Recipient Organization
UNIV OF NORTH CAROLINA
(N/A)
CHAPEL HILL,NC 27514
Performing Department
(N/A)
Non Technical Summary
As the global population continues to grow, so do our needs for resources and food. The cereal crop rice provides ~30% of the calories consumed by humans globally. Increasing rice robustness and yield (number of grains) would help to meet these global food needs. Previous work has demonstrated that higher levels of the plant hormone cytokinin positively influence grain production in rice, specifically by increasing the number of grains produced by each panicle (inflorescence). This is a very promising lead to follow, as manipulation of this process would produce high-yield rice. However, the regulatory mechanism behind this phenomenon is not yet understood. By using the popular gene editing system CRISPR/Cas9, we have developed an extensive collection of rice lines with precise edits disrupting a group of genes (called the type-A Response Regulators) known to inhibit cytokinin response--meaning these plants should have heighted response to cytokinin and increased grain yield. Further, by using traditional breeding techniques, we are crossing these lines to wild type plants and select those individuals that retain the precise mutation(s) of interest but lack transgenic material. We are studying these lines closely to identify plants with better performance by measuring important traits such as plant size, number of days to flower, number of panicles/inflorescences per plant, ability to efficiently harvest light energy (photosynthesis), and grain yield. Using these data, we will identify the most productive lines and study gene expression via RNA-sequencing in their inflorescence meristems--the developmental tissue that gives rise to the mature panicle and, ultimately, grains--as compared to wild type (un-edited) plants. From these data, genes targeted by the type-A RRs will be identified and studied further; thus, shedding light on the mechanism of type-A RR action. Overall, this work will bring us much closer to establishing highly productive field-ready rice lines.
Animal Health Component
25%
Research Effort Categories
Basic
50%
Applied
25%
Developmental
25%
Goals / Objectives
The primary focus of this work is to assess the roles of the type-A RRs (players in the cytokinin signaling pathway in plants) in various aspects of plant development, including grain yield, individual grain size, fitness, and the rate of photodamage. The direct outcomes of this work will be to identify rice lines with alterations to grain yield and rates of photosynthesis as compared to the wild type and identify specific downstream genes determining these traits regulated by the type-A RRs from transcriptional data in wild type and mutant lines. The proposed research will elucidate the role of the type-A RRs in regulating gene expression and growth and development in rice, furthering our understanding of the downstream targets and mechanisms of this signaling pathway.Objectives:(1) Finalize multiple knockout mutants in rice for the four type-A RR clades.CRISPR/Cas9-generated multiple gene knockout lines (by clade) for the type-A RRs (negative regulators of the cytokinin signaling pathway) will be finalized. These lines will be genotyped by sequencing and checked for presence of the CRISPR/Cas9 transgene via PCR screening. Where possible, secondary alleles and lower-order mutants within each clade will be identified and screened in the same manner. Lines lacking the CRISPR/Cas9 transgene will be isolated for downstream experiments.(2) Characterize plant morphology and productivity in the four type-A rr lines.Using the type-A rr mutant lines lines generated in (1), growth and yield-relevant traits will be measured at various plant growth stages in order to identify the effects of disruption of these genes on grain yield. A large-scale greenhouse morphological screen will be carried out to gather data such as tiller number, floral morphology, panicle number, and grain yield in these lines. Seedling assays will also be conducted to characterize seedling morphology, and cytokinin response assays will be done to measure sensitivity to this hormone in the lines. The data generated from these experiments will be used to identify possible clade-specific regulated traits and guide future experiments. This work will also determine if any of these lines exhibit increased grain yield.(3) Characterize gene expression patterns and identify probable transcriptional targets of the type-A RRs in developing rice panicles.Transcriptome sequencing will be done with developing panicles (inflorescences) from the most promising mutant line identified in (2) (i.e., the line with the greatest increase in yield) and wild type plants. Differential gene expression analysis will be conducted to (a) characterize broad changes in the pattern of gene expression between wild type and the mutant, and (b) identify genes targeted by the type-A RRs. Together with the data generated in (2), a better mechanism for the roles of the type-A RRs in cytokinin signaling in rice will be established.
Project Methods
EffortsThe aims of this project are: 1) finalize multiple knockout mutants in rice for the four type-A RR clades, 2) characterize plant morphology and productivity in the four type-A rr lines, and 3) identify identify probable transcriptional targets of the type-A RRs in the developing rice panicle. Knockout rice lines for the type-A RRs are being made using CRISPR/Cas9, and mutations identified through a combination of sanger sequencing and PCR. Back crosses (to wild type) are being done with established mutant lines to remove the transgenic CRISPR/Cas9 cassette, producing stable non-transgenic lines. Plant morphology and productivity will be assessed from a large-scale screen carried out in the green house. Many traits will be measured--grain yield will be determined by counting the number of filled seeds/panicle in matured plants, and photosynthesis traits measured using a portable spectrophotometer device, to name a couple. Developing panicles will be isolated from the most promising mutant line identified in this screen (e.g., the line with the greatest increase in yield as compared to wild type) and wild type, RNA extractions done, and RNAseq performed to generate transcriptome data. These data will be analyzed following a well-established differential expression analysis pipeline to identify key genes of interest that are expressed differentially in wild type versus type-A rr during panicle development. EvaluationThe direct outcomes of this work include a collection of stable non-transgenic type-A rr knockout rice lines, measurements of important agricultural traits in these lines, and isolation of lines that are more productive compared to wild type (i.e., with increased grain yield and with altered rates of photosynthesis). Transcriptome data will be generated from the developing panicles of wild type and the most interesting type-A rr line identified, and a list of genes potentially targeted by the type-A RRs produced. Research findings are anticipated to be published as 1-2 journal articles and presented at at least 2 conferences in either the form of posters or talks. A public GitHub repository will be established for all code and data analysis methods associated with the project. At least 2 undergraduate students will be recruited to work on the project (as mentees to the PD) and present their findings in papers and/or posters each semester. The broader significance of the project and its potential societal contributions will be conveyed to the public at the UNC Science Expo each April.