Source: UNIVERSITY OF TENNESSEE submitted to NRP
MECHANISMS OF RESISTANCE TO VIP3AA PROTEINS AS PLANT INCORPORATED PROTECTANTS IN GE CORN AND COTTON
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1024833
Grant No.
2021-67013-33567
Cumulative Award Amt.
$498,580.00
Proposal No.
2020-05843
Multistate No.
(N/A)
Project Start Date
Jan 1, 2021
Project End Date
Dec 31, 2024
Grant Year
2021
Program Code
[A1112]- Pests and Beneficial Species in Agricultural Production Systems
Recipient Organization
UNIVERSITY OF TENNESSEE
2621 MORGAN CIR
KNOXVILLE,TN 37996-4540
Performing Department
Entomology & Plant Path - RES
Non Technical Summary
Genetically engineered (GE) crops producing insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) as plant incorporated protectants (PIPs) are the most important contribution of biotechnology to pest control in US agriculture. The most serious threat to the sustainable use and public benefits of Bt crops is the evolution of resistance in target pests. The Vip3Aa toxin is currently an essential Bt PIP in GE corn and cotton to control devastating Helicoverpa zea (corn earworm, CEW) and Spodoptera frugiperda (fall armyworm, FAW) larvae. Emerging evidence supports imminence of CEW and FAW resistance to Vip3Aa PIPs, yet information on field resistance mechanisms or genes involved is scarce.The goal of this project is to resolve this knowledge gap by capitalizing on availability of field derived Vip3Aa-resistant CEW and FAW strains to understand resistance mechanisms and identify resistance genes. Objectives 1 and 2 will test association of Vip3Aa resistance with altered toxin processing in the gut and binding to receptors, respectively. Objectives 3 and 4 will perform a comprehensive transcriptomic analysis (RNAseq) to identify Vip3Aa resistance alleles in CEW and FAW and advance estimation of their frequency. Expected outcomes will identify mechanisms and genes involved in resistance to Vip3Aa PIPs as crucial information to evaluate resistance management tools that safeguard public benefits of Bt PIPs for US agriculture and guide the improvement of Bt crop technologies.
Animal Health Component
55%
Research Effort Categories
Basic
25%
Applied
55%
Developmental
20%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2113110113020%
2113110108020%
2113110115020%
2113110104020%
2113110100020%
Knowledge Area
211 - Insects, Mites, and Other Arthropods Affecting Plants;

Subject Of Investigation
3110 - Insects;

Field Of Science
1040 - Molecular biology; 1130 - Entomology and acarology; 1000 - Biochemistry and biophysics; 1080 - Genetics; 1150 - Toxicology;
Goals / Objectives
The goal of this project is to resolve the current knowledge gap on field resistance mechanisms to the Vip3Aa insecticidal protein from Bacillus thuringiensis (Bt) as plant incorporated protectant (PIP) in genetically engineered (GE) crops. These GE crops are the most important contribution of biotechnology to pest control in US agriculture, and the most serious threat to the sustainable use and public benefits is the evolution of resistance in target pests. The Vip3Aa toxin is currently an essential Bt PIP in GE corn and cotton to control devastating Helicoverpa zea (corn earworm, CEW) and Spodoptera frugiperda (fall armyworm, FAW) larvae. While emerging evidence supports imminence of CEW and FAW resistance to Vip3Aa PIPs, information on field resistance mechanisms or genes involved is scarce. This information is critical to evaluate resistance management tools that safeguard public benefits of Bt PIPs for US agriculture and guide the improvement of Bt crop technologies.The project capitalizes on available Vip3Aa-resistant strains of CEW and FAW and we plan to achieve our goal by completing the following objectives:Objective 1: Testing role of Vip3Aa processing and stability in resistance. Available data suggests that gut pH conditions and processing of Vip3Aa may be altered in resistant insects. We plan to test this hypothesis by comparing gut pH and processing of Vip3Aa toxin in susceptible and resistant CEW and FAW strains. Any differences detected will be further evaluated in cytotoxicity assays with cultured insect cells.Objective 2: Testing binding of Vip3Aa to receptors. Alteration in binding to receptors is the most common mechanism to Bt PIPs and preliminary evidence suggests that this mechanism may be involved in resistance to Vip3Aa. We will test this hypothesis by measuring and comparing binding of radiolabled Vip3Aa to midgut proteins from susceptible and resistant CEW and FAW larvae. Bingin will also be tested using biotinylated Vip3Aa toxin and Western blotting and purification of Vip3Aa-binding proteins from guts of susceptible and resistant CEW and FAW larvae.Objective 3: Identification of candidate Vip3Aa resistance genes in CEW and FAW. We will use RNAseq in dentifying candidate Vip3Aa resistance genes and alleles in strains of CEW and FAW. Detected indels/mutations or altered gene expression detected in RNAseq will be confirmed by re-sequencing or quantitative PCR (qPCR), respectively.Objective 4: Linkage of candidate resistance alleles with resistance to Vip3Aa and estimation of frequency. Candidate resistance alleles identified in Objective 3 will be tested for linkage with resistance to Vip3Aa using backcrosses and genotyping of F2 subfamilies and determining statistical deviation from expected genotype proportions. In estimating the frequency of identified Vip3Aa resistance alleles, we will genotype an archived collection of >13,800 genomic DNA samples from individual FAW collected in the US and the Caribbean.
Project Methods
Objective 1: Testing role of Vip3Aa processing and stability in resistance.Three strains of corn earwom (Helicoverpa zea, CEW) and five of fall armyworm (Spodoptera frugiperda, FAW) differing in their susceptibility to Vip3Aa and Cry toxins will be used in this project too establish comparisons and detect the mechanism explaining the resistance phenotype. Vip3Aa and Cry1 toxins will be produced and purified from bacterial cultures. The mean pH in the three gut regions (foregut, midgut and hindgut) of actively feeding early 4th instar CEW and FAW larvae (15 biological replicates) will be measured in situ using a microelectrode probe. If significant pH differences are detected between susceptible and Vip3Aa-resistant strains, we will test Vip3Aa binding (as described in Objective 2) under these pH conditions to test its effect on binding and resistance. Gut proteases will be extracted from the considered CEW and FAW strains and used for in vitro Vip3Aa digestion assays. Relevance of Vip3Aa processing in resistance will be tested in cytotoxicity assays with cultured insect cells exposed to the Vip3Aa protein processed by the CEW and FAW strains considered. Significant pH differences between susceptible and resistant strains of each species for each gut region will be tested using repeated measures analysis with the pH values as the response variable, susceptibility as the between subject variable, and the gut regions as the within subject variable. Differences in relative levels of processed Vip3Aa and Cry1Fa toxin from densitometry measurements will be tested using one-way ANOVA at the significance level of 0.05. Cytotoxicity assays will be analyzed using probit regression for randomized block design, with percentage of death as the response variable, and treatment and dose as the between-subject effects. Ranked transformation will be applied if residuals show unequal variance and non-normality. Post hoc multiple comparisons will be conducted with Tukey's adjustment. Statistical significance will be identified at the significance level of 0.05.Objective 2: Testing binding of Vip3Aa to receptors.Purified VIp3Aa and Cry toxins will be labeled with biotin or iodine-125 isotope, and midgut brush border membrane vesicles (BBMVs) from larvae of the considered CEW and FAW strains will be prepared using differential centrifugation. Binding assays will use labeeld toxins and BBMVs and include detection by Western blotting (for biotinylated toxins) or quantification in a gamma counter (for radiolabeled toxins). Specific binding will be determined by subtracting non-specific binding, detected in the presence of 300-fold excess of unlabeled homologous toxin, from total binding. Identification of Vip3Aa receptors in BBMVs from the susceptible and resistant CEW and FAW strains considered will be performed in triplicate using a combined pull down and proteomic approach previously used with cultured insect cells. Tandem mass spectra will be analyzed using Mascot (Matrix Science, London, UK) and queried against the annotated genes of reference genomes for CEW and FAW and transcriptome databases developed in Objective 3. Significance of differences in Vip3A binding parameters between susceptible and resistant strains will be tested using a t-test and post-hoc Mann Whitney Rank Sum test when normality tests fail, with p <0.05. Normalized protein abundance factors from NSAF of three biological replicates will be compared using an ANOVA with a Benjamini-Hochberg correction at the p < 0.01 significance level in Scaffold to increase the probability of accurate detection of differences in protein levels between susceptible and resistant strains for each of the identified proteins.Objective 3: Identification of candidate Vip3Aa resistance genes in CEW and FAW. Illumina TruSeq STranded mRNA libraries will be prepared for three biological replicate pools containing RNA from guts of 15 individuals each (total 45 individuals from each strain). The libraries will be sequenced on an Illumina NovaSeq instrument to generate ~75 million reads per 15-sample pooled library. Reads will be first filtered for quality and adapter sequence, and then mapped with HISAT2 to the appropriate genome. We will examine the data for transcript sequence variation by reconstructing the transcripts present in each pool with StringTie. Variation in transcripts within and across each pool will be compared at each gene locus, especially focusing on genes encoding Vip3Aa binding proteins. The susceptible and Cry1-resistant strains will both act as a control for comparisons of gene expression and allelic composition. Functional impact of identified SNPs/indels will be called using GATK and assessed with SNPEff. Candidate alleles for Vip3Aa resistance will be confirmed using Sanger sequencing (indels/mutations) or quantitative real-time PCR (qRT-PCR, for genes with altered expression) using RNA samples used for preparation of sequencing libraries. All comparisons will be tested for significance at the p < 0.05 level between susceptible and resistant strains using t-test (for a single transcript) or a one-way ANOVA (multiple transcripts).Objective 4: Linkage of candidate resistance alleles with resistance to Vip3Aa and estimation of frequency. Candidate resistance alleles from Objective 3 will be tested for co-segregation with resistance by targeted sequencing of F2 backcross subfamilies. A heterozygous F1 generation (Sr) from crossing susceptible and Vip3Aa-resistant CEW and FAW strains (SS x rr), will be backcrossed with resistant individuals (rr) to yield an F2 consisting of 50% heterozygotes (Sr) and 50% homozygous resistant (rr) individuals. F2 backcross subfamilies (100 larvae each) will be exposed to either a control diet or a discriminatory Vip3Aa dose killing heterozygote (Sr) individuals. Survivors in both subfamilies will be genotyped using PCR amplification and sequencing (for mutations and indels) or qRT-PCR (for altered expression levels). The Chi-square (χ2) test for goodness of fit will evaluate cosegregation of survival to Vip3Aa with the presence in homozygosity of the candidate resistance allele. Statistical deviation of observed from expected proportions of heterozygote (Sr) and homozygote (rr) individuals in the F2 subfamilies will be evaluated using the Chi-square test (p <0.05). Estimation of resistance allele frequency will only be performed for alleles involving mutations/indels in FAW. Based on the nature and type of allele we will use Taqman or discriminatory PCR reactions for allele detection. As template we will capitalize on availability of a collection of 13,800 FAW genomic DNA (gDNA) samples already processed and archived. The genotype of each tested samples will be used to estimate allele frequency using the Hardy-Weinberg equation.

Progress 01/01/21 to 12/31/24

Outputs
Target Audience:The target audience for this project included scientists and students in academia, government, and industry laboratories working on the control and monitoring of resistance in fall armyworm and corn earworm to Vip3Aa as a plant incorporated protectant. Additionally, audiences reached include farmers in the USA and South America, where Vip3Aa is an available plant-incorporated protectant in transgenic crops, and corn farmers, undergraduate and graduate students, and researchers in Asia (Cambodia and the Philippines) and Africa (Kenya) where fall armyworm is a significant invasive pest. A third group reached by our project communications includes students, postdocs, and technical personnel worldwide who are interested in acquiring knowledge and research tools developed through the project's objectives. Information from the project has been shared with these target audiences through invited and contributing presentations at national and international scientific conferences, peer-reviewed publications, and workshops and training sessions. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project has provided training activities on insect rearing and bioassays, bioinformatic analysis of RNAseq and bulk segregant analysis, digestive tract pH measurements, bacterial protein production and purification, labeling of proteins and binding assays, bioassays, and quantitative PCR. How have the results been disseminated to communities of interest?Information from the project has been shared with target audiences through invited and contributing presentations at scientific conferences, including the Beltwide Cotton conference, the Southeastern branch of the Entomological Society of America, the Society for Invertebrate Pathology, the International Congress of Entomology, the International Working Group on Ostrinia and other pests of corn (IWGO), the Plant and Animal Genome (PAG) Conference, and the Entomological Society of America. Outcomes from the project were also shared with multidisciplinary audiences through invited presentations at the International Center for Insect Physiology and Ecology (ICIPE, Kenya), the University Institute of Biotechnology and Biomedicine of the University of Valencia (BIOTECMED, Spain), the International Conference on Insect pest Management (ICIPM, Nanjing, China), and the International Symposium on Plant Biosafety (Kunming City, China). A synthesis of the project goals and research outcomes is shared with the public through a dedicated link on our laboratory webpage (http://juratfuenteslab.utk.edu/Btresistance.html; up to 15K impressions per month from 21 countries), the laboratory Facebook group page (https://www.facebook.com/groups/614669982344958;183 members from 7 countries), and the PI's Linked In account (875 followers, 5,960 annual post impressions). What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Impact description Insecticidal proteins from Bacillus thuringiensis (Bt) protect >85% of the cotton and corn acreage in the USA from caterpillar pests, providing valuable economic and environmental benefits to US agriculture. Corn producing Bt proteins (Bt corn) alone contributed $7 billion to the US economy, including $4 billion in benefits from pest suppression for non-Bt growers. However, insect resistance to Bt proteins, particularly in the corn earworm (CEW) and fall armyworm (FAW), threatens these benefits. The corn earworm causes 2.5% annual grain losses, and damage to sweet corn can reach 50% in unsellable product. The fall armyworm, a rising global pest, cost farmers $10 billion annually in control and yield losses. Vip3Aa is currently the only highly effective Bt protein in transgenic corn and cotton against these pests, but resistance has been isolated from field populations. This project investigated the mechanisms and genes involved in Vip3Aa resistance in CEW and FAW, using field-derived populations. Key findings include reduced binding of Vip3Aa to gut receptors as the main mechanism of resistance, with altered processing of Vip3Aa also associated with resistance in FAW. Genomic studies identified genes linked to these changes. This change in knowledge aids scientists in academia, government, and industry in developing of more cost-effective and sensitive resistance monitoring tools and improved insecticidal proteins. These advancements support a change in action through management tools to safeguard the long-term benefits of Bt proteins for US agriculture and provide a change in condition by extending higher and safer food and fiber production. Objective 1: Testing role of Vip3Aa processing and stability in resistance. 1) Major activities: Produced and purified Vip3Aa protoxin; measured pH along the digestive canal of CEW and FAW larvae; analyzed Vip3Aa processing by midgut fluids; quantified specific digestive enzymatic activities in gut fluids; tested activity of pre-processed Vip3Aa in resistant FAW larvae. 2) Data collected: Chromatograms of Vip3Aa purification, pH recordings, images of electrophoretic gels and densitometry measurements, serine protease activity, susceptibility and stunting of FAW larvae. 3) Summary statistics and discussion of results: No significant differences (ANOVA; P<0.05) in gut pH in relation to resistance. Midgut pH drop studies supported altered Vip3Aa processing only in a strain of FAW from Louisiana. In this strain, gut serine protease activity was significantly reduced (ANOVA; P<0.05). Pre-processing of Vip3Aa with gut fluids from susceptible FAW increased susceptibility in resistant larvae. 4) Key outcomes or other accomplishments realized: Processing or midgut pH conditions are not associated with resistance to Vip3Aa in CEW or FAW, except a strain of FAW from Louisiana where resistance is associated with reduced Vip3Aa processing from lower serine protease activity in gut fluids. Objective 2: Testing binding of Vip3Aa to receptors 1) Major activities: Prepared midgut brush border membrane vesicle (BBMV) proteins, labeled Vip3Aa toxin with biotin, fluorescence, and radioactive iodine, and conducted binding assays. Bound Vip3A toxin was quantified from radioactivity, densitometry in Western blots (biotinylated Vip3Aa), and fluorescence detection. 2) Data collected: Disintegrations per minute for radiolabeled Vip3Aa, densitometry of chemiluminescence, fluorescence units. Specific binding estimated as total (no competitor present) minus non-specific ( >100-fold unlabeled toxin) binding. 3) Summary statistics and discussion of results: Significantly more Vip3Aa bound to BBMV from susceptible than to Vip3A-resistant CEW and FAW, independently of labeling used. 4) Key outcomes or other accomplishments realized: Reduced binding is associated with resistance to Vip3Aa in CEW and FAW. Objective 3: Identification of candidate Vip3Aa resistance genes in CEW and FAW (80% completed). 1) Major activities: Conducted RNAseq and bulk segregant analysis (BSA) with susceptible and Vip3Aa-resistant CEW and FAW. Bioinformatic identification of differentially expressed genes (DEGs) and loci containing genetic variation statistically associated resistance to Vip3Aa. Quantitative PCR to confirm selected DEGs. 2) Data collected: QC (quantity, quality, and purity) of purified RNA and DNA; raw Illumina short-read sequencing reads (150 bp paired-end read configuration); relative transcript levels for DEGs. 3) Summary statistics and discussion of results: The percentage of effective reads used for analysis from total and Q30 was >95%. PCA plots detected separate groupings of susceptible and Vip3Aa-resistant samples. Changes in gene expression (adjusted P<0.05 and >1 log2 fold) confirmed by quantitative PCR. BSA identified loci for each CEW and FAW strain associated (P<0.05) with resistance to Vip3Aa. 4) Key outcomes or other accomplishments realized: Candidate resistance genes to Vip3Aa in CEW and FAW. List includes potential Vip3Aa receptor genes that would explain reduced Vip3Aa binding and regulatory genes that could trans-regulate Vip3Aa binding and/or processing. Objective 4: Linkage of candidate resistance alleles with resistance to Vip3Aa and estimation of frequency 1) Major activities: Segregating subfamilies for linkage analysis. Developed a panel of genes for highly multiplexed targeted sequencing. 2) Data collected: Percentage mortality in F2 subfamilies exposed to control or diet containing Vip3Aa protein, raw sequences from highly multiplexed targeted next generation sequencing of 274 fall armyworm samples collected from 14 locations in 8 US states and Puerto Rico. 3) Summary statistics and discussion of results: Resistance to Vip3Aa in FAW and CEW follows Mendelian transmission of an autosomal recessive trait. Highly multiplexed exonic amplicon sequencing detected resistance alleles in multiple genes across different locations. 4) Key outcomes or other accomplishments realized: Highly multiplexed targeted sequencing provides a valid method for monitoring Vip3Aa resistance, even at low frequencies.

Publications

  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Ross S, Yang F, Santiago-Gonz�lez JC, Abdelgaffar H, Kerns DD, Jurat-Fuentes JL, Sun X, Collett D, Kerns DL. Evaluation of GS-omega/kappa-Hxtx-Hv1a and Bt toxins against Bt-resistant and -susceptible strains of Helicoverpa zea (Boddie) and Spodoptera frugiperda (J.E. Smith). Pest Manag Sci. 2025 Feb 18. doi: 10.1002/ps.8725. Epub ahead of print. PMID: 39964042.
  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Yang F, Head GP, Kerns DD, Jurat-Fuentes JL, Santiago-Gonz�lez JC, Kerns DL. Diverse genetic basis of Vip3Aa resistance in five independent field-derived strains of Helicoverpa zea in the US. Pest Manag Sci. 2024 Jun;80(6):2796-2803. doi: 10.1002/ps.7988. Epub 2024 Feb 7. PMID: 38327120.
  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Silva, T., G.A. Sword, A. Miller, J.A. Qureshi, G.P. Head, D.D. Kerns, J-L. Jurat-Fuentes, J. Villegas, T.B. Towles, X. Ni, F.P. F. Reay-Jones, D. Carrillo, D.R. Cook, C. Daves, M. J. Stout, B. Thrash, S. V. Paula-Moraes, S. Lin, B. Patla, Y. Niu, C. I.R. Sakuno, and F. Huang. 2024. Reversal of practical resistance in fall armyworm to Cry1F maize: A case report on the resistance to susceptibility in Bt crops from the southeastern United States. J. Pest Sci. https://doi.org/10.1007/s10340-024-01804-y.
  • Type: Peer Reviewed Journal Articles Status: Accepted Year Published: 2025 Citation: Roy, R., H. Abdelgaffar, D. Kerns, M. Huff, M. Staton, F. Yang, F. Huang, J. L. Jurat-Fuentes. 2025. Reduced processing and toxin binding associated with resistance to Vip3Aa in a strain of fall armyworm (Spodoptera frugiperda) from Louisiana. Pest Manag. Sci. (Accepted, in press).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Invited symposium presentation by Juan Luis Jurat-Fuentes, Dawson Kerns, Rajeev Roy, Fei Yang, David Kerns, and Fangneng Huang. Common and distinct mechanisms of resistance to Vip3Aa in pests of transgenic corn. XXVII International Congress of Entomology, Kyoto, Japan, August 2024.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Contributing oral presentation by Dawson Kerns, Matthew Huff, Ryan Kuster, Fangneng Huang, David Kerns, Margaret Staton, and Juan Luis Jurat-Fuentes. Mechanistic and genetic characterization of Vip3Aa resistance in Helicoverpa zea, Cotton Beltwide Conference, Fort Worth, TX, January 2024.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Contributing oral presentation by Jos� Santiago-Gonz�lez, Fei Yang, David Kerns, Dawson Kerns, and Juan Luis Jurat-Fuentes. Effective dominance and cross-resistance of Vip3Aa39 resistance in the tobacco budworm Chloridea virescens (Lepidoptera: Noctuidae). 72nd Annual Meeting of the Southwestern Branch of the Entomological Society of America, Albuquerque, NM, April 2024.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Silva, T., G. Sword, A. Miller, J.A. Qureshi, G. P. Head, D.D. Kerns, J.L. Jurat-Fuentes, J. Villegas, T. Towels, X. Ni, D. Carrillo, D. Cook, C. Daves, F. Reay-Jones, M. Stout, B. Thrash, S.V. Paula-Moraes, S. Lin, B. Patla, Y. Niu, C.R. Sakuno, and F. Huang. 2023. No resistance to Cry1F detected in fall armyworm from the southeastern U.S. after a decade since practical resistance to Cry1F corn was documented. Annual Meeting of Entomological Society of America, National Harbor, MD, Nov 4-9, 2023.


Progress 01/01/23 to 12/31/23

Outputs
Target Audience:The target audience reached during the current period includes scientists and students in academia, government and industry working on control and monitoring of resistance in fall armyworm and corn earworm to Vip3Aa as a plant incorporated protectant. Additionally reached audiences include farmers in the USA and South America where Vip3Aa is an available plant incorporated protectant in transgenic crops, and corn farmers and undergraduate and graduate students in Cambodia and researchers in Kenya (Africa), where fall armyworm is a devastating invasive pest. A third audience group reached by our project communications are students, postdocs and technical personnel globally that are interested in acquiring the research techniques used in attaining objectives in the project. Available data from the project was shared during this performance period with target audiences during contributing presentations at annual meetings of the Beltwide Cotton conference (January 2023, New Orleans, LA), Southeastern branch of the Entomological Society of America (March 2023, Little Rock, AR), the Society for Invertebrate Pathology (August 2023, Maryland, MD) and the Entomological Society of America (November 2023, National Harbor, MD). Project results were also shared in invited presentations at the Plant and Animal Genome (PAG) conference (January 2023, San Diego, CA), the meeting of the International Working Group of Pests of Corn and Ostrinia (IWGO, May 2023, Kenya), the International Center for Insect Physiology and Ecology (ICIPE, May 2023, Kenya), the University Institute of Biotechnology and Biomedicine of the University of Valencia (BIOTECMED, July 2023), the 3rd International Conference on Insect pest Management (ICIPM, October 2023, Nanjing, China), and the International Symposium on Plant Biosafety (October 2023, Kunming City, China). A synthesis of the project goals and research outcomes are shared with the public through a dedicated site on our laboratory webpage (http://juratfuenteslab.utk.edu/Btresistance.html; 6 to 12K impressions per month), the laboratory Facebook group page (https://www.facebook.com/groups/614669982344958;165 members from 7 countries), and the PI's Linked In account (647 followers, 3,909 post impressions). Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?During the current reporting period, a postdoctoral research associate in the project has provided training in bioinformatics for two PhD students on analysis of RNAseq and bulk segregant analysis data. The two PhD students also attended international (Society for Invertebrate Pathology) and national (Entomological Society of America) conferences to present their results and learn about new developments in the field of research. How have the results been disseminated to communities of interest?Current results have been shared with scientists, students and postdocs from laboratories in academia, government, and industry through invited symposium presentations at scientific institutions (International Center for Insect Physiology and Ecology) and conferences. Results were also shared through peer-reviewed publications. Updates on results and publications are also shared with the a broader general public audience through postings on social media: Facebook group for the PI's laboratory with 170 members from 8 countries (https://www.facebook.com/groups/614669982344958) and the PI's LinkedIn page with 689 connections, 753 followers and 19,833 impressions (https://www.linkedin.com/in/juan-luis-jurat-fuentes-6ab0245/). What do you plan to do during the next reporting period to accomplish the goals?Objective 2: Preparation and submission of manuscript describing mechanism of resistance to Vip3Aa in strains of fall armyworm from LA and TX for publication in peer-reviewed journal. Objective 3: We have identified a single genetic locus associated with resistance to Vip3Aa in corn earworm. We plan to perform long read sequencing to identify the specific genomic alteration resulting in resistance to Vip3Aa in corn earworm. The same process will be followed for genetic loci associated with resistance to Vip3Aa in fall armyworm. In this case, our genetic analyses (RNAseq and BSA) suggest the resistance gene affects multiple steps in the mode of action of the toxin. We have also have started analysis of an alternative fall armyworm strain originating from Texas that seems to have a clearer resistant phenotype involving reduced Vip3Aa binding, like our findings with corn earworm. We expect that we should be able to identify the resistance gene in at least one of these strains. Multiplexed targeted sequencing panels will be developed to detect these resistance alleles. Objective 4: Use the multiplexed targeted sequencing panel from Objective 3 in linkage tests and testing archived insect genetic materials.

Impacts
What was accomplished under these goals? Impact description Insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) protect >85% of the cotton and corn acreage in the USA from damage by devastating caterpillar pests. Plants producing these Bt proteins provide valuable economic and environmental benefits. Cumulative benefits of corn producing Bt proteins (Bt corn) in the Corn Belt 9 years after commercialization reached ~$7 billion. During this period, benefits were not limited to growers planting Bt crops, as areawide pest suppresion by Bt corn provided $4 billion in benefits to farmers not growing Bt corn. All these benefits are now seriously threatened by insect resistance to Bt proteins, especially in the corn earworm and fall armyworm, the two pest models used in this project. The corn earworm causes grain losses estimated at 2.5% annually and damage to sweet corn can reach 50% in unsellable product. On the other hand, the fall armyworm is quickly becoming a global pest estimated to cost farmers $10 billion per year. The Vip3Aa protein is currently the only highly effective Bt protein produced in transgenic corn and cotton against both pests, yetresistant insects have been isolated from screens of field populations. This project capitalizes on available field-derived Vip3Aa-resistant strains of both corn earworm and fall armyworm in identifying mechanisms of resistance and genes involved. Data obtained so far supports that reduced binding of Vip3Aa to receptors in the gut of the caterpillars is responsible for resistance, although resistant fall armyworm caterpillars also seem to process the Vip3Aa protein differently than susceptible caterpillars. Genetic studies identify mutations in genes that are behind the reduced Vip3Aa processing and binding observed in resistant caterpillars. This information induces a change in knowledge for scientists and students in academia, government and industry working on control and monitoring of resistance in fall armyworm and corn earworm to Vip3Aa on how insects become resistant to Vip3Aa. This knowledge allows the development of more cost-effective and sensitive resistance detection methods, as well as guiding the design of improved insecticidal proteins. Availability of improved resistance screening tools in turn facilitates a change in action using improved detection and management tools by regulators, industry and farmers to better safeguard public benefits of Bt insecticidal proteins for US agriculture. Continuation of these benefits provides a change in condition by extending higher and safer food and fiber productivity, making US farmers more competitive in the global market while reducing environmental pollution with pesticides. Specific to this reporting period: Objective 1: Testing role of Vip3Aa processing and stability in resistance (100% completed) 1) Major activities completed / experiments conducted: Quantification of specific enzymatic activity in midgut fluids form susceptible and Vip3A-resistant fall armyworm and corn earworm. 2) Data collected: Specific activity values from reactions using larval midgut fluids and fluorescent substrates for trypsin and chymotrypsin activities. Susceptibility of fall armyworm larvae to Vip3Aa processed with midgut fluids from susceptible or resistant larvae. 3) Summary statistics and discussion of results: We detected reduced trypsin and chymotrypsin activities in larval gut fluids from Vip3Aa-resistant fall armyworm from Louisiana, while in a population from Texas protease activities are increased (ANOVA; P<0.05). Results from bioassays detected increased activity of Vip3Aa when processed with midgut fluids from susceptible than resistant fall armyworm larvae. Reduced processing was not observed in Vip3A-resistant corn earworm, but a reference Cry-resistant strain displayed rate of processing of Cry1Ac protein. 4) Key outcomes or other accomplishments realized: Differential processing of Vip3Aa by midgut fluids explains part of the resistance to this protein in fall armyworm from Louisiana, but not from Texas. Differential processing is not associated with resistance to Vip3Aa in corn earworm but may explain resistance to Cry1Ac. Objective 2: Testing binding of Vip3Aa to receptors (95% completed) 1) Major activities completed / experiments conducted: Binding of labeled Vip3A toxin was tested and compared between brush border membrane vesicles (BBMV) from susceptible and resistant fall armyworm larvae from Texas using fluorescently labeled or biotinylated Vip3Aa detection. 2) Data collected: Chemiluminescence detection of biotinylated Vip3Aa bound to BBMV in Western blots. Detection of fluorophore-labeled Vip3Aa bound to BBMV. Specific binding was estimated as the difference between total binding (with no competitor present) and non-specific binding (in the presence of a >100-fold excess of unlabeled Vip3Aa). 3) Summary statistics and discussion of results: More Vip3Aa bound to BBMV from susceptible than to Vip3A-resistant fall armyworms form Texas. The same result was observed in resistant fall armyworm from Louisiana. 4) Key outcomes or other accomplishments realized: Reduced Vip3Aa binding to receptors in the midgut is associated with resistance in both corn earworm and fall armyworm. Objective 3: Identification of candidate Vip3Aa resistance genes in CEW and FAW (80% completed). 1) Major activities completed / experiments conducted: Bulk segregant analysis was performed for susceptible and Vip3Aa-resistant strains of corn earworm and fall armyworm, and a Cry1Ac/Cry2Ab-resistant strain of corn earworm. Bioinformatic analyses identified genome regions with variation associated with the Vip3Aa resistant phenotype. 2) Data collected: Raw reads were trimmed, and high-quality reads mapped to reference genome for each species. Detection of variants (polymorphisms) mapped to the reference genome. 3) Summary statistics and discussion of results: One single significant peak (P<0.05) was detected in Vip3Aa-resistant corn earworm when compared to susceptible or Cry1AC/Cry2A resistant populations. Genes in this locus display high number of polymorphisms and provide a short list of candidate genes to test for linkage with resistance. One relevant peak was also found for Vip3Aa-resistant fall armyworm from Louisiana. This locus includes multiple genes involved in regulatory processes, suggesting the possibility of trans-regulation of genes as a mechanism of resistance in this case. 4) Key outcomes or other accomplishments realized: List of candidate genes in Vip3A-resistant insects obtained. This list includes potential Vip3Aa receptors in corn earworm, and proteases and regulatory genes that may simultaneously affect Vip3Aa processing and binding in fall armyworm, in agreement with observations from Objective 3. Objective 4: Linkage of candidate resistance alleles with resistance to Vip3Aa and estimation of frequency (40% completed) 1) Major activities completed / experiments conducted: Developed a panel of multiple target genes for highly multiplexed targeted sequencing. 2) Data collected: Raw sequences from next generation sequencing of amplicons from highly multiplexed PCR reactions of 274 samples of fall armyworm collected from 14 locations in 8 US states and Puerto Rico. 3) Summary statistics and discussion of results: We developed a panel of targets amplifying exonic regions and intron-exon boundaries in 7 candidate resistance genes to Bt proteins and pesticides in fall armyworm. Sequence data was parsed and mapped to reference gene sequences to identify and quantify frequency of resistance mutations in these genes across distinct locations. 4) Key outcomes or other accomplishments realized: Highly multiplexed targeted sequencing can be used to detect resistance alleles in multiple genes, as a strategy to monitor for Vip3Aa resistance.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Contributing presentation by Kerns D, Yang F, Kerns D, and Jurat-Fuentes JL: "Role of midgut processing and receptor binding in Helicoverpa zea Vip3Aa resistance", Beltwide Cotton Conference, New Orleans, LA, January 2023.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Contributing presentation by Roy R, Kerns D, Kerns D, Yang F, Huang F and Jurat-Fuentes JL: "Testing Vip3Aa binding in susceptible and resistant Spodoptera frugiperda (Lepidoptera: Noctuidae) strains", annual meeting of the Southeastern Branch of the Entomological Society of America, Little Rock, AR, March 2023
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Invited workshop presentation by Jurat-Fuentes JL, Schlum K, Mishra S, Tandi P, de Bortoli, CP, Lamour K, and Emrich S: "Targeted sequencing in screening for resistance to transgenic corn and insecticidal RNAi", Plant and Animal Genome Conference 2023 (PAG 30), San Diego, CA, January 2023.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Invited symposium presentation by Jurat-Fuentes, J.L.: "Monitoring for resistance to Bt maize using targeted sequencing", International Working Group on Ostrinia and Other Pests of Corn (IWGO), Nairobi, Kenya, May 2023.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Contributing presentation by Kerns D, Yang F, Kerns D, Stewart S, and Jurat-Fuentes JL: "Resistance to Vip3Aa in Helicoverpa zea is associated with reduced toxin binding", annual meeting of the Society for Invertebrate Pathology, College Park, MD, August 2023.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Contributing presentation by Roy R, Yang F, Kerns D, Stewart S, and Jurat-Fuentes JL: "Vip3Aa resistance mechanism in a strain of Spodoptera frugiperda (Lepidoptera: Noctuidae) from Louisiana", annual meeting of the Society for Invertebrate Pathology, College Park, MD, August 2023.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Invited symposium presentation by Jurat-Fuentes, J.L.: "Targeted sequencing in monitoring and forecasting fall armyworm resistance", 3rd International Conference on Insect Pest Management (ICIPM), Nanjing, China, October 2023.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Invited symposium presentation by Jurat-Fuentes, J.L.: "Tracking practical resistance to transgenic maize across boundaries", International SYmposium on Plant Biosafety (ISPB)), Kungming, China, October 2023.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Invited symposium presentation by Jurat-Fuentes JL: "Mechanisms and dispersal of practical resistance to Bt corn in the fall armyworm (Spodoptera frugiperda)", Annual Meeting of the Entomological Society of America, National Harbor, MD, November 2023.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Contributing presentation by Huang F, co-authors: Silva T, Sword G, Miller A, Qureshi J, Head GH, Kerns D, Jurat-Fuentes JL, Villegas J, Ni X,: "No resistance to Cry1F detected in fall armyworm from the southeastern US after a decade since practical resistance to Cry1F corn was documented", Annual Meeting of the Entomological Society of America, National Harbor, MD, November 2023.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Invited symposium presentation by Kerns, D: "Characterizing Vip3Aa resistance mechanism in a field-derived Helicoverpa zea strain?", Annual Meeting of the Entomological Society of America, National Harbor, MD, November 2023.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Invited symposium presentation by Roy, R.: "Differential processing associated with resistance to Vip3Aa in Spodoptera frugiperda Lepidoptera: Noctuidae", Annual Meeting of the Entomological Society of America, National Harbor, MD, November 2023.
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Kerns DD, Yang F, Kerns DL, Stewart SD, Jurat-Fuentes JL. Reduced toxin binding associated with resistance to Vip3Aa in the corn earworm (Helicoverpa zea). Appl Environ Microbiol. 2023 Dec 21;89(12):e0164423. doi: 10.1128/aem.01644-23. Epub 2023 Nov 28. PMID: 38014960; PMCID: PMC10734485.
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Huang F, Niu Y, Silva T, Brown S, Towles T, Kerns D, Jurat-Fuentes JL, Head GP, Carroll M, Walker W, Lin S. An Extended Investigation of Unexpected Helicoverpa zea (Boddie) Survival and Ear Injury on a Transgenic Maize Hybrid Expressing Cry1A/Cry2A/Vip3A Toxins. Toxins (Basel). 2023 Jul 22;15(7):474. doi: 10.3390/toxins15070474. PMID: 37505743; PMCID: PMC10467152.
  • Type: Journal Articles Status: Accepted Year Published: 2023 Citation: Tandy P, Lamour K, Placidi de Bortoli C, Nagoshi R, Emrich SJ, Jurat-Fuentes JL. Screening for resistance alleles to Cry1 proteins through targeted sequencing in the native and invasive range of Spodoptera frugiperda (Lepidoptera: Noctuidae). J Econ Entomol. 2023 Jun 13;116(3):935-944. doi: 10.1093/jee/toad061. PMID: 37311017.
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Santiago-Gonz�lez JC, Kerns DL, Yang F. Resistance Allele Frequency of Helicoverpa zea to Vip3Aa Bacillus thuringiensis Protein in the Southeastern U.S. Insects. 2023 Feb 7;14(2):161. doi: 10.3390/insects14020161. PMID: 36835730; PMCID: PMC9958976.


Progress 01/01/22 to 12/31/22

Outputs
Target Audience:The target audience reached during the current period includes scientists and students in academia, government and industry working on control and monitoring of resistance in fall armyworm to Vip3Aa as a plant incorporated protectant. Additionally reached audience includes farmers in the USA and South America where Vip3Aa is an available plant incorporated protectant in transgenic crops. A third audience group reached by our project communications are students, postdocs and technical personnel interested in acquiring and using the research techniques used in completion of the project. Available data from the project was shared with target audiences during invited seminars at research institutions, contributing presentations at the annual meetings of the Society for Invertebrate Pathology (August 2022, virtual) and the Entomological Society of America (November 2022, Vancouver BC), and a presentation at the meeting of the International Working Group of Pests of Corn and Ostrinia (IWGO, May 2022, virtual). A synthesis of the project goals and research outcomes is shared with the public through a dedicated site on our laboratory webpage (http://juratfuenteslab.utk.edu/Btresistance.html; 6 to 12K impressions per month) and the laboratory Facebook group page (https://www.facebook.com/groups/614669982344958; 165 members from 7 countries). Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?During the current reporting period, the project has provided training to two graduate students (PhD) on insect husbandry, bioassay and dissection, production, and purification of insecticidal proteins from bacterial cultures, isolation and quality evaluation of genetic material (RNA), binding assays with labeled insecticidal proteins, measurement of gut digestive activity and pH, and bioinformatic analysis of RNAseq data. A postdoctoral research associate also participated in training for bioinformatic analysis of RNAseq data. An undergraduate student participating in the project has been trained in insect husbandry and performance of bioassays. How have the results been disseminated to communities of interest?Current results have been shared with scientists from academia, government, and industry as well as students and postdocs through invited presentations at academic groups and contributing presentations at scientific meetings. Data generated so far in the project has been also shared in peer-reviewed publications. Updates on results and publications are also shared with the general public through postings on social media through a Facebook page for the PI's laboratory with 170 members from >7 countries: https://www.facebook.com/groups/614669982344958. What do you plan to do during the next reporting period to accomplish the goals?Objective 1- Compile data comparing Vip3Aa processing and gut pH in susceptible and Vip3Aa-resistant fall armyworm and corn earworm for publication in peer-reviewed journal. Objective 2- Compile Vip3Aa binding data comparing susceptible and Vip3Aa-resistant fall armyworm and corn earworm for publication in peer-reviewed journal. Objective 3- Analyse RNAseq data for nucleotide polymorphisms (SNPs) in putative Vip3Aa-receptor genes. Confirm differentially expressed genes by quantitative PCR and Sanger sequencing (for SNPs). Objective 4- Initiate linkage tests with candidate resistance genes from Objective 3 and stored genetic materials.

Impacts
What was accomplished under these goals? Impact Public, economic, and environmental benefits of insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) as plant incorporated protectants (PIPs) are seriously threatened by the evolution of resistance in targeted pests. This technology represents >85% of the corn and cotton grown in the USA and its cumulative benefits for corn in the Corn Belt since commercialization amounts to almost $1 billion/year. Areawide pest control provided by Bt corn also benefits to farmers not growing Bt corn. The two pest models in this project are the corn earworm and the fall armyworm. Larvae of corn earworm cause grain losses estimated at 2.5% annually, and damage to sweet corn can reach 50% in unsalable product. The fall armyworm is quickly expanding as a global pest estimated to cost farmers $10 billion/year. The Vip3Aa protein from Bt is currently an essential PIP in Bt corn and cotton against both of these pests, yetemerging evidence supports imminence of resistance in both cases. This project capitalizes on available field-derived Vip3Aa-resistant strains of both corn earworm and fall armyworm in identifying mechanisms of resistance. Data obtained support reduced binding to Vip3Aa receptors associated with resistance, although alterations in Vip3Aa processing in the larval gut are also detected. This information represents a change in knowledge on mechanism of resistance to Vip3Aa, which in turn advances a change in action involving adoption of improved detection and management tools to safeguard public benefits of Bt PIPs. Continuation of these benefits provides a change in condition by allowing higher food productivity while reducing environmental pollution with pesticides. Progress during reporting period Objective 1: Testing role of Vip3Aa processing and stability in resistance (95% completed) Major activities completed / experiments conducted: Measurement of digestive canal pH in susceptible and Vip3Aa-resistant corn earworm and fall armyworm larvae under control conditions or after exposure to Vip3Aa. Electrophoretic analysis and quantitation by densitometry of Vip3Aa protoxin processing by midgut fluids from susceptible and Vip3Aa-resistant corn earworm and fall armyworm larvae. Data collected: pH values from the digestive tube (foregut, midgut and hindgut) of larvae after feeding on buffer or Vip3Aa solution; images of electrophoretic gels stained for total protein detecting processing of Vip3Aa protoxin by midgut fluids; densitometry measurements of Vip3Aa protoxin and toxin bands in electrophoresis gels. Summary statistics and discussion of results: We detected no significant differences (ANOVA; P<0.05) in the gut pH of larvae from susceptible and Vip3Aa-resistant strains under control conditions. The midgut pH dropped from alkaline to neutral in susceptible insects after treatment with Vip3Aa, as expected from midgut damage as presented in previous reports. In contrast, for some Vip3Aa-resistant larvae this drop was not observed independently of using Vip3Aa protoxin or processed to toxin, suggesting lack of midgut damage in both cases. No significant differences (ANOVA; P<0.05) in the rate of Vip3Aa processing detected between midgut fluids from susceptible and Vip3Aa-resistant corn earworm. Processing of Vip3Aa occurred by Vip3Aa-resistant fall armyworm appeared reduced compared to susceptible larvae. Key outcomes or other accomplishments realized: Processing or pH conditions are not associated with resistance to Vip3Aa in corn earworm. Potential involvement of reduced processing in resistance to Vip3Aa in fall armyworm, which is not associated to changes in gut pH. Objective 2: Testing binding of Vip3Aa to receptors (85% completed) Major activities completed / experiments conducted: Binding of labeled Vip3A toxin was tested and compared between brush border membrane vesicles (BBMV) from susceptible and resistant corn earworm and fall armyworm larvae using direct quantification of radiolabeled Vip3Aa, Western blotting, and/or fluorescently labeled Vip3Aa detection. Data collected: Disintegrations per minute (dpm) for radiolabeled Vip3Aa bound to BBMV. Enhanced chemiluminescence detection of biotinylated Vip3Aa bound to BBMV in Western blots. Detection of fluorescence from fluorophore-labeled Vip3Aa bound to BBMV. Specific binding was calculated as the difference between total binding (with no competitor present) and non-specific binding (in the presence of a >100-fold excess of unlabeled toxin). Saturation binding assays were performed with constant BBMV and increasing ligand and unlabeled competitor amounts. Binding competition experiments were performed with constant BBMV and ligand inputs but increasing unlabeled competitor amounts. Summary statistics and discussion of results: Significantly more radiolabeled Vip3Aa bound to BBMV from susceptible than to Vip3A-resistant corn earworm. The same result was observed using biotinylated Vip3Aa for corn earworm and fall armyworm in Western blots. Key outcomes or other accomplishments realized: Reduced Vip3Aa binding to receptors in the midgut is associated with resistance in both corn earworm and fall armyworm. Objective 3: Identification of candidate Vip3Aa resistance genes in CEW and FAW (50% completed) Major activities completed / experiments conducted: RNAseq was performed for susceptible and Vip3Aa-resistant strains of corn earworm and fall armyworm, including a Cry1Ac/Cry2Ab-resistant strain of CEW. Six biological replicates were used per strain. Bioinformatic analyses resulted in list of differentially expressed genes and pathways enriched in differentially expressed genes. Data collected: Raw reads were trimmed, and high-quality reads mapped to reference genome for each species. Estimation of read counts and significantly differentially expressed genes across samples. Detection of variants (polymorphisms) ongoing. Summary statistics and discussion of results: Percentage of effective reads used for analysis from total and Q30 was >95%. For corn earworm, unique mapping sequences was >84%, and PCA plots detected separate groupings of susceptible and Vip3Aa-resistant samples. Grouping in fall armyworm samples was also observed but not as clear, due to samples from resistant strain having a broader distribution. Changes in gene expression were analyzed with P<0.05 and >1 log2 fold change. In corn earworm, a total of 224 and 212 genes were up- and down-regulated, respectively, in Vip3Aa-resistant when compared to Cry-resistant and susceptible samples. Down-regulated genes included membrane receptors, in agreement with reduced Vip3Aa binding detected in Objective 2. In fall armyworm, 1,638 and 1,308 genes were up- and down-regulated, respectively, in Vip3Aa-resistant when compared to susceptible samples. Further testing by quantitative PCR, and potentially bulk segregant analysis, will help confirm list of candidate resistance genes. Key outcomes or other accomplishments realized: List of differentially expressed genes in Vip3A-resistant insects obtained. In agreement with observations from Objective 3, this list includes potential Vip3Aa receptors (corn earworm) as well as proteases that may affect Vip3Aa processing (fall armyworm). These genes will be further confirmed by quantitative PCR before testing for cosegregation in Objective 4. Objective 4: Linkage of candidate resistance alleles with resistance to Vip3Aa and estimation of frequency (20% completed) Major activities completed / experiments conducted: Crosses and backcrosses performed to obtain segregating subfamilies for linkage analysis, and midgut tissues dissected. Data collected: None this period. Summary statistics and discussion of results: Noen this period Key outcomes or other accomplishments realized: Midgut tissue samples prepared will be used with candidate resistance genes from Objective 3 in testing cosegregation with resistance.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Kerns, D., Yang, F., Kerns, D., Stewart, S., and Jurat-Fuentes, J.L. "Binding of Vip3A toxin to resistant and susceptible Helicoverpa zea brush border membrane vesicles". Beltwide Cotton Conference, San Antonio, TX (January 4-6)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Kerns, D., Yang, F., Kerns, D., and Jurat-Fuentes, J.L. "Is Vip3Aa binding altered in resistant corn earworm (Helicoverpa zea)?". Annual Meeting Entomological Society of America, Vancouver, B.C. (November 13-16)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Kerns, D., Yang, F., Kerns, D., and Jurat-Fuentes, J.L. "Is Vip3Aa binding altered in resistant corn earworm (Helicoverpa zea)?". Annual Meeting Tennessee Entomological Society, Knoxville, TN (November 13-16)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Silva, T., G. Sword, G.P. Head, D. Kerns, J.J. Jurat-Fuentes, J. Qureshi, X. Ni, T. Towels, J. Villegas, D. Carrilo, S. Paula-Moraes, Y. Niu, C. Sakuno, S. Lin, B. Plata, A. Cerutti, and F. Huang. 2022. Susceptibility of fall armyworm populations from the southern region of the United States to four common Bacillus thuringiensis proteins. 2022 LSU Department of Entomology Graduate Student Symposium. Oct 20, 2022.
  • Type: Other Status: Published Year Published: 2022 Citation: Huang, F. 2022. Invited presnetation: "Resistance management: lessons from 25 years of global Bt crop use." University of S�o Paulo (ESALQ-USP), Piracicaba, Sao Paulo, Brazil, April 6, 2022.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Tiago, S., Y. Niu, G. Sword, J. Qureshi, G.P. Head, D. D. Kerns, J. L. Jurat-Fuentes, X. Ni, D. Carrillo, C. R. Sakuno, and F. Huang. 2022. Extended investigation of fall armyworm resistance in the southern United States to four common Bt proteins. Annual meeting of Entomological Society of America, Vancouver, Canada, Nov 12-16, 2022.
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Yang, F., Wang, Z., and D.L. Kerns. 2022. Resistance of Spodoptera frugiperda to Cry1, Cry2, and Vip3Aa Proteins in Bt Corn and Cotton in the Americas: Implications for the Rest of the World. J. Econ. Entomol. 115(6): 17521760.
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2022 Citation: Santiago-Gonzalez, J.C., Kerns, D., Head, G.P., and F. Yang. 2022. A modified F2 screen for estimating Cry1Ac and Cry2Ab resistance allele frequencies in Helicoverpa zea (Lepidoptera: Noctuidae). J. Econ. Entomol.


Progress 01/01/21 to 12/31/21

Outputs
Target Audience:The target audience reached during the current period includes scientists and students in academia, government and industry involved in control and monitoring of resistance in fall armyworm to Vip3Aa as plant incorporated protectant. Additional reached audience includes farmers in the USA and South America, where Vip3Aa is an available plant incorporated protectant in transgenic crops. A third audience group reached by our project communications are students, postdocs and technical personnel interested in acquiring and using the research techniques used in completion of the project. Available data from the project was shared with target audiences during an invited program symposium presentation at the annual meeting of the Society for Invertebrate Pathology (June 2021). A synthesis of the project goals and research outcomes are shared with the general public through a dedicated page on our laboratory webpage (http://juratfuenteslab.utk.edu/Btresistance.html) and the laboratory Facebook page (https://www.facebook.com/groups/614669982344958; 165 members from 7 countries) Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?During the current reporting period, the project provided training to two graduate students (PhD) on insect husbandry, bioassay and dissection, production and purification insecticidal proteins from bacterial cultures, labeling of proteins and performance of binding assays, electrophoresis and Western blotting, and isolation and quality evaluation of genetic material (RNA). An undergraduate student participating in the project has been trained in insect husbandry and performance of bioassays. How have the results been disseminated to communities of interest?Current results have been shared with scientists from academia, government and industry as well as students and postdocs through an invited program symposium talk at a scientific congress. Preliminary results were also shared during an invited public WebCast seminar in Spanish. This presentation is archived at YouTube and has received 155 views (3/4/2022). Preliminary results and publications are shared regularly with the general public through postings in social media through a Facebook page for the PI's laboratory with 139 members from >7 countries: https://www.facebook.com/groups/614669982344958. What do you plan to do during the next reporting period to accomplish the goals?Objective 1- Compare processing of Vip3Aa protoxin by midgut fluids from susceptible and Vip3Aa-resistant fall armyworm (FAW) and corn earworm (CEW) larvae. If quantitative or qualitative differences are detected we will test the activity of processed Vip3Aa against cultured insect cells that are naturally susceptible to the Vip3Aa toxin. Objective 2- Finalize replication of binding assays and preparation of data for publication in peer-reviewed journal. If differences in Vip3Aa binding between susceptible and resistant FAW and CEW are confirmed we will proceed to identification of candidate Vip3Aa receptors in midgut brush border membranes. Objective 3- Sequence and analyze RNAseq data to identify candidate resistance genes in both FAW and CEW strains. Depending on the results, if no clear candidate genes are identified we will follow up with additional RNAseq sequencing and/or bulk segregant analysis using genetic materials from F2 individuals from backcrosses. Objective 4- Processing of collected tissues for purification of genetic materials. If candidate genes/alleles are identified in Objective 3we will initiate linkage tests with the prepared genetic materials. Collections of field FAW and CEW will continue for future genotyping and estimation of resistance allele frequency.

Impacts
What was accomplished under these goals? Impact The most serious threat to the sustainable use and continued public and environmental benefits of genetically engineered (GE) crops producing insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) as plant incorporated protectants (PIPs), is the evolution of resistance in targeted pests. The Vip3Aa Bt protein is currently an essential PIP in GE corn and cotton, yetemerging evidence supports imminence of resistance in two devastating caterpillar pests: Helicoverpa zea (corn earworm, CEW) and Spodoptera frugiperda (fall armyworm, FAW). This project capitalizes on uniquely available field-derived Vip3Aa-resistant strains of CEW and FAW in identifying mechanisms conducive to resistance to Vip3Aa. Information from this project is critical to advance a change in knowledge on resistance to Vip3Aa to lead a change in action involving improved monitoring and management tools that safeguard public benefits of Bt PIPs for US agriculture. Progress during reporting period Objective 1: Testing role of Vip3Aa processing and stability in resistance (15% completed) 1) Major activities completed / experiments conducted: Produced and purified Vip3Aa protoxin and examined processing by midgut fluids from reared corn earworm (CEW) and fall armyworm (FAW) and commercial trypsin. 2) Data collected: Chromatograms of Vip3Aa purification, images of electrophoretic gels stained for total protein detecting Vip3Aa protoxin and processed toxin. 3) Summary statistics and discussion of results: We identified insect midgut fluids and commercial trypsin amounts needed for effective processing the Vip3Aa protoxin (~90 kDa ) to the activated toxin form (~62 kDa) after incubation for 1 hour at room temperature, 30 and 37°C. 4) Key outcomes or other accomplishments realized: Conditions for Vip3Aa activation allows performance of comparative processing studies between midgut fluids from susceptible and Vip3Aa-resistant CEW and FAW lines. Objective 2: Testing binding of Vip3Aa to receptors (70% completed) 1) Major activities completed / experiments conducted: Produced and purified Vip3Aa activated toxin and labeled with, radioactive iodine (I-125), biotin or a fluorescent tag. Backcrosses and re-selection were used to obtain susceptible and Vip3A-resistant strains of CEW and FAW with a shared genomic background. Midgut samples of susceptible and Vip3A-resiustant CEW and FAW strains, as well as of F1 larvae from crosses between susceptible and resistant moths and a Cry1Ac/Cry2Ab-resistant strain of CEW, were prepared. Dissected tissues were used to prepare brush border membrane vesicle (BBMV) proteins. Binding of labeled Vip3A toxin was tested and compared between BBMV from susceptible and resistant larvae using direct quantification of radiolabeled Vip3Aa, Western blotting, or fluorescence detection. 2) Data collected: Enrichment of BBMV marker enzymes as evidence for quality of BBMV preparation. Disintegrations per minute (dpm) for radiolabeled Vip3Aa bound to BBMV. Enhanced chemiluminescence detection of biotinylated Vip3Aa bound to BBMV in Western blots. Detection of fluorescence from fluorophore-labeled Vip3Aa bound to BBMV. Specific binding was calculated as the difference between total binding (with no competitor present) and non-specific binding (in the presence of a >100-fold excess of unlabeled toxin). 3) Summary statistics and discussion of results: Significantly more radiolabeled Vip3Aa bound to BBMV from susceptible to Vip3A-resistant FAW from Louisiana, while binding in FAW from Texas does not appear altered. The same result was observed using biotinylated Vip3Aa. Binding of radiolabeled Vip3Aa was also reduced in BBMV from Vip3Aa-resistant compared to susceptible or Cry1Ac/Cry2Ab-resistant CEW. The same results were observed with biotinylated Vip3Aa in Western blots. Preliminary experiments with fluorescent Vip3Aa yielded inconclusive results for both FAW and CEW and are being replicated. 4) Key outcomes or other accomplishments realized: Reduced binding is associated with resistance to Vip3Aa in FAW from Louisiana but not from Texas. The Vip3Aa-resistant strain of CEW from Texas also displayed reduced Vip3Aa binding. Objective 3: Identification of candidate Vip3Aa resistance genes in CEW and FAW (25% completed). 1) Major activities completed / experiments conducted: Individual midguts from susceptible and Vip3Aa-resistant strains of FAW (Louisiana) and CEW (Texas), and a Cry1Ac/Cry2Ab-resistant strain of CEW were dissected and used to purify total RNA. Quality control (QC) for purified RNA samples identified samples amenable to sequencing library preparation for RNAseq. 2) Data collected: Quality control data for RNA samples included evaluating quantity (using Qubit Fluorometer), quality (using agarose electrophoresis) and purity (using NanoDrop Spectrophotometer). Data collected include RNA amounts, presence of expected 18S and 28S bands in electrophoretic gels, and A260/A280 (detecting contamination with DNA or protein) and A260/A230 (detects contamination with wash solutions, chaotropic salts, phenols or proteins) ratios for purity. 3) Summary statistics and discussion of results: None during this period. 4) Key outcomes or other accomplishments realized: Samples with passing QC identified and used for mRNA enrichment and library preparation for RNAseq. Objective 4: Linkage of candidate resistance alleles with resistance to Vip3Aa and estimation of frequency (20% completed) 1) Major activities completed / experiments conducted: Crosses and backcrosses to obtain segregating subfamilies for linkage analysis were performed, and midgut tissues dissected. Near isogenic susceptible and Vip3A-resistant FAW and CEW strains were crossed to generate F1 hybrids, which were sibmated or backcrossed to resistant moths. Neonates from sibmating and backcrossing were reared on either control diet or diet containing a discriminatory dose of Vip3Aa protein killing heterozygotes for resistance. After reaching 5th instar, midgut tissues from survivors were dissected and individually flash frozen. Field-collected FAw and CEW samples were stored frozen for future genotyping for candidate resistance alleles. 2) Data collected: Percentage mortality in F2 subfamilies exposed to control or diet containing Vip3Aa protein. 3) Summary statistics and discussion of results: Mortality observed in FAW and CEW F2 subfamilies when exposed to diet containing Vip3Aa reached almost 75%. This percentage is expected from presence of 25% of homozygous resistant individuals in the F2 generation as predicted from Mendelian transmission of a recessive genetic trait. 4) Key outcomes or other accomplishments realized: Midgut tissue samples prepared will be used for testing presence of candidate resistance alleles and cosegregation with resistance.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Invited Program Symposium presentation by Jurat-Fuentes, JL: "Challenges and opportunities for bacterial control of Spodoptera frugiperda", Annual meeting of the Society for Invertebrate Pathology, virtual meeting (June 20201).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Invited WebCast by the Institute of Biotechnology of the Autonomous University of Mexico presented by Jurat-Fuentes, J.L.: "Development of technologies for the detection of resistance to Bacillus thuringiensis" https://www.youtube.com/watch?v=ZS2UhM4gMzE, 155 views as of 3/4/2022.