Source: TEXAS A&M UNIVERSITY submitted to NRP
MODIFYING TERPENE BIOSYNTHESIS IN COTTON TO ENHANCE INSECT RESISTANCE USING A TRANSGENE-FREE CRISPR/CAS9 APPROACH
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1028362
Grant No.
2022-67013-36898
Cumulative Award Amt.
$294,000.00
Proposal No.
2021-11396
Multistate No.
(N/A)
Project Start Date
Mar 15, 2022
Project End Date
Mar 14, 2026
Grant Year
2022
Program Code
[A1811]- AFRI Commodity Board Co-funding Topics
Recipient Organization
TEXAS A&M UNIVERSITY
750 AGRONOMY RD STE 2701
COLLEGE STATION,TX 77843-0001
Performing Department
Entomology
Non Technical Summary
Insect pests pose a major threat to United States agriculture and many pest management tools, such as pesticides are losing their effectiveness as pests become resistant. This contributes to lost agricultural productivity, financial burdens for growers and consumers, and environmental burdens through increased pesticide inputs. A major goal of this project is to develop new targeted agricultural pest management technologies that reduce crop losses and production costs, while improving environmental safety.We will use cutting-edge genome-editing technology to create cotton plants with modified production of ecologically important defense compounds called terpenoids. Terpenoids play important roles in plant defense against insects and pathogens and by altering plant production of these compounds, we expect to boost plant resistance to key insect pests. We will characterize the defense chemistry of genome-edited cotton plants and evaluate their resistance against a suite of key pest species, including cotton aphids and bollworm caterpillars. The findings from this research will increase our knowledge about terpenoid biosynthesis in cotton plants and the role of terpenoid compounds in cotton resistance against insect pests, which will advance the fields of biochemistry, chemical ecology, and plant biology. This research will also contribute to improving sustainable and effective pest management strategies by identifying new technologies that can reduce pesticide inputs and improve productivity.
Animal Health Component
10%
Research Effort Categories
Basic
50%
Applied
10%
Developmental
40%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2011710108050%
2011710113050%
Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1710 - Upland cotton;

Field Of Science
1130 - Entomology and acarology; 1080 - Genetics;
Goals / Objectives
The major goal of our project is to enhance plant resistance to insect pests using a targeted gene-editing approach to modify ecologically important plant metabolites. For this project co-funded by The Cotton Board, we will work with the economically important crop, upland cotton (Gossypium hirsutum), and a selection of its key insect pests as a model system, using a transgene-free genome-editing approach to modify cotton terpene biosynthesis. We will achieve our goal by pursuing three specific objectives: 1. Use CRISPR/Cas9 genome editing and carbon nanotube delivery technologies to rapidly develop cotton plants with a stably modified terpene synthesis pathway. 2. Quantify the phenotypic effects of terpene synthase modification on plant terpenoid production, growth, development, and whole-plant gene-expression patterns. 3. Assess the effects of terpene pathway modification on plant resistance to key insect pests. We expect that removing select monoterpenes will shift overall terpene biosynthesis, increasing plant resistance against insects without negative consequences for plant primary metabolism or recruiting natural enemies. This new technology will contribute to improved cotton insect pest management that reduces pesticide use, preserves current technologies, and maintains yields.
Project Methods
Objective 1. Use CRISPR/Cas9 genome editing and carbon nanotube delivery technologies to rapidly develop cotton plants with a stably modified terpene synthesis pathway. We will electrostatically attach plasmid DNA (pDNA) expressing CRISPR/Cas9 and their corresponding guide RNAs (gRNAs) to single-walled carbon nanotubes (SWCNTs) to create in planta targeted knockouts in cotton plants. We will develop guide RNAs (gRNAs) by sequencing the monoterpene synthase gene GhTPS3 and phytoene desaturase (GhPDS) as a control gene from the cotton cultivar TAMCOT73. CRISPR/Cas9 cassettes will be designed using gRNAs for the target loci and will be assembled into vectors. In vivo validation of the editing efficiency of gRNAs will be performed using isolated cotton protoplasts. We will test for differentiate gene copies and amplified fragments of edited and non-edited protoplasts will undergo Amplicon Next Generation Sequencing (MiSeq platform). In planta transformation approaches will be tested using fluorescent reporter genes (GFP and YFP) to determine efficiency levels of delivery of pDNA-SWCNT complexes in leaves. The optimized method will be applied to excised shoot apical meristem (SAM) tissue, followed by fluorescent imaging and GUS expression will be determined (Jefferson et al. 1987). Next, whole seeds will be imbibed directly in a pDNA-SWCNT solution and tested for transient expression of the fluorescent reporter gene to see if the carbon nanotubes can enter the seed during germination. We will prepare pDNA-SWCNTs with a plasmid expressing Cas9 and gRNAs targeting GhTPS3 and GhPDS; to determine if gene editing can transiently occur in germline cells and be passed on to the growing seedling as a stably inherited mutation. As the GhPDS knock-out shows a visible phenotype, large batches of samples can be treated with the plasmid pDNA-SWCNTs and grown into seedlings to test for successful gene editing. Plants with potential knock-out of GhTPS3 will be screened to identify candidates for further advancement. Selfing for two generations will be performed to generate homozygous lines and subsequent seed. The T1 plants will be characterized to select for homozygous mutations at the target loci. Objective 2: Quantify the phenotypic effects of terpene synthase modification on 1) plant terpene profiles, 2) growth and development, and 3) whole-plant gene-expression patterns. 1) To determine whether silencing GhTPS3 alters G. hirsutum terpenoid production, terpene compounds from different tissues and developmental stages of genome-edited and control plants will be collected and analyzed using 2 complementary methods: 1) dynamic headspace sampling to characterize the volatile compounds emitted (constitutive and herbivore-induced) from intact plants, which are important cues for host location or repelling insect herbivores and attracting natural enemies (Helms et al., 2014), and 2) microwave extraction to analyze terpenoids present in different tissues of cotton plants (Yip et al., 2019). Headspace collections will be conducted at 2 weeks (seedlings) and 8 weeks (early maturity) post germination. Intact leaves and stems of plants will be enclosed in individual glass chambers. Filtered air will be forced into the chambers and air will be pulled from chambers through a volatile filter trap using vacuum. One group of plants of each edited and wildtype genotype will be infested with herbivores. In separate experiments, plants will be infested with either 2 second instar Helicoverpa zea, 30 adult Aphis gossypii, or 45 adult Lygus hesperus or will remain undamaged as controls. For microwave extraction, tissues from 2-week and 8-week-old plants (e.g., leaves, squares, and roots) will be analyzed. Samples will be collected from each tissue type and solvent extracted following previously described methods (Gomez et al., 2001). For both methods, terpenoid compounds will be analyzed (quantified and identified) using a gas chromatograph-mass spectrometer (Helms et al., 2014, 2019).2) To evaluate plant growth, development, and potential agronomic traits, we will quantify and compare the growth of genome-edited and wildtype control plants across development. We will measure germination success (%), plant height, the number of nodes, the first fruiting node (FFN), seed numbers, and fiber quantity/quality.3) To determine whether silencing GhTPS3 in G. hirsutum alters whole plant gene expression patterns, we will perform a RNA-Seq experiment comparing the transcriptomes of genome-edited and control plants at two developmental stages. Leaves from 2-week-old seedlings and 8-week-old early reproductive maturity plants of Wildtype (control) and GhTPS3-silenced plants will be used. RNA extraction from plant tissues will use a modified TRIzol protocol (Invitrogen). Samples will be and NextGen Illumina-based RNA-Seq data generation, de novo transcriptome backbone assembly, read mapping, data acquisition and statistical data analysis to identify differentially expressed candidate genes will be performed. RT-qPCR will be used for validation (Artico et al., 2010).Objective 3: Assess the effects of terpene pathway modification on plant resistance to three key insect pests. 1) To measure pest performance, we will conduct no-choice feeding experiments on genome-edited and control plants, infesting individual plants with either 10 adult aphids, 5 lygus nymphs, or 10 bollworm neonates and quantifying insect numbers, mass, and plant damage. 2) To measure insect preference, we will conduct 2-choice assays with pairs of edited and control plants, releasing insects in the cage and determining where they settle or lay eggs.The overall project success will be evaluated on 1) The generation of cotton plants with stably modified terpene biosynthesis (sequencing and phenotyping experiments). 2) The generation of new knowledge about terpene biosynthesis in cotton and the role of terpenes in cotton resistance against insect pests. This will be evidenced through phenotyping and resistance assays and ultimately through scientific publications, citations, and presentations. 3) Creating an impact on insect pest management in cotton as measured by results from resistance assays and ultimately through adoption of our technologies by the cotton industry.

Progress 03/15/24 to 03/14/25

Outputs
Target Audience:The target audience for this project includes scientists, farmers, industry, students, and members of the broader public. Our efforts to reach these groups during the 2024 reporting period included the following: We communicated our results on genetic modification of cotton terpene production with members of the scientific community and industry groups through presentations at the 2024 Entomological Society of America Annual Meeting and 2025 Cotton Beltwide Annual Meeting. PI Sword also presented project results as part of invited departmental seminars at the University of Arizona, North Carolina State University and Texas A&M University. These activities allowed us to receive valuable feedback on our project. The PI and Co-PIs engaged in classroom teaching (an undergraduate insect ecology course and a field crop entomology course and a graduate course on genome editing in crop plants), where this research was discussed. The PI and Co-PIs also mentored graduate and undergraduate research students, and a postdoctoral researcher. Changes/Problems:The original timeline for the development and subsequent use of transformed CRISPR-edited knockout plants was delayed due to a combination of unforeseen personnel changes and technical challenges. These issues have been resolved and work is now in progress. What opportunities for training and professional development has the project provided?PI Helms and Co-PI Sword mentored two Ph.D. students, a postdoc, and an undergraduate student conducting research for and related to this project. Co-PI Thomson is advising a postdoctoral research associate. Students are gaining technical training in designing and conducting experiments and analyzing and interpreting data. They are also gaining professional development opportunities through participation in seminars and workshops in teaching/mentoring and statistical methods. How have the results been disseminated to communities of interest?We communicated preliminary results with members of the scientific community and industry groups through presentations at the Cotton Beltwide Annual Meeting and Entomological Society of America Meeting and university departmental seminars. We also shared our findings with Cotton Incorporated. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, we will continue our efforts to establish stably transformed plants with silenced TPS3. We will continue conducting phenotyping experiments, including growing plants to maturity for plant mapping, seed production, and lint quality analyses. We will also continue with the remaining insect resistance bioassays.

Impacts
What was accomplished under these goals? Impact The major goal of our project is to enhance cotton plant resistance to insect pests using a targeted gene-editing approach to modify ecologically important plant metabolites. This research will contribute to more sustainable pest management by creating new lines of cotton plants with altered defense metabolites that enhance their resistance to insect pests. Another long-term goal is making these lines available to farmers. This research will also increase our fundamental knowledge about terpene biosynthesis in cotton and the role of terpenes in cotton resistance against insect pests. Objective 1. Use CRISPR/Cas9 genome editing and carbon nanotube delivery technologies to rapidly develop cotton plants with a stably modified terpene synthesis pathway.: In 2024, our collaborators at the University of North Texas were able to generate plants using Agrobacterium-mediated plant transformations. We are now awaiting the genotyping results to generate lines of stably transformed cotton plants with knocked-out TPS3. As an alternative approach, we developed a virus-induced gene silencing (VIGS) method to create cotton plants with transiently silenced genes to use for plant phenotyping and insect bioassays while we wait for the genome-edited plants to be available for experiments. We developed a seed-soak method for VIGS delivery to cotton seeds and optimized several parameters (e.g., concentration, temperature, duration) to achieve efficient and reliable knockdown of TPS3 gene expression. Objective 2: Quantify the phenotypic effects of terpene synthase modification on 1) plant terpene profiles, 2) growth and development, and 3) whole-plant gene-expression patterns.: In 2024, we continued phenotyping our VIGS-generated TPS3-deficient plants. We quantified expression levels of our target and control genes using qPCR and found that TPS3 expression is significantly reduced in treatment plants compared to controls. We quantified terpene levels in cotton leaves and found significantly lower quantities of the monoterpenes, a-pinene and b-pinene, which are synthesized by TPS3. We also worked to characterize the volatile emissions from VIGS TPS3-silenced plants to determine how silencing this gene affects the emission of plant volatiles. Our findings indicate that VIGS TPS3-silenced plants also have altered volatile emissions, including for the target monoterpenes, a-pinene and b-pinene. We assessed plant growth and development parameters and found that the TPS3 VIGS treatment did not have negative effects on plant growth and development as we noted similar growth patterns and development times across our treatment and negative control plants. However, our CLA1 visual control plants were notably stunted in their growth and development, which was expected due to a lack of photosynthetic ability. Objective 3: Assess the effects of terpene pathway modification on plant resistance to three key insect pests.: In 2024, we conducted insect resistance bioassays, focusing on how VIGS silencing of TPS3 affects insect preference and performance. We found that fourth-instar Helicovera zea larvae had lower relative growth rates when feeding on VIGS TPS3-silenced plants compared to control plants and H. zea neonates consumed less leaf tissue on TPS3-silenced plants compared to control plants. However, in a two-choice feeding experiment, H. zea larvae did not show a clear preference for either treatment or control plants. These findings indicate that silencing the TPS3 gene increased cotton resistance to H. zea by decreasing larval performance. In contrast, we found that Aphis gossypii aphids prefer control plants over TPS3-silenced plants, indicating that silencing the TPS3 gene increases plant resistance by reducing aphid attraction and/or host finding ability.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Clark, M.C., Behmer, S.T., and Sword, G.A. Modifying phytosterol profiles in upland cotton reduces the performance of cotton aphids. Entomological Society of America Annual Meeting. November 2024. Phoenix, AZ
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Sword, G.A. From Locusts to Cotton and Back on the Shoulders of Giants. University of Arizona, Department of Entomology Seminar, October 2024, Tucson, AZ
  • Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: Sword, G.A. From Cotton to Locusts and Back: Tales of Edited Genomes and Invasive Swarms. Texas A&M Department of Entomology Seminar. January 2025, College Station, TX
  • Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: Sword, G.A. From Locusts to Cotton and Back: Tales of Edited Genomes and Invasive Swarms. Colorado State University, Department of Agricultural Biology seminar, February 2025, Ft. Collins, CO
  • Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: Sword, G.A. From Locusts to Cotton and Back: Tales of Edited Genomes and Invasive Swarms. North Carolina State University, Department of Agricultural Biology seminar, February 2025, Raleigh, NC
  • Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: Clark, M.C., Rathore, K., Helms, A.M., Kurtz, R., Zhu-Salzman, K., Behmer, S.T., Sword, G.A. Exploiting a Metabolic Constraint in Herbivorous Pests May Provide a Novel Pest Management Strategy in Cotton. Beltwide Cotton Conference, Cotton Insect Research and Control conference. January 2025. New Orleans, LA


Progress 03/15/23 to 03/14/24

Outputs
Target Audience:The target audience for this project includes scientists, farmers, industry, students, and members of the broader public. Our efforts to reach these groups during this reporting period included the following: We communicated our results on genetic modification of cotton terpene production with members of the scientific community and industry groups through presentations at the 2024 Cotton Beltwide Annual Meeting and 2023 Entomological Society of America Annual Meeting. This allowed us to receive valuable feedback on our project. The PI and Co-PIs engaged in classroom teaching (an undergraduate insect ecology course and a field crop entomology course and a graduate course on genome editing in crop plants), where this research was discussed. The PI and Co-PIs also mentored graduate and undergraduate research students, including students from underrepresented groups in STEM, and a postdoctoral researcher. Changes/Problems:One of the significant challenges has been variability in the efficiency of VIGS across different batches of plants. This variability can lead to uneven gene silencing. We have taken steps to address this issue by optimizing several aspects of our delivery protocol and growing methods. The original timeline for the development and subsequent use of transformed CRISPR-edited knockout plants was delayed due to a combination of unforeseen personnel changes and technical challenges. These issues have been resolved and work is now in progress. What opportunities for training and professional development has the project provided?PI Helms and Co-PI Sword are mentoring two Ph.D. students and two undergraduate students conducting research for and related to this project. Co-PI Thomson is advising a postdoctoral research associate. Students are gaining technical training in designing and conducting experiments and analyzing and interpreting data. They are also gaining professional development opportunities through participation in seminars and workshops in teaching/mentoring and statistical methods. How have the results been disseminated to communities of interest?We communicated preliminary results with members of the scientific community and industry groups through presentations at the Cotton Beltwide Annual Meeting and Entomological Society of America Meeting. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, we will continue optimizing the VIGS protocols to maximize the silencing efficiency and duration. We will then use these plants for additional phenotyping experiments (as outlined in objective 2) and in bioassays with insects (as outlined in objective 3). This will provide an initial investigation into how modifying plant terpene profiles affects plant resistance against insect pests. We hope to receive agrobacterium transformed plants in late 2024 and will conduct phenotyping experiments (objective 2) for these plants, to be followed by insect assays (objective 3) with the next generation of plants.

Impacts
What was accomplished under these goals? Impact The major goal of our project is to enhance plant resistance to insect pests using a targeted gene-editing approach to modify ecologically important plant metabolites. During the second year of this project, we mentored key personnel and conducted experiments for the proposed objectives. This research will contribute to more sustainable pest management by creating new lines of cotton plants with altered defensive metabolites and making lines with enhanced pest resistance available to farmers for their use. This research will also increase our fundamental knowledge about terpene biosynthesis in cotton and the role of terpenes in cotton resistance against insect pests. Objective 1. Use CRISPR/Cas9 genome editing and carbon nanotube delivery technologies to rapidly develop cotton plants with a stably modified terpene synthesis pathway. In the previous reporting period (2022), we made several attempts at in planta transformation using carbon nanotube delivery for plant infiltrations. Unfortunately, we were unable to achieve stably transformed plants using this approach. In 2023, we proceeded with our proposed contingency plan, to work with University of North Texas on Agrobacterium-mediated plant transformations. This work is ongoing with transformation constructs prepared and delivered to UNT, with the initial the attempts to obtain transformed plant material underway. We also developed a virus-induced gene silencing (VIGS) approach to create cotton plants with transiently silenced genes to use for plant phenotyping and insect bioassays while we wait for the genome-edited plants to be available for experiments. We developed a seed-soak method for VIGS delivery to cotton seeds. This included optimization of several parameters (e.g., concentration, temperature, duration) to achieve efficient and reliable knockdown of TPS3 gene expression. Objective 2: Quantify the phenotypic effects of terpene synthase modification on 1) plant terpene profiles, 2) growth and development, and 3) whole-plant gene-expression patterns. In the 2023 reporting period, we tested our phenotyping approaches using the VIGS plants. This included quantifying expression levels of our target and control genes using qPCR, quantifying terpene levels, and assessing plant growth and development parameters. We have successfully shown knockdown of TPS3 gene expression using our VIGS approach in qPCR experiments following cotyledon leaf infiltrations and are working to measure TPS3 expression levels in plants treated with the VIGS seed-soak method. We also found significant changes in the terpene profiles of our VIGS TPS3 plants. The silenced plants produced significantly lower quantities of the monoterpenes, a-pinene and b-pinene, which are synthesized by TPS3. However, the abundances of the other two terpenes produced by TPS3, b-phellandrene, and g-terpinene, were below our instrument's detection limit and could not be quantified. Notably, we also saw significant increases in the production of several sesquiterpenes in our TPS3-silenced plants. These included b-caryophyllene, a-bergamotene, a-caryophyllene, g-bisabolene, and b-bisabolol. Together, these findings support our hypotheses that silencing TPS3 will reduce key monoterpenes and result in an overall change in the plants' terpenoid profiles. We are now working to characterize the volatile emissions from TPS3-silenced plants to determine how silencing this gene affects the emission of plant volatiles. Our findings also indicate that the TPS3 VIGS treatment did not have any negative effects on plant growth and development as we noted similar growth patterns and development times across our treatment and negative control plants. However, our CLA1 visual control plants were notably stunted in their growth and development, which was expected due to a lack of photosynthetic ability. Objective 3: Assess the effects of terpene pathway modification on plant resistance to three key insect pests. Nothing to report.

Publications

  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Clark, M.C., Behmer, S.T., Zhu-Salzman, K. and Sword, G.A. Modifying phytosterol production in upland cotton reduces the performance of insect herbivores. Entomological Society of America Annual Meeting. November 2023. *1st Place in the 10 minute student paper competition, Graduate PBT: Biocontrol and IPM section)
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Sword, G.A. Cotton genome editing for improved insect pest management. ESA Workshop: Regulatory Considerations in Developing Biotech Insect Protection Products, Entomological Society of America Annual meeting. November 2023.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Sword, G.A., Thomson, M., Clark, M.C. & Allen, H. & Helms, A. Modifying volatile emissions from cotton to enhance insect resistance using a transgene-free CRISPR/Cas9 approach. USDA-NIFA Commodity Board Co-Funding Program Project Directors meeting, August 2023.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Allen, H., Sword, G.A. & Helms, A. Evolving Pest Management in Cotton Production through Targeted Terpene Synthesis Modulation via VIGS and CRISPR/Cas9 Technologies. Beltwide Cotton Conference, Cotton Insect Research and Control conference. January 2024.


Progress 03/15/22 to 03/14/23

Outputs
Target Audience:The target audience for this project includes scientists, farmers, industry, students, and members of the broader public. Our efforts to reach these groups during the previous reporting period included the following: We communicated preliminary results and related research on cotton terpene emissions with members of the scientific community and industry groups through presentations at the Cotton Beltwide Annual Meeting. This allowed us to receive valuable feedback on our project. The PI and Co-PIs engaged in classroom teaching (an undergraduate insect ecology course and a field crop entomology course and a graduate course on genome editing in crop plants), where this research was discussed. The PI and Co-PIs also mentored graduate and undergraduate research students, including students from underrepresented groups in STEM. PI Helms shared information about this project through an extension radio program on public radio called "Garden Success". This research was also featured in a press release article produced by Texas A&M AgriLife that was widely distributed online. Changes/Problems:During this reporting period (2022), we made several attempts at in planta transformation using carbon nanotube delivery for plant infiltrations. Unfortunately, we were unable to achieve stably transformed plants using this approach. Following our proposed contingency plan, we initiated a collaboration with University of North Texas to begin Agrobacterium-mediated plant transformations, which are now ongoing. We also developed a virus-induced gene silencing (VIGS) approach to create cotton plants with transiently silenced genes to use for insect bioassays while we wait for the genome-edited plants to be available for experiments. We tested multiple VIGS infiltration methods, including direct infiltrations through cotyledon wounding, and a seed-soaking method and are moving forward with these plants until our stably transformed plants are ready. What opportunities for training and professional development has the project provided?PI Helms and Co-PI Sword are mentoring two Ph.D. students and two undergraduate students conducting research for and related to this project. Co-PI Thomson is advising a postdoctoral research associate. A new Ph.D. student was recruited to work on this project starting in fall 2022. Students are gaining technical training in designing and conducting experiments and analyzing and interpreting data. They are also gaining professional development opportunities through participation in seminars and workshops in teaching/mentoring and statistical methods. How have the results been disseminated to communities of interest?We communicated preliminary results and related research on cotton terpene emissions with members of the scientific community and industry groups through presentations at the Cotton Beltwide Annual Meeting and local Texas A&M research symposia. PI Helms shared information about this project through an extension radio program on public radio called "Garden Success". This research was also featured in a press release article produced by Texas A&M AgriLife that was widely distributed online. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, we will continue optimizing the VIGS protocols to maximize the silencing efficiency and duration. We will then use these plants in bioassays with insects (as outlined in objective 3) as an initial investigation into how modifying plant terpene profiles affects plant resistance against insect pests. We also expect to receive our first batch of agrobacterium transformed plants in late 2023 and will conduct phenotyping experiments (as outlined in objective 2) for these plants, to be followed by insect assays with the next generation of plants.

Impacts
What was accomplished under these goals? Impact The major goal of our project is to enhance plant resistance to insect pests using a targeted gene-editing approach to modify ecologically important plant metabolites. During the first year of this project, we recruited and trained key personnel and began conducting experiments for the proposed objectives. This research will contribute to more sustainable pest management by creating new lines of cotton plants with altered defensive metabolites and making lines with enhanced pest resistance available to farmers for their use. This research will also increase our fundamental knowledge about terpene biosynthesis in cotton and the role of terpenes in cotton resistance against insect pests. Objective 1. Use CRISPR/Cas9 genome editing and carbon nanotube delivery technologies to rapidly develop cotton plants with a stably modified terpene synthesis pathway. During this reporting period (2022), we made several attempts at in planta transformation using carbon nanotube delivery for plant infiltrations. Unfortunately, we were unable to achieve stably transformed plants using this approach. Following our proposed contingency plan, we initiated a collaboration with University of North Texas to begin Agrobacterium-mediated plant transformations. We also developed a virus-induced gene silencing (VIGS) approach to create cotton plants with transiently silenced genes to use for insect bioassays while we wait for the genome-edited plants to be available for experiments. We tested multiple VIGS infiltration methods, including direct infiltrations through cotyledon wounding, and a seed-soaking method. Objective 2: Quantify the phenotypic effects of terpene synthase modification on 1) plant terpene profiles, 2) growth and development, and 3) whole-plant gene-expression patterns. In the current reporting period, we tested our phenotyping approaches using the VIGS plants. This included quantifying expression levels of our target and control genes using qPCR, quantifying terpene levels, and assessing plant growth and development parameters. We have successfully shown knockdown of TPS3 gene expression using our VIGS approach in qPCR experiments following cotyledon leaf infiltrations. Objective 3: Assess the effects of terpene pathway modification on plant resistance to three key insect pests. Nothing to report

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Clark, Mason, Spencer T. Behmer, Anjel Marie Helms, Michael J. Thomson, Keyan Zhu Salzman, Ryan Kurtz, Kater Hake, Kent Chapman, and Gregory A. Sword. Gene Editing Approaches for Cotton Pest Management. Beltwide Cotton Conference. January 2022.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Alwedyan, Malek, Anjel Marie Helms, and Michael J. Brewer. Cotton Volatiles and Injury Response Stimulated by Feeding of Sucking Bugs. Beltwide Cotton Conference. January 2022.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Clark, M. "EcoVIGS: Virus-induced gene silencing (VIGS) as a promising tool for investigating cotton-herbivore interactions." Texas A&M University Ecology and Evolutionary Biology Program Seminar, College Station, TX. Oct. 31, 2022.