Progress 03/15/18 to 03/14/23
Outputs
Target Audience:Target audience reached during this reporting period includes scientific groups in academia, government and industry involved in control of fall armyworm with insecticidal proteins from entomopathogenic bacteria. Our target audience also included farmers in the USA, Brazil, Argentina, Sub-Saharan Africa, Southeastern Asia, ansd Australia where the fall armyworm is a significant pest and/or has developed resistance to bacterial insecticidal proteins and small molecule pesticides. A third audience reached are students and technical personnel interested in acquiring and using the resistance genotyping methods developed in the project. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?During the current reporting period, training activities have included collaborations with computer scientists and bioinformaticians to design and implement a multiplexed targeted sequencing tool and conduct analyses of the resulting data. The project provided training on molecular biology methods (cloning, subcloning, PCR), insect rearing, performance of bioassays, insect cell culture and transformation, and bioinformatics for a graduate (PhD) student and a postdoc. How have the results been disseminated to communities of interest?Results were shared in peer-reviewed publications, invited and contributing seminars, and postings in social media (public Facebook page for the PI's laboratory with 169 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?
Nothing Reported
Impacts
What was accomplished under these goals?
IMPACT: The fall armyworm (Spodoptera frugiperda) is one of the most economically relevant pests of corn and other crops in the Western Hemisphere that in the last decade has raise as a threat to global food security due to its expansion into Sub-Saharan Africa, India, Southeast Asia and Australia. Importantly, this pest is notorious for its ability to develop resistance to insecticides, hindering its control. This project aimed at identifying mutations in fall armyworm genes leading to resistance against biopesticides and insecticidal transgenic crops, and then capitalize on this information in developing a DNA-based tool amenable to high throughput detection of insecticide resistance in field fall armyworm populations. This tool allows detection and estimation of resistance frequency in field fall armyworm populations, guiding the choice of insecticides that should be used for effective control and to delay the onset of resistance. Genetic information gathered through this project leads a fundamental change in knowledge by describing significant genetic exchange of material between populations in the Americas, highlighting the risk for spread of resistance across large geographies. Analysis of genetic information also resulted in a change in knowledge by detecting two maternal lineages corresponding to host strains (corn versus rice) of fall armyworm, suggesting potential reproductive preference or isolation between these host strains, which has consequences for crops at risk of fall armyworm attack. A change in knowledge relevant to decision-making in fall armyworm control included confirmation that mutations causing fall armyworm resistance to transgenic corn do not affect susceptibility to synthetic pesticides. This conclusion supports the combined use of these pest control tools. Development of a DNA-based tool in detecting and quantifying frequency of candidate resistance mutations leads a change in action by providing a method for effective resistance screening. Information from these DNA-based screens will lead decisions on pest control tools to optimize fall armywom control and delaying resistance evolution, leading to a change in current conditions on the impact of fall armyworm on food security. Objective 1- Elucidate regions of ABCC2-Cry toxin interaction that are critical to insect susceptibility (2018-2022) 100% completed (see key outcomes below). 1) Major activities completed: No activities on this objective during this period. 2) Data collected: No data collected for this objective during this period. 3) Discussion of results: No data collected for this objective during this period. 4) Key outcomes or other accomplishments realized: Earlier work on this objective demonstrated gene editing by CRISPR/Cas9 as an effective tool in generating strains of fall armyworm harboring an edited genotype resulting in a desirable phenotype. During performance of this objective, reports from other groups identified the Cry1F-binding regions in SfABCC2 using CRISPR-Cas9 and other approaches, which was the main goal in this objective. Consequently, we shifted efforts to additional goals in other objectives. Objective 2- Develop a highly sensitive, high throughput, multiplexed targeted sequencing method for screening of resistance to Cry toxins (2018-2023). 100% completed. 1) Major activities completed: During this period, we completed and tested the bioinformatic pipeline for use in analyzing raw sequence reads from our multiplex targeted sequencing approach to resistance screening. This pipeline can detect candidate variations in the SfABCC2 gene predicted to result in resistance to the Cry1F plant incorporated protectant in corn and cotton. We used this tool in detecting known and finding new candidate resistance alleles in 118 sequenced fall armyworm samples including 17 from South Africa, 15 from Ghana, 14 from Togo, 18 from Myanmar, 32 from Puerto Rico, and 22 from the continental USA (7 from Florida, 7 from South Carolina, and 2 each from Texas, Tennessee, Maryland, and Minnesota. We further extended multiplexed targeted sequencing as a screening tool for 9 resistance genes for multiple plant incorporated protectants and small molecule pesticides in 288 fall armyworm samples from the continental USA. 2) Data collected: Genomic DNA was purified from individual fall armyworm samples and used to amplify 571 exonic fragments from 9 target genes using overlapping TILING primers. Raw small amplicon sequencing reads were quality-trimmed, filtered for adapter sequences and high quality, and mapped to reference gene models using default settings in CLC Genomics Workbench. To reduce false-positive genotype assignment, we required at least 10X sequence coverage in the PCR targeted region. Variant calling was performed using default settings in CLC Genomics Workbench. Missense, frameshift and nonsense mutations for all samples were collected into a single csv file with a custom script written for Python 3.8 to identify variants and to determine if the detected variants would induce frame shifts or mutations in predicted binding regions for plant incorporated protectants or target sites for pesticides. Any samples with missing data across all loci were excluded from analysis when detecting frameshift and premature stop codons. Mutations of interest were confirmed using amplification by PCR followed by Sanger sequencing. 3) Discussion of results: Multiplexed targeted sequencing detected a total of 2,694 mutations, most of them (1990, 73.8%) were in predicted intronic regions. Out of the 744 mutations detected in exonic regions, there were 54 nonsynonymous mutations. Filtering of detected frameshift mutations for a minimum 35% representation in the total number of reads mapping (minimum estimated for a heterozygote), and Sanger sequencing validated two mutations. One of these mutations was a resistance allele to Cry1F we previously reported from Puerto Rico, and the other a new candidate resistance allele to Cry1F and Cry1A proteins in fall armyworm from Puerto Rico and South Carolina. Estimates of resistance allele frequency identified populations in Puerto Rico as harboring highest frequency of resistance alleles compared to populations in continental USA. Mutations previously reported to be responsible to fall armyworm resistance to pesticides and plant incorporated protectants were detected by our multigene multiplexed targeted sequencing approach in fall armyworm samples from diverse locations in the continental USA and Puerto Rico. Relative frequency of resistance mutations was higher for fall armyworm of the "corn" versus "rice" race and were more common in Puerto Rico. 4) Key outcomes or other accomplishments realized: Our results detected, for the first time, a resistance allele in fall armyworm form Puerto Rico and South Carolina, evidence that the expected migratory route of fall armyworm may help disperse resistance alleles from the Caribbean into continental USA. This change in knowledge demonstrates the use of our targeted approach to test the spread of relevant genetic mutations through migration. This multigene targeted approach to genotyping genes involved in resistance to plant incorporated protectants and pesticides facilitates a change in action by providing a DNA-based screening tool for use in resistance monitoring programs. The approach is amenable to high throughput screening needed for detailed characterization of resistance status in fall armyworm populations, which can lead a change in action by allowing selection of best pesticidal tools for specific locations. Multiplexed targeted sequencing is also easily extendable to alternative pests and allows inclusion of additional resistance genes. Availability of this highly sensitive, high throughput resistance monitoring tool is critical to improve insect resistance management outcomes and thus facilitates a change in conditions by improving control of devastating pests of crops.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Contributing presentation by Kerns, D., co-authors: F. Yang, D. Kerns, S. Stewart and J. L Jurat-Fuentes. 'Binding of Vip3A toxin to resistant and susceptible Helicoverpa zea brush border membrane vesicles', Beltwide Cotton Conference, San Antonio, TX, January 2022.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Contributing presentation by Jurat-Fuentes, J.L.: 'DNA-based screening and mechanisms of resistance to Bt corn in fall armyworm and corn earworm', International Working Group in Ostrinia and Pests of Corn (IWGO), virtual meeting, May 2022.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Invited presentation at 'Plant Biotechnology' symposium by Jurat-Fuentes, J.L.: 'Field-evolved resistance to transgenic maize in fall armyworm:local or migrant?', BioCubaAgro 2022, virtual presentation, June 2022.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Invited presentation at 'Pathogens and Pest Control in Agriculture' In-depth Symposium by Jurat-Fuentes, J.L.: 'Entomopathogenic bacteria against an emerging global superpest: Bt and the fall armyworm', American Society for Microbiology Microbe meeting, Washington, DC, June 2022.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Invited presentation by Jurat-Fuentes, J.L.: 'Insect Molecular Pathology and Resistance', Estacion Experimental Obispo Coimbres, Tucuman, Argentina, June 2022.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Contributing poster by Placidi de Bortoli, C., presented by Jurat-Fuentes, J.L., co-authors: C. Placidi de Bortoli, P. Tandy, K. Lamour, S. Emrich, and R. Nagoshi. 'Targeted sequencing to screen for resistance to Cry proteins from Bacillus thuringiensis in the fall armyworm, Spodoptera frugiperda (J. E. Smith)', Annual Meeting of the Entomological Society of America, Vancouver, BC, Canada, November 2022.
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Banerjee, R., C. P. de Bortoli, F. Huang, K. H. Lamour, R. Meagher, D. Buntin, X. Ni, F. P. Reay Jones, S. D. Stewart, and J. L. Jurat-Fuentes. 2022. Large genomic deletion linked to field-evolved resistance to Cry1F corn in fall armyworm (Spodoptera frugiperda) from Florida. Scientific Reports, 12(1):13580 doi: 10.1038/s41598-022-17603-3.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Invited presentation at 'Genetic Architecture in Arthropods from Insecticide Exposure'symposium by Jurat-Fuentes, J.L.: 'Targeted sequencing in screening of insect resistance to plant incorporated protectants and RNAi', Plant and Animal Genome (PAG30) meeting, San Diego, CA, January 2023.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Invited presentation at 'Genomcis in current and future maize pest research' symposium by Jurat-Fuentes, J.L.: 'Monitoring for resistance to Bt corn using targeted sequencing', Meeting of the International Working Group on Ostrinia and other petss of corn (IWGO), Nairobi (Kenya), May 2023.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Invited presentation at the International Center for Insect Physiology and Ecology (ICIPE) by Jurat-Fuentes, J.L.: 'Novel approaches for control and detection of resistance to Bt proteins in fall armyworm (Spodoptera frugiperda)', Nairobi (Kenya), May 2023.
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2023
Citation:
Tandy, P., K. Lamour, C. P. de Bortoli, R. Nagoshi, S. J. Emrich, and J. L. Jurat-Fuentes. 2023. Screening for resistance alleles to Cry1 proteins through targeted sequencing in the native and invasive range of Spodoptera frugiperda (Lepidoptera: Noctuidae). Journal of Economic Entomology
|
Progress 03/15/21 to 03/14/22
Outputs
Target Audience:Target audience reached during this reporting period includes scientific groups in academia, government and industry involved in control of fall armyworm with insecticidal proteins from entomopathogenic bacteria. Our target audience also included farmers in the USA, Brazil, Argentina, Sub-Saharan Africa, and Southeastern Asia were the fall armyworm is a significant pest and/or has developed resistance to bacterial insecticidal proteins. A third audience reached are students and technical personnel interested in acquiring and using the resistance genotyping methods developed in completion of the project. Changes/Problems:A publication from a peer group (Liu et al, 2021, SfABCC2 transporter extracellular loops 2 and 4 are responsible for the Cry1Fa insecticidal specificity against Spodoptera frugiperda. Insect Biochem Molec Biol 135:103608) identified the extracellular loop region in SfABCC2 that is critical to Cry1F binding and toxicity. Identification and functional validation of this region was our goal in Objective 1. In avoiding duplication of work, for the remaining of the project we plan to focus Objective 1 on cadherin as an alternative Cry toxin receptor known to be important in resistance to plant incorporated protectants (PIPs). The goal is to determine PIPs that bind to fall armyworm cadherin and incorporate it as target in Objective 2 to determine the level of polymorphism in the cadherin gene across fall armyworm populations as a proxy for the potential for resistance evolution. What opportunities for training and professional development has the project provided?During the current reporting period training activities have included collaborations with computer scientists to design and implement genomic data analysis and design of a targeted sequencing pipeline. The project provided training on molecular biology methods (cloning, subcloning, PCR...), isolation of genetic materials, and bioinformatics for graduate (PhD) students and a postdoc. How have the results been disseminated to communities of interest?Results were shared in peer-reviewed publications, invited symposium and seminar presentations and postings in social media (public Facebook page for the PI's laboratory with 165 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- Testing of cadherin binding to Cry proteins in use or projected to be commercialized as plant incorporated protectants (PIPs) in Bt crops. Comparison of cadherin proteins from multiple fall armyworm strains resistant to PIPs. Objective 2- Incorporatin cadherin gene in targeted sequencing efforts to examine polymorphism levels (diversity) present in fall armyworm populations from diverse locations. Sequencing and genotyping of fall armyworm samples for known and candidate resistance SfABCC2 alleles from regions with low or undetected resistance to PIPs, including the invasive range of fall armyworm (Africa and Asia).
Impacts
What was accomplished under these goals?
IMPACT: Development of a pipeline for genotyping of fall armyworm (Spodoptera frugiperda) samples reveal a fundamental change in knowledge by describing a high throughput DNA-based method to detect resistance alleles to insecticidal proteins and plant incorporated protectants. Availability of these methods can lead a change in action by replacement or implementation with currently mandated bioassay-based resistance monitoring efforts for plant incorporated protectants. These impacts are expected to lead a change in current conditions of the impact of fall armyworm on food and fiber security. Objective 1- Elucidate regions of ABCC2-Cry toxin interaction that are critical to insect susceptibility (2018-2023) 80% completed (see Project Change). Major activities completed: As described in the Project Changes section of this report, a published work from a peer group identified the functional Cry1F receptor domains in SfABCC2, which was the main goal of our Objective 1. In avoiding duplication of efforts, we have concentrated our research on another Cry protein receptor relevant for resistance in fall armyworm and other insects, cadherin. We cloned the full-length transcript for cadherin in susceptible and Cry1F-resistant fall armyworm strains, and subcloned it in a vector for expression and testing of Cry1F binding. Data collected: Sequencing and cloning of cadherin full length transcript and cloning into expression vector. Testing of cadherin binding to Cry1F protein. Discussion of results: Cadherin proteins are functional receptors for Cry plant incorporated protectants (PIPs) against lepidopteran larvae, yet the role of cadherin in Cry intoxication and resistance in fall armyworm is not well established. Our focus in cadherin is fueled by the possibility of expanding the genotyping pipeline developed in Objective 2 to cadherin as an additional target resistance gene. Binding of Cry1F to cadherin cloned from susceptible and resistant fall armyworm strains determined no differences in binding, supporting that this protein may not be relevant to Cry1F resistance in fall armyworm. However, its role as receptor for other PIPs or its level ofpolymorphism in field fall armywom populations are not known. Key outcomes or other accomplishments realized: A change in knowledge was realized by the discovery that binding of Cry1F to cadherin is not altered in Cry1F-resistant fall armyworms. Objective 2- Develop a highly sensitive, high throughput, multiplexed targeted sequencing method for screening of resistance to Cry toxins (2018-2022). 90% completed. Major activities completed: Development of a targeted sequencing pipeline to genotype individual fall armyworm samples for known and candidate resistance alleles. Continued field collection, purification of genetic material and targeted and whole genome sequencing of fall armyworm samples. Genotyping for Cry1F resistance of field collected fall armyworm samples from Puerto Rico focused on SfABCC2 mutations. Data collected: Taqman PCR genotyping of fall armyworm samples for known resistance allele from Puerto Rico (SfABCC2mut). Raw whole genome and targeted sequencing reads quality-trimmed and filtered for adapter sequences and errors. Cleaned reads were mapped to the fall armyworm corn reference genome (v6.0) and reference SfABCC2 gene model. Custom k-mer and bioinformatic pipeline were used to identify synonymous and non-synonymous mutations in SfABCC2. Discussion of results: We evaluated targeted sequencing as a method allowing detection of known and novel candidate resistance alleles to Cry proteins. As a model, we targeted a Cry1F receptor gene (SfABCC2) in fall armyworm moths from Puerto Rico, a location reporting continued practical field resistance to Cry1F-producing corn. Targeted sequencing detected high frequency of a previously reported Cry1F resistance allele (SfABCC2mut), which was present in 34% of the samples tested. In addition, targeted sequencing also detected a resistance allele originally described in fall armyworm populations from Brazil, and one nonsense and nine frameshift SfABCC2 mutations as novel candidate Cry1F resistance alleles. Key outcomes or other accomplishments realized:These results support targeted sequencing as effective for monitoring for known resistance alleles and to uncover candidate resistance alleles to Bt crops for cases in which relevant Cry protein receptors are known. Results provide evidence for high frequency of truncating mutations in SfABCC2 in Puerto Rico, explaining high levels of resistance to Cry1F corn in that region. We also found unexpected evidence for common resistance allelesto Cry1F corn in fall armyworm from Brazil and Puerto Rico, which could be explained by genetic flow between these regions. Taken together, these results represent a change in knowledge by validating targeted sequencing for resistance monitoring to Bt crops, which is expected to facilitate a change in action by adoption of this procedure for monitoring of resistance-relevant mutations of selected genes in different insects.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Schlum K, Lamour K, Tandy P, Emrich SJ, de Bortoli CP, Rao T, Viteri Dillon DM, Linares-Ramirez AM, Jurat-Fuentes JL (2021) Genetic screening to identify candidate resistance alleles to Cry1F corn in fall armyworm using targeted sequencing. Insects 12(7): 618.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Xie, Y, Xu, C, Gao, M, Zhang, X, Lu, L, Hu, X, Chen, W, Jurat-Fuentes, JL, Zhu, Q, Liu, Y, Lin, M, Zhong, J, and Liu, X (2021). Docking-based generation of antibodies mimicking Cry1A/1B protein binding sites as potential insecticidal agents against diamondback moth (Plutella xylostella). Pest Manag. Sci. 77(10): 4593-4606.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Tessnow, AE, Gilligan, TM, Burkness, E, Placidi de Bortoli, C, Jurat-Fuentes, JL, Porter, P, Sekula, D, Sword, GA (2021) Novel real-time PCR based assays for differentiating fall armyworm strains using four single nucleotide polymorphisms. PeerJ 9: e12195.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Perera, OP, Little, NS, Abdelgaffar, H, Jurat-Fuentes, JL, Reddy, GVP (2021) Genetic knockouts indicate that the ABCC2 protein in the bollworm Helicoverpa zea is not a major receptor for the Cry1Ac insecticidal protein. Genes 12(10): 1522.
- 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).
|
Progress 03/15/20 to 03/14/21
Outputs
Target Audience:Target audience for the project includes scientific groups in academia, government and industry involved in control of fall armyworm with insecticidal proteins from entomopathogenic bacteria, population genomics of fall armyworm and the development of sequencing technologies to detect and monitor the dispersal of resistance genes among fall armyworm populations. Our target audience also includes farmers in the USA, Brazil, Argentina, Sub-Saharan Africa, and Southeastern Asia were the fall armyworm is a significant pest and/or has developed resistance to insecticidal proteins. A third audience group targeted by our project are students and technical personnel interested in acquiring and using the resistance genotyping methods developed in completion of the project. During this time period, project progress has been shared with target audiences in invited symposium and contributing presentations at scientific congresses, seminars and peer-reviewed publications. Changes/Problems:In Objective 1 we plan testing the use of nano straws and electroporation as alternatives to overcome lower than desired transformation rates obtained using lipofection of insect cell cultures. What opportunities for training and professional development has the project provided?During the current reporting period, training activities have included collaboration with computer scientists to design and implement genomic data analysis and design of a targeted sequencing pipeline. The project provided training on molecular biology methods (cloning, subcloning, PCR...), insect rearing, performance of bioassays, insect cell culture and transformation, and bioinformatics for graduate (PhD) students. In addition, 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?Results were shared in publications, seminars and postings in social media (public 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- Optimization of expression of cloned truncated SfABCC2 fragments in insect cell cultures by using alternative transformation procedures (nanostraws and electroporation). Testing Cry1F binding and fucntional receptor properties for expressed SfABCC2 fragments. The region involved in binding will be targeted for editing using CRISPR/Cas9 to test functionality in vivo. Objective 2- Testing of bioinformatics pipeline to identify indels and mutations predicted to result in truncated SfABCC2 proteins from multiplexed targeted sequencing data.
Impacts
What was accomplished under these goals?
IMPACT: Comparisons of fall armyworm populations at the whole genome level reveal a fundamental change in knowledge by describing significant gene flow between populations in the Americas, highlighting the risk for spread of resistance across large geographies. Mitochondrial genome comparisons result in a change in knowledge by detecting two maternal lineages corresponding to host strains (corn versus rice) of fall armyworm, suggesting potential reproductive preference or isolation between these host strains. A change in knowledge relevant to decision-making in fall armyworm control include confirmation that mutations causing fall armyworm resistance to transgenic corn producing the Cry1F insecticidal protein do not affect susceptibility to synthetic pesticides. These impacts are expected to lead a change in actions for more effective control of fall armyworm and delaying resistance, which would lead to a change in current conditions of the impact of fall armyworm on food security. Objective 1- Elucidate regions of ABCC2-Cry toxin interaction that are critical to insect susceptibility (2018-2022) 35% completed. 1) Major activities completed: Developed and tested gene editing in fall armyworm usign the CRISPR/Cas9 system. Edited fall armyworms were tested for susceptibility to Cry1F and selected synthetic pesticides. Assembly of the SfABCC2 gene allowing identifycation of Protospacer Adjacent Motifs (PAMs) to target in designing guide RNAs for further gene editing work in the S. frugiperda strain used for the project. Expression vectors for truncated fragments of the ABCC2 protein from fall armyworm (SfABCC2) generated and used to transform insect cell cultures. 2) Data collected: Genotyping of gene-edited larvae to confirm strain homozygosity for indel generated by CRISPR/Cas9. Dose bioassays with Cry1F, Black Flag Extreme Insect Control (deltamethrin active ingredient at 0.32%), Prevathon (chlorantraniliprole active ingredient at 0.43 lb/gal), Brigade (bifenthrin active ingredient at 2 lb/gal), Radiant SC (spinetoram active ingredient at 1 lb/gal), and Orthene 97 WSP (acephate active ingredient at 97%). Sequencing and assembly of SfABCC2 gene and expression vectors harboring the wild type transcript and truncated forms for expression in insect cells. Levels of fluorescence from marker gene (greenfluorescent protein) to determine transformation efficiency. 3) Discussion of results: Fall armyworm originally collected from Puerto Rico displayed high levels of resistance to Cry1F and lower levels of resistance to a pyrethroid. Genotyping confirmed generation of a homozygous SfABCC2 knockout strain of fall armyworm. The knockout involved a 2 bp deletion in exon 13 of SfABCC2, resulting in a premature stop codon. When compared to the parental (non-edited) strain, the SfABCC2 knockout strain was 25-fold resistant to Cry1F but was not resistant to any of the tested synthetic and semisynthetic pesticides. Resistance to deltamethrin was associated with increased cytochrome P450 gene copy number in fall armyworm from Puerto Rico compared to Mississippi. Lipofection resulted in low levels (<30%) of insect cell transformants (based on marker gene expression) using the prepared SfABCC2 expression constructs. 4) Key outcomes or other accomplishments realized: Gene editing by CRISPR/Cas9 is effective in generating strains of fall armyworm harboring a desired genotype and allowed a change in knowledge over nonsense mutations in the SfABCC2 gene causing resistance to transgenic corn producing Cry1F but not affecting susceptibility to other pesticides. A change in knowledge was also realized by identifying Cytochromre P450 gene copy number variation as mechanisms for reduced susceptibility to pesticides in fall armyworm. Objective 2- Develop a highly sensitive, high throughput, multiplexed targeted sequencing method for screening of resistance to Cry toxins (2018-2022). 70% completed. 1) Major activities completed: Comparison at the whole and mitochondrial genome levels of 55 fall armyworm samples collected from the USA, Puerto Rico, Brazil, Argentina and Kenya. Development of an SfABCC2 gene reference model for a targeted sequencing pipeline. Continued field collection, purification of genetic material and sequencing of fall armyworm samples. 2) Data collected: Raw whole genome sequencing reads were quality-trimmed and filtered for adapter sequences and errors. Remaining reads were mapped to the fall armyworm corn reference genome (v3.1). Host strain (corn or rice) was determined using mitochondrial and nuclear markers. Variants were called resulting in detection of 126,977,977 single nucleotide polymorphisms (SNPs) and indels. Further quality filtering resulted in a dataset of 2,762,958 SNPs that was used for nuclear SNP analyses and diversity estimates. Mitochondrial sequences were extracted from the whole genome sequence reads with a mitochondrial gene sequence as the seed, resulting in a continuous complete mitochondrial chromosome of around 15 kbp. The population structure of the 55 genomic samples was surveyed using parametric and non-parametric methods. 3) Discussion of results: Findings from genomic comparisons support lack of clear fall armyworm population structure among the locations sampled. Only mitochondrial genomes indicated two different maternal lines mostly separating host strains. All phenotyped strains that were resistant to transgenic corn producing the Cry1F toxin belonged to the corn maternal line, suggesting that this strain and probably not the rice host strain is under selection pressure for resistance evolution to Cry1F. Laboratory-reared strains displayed comparable levels of genetic variability when compared to field-collected samples. 4) Key outcomes or other accomplishments realized: The genomic resources generated represenet a change in knowledge on fall armyworm population genetics and allow further exploration of gene flow and how it may impact a change in management of fall armyworm as an expanding global superpest. Information also leads to a change in knowledge by validating the use of laboratory-reared insects as genetic models and proxy to field-collected individuals. Previously available SfABCC2 gene models were fragmented and did not contain a substantial intronic region detected and identified by whole genome sequencing. This validated SfABCC2 gene model represents a change in knowledge identifying a valid reference for genotyping by targeted sequencing.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Jurat-Fuentes JL, Heckel DG, Ferr� J. Mechanisms of Resistance to Insecticidal Proteins from Bacillus thuringiensis. Annu Rev Entomol. 2021 Jan 7;66:121-140. doi: 10.1146/annurev-ento-052620-073348. PMID: 33417820.
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Gimenez S, Abdelgaffar H, Goff GL, Hilliou F, Blanco CA, H�nniger S, Bretaudeau A, Legeai F, N�gre N, Jurat-Fuentes JL, d'Alen�on E, Nam K. Adaptation by copy number variation increases insecticide resistance in the fall armyworm. Commun Biol. 2020 Nov 12;3(1):664. doi: 10.1038/s42003-020-01382-6. PMID: 33184418; PMCID: PMC7661717.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Abdelgaffar H, Perera OP, Jurat-Fuentes JL. ABC transporter mutations in Cry1F-resistant fall armyworm (Spodoptera frugiperda) do not result in altered susceptibility to selected small molecule pesticides. Pest Manag Sci. 2021 Feb;77(2):949-955. doi: 10.1002/ps.6106. Epub 2020 Oct 10. PMID: 32985759.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Invited Symposium presentation by Jurat-Fuentes, J.L., co-authors: Banerjee, R., Schlum, K., Lamour, K., Emrich, S., Placidi de Bortoli, C., 'Resistance to Bt corn in fall armyworm: Local or migratory?' Annual Meeting of the Entomological Society of America, November 2020 (virtual meeting).
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Schlum KA, Lamour K, de Bortoli CP, Banerjee R, Meagher R, Pereira E, Murua MG, Sword GA, Tessnow AE, Viteri Dillon D, Linares Ramirez AM, Akutse KS, Schmidt-Jeffris R, Huang F, Reisig D, Emrich SJ, Jurat-Fuentes JL. Whole genome comparisons reveal panmixia among fall armyworm (Spodoptera frugiperda) from diverse locations. BMC Genomics. 2021 Mar 12;22(1):179. doi: 10.1186/s12864-021-07492-7. PMID: 33711916; PMCID: PMC7953542.
- Type:
Book Chapters
Status:
Published
Year Published:
2020
Citation:
Rao, T, Jurat-Fuentes, JL. Advances in the use of entomopathogenic bacteria/microbial control agents (MCAs) as biopesticides in suppressing crop insect pests. Chapter in Biopesticides for Sustainable Agriculture, Edited by N. Birch and T. Glare, Burleigh Dodds Science Publishing, Cambridge, UK, 2019, ISBN: 978 1 78676 356 3.
|
Progress 03/15/19 to 03/14/20
Outputs
Target Audience:Target audience for the project includes scientific groups in academia, government and industry involved in control of fall armyworm with insecticidal proteins from entomopathogenic bacteria, and the development of sequencing technologies to detect and monitor the dispersal of resistance genes. Our target audience also includes farmers in the USA, Brazil, Argentina, Sub-Saharan Africa, and Southeastern Asia were the fall armyworm is a significant pest and/or has developed resistance to insecticidal proteins. A third audience group targeted by our project are students and technical personnel interested in acquiring the diverse research methods developed during completion of the project. Project progress and results have been shared with target audiences in invited symposium and contributing presentations at scientific congresses, seminars and peer-reviewed publications. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project is providing training on molecular biology methods (cloning, subcloning, PCR...), insect rearing, performance of bioassays, and insect cell culture and transformation, for a graduate (PhD) student and two postdocs. Another graduate (PhD) student if receiving training in bioinformatics and analysis of massive parallel sequencing projects. An undergraduate student participating in the project has been trained in insect husbandry, purification of insecticidal proteins and performance of bioassays. How have the results been disseminated to communities of interest?Data and results from the project have been disseminated to academic scientists and students in invited seminar presentations at university departments and symposium and contributing presnetations at national and international scientific congreses. Audiences in these venues also included scientists from private industry and government. Information has also been included in website featuring research from the group (http://juratfuenteslab.utk.edu/) What do you plan to do during the next reporting period to accomplish the goals?Objective 1- We will transform cultured cell lines with truncated SfABCC2 fragments and to test their Cry1F binding. Once the region of SfABCC2 involved in Cry1F binding is identified, we will test its receptor functionality in cytotoxicity assays. The region responsible for productive binding (i.e. leading to toxicity) will be targeted for editing using CRISPR/Cas9 to test functionality in vivo. Objective 2- The prototype bioinformatic pipeline created to automate analysis of raw targeted sequencing data will be tested using fall armyworm samples from Puerto Rico containing known Cry1F-resistance alleles as reference. Both detection of candidate resistance alleles and estimation of their frequency will be performed. Candidate Cry1F-resistance alleles in resistant S. frugiperda lines will be identified and tested for linkage. Targetd sequencing will also be adapted to amplify genes involved in resistance to Cry2Ab and Vip3Aa toxins, as they become available in the literature or are idenified in parallel projects.
Impacts
What was accomplished under these goals?
Objective 1.- Elucidate regions of ABCC2-Cry toxin interaction that are critical to insect susceptibility (2018-2020) 30% completed. Progress: Expression vectors for ABCC2 truncated proteins created and purified. Cell transformation using lipofection was not succesful and electroporation is being optimized. Succesful editing of SfABCC2 gene using CRISPR/Cas9 to introduce a mutation resulting in a truncated SfABCC2 protein and resistance to Cry1F. Used edited line to demonstrate that mutations in SfABCC2 do not affect susceptibility to synthetic pesticides. Objective 2.- Develop a highly sensitive, high throughput, multiplexed targeted sequencing method for screening of resistance to Cry toxins and use it to screen fall armyworm collected in the USA, Brazil, Colombia, and African countries (2018-2022). Progress: Sequenced >93% of the full lentgh SfABCC2 gene using targeted sequencing in fall armyworm samples from USA, Kenya, Argentina and Brazil. Developed protoype pipeline to automate targeted sequencing data analysis. Develop reference SfABCC2 gene sequence to use in automated pipeline to detect candidate resistance alleles. The whole genome of S. frugiperda has been sequenced for 55 individuals from the USA, Brazil, Argentina, Puerto Rico and Kenya. Results from bioinformatic whole genome comparisons identify no apparent population structure for all the samples, separation of corn-straina nd rice-strain samples, similar diversity between laboratory-reared and field fall armyworm, and the existence of two main maternal S. frugiperda lineages. Mutations in the SfABCC2 gene that appear linekd to resistance to Cry1F detected in samples from Brazil and Argentina. Identification of Cry1F-resistance alelle in a population from Florida, which entails a gene fusion event in the SfABCC2 locus.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Gomis-Cebolla, J., Ferreira Dos Santos, R., Wang, Y., Caballero, J., Caballero, P., He, K., Jurat-Fuentes, J. L., and J. Ferr� (2020) "Domain shuffling between Vip3Aa and Vip3Ca: Chimera stability and insecticidal activity against European, American, African, and Asian Pests" Toxins 12(2): E99.
- Type:
Other
Status:
Published
Year Published:
2019
Citation:
Invited seminar: "Mechanisms and dispersal of resistance to Bt corn in the fall armyworm", presented by Juan Luis Jurat-Fuentes at the Department of Entomology, Texas A&M University, College Station (TX), March 2019.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Invited presentation by Jurat-Fuentes, J. L. 'Cry toxins for ecofriendly pest control: Mechanisms of action and field-evolved resistance', Genomics and Systems Biology IX, Abu Dhabi (UAE), February 2019.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Contributing presentation by Jurat-Fuentes, J. L., co-authors: Placidi de Bortoli, C., Banerjee, R., Abdelgaffar, H., Perera, O. P., Assirati, G. J. 'Mechanisms, frequency and biological implications of resistance to transgenic corn in Spodoptera frugiperda', Eighth International Symposium on Molecular Insect Science, Barcelona (Spain), July 2019.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Contributing presentation by Jurat-Fuentes, J. L. 'Mechanisms, frequency and dispersal of resistance to transgenic corn in fall armyworm (Spodoptera frugiperda)', Meeting of the International Working Group on Ostrinia and Other Pests of Corn, Engelberg (Switzerland), October 2019.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Invited symposium presentation by Rao, T., co-authors: Huff, M., Placidi de Bortoli, C., Lamour, K., Staton, M., Jurat-Fuentes, J. L. 'Targeted next generation sequencing to monitor for resistance to Bt corn in Spodoptera frugiperda' Annual Meeting of the Entomological Society of America, St. Louis (MO), November 2019.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Contributing presentation by Placidi de Bortoli, C., co-authors: Banerjee, R., Huang, F., Hasler, J., Reay-Jones, F., Meagher, R., Stewart, S., Viteri, D., Ni, X., Linarez, A., Buntin, D., Narva, K., Jurat-Fuentes, J. L. 'Identification and frequency of an allele linked to resistance against Cry1Fa corn in Spodoptera frugiperda from Florida', Annual Meeting of the Entomological Society of America, St. Louis (MO), November 2019.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Contributing presentation by Ferreira dos Santos, R., co-authors: Assirati, G. J., Placidi de Bortoli, C., Hietala, L., Jurat-Fuentes, J. L. ' Effects of Bt toxins on flight activity of Spodoptera frugiperda (Lepidoptera: Noctuidae)', Annual Meeting of the Entomological Society of America, St. Louis (MO), November 2019.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2020
Citation:
Abdelgaffar, H., Perera, O.P., and Jurat-Fuentes, J.L. "ABC transporter mutations in Cry1F-resistant fall armyworm (Spodoptera frugiperda) do not result in altered susceptibility to synthetic pesticides" Pest Management Science.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Plenary symposium presentation by Jurat-Fuentes, J. L. 'Mechanisms of practical resistance to commercially relevant entomopathogenic bacteria', Annual Meeting of the Society for Invertebrate Pathology, Valencia (Spain), August 2019.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Contributing presentation by Jurat-Fuentes, J. L., co-authors: Placidi de Bortoli, C., Banerjee, R., Meagher, R., Abdelgaffar, H., Yang, F., Kerns, D., Huang, F., Akutse, K., Rao, T. 'Mechanisms and spread of resistance to transgenic corn in fall armyworm (Spodoptera frugiperda)', Annual Meeting of the Society for Invertebrate Pathology, Valencia (Spain), August 2019.
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Progress 03/15/18 to 03/14/19
Outputs
Target Audience:The project aims at identifying functional receptor sites and developing technology for detection of resistance alleles to insecticidal proteins against the fall armyworm (Spodoptera frugiperda). Consequently, our target audience includes scientific groups in academia, government and industry involved in control of fall armyworm with insecticidal proteins from entomopathogenic bacteria, and the development of sequencing technologies to detect and monitor the dispersal of resistance genes. Our target audience also includes farmers in the USA, Brazil, Argentina and Sub-Saharan Africa, were the fall armyworm is a significant pest and/or has developed resistance to insecticidal proteins. A third audience group targeted by our project are students and technical personnel interested in acquiring the diverse research methods developed during completion of the project. Since project progress has not resulted in significant results amenable to data presentations, outreach efforts during this reporting period have been limited to presentations for diverse target audiences on the rationale for the project. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?During the current reporting period, the PI (Jurat-Fuentes) has received practical training on genetic insect transformation methods through a workshop at the Insect Transformation Facility at the University of Maryland. The project is providing training on molecular biology methods (cloning, subcloning, PCR...), insect rearing, performance of bioassays, and insect cell culture and transformation, for a graduate (PhD) student. In addition, 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?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?Objective 1- We plan to express the cloned truncated SfABCC2 fragment in insect cell cultures to test their Cry1F binding properties. Fragments for which Cry1F toxin binding is confirmed will be tested for receptor functionality in toxicity assays. The region involved in binding will be targeted for editing using CRISPR/Cas9 to test functionality in vivo. Objective 2- Primers for TILING PCR will be optimized to increase coverage of the SfABCC2 gene. Genetic markers will be selected from the S. frugiperda genomes sequenced to date and used to perform population genetic studies. A bioinformatics pipeline to identify indels and mutations predicted to result in truncated SfABCC2 proteins from multiplexed NGS data will be designed and tested.
Impacts
What was accomplished under these goals?
Objective 1- Elucidate regions of ABCC2-Cry toxin interaction that are critical to insect susceptibility (2018-2020) 25% completed. Progress: We have designed primers and cloned truncated fragments of the ABCC2 protein from fall armyworm (SfABCC2) into vectors for their expression in insect cell cultures. In preparation for editing the SfABCC2 gene using the CRISPR/Cas9 system, we have sequenced the genome of the S. frugiperda strain to be used for editing, to identify Protospacer Adjacent Motifs (PAMs) to target in designing guide RNAs. Pesonnel assigned to the project attended Objective 2- Develop a highly sensitive, high throughput, multiplexed targeted sequencing method for screening of resistance to Cry toxins and use it to screenfall armyworm collected in the USA, Brazil, Colombia, and African countries (2018-2022). Progress: Overlapping primers were designed to amplify the full length SfABCC2 gene and used to amplify >93% of the gene using TILING PCR. The genome of S. frugiperda from diverse locations has been sequenced to determine genetic variability between populations of this insect and polymorphisms in the SfABCC2 gene.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Dominant point mutation in a tetraspanin gene associated with field-evolved resistance of cotton bollworm to transgenic Bt cotton.
Jin L, Wang J, Guan F, Zhang J, Yu S, Liu S, Xue Y, Li L, Wu S, Wang X, Yang Y, Abdelgaffar H, Jurat-Fuentes JL, Tabashnik BE, Wu Y
Proc Natl Acad Sci U S A. 2018 Nov 13;115(46):11760-11765. doi: 10.1073/pnas.1812138115. Epub 2018 Oct 31.
PMID: 30381456
- Type:
Journal Articles
Status:
Under Review
Year Published:
2019
Citation:
Mechanisms for high levels of resistance to commercially-relevant entomopathogenic bacteria
Placidi de Bortoli C, Jurat-Fuentes JL
Current Opin Insect Sci
- Type:
Journal Articles
Status:
Submitted
Year Published:
2019
Citation:
Identification of a native Bacillus thuringiensis strain from Sri Lanka active against Dipel-resistant Plutella xylostella
Baragamaarachchi R, Samarasekera JKR, Weerasenab OVSJ, Lamour K, Jurat-Fuentes JL
Biological Control
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