Recipient Organization
University of Utah
201 S President Circle RM 408
Salt Lake City,UT 84112-9023
Performing Department
(N/A)
Non Technical Summary
By consuming crop plants, herbivores (which are animals that feed on plant tissues) cause major losses in crop yields, which in turn negatively impacts farmers and human welfare more generally. Herbivores are diverse and include insects like caterpillars and aphids that damage a range of dicot crop plants (like soybean and tomato) as well as cereal crop plants (like maize, wheat, barley, and sorghum). While many insect herbivores have been extensively studied, less is known about spider mite pests of crops plants. Spider mites are only distantly related to insects and are pests of cereal crops including maize and sorghum, especially under conditions of high heat, aridity, and drought. In fact, during such conditions, the generalist two-spotted spider mite Tetranychus urticae (which feeds on diverse dicot and cereal crop plants) and the grass specialist Banks grass mite Oligonychus pratensis (which feeds mainly on cereals such as maize, sorghum and wheat) can become significant pests of cereal crops in many agricultural regions. This project will investigate the genomic, genetic, and molecular bases of both mite species' ability to interact with and damage maize, sorghum, and barley. While both the two-spotted spider mite and the Banks grass mite are morphologically similar and feed on plants in a similar way, because they differ in the range of host plants they feed upon, they may therefore interact differently with the cereal species on which they are crop pests. Hence, the project will assess similarities and differences in the two mite species' molecular responses to cereal hosts and test the roles of specific mite genes in mediating the herbivore-plant interactions. To accomplish the project objectives the genome of the Banks grass mite will be sequenced and compared to that of the already existing genome of the two-spotted spider mite, and the transcriptional responses (which genes are turned on or off) of both mite species to multiple cereal plant hosts (maize, sorghum, and barley) will also be assessed. The comparative genomic and gene expression studies will identify shared genes in both species that may be important for mediating their ability to feed on cereal hosts, as well as genes in each species that may have more species-specific roles. Further, specific genes identified as candidates for allowing spider mites to colonize, feed upon, and damage cereal crops will be functionally tested using CRISPR-Cas9 based gene editing techniques that have recently been developed for spider mites. The knowledge and genetic and genomic resources to be generated by the project will inform genome-enabled approaches to control these two destructive pests of cereal crops for which economically important outbreaks typically occur during conditions of drought when plants are already experiencing a major stress that reduces crop yields. Additionally, educational materials to be developed and released via the Learn.Genetics web resource will inform a diverse audience including K-12 and undergraduate students and educators, and the public, on the importance of heat and drought stress on crops in the context of herbivores and herbivore-induced impacts on crop yields. Finally, undergraduate and graduate students will be trained in molecular, genetic, genomic, and bioinformatic methods of importance for developing modern and sustainable agricultural practices.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
Among arthropod pests of cereals in the grass family (Poaceae), little is known about the commonalities and differences in how generalist and specialist spider mites persist on and damage cereal hosts. Therefore, we will identify the underpinnings of cereal-host-plant use by the extreme generalist two-spotted spider mite Tetranychus urticae (feeds on many plant species in many plant families) and the grass specialist Banks grass mite Oligonychus pratensis (feeds on plants mainly within the grass family). Elucidating genomic, intra-specific genetic, and transcriptomic features of T. urticae and O. pratensis that are in sister genera, and that cause essentially identical patterns of damage to grass leaf cells, will allow factors associated with long-term evolution of host plant ranges to be disentangled absent the confounders associated with many existing comparisons of generalistto specialist herbivores. The work will hence address important gaps in our knowledge by providing critical information about how gene families involved in cereal-host-plant use differ (i.e., in numbers and gene expression) across the generalist-to-specialist spectrum in a group of herbivores that is relatively understudied compared to insects, but that causes economic damage to multiple and major cereal crops (especially under conditions of high heat, aridity, and plant water deficit stresses). A component of the project will also be to develop and/or extend gene editing methods in spider mites in the context of assessing the function of specific genes in mite-plant interactions. We will also develop educational materials that will be freely available to K-12 students, undergraduates, student educators, and the public on the impacts of heat and drought stress on plants and herbivores and the associated impact on agriculture (e.g., reduced crop yields). The project will train undergraduate students and at least one graduate student in the field of plant-herbivore interactions.Objectives:1. Produce O. pratensis and T. urticae inbred lines to serve as the biological basis for downstream genetic and genomic studies (see objectives below).2. Starting with a highly inbred O. pratensis strain (see Objective 1 above) generate a highly contiguous reference genome sequence for O. pratensis, annotate the genome, and perform comparative genomic studies with the existing T. urticae genome.3. Collect a population sample ofO. pratensis strains from within the state of Utahand assess genetic variation in the species sample (including comparing patterns of genetic variation to that observed in the generalist T. urticae for which comparable data is already publicly available).4. Assess transcriptomic plasticity to cereal hosts for T. urticae and O. pratensis to test hypotheses that generalist and specialist herbivores will have differing extents of transcriptomic reprogramming upon host shifts, and also identify candidate genes in both species that may mediate the two species' respective abilitiesto be major pests on cereal crop plants. As part of this objective, cereal plant hosts that will be used will include inbred strains of maize (Zea mays, including lines differing in benzoxazinoid specialized compound levels), sorghum (Sorghum bicolor), and barley (Hordeum vulgare). For reference for the generalist T. urticae, two non-cereal hosts will also be included (tomato, Solanum lycopersicum, and soybean, Glycine max).5. Based on findings resulting from the above objectives, multiple follow-up studies are possible, but a major anticipated direction of experimentation is functional testing of candidate genes identified as potentially important in mediating mite-plant interactions by gene-editing (using CRISPR-Cas9 inactivation). The genes to be inactivated will be selected based on the above comparative genomic and gene expression studies, and the impact of knockouts of mite genes on interactions with host plants will be evaluated. While gene editing methods have already been reported in T. urticae, an objective is to extend/develop the methods for O. pratensis.6. In one or more semesters in each of two years of the project, the PDwill run Science Research Initiative streams to introduce undergraduate students to research. Students involved in the Science Research Initiativestreams will participate in both bioinformatic and laboratory aspects of the project.7. Working with the Genetic Science Learning Center of the University of Utah, educational materials will be developed suitable for K-12 students, undergraduates, teachers, and the public, and will be housed on the Learn.Genetics public educational resource overseen by the Genetic Science Learning Center. The materials the PD will develop will introduce the target audiences to the impact of high heat, aridity, and drought on agriculture with a specific focus on how herbivores and plant-herbivore interactions are impacted (including increased crop losses associated with spider mite outbreaks under these abiotic stresses that are predicted tobecome more severe in many regions in coming years and decades).8. Undergraduate students and at least one graduate student will be trained in the areas of plant biology, arthropod herbivore biology, genetics, molecular biology, and bioinformatics. An objective of this training, along with the undergraduate educational experiences to be delivered through the Science Research Initiative program (see Objective 6), is to enhance retention and performance in science, technology, engineering, and mathematics (STEM) fields.
Project Methods
Description of methods for the project:1. Strains of O. pratensis and T. urticae will be collected and inbred by sequential rounds of mother-son inbreeding to create isogenic lines (a larger population sample of O. pratensis strains will also be collected and expanded from single females). Genomic DNA-seq data for the strains will be produced and evaluated for the expected outcomes (for instance, variant prediction and determination of the absence of segregating genetic variation as predicted if lines are truly inbred). If and as needed, specific variants will be evaluated by PCR and Sanger sequencing.2. Starting with an inbred line of O. pratensis, long DNA sequence readswill be generated and used for genome assembly to create a reference O. pratensis genome sequence (PacBio and/or Nanopore long reads will be used). The resulting assembly will then be structurally and functionally annotated (e.g., by using BRAKER and InterProScan, respectively). The quality of the assembly will be assessed by standard metrics such as N50 values and contig numbers, and the quality of both the assembly and annotation will be assessed with tools such as BUSCO.3. Comparative genomic analyses will be performed between the O. pratensis reference genome, the existing reference genome for T. urticae, and select other arthropod genomes to identify gene family expansions and/or contractions in the two mite species. The extent to which such families also harbor intra-specific variation in the mite species will also be evaluated using variant predictions (see above). Methods to be used will include, for example, automated construction of alignments and phylogenetic trees for gene families of interest, for which manual inspection of alignments will be used for quality assessment and validation. Genes in families that are specifically expanded in, for instance, O. pratensis, will be identified as candidates to underlie the grass specialist's interactions with its hosts like maize, sorghum, and barley, and will also be candidates for follow-up studies (see below).4. Gene expression data (in the form of RNA-seq reads) will be collected for strains of O. pratensis and T. urticae upon host plant shifts from a maize line to multiple maize lines with known variation in arthropod defenses (varying levels of benzoxazinoid specialized compounds) as well as sorghum and barley (and for T. urticae, two dicot species will be included as outgroups, tomato and soybean). The downstream analyses will be, for given mite strains, pair-wise detection of differentially expressed genes as a function of host plants, as well as cluster-based analyses that will examine broader patterns across multiple plant hosts. Genes that show specific and/or dramatic expression changes among hosts will be identified as candidates to mediate interactions between the two spider mite species and the cereal crop plants included in the project. In combination with the comparative genomic analyses (see above), candidate genes to be tested in functional studies (gene editing) will be identified.5. Candidate genes for mediatingmite-cereal interactions identified as described above in O. pratensis and/or T. urticae will be inactivated using CRISPR-Cas9 based gene editing. After recovering stable mutant lines, the impacts on specific hosts will be assessed (for instance, by determining mite reproductive rates or other performance measures for mutant strains as compared to isogenic wildtype controlstrains across different host plants). Significant effects of given knockouts will be assessed statistically, for example, by using two-way ANOVA tests (a significant test result for a given gene would indicate a role for the respective gene in the colonization and proliferation of a mite strainon a given host plant or set of host plants).6. Materials on the impact of climate change on herbivores and plant-herbivore interactions (especially high heat, aridity, and drought) will be developed by the PD, in consultation with staff at the Genetic Science Learning Center, for public release on the Learn.Genetics educational resource. Writing evaluation tools and/or Genetic Science Learning Centerstaff feedback will be used to adjust wording on written materials to a middle to high school reading level that is appropriate for a large audience of students and the public.7. Students participating in Science Research Initiative streams to be run by the PD will receive training in various aspects of the project and directly participate in the project's research. Best practice methods for involving undergraduate students in science will be followed. For instance, the PD will prioritize student input and research to answer questions unknown by both the PD and students (factors that have been associated with greater student gains in programs at other institutions that are similar in design and purpose to the University of Utah's Science Research Initiative). Beyond specific undergraduate student involvement in Science Research Initiative streams, mentoring of other students (a graduate student and undergraduates working directly in the laboratory on the project) will follow similar best practices.Additional evaluation and impact assessment for the research aspects of the project (Points 1-5 above) will be based on publications and the public release of datasets to be generated by the project (as well as by citations of published work). Page views will be recorded by the Genetic Science Learning Center to assess the impact of the educational materials to be released on the Learn.Genetics web resource (Point 6 above). For Point 7 above, the Science Research Initiative program is assessed by University of Utah College of Science staff members. At the beginning, middle, and end of each semester, students will answer online questionaries about attitudes toward science and challenges and experiences in the Science Research Initiative program. Additionally, data will be collected about retention in science, technology, engineering, and math (STEM) majors and performance in introductory science courses (this will be evaluated in reference to students not participating in the Science Research Initiative program).