembled genomic contig from the resistant T. ni genome have been verified by sequencing of the genomic DNA fragments in the mutation regions amplified by PCR from both the susceptible and resistant T. ni. To verify the association of mutations with resistance, T. ni from a different genetic background has also been additionally examined for the mutations in candidate genes. Candidate resistance-associated mutations identified in the Cry1Ac-R2 locus have been functionally studied and their roles in resistance to Cry1Ac been analyzed. In the resistance gene locus, the molecular genetic mechanisms for the resistance associated expression levels of APN genes have been identified. To functionally examine and understand the identified mutations associated with resistance, T. ni strains carrying various mutations in the candidate resistant genes have been generated by CRISPR/Cas9 mutagenesis. Additional mutant T. ni strains are continuing to be generated and used to functionally analyze their roles in Bt resistance in T. ni. With the identification of mutations associated with Cry1Ac resistance in T. ni, diagnostic PCR assays have been developed to detect and sequence resistance-associated mutant alleles from T. ni specimens collected from the field. We are continuing the analysis of the T. ni samples for the resistance-associated mutations.

Publications

  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Cotto-Rivera, R.O., Joya, N., Hern�ndez-Mart�nez, P., Ferr�, J. and Wang, P. (2024) Downregulation of APN1 and ABCC2 mutation in Bt Cry1Ac-resistant Trichoplusia ni are genetically independent. Applied and Environmental Microbiology, 90 (10), e00742-24.
  • Type: Theses/Dissertations Status: Published Year Published: 2024 Citation: Cotto-Rivera, R.O. (2024) Understanding insect midgut receptors for Bt protein Cry1Ac and mechanism of resistance in the cabbage looper, Trichoplusia ni. PhD dissertation, Cornell University.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Wang, P. (2024) Mechanisms of resistance to Bacillus thuringiensis Cry proteins in a generalist insect Trichoplusia ni. XXVII International Congress of Entomology. Aug. 25-30, 2024. Kyoto, Japan.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Wang, P. (2024) Understanding the molecular genetic basis of a novel mechanism of Bt resistance in insects. USDA Biotechnology Risk Assessment Grants Program, 2024 Annual Project Directors Meeting, May 21, 2024.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Shao, E., Cotto-Rivera, R., Tetreau, G. and Wang, P. (2023) Functional examination of aminopeptidase N1 in resistance to Bt toxins in Trichoplusia ni. Annual Meeting of the Entomological Society of America, Nov. 5-8, 2023. National Harbor, MD.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Kihata, N. (2024) Investigating the role of the APN gene in resistance to Bt toxin Cry1Ac in Trichoplusia ni. Cornell AgriTech Research Symposium, Jun 12, 2024. Geneva, NY.


Progress 09/01/22 to 08/31/23

Outputs
Target Audience:The target audience of this project are researchers and technologists from both academia and industry in the areas of agricultural biotechnology, insect pathology, insect pest control and management of insect resistance to Bt toxins. Audience of this project also include insect pest management professionals and crop growers, and students, postdocs and other trainees in relevant areas, relevant government agencies, and the general public. During this project period, target audience are reached through our publications in scientific journals and presentations of research our findings in scientific meetings, and in classroom lectures, laboratory training of graduate students, undergraduate research interns and other researchers, by presentations in academic workshops and in educational open house and other outreach events to the general public. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided training opportunities to 2 graduate students and 2 undergraduate students, and other trainees who obtained knowledge and skills for career development from the research. How have the results been disseminated to communities of interest?During this project period, results from the project have been delivered to the audience through our publications in scientific journals and presentations of research our findings in scientific meetings, and in classroom lectures, laboratory training of graduate students, undergraduate research interns and other researchers, and by presentations in academic workshops and educational open house events to the general public. What do you plan to do during the next reporting period to accomplish the goals?The project will continue as proposed to identify the resistance gene Cry1Ac-R2 in Trichoplusia ni and functionally understand the resistance conferring mutation in the insects from the field.

Impacts
What was accomplished under these goals? This project aims to identify a novel Cry1Ac resistance gene Cry1Ac-R2 in Trichoplusia ni, and to functionally understand the Cry1Ac-R2 gene and its mutation in Cry1Ac resistance in insects. The project is designed and has been carried to map the genetic locus of the novel gene Cry1Ac-R2 in T. ni genome, identify the Cry1Ac-R2 gene and associated mutation(s) in T. ni genome, and functionally verify and understand the gene mutation(s) conferring the novel resistance to Bt toxins. 1) We have identified the linkage group of Cry1Ac-R2 gene by genetic linkage analysis of the resistance using bulked segregant analysis sequencing (BSA-seq) and the resistance locus has been further localized in the linkage group using PCR-amplicon sequencing (AmpSeq). The genes in the resistance locus have been annotated, and the expression of those genes in the midgut of the resistant T. ni has been comparatively analyzed with the susceptible T. ni, using data from RNA-seq of the larval midgut from susceptible and resistant T. ni. 2) To identity mutations in the candidate resistance genes in the Cry1Ac-R2 locus, a genomic DNA contig in the resistance gene locus from the resistant T. ni has been assembled de novo, using whole genome sequencing data from the resistant T. ni, and candidate resistance associated mutations have been identified. For the major genes differed in DNA sequence between the susceptible and the resistant strains in the resistance locus, their cDNAs have been sequenced in full length to confirm the mutations in the CDS. Furthermore, the gene mutations found in the de novo assembled contig from the resistant T. ni genome have been verified by sequencing of the genomic DNA fragments in the mutation regions amplified by PCR from the resistant T. ni. 3) To functionally confirm and understand the newly identified resistance associated genes, T. ni strains carrying various mutations in the candidate resistant genes have been generated by CRISPR/Cas9 mutagenesis. Additional mutant T. ni strains are continuing to be generated. The mutant T. ni strains generated are being used to functionally analyze their roles in Bt resistance in T. ni.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Wang, P. (2023) Bt resistance in Trichoplusia ni: mechanisms of resistance to multiple Bt toxins in a generalist insect. Second International Molecular Plant Protection Congress. May 15-18, 2023. Bursa, Turkey.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Wang, P. (2023) Understanding the molecular genetic basis of a novel mechanism of Bt resistance in insects. USDA Biotechnology Risk Assessment Grants Program, 2023 Annual Project Directors Meeting, May 23-24, 2023.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Joya, N., Cotto-Rivera, R. O., Hern�ndez-Mart�nez, P., Wang, P. and Ferr�, J. (2023) Alterations of toxin binding sites associated to Cry1Ac and Cry1F resistance in Trichoplusia ni. Annual Meeting of the Society for Invertebrate Pathology. July 30 - August 3, 2023. College Park, MD.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Cotto, R., Joya, N., Hern�ndez-Mart�nez, P. and Wang, P. (2022) Carbohydrate moieties play an important role in binding and toxicity of Bt toxin Cry1Ac in the midgut of cabbage loopers. The Entomological Society of America, Entomological Society of Canada and the Entomological Society of British Columbia Joint Annual Meeting, Nov. 13-16, 2022. Vancouver, BC. Canada.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Yang, X., Pfannenstiel, L. and Wang, P. (2022) Genetic mapping of a second resistance gene to Bt Cry1Ac in the cabbage looper, Trichoplusi ni. The Entomological Society of America, Entomological Society of Canada and the Entomological Society of British Columbia Joint Annual Meeting, Nov. 13-16, 2022. Vancouver, BC. Canada.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Leal, A., Wang, S. and Wang, P. (2023) A preliminary study of physiological functions of the kynurenine pathway in Trichoplusia ni. Summer Research Scholars Program of Cornell AgriTech, Cornell University. Aug 4, 2023.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Ziemke, T., Wang, P. and Duplais, C. (2023) The fate of defensive potato toxin in the cabbage looper (Trichoplusia ni): Understanding detoxification and excretion mechanisms. Summer Research Scholars Program of Cornell AgriTech, Cornell University. Aug 4, 2023.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Cotto-Rivera, R. and Wang, P. (2023) Understanding the role of multiple gene mutations in Bt-Cry1Ac resistance in the cabbage looper, Trichoplusia ni. Cornell AgriTech Research Symposium, Aug 8, 2023. Geneva, NY.


Progress 09/01/21 to 08/31/22

Outputs
Target Audience:The target audience of this project are researchers and technologists from both academia and industry in the areas of agricultural biotechnology, insect pathology, insect pest control and management of insect resistance to Bt toxins. Audience of this project also include insect pest management professionals and crop growers, and students, postdocs and other trainees in relevant areas, relevant government agencies, and the general public. During this project period, target audience are reached through our publications in scientific journals and presentations of research our findings in scientific meetings, and in classroom lectures, laboratory training of undergraduate research interns and researchers and graduate students, presentations in academic workshops and in educational open house events to the general public. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided training opportunities to 2 graduate students and 1 undergraduate student, and other trainees who obtained knowledge and skills for career development from the research. How have the results been disseminated to communities of interest?During this project period, results from the project have been delivered to the audience through our publications in scientific journals and presentations of research our findings in scientific meetings, and in classroom lectures, laboratory training of graduate students, undergraduate research interns and other researchers, and by presentations in academic workshops and educational open house events to the general public. What do you plan to do during the next reporting period to accomplish the goals?The project will continue as proposed to identify the resistance gene Cry1Ac-R2 in Trichoplusia ni and functionally understand the resistance conferring mutation in the insects from the field.

Impacts
What was accomplished under these goals? This project aims to identify the novel Cry1Ac resistance gene Cry1Ac-R2 from Trichoplusia ni, functionally understand the Cry1Ac-R2 gene and its mutation in Cry1Ac resistance in the insect. To reach the research aims, the project is designed and has been carried to map the genetic locus of the novel gene Cry1Ac-R2 in T. ni genome, identify the Cry1Ac-R2 gene and associated mutation(s) in T. ni genome, and functionally verify and understand the gene mutation(s) conferring the novel resistance to Bt toxins. We have identified the linkage group of Cry1Ac-R2 gene by genetic linkage analysis of the resistance with molecular markers and the resistance locus has been further localized in the linkage group. The genes in the resistance locus have been annotated and the expression of these genes in the midgut of T. ni larvae has been analyzed. Using the T. ni genome sequence and resequencing data, we have tentatively identified the mutations in the candidate resistance genes. With the progress of our research, the research objectives proposed for the first year of the project have been reached.

Publications

  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Ma, X., Shao, E., Chen, W., Cotto-Rivera, R. O., Yang, X., Kain, W., Fei, Z. and Wang, P. (2022) Bt Cry1Ac resistance in Trichoplusia ni is conferred by multi-gene mutations. Insect Biochemistry and Molecular Biology, 140, 103678.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Cotto, R., Shao, E., Yang, X. and Wang, P. (2022) Roles of candidate Bt receptors in susceptibility of Trichoplusia ni to Bt toxin Cry1Ac. 2022 International Congress on Invertebrate Pathology and Microbial Control, held virtually from Aug 1-4, 2022 in Nelson Mandela Bay, Eastern Cape South Africa.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Kain, W., Cotto, R. and Wang, P. (2022) Resistance of cabbage loopers to Bt toxin Cry1F and to Cry1Ac+Cry1F cotton plants. 2022 International Congress on Invertebrate Pathology and Microbial Control, held virtually from Aug 1-4, 2022 in Nelson Mandela Bay, Eastern Cape South Africa.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Cotto, R. (2022) To understand insect midgut receptors for Bt toxins and receptor-mediated Bt resistance mechanisms. Summer Research Scholars Program of Cornell AgriTech, Cornell University. Jun 30, 2022.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Gutierrez, J.B., Cotto, R. O., Kain, W. and Wang, P. (2022) Fitness costs associated with Bt resistance-conferring mutations in Trichoplusia ni. Summer Research Scholars Program of Cornell AgriTech, Cornell University. Jul 29, 2022.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Wang, P. (2021) Genetically engineered insects and viruses for pest control. Annual Meeting of the Entomological Society. October 30 - November 3, 2021. Denver, CO.