Progress 05/01/23 to 04/30/24
Outputs
Target Audience:The target audience includes the plant science community at large, plant biologists, and wheat scientists and wheat growers. Changes/Problems:While we are purifying genetic background of the S1 and S3 KO mutants, a Chinese group most recently published a paper in the journal Plant Cell and Environment (https://onlinelibrary.wiley.com/doi/epdf/10.1111/pce.14890) and reported the results using a set of S1 and S3 KO mutants on phenotypes including agronomical traits, plant types, grain size, physiology (drought and heat tolerance). To avoid repeat, we will minimize the effort in the Objective 1a for further evaluation of the S1 and S3 KO mutations. Instead, we will focus on fine-tuning the expression dosage of the wheat BIN2 homologs and development mutation of TaHLH489 in the S-1Db background. What opportunities for training and professional development has the project provided?This project trained three research scientists, an undergraduate student, and a technician in genome editing, wheat genetics, and molecular biology. At SDSU, post-doctoral scientist Dr. Ming Ma and Technician Yanhang Zhang originally worked on other projects. They were reassigned to work on this project, but they were supported by other funds as in-kind contribution while we are recruiting a postdoc and graduate student. Ming Ma, previously trained in rice biology, joined the project in Nov 2023 and worked on biotin labelling, protein extraction, protein detection by Western Blot, protein purification, and protoplast isolation and transformation. Yanhang Zhang worked on wheat transformation. William Hummel, a sophomore in plant science major, is training in DNA extraction and PCR genotyping. At Carnegie Institute, early career researcher Andres Reyes with a background in molecular biology and bioinformatics worked on protein digestion and LC-MS/MS profiling, and Research assistant Tarabryn Grismer with a background in Biochemistry and Bioinformatics worked on data analysis. How have the results been disseminated to communities of interest?The protocols developed, including extraction of wheat proteins, Western blotting, and proxy labeling, are posted on the lab webpage (https://www.sdstate.edu/li-lab). What do you plan to do during the next reporting period to accomplish the goals?Objective 1. Fine-tune S1 expression dosage Continue the genotyping effort for identifying double KO mutations combined with the S-D1b allele. Phenotype homozygous S1 and S3 KO mutations in the S-D1b background. Screen the CRISPR-mutagenesis populations for TaHLH489 KO mutations. Objective 2. Identify S1-interacting proteins Compare the phosphoproteome between Fielder, s-ad1 mutant, and S-D1b-YFP-TbID transgenic plants. Determine S1 expression pattern in wheat tissues using a confocal fluorescence microscope. Identify S1-interacting proteins (SIPs) from primary roots and coleoptiles by LC-MS/MS profiling YFP-YFP-TbID and S-D1b-YFP-TbID. Validate the top SIP candidates by Y2H, BIFC, and phosphorylation assay. Objective 3. Identify TaBZR partners and targets Identify BZR1-interacting proteins (BIPs) from primary roots and coleoptiles by LC-MS/MS profiling YFP-YFP-TbID and BZR1- YFP-TbID. Validate the top BIP candidates by Y2H, BIFC, and phosphorylation assay. Determine BZR1 expression pattern in wheat tissues using a confocal fluorescence microscope
Impacts
What was accomplished under these goals?
Goal One: Fine-tune S1 expression dosage (10% Accomplished) Purifying the genetic background of S1 and S3 knockout mutants. We previously showed that the sphaerococcum syndrome is controlled by gain-of-function mutations at Sphaerococcum 1 (S1) locus on chromosome 3D, i.e., the dwarf allele S-D1b and S-D1c. In addition to the homoeologs in the A and B genome, namely S-1A and S-1B, there are also homologs with ~95% similarity to the S1 proteins in the wheat genome located on the group-1 chromosomes named S-A3, S-B3, and S-D3. S1 and S3 are the wheat homologs of the Arabidopsis BIN2 protein. We have developed knockout (KO) mutants s-a1, s-d1, s-a3, s-b3, s-d3, and double mutants s-ad1 and triple mutant s-abd3. For phenotyping these KO mutants, we first cleaned their genetic background by backcrossing the double and triple mutants with wild-type (WT) Fielder and selected the non-transgenic mutants from the F2 populations by genotyping. We have obtained five transgene-free single mutants (s-a1, s-d1, s-a3, s-b3, and s-d3) and 4 double mutants (s-ad1, s-ab3, s-ad3, and s-bd3). Integrating S1 and S3 knockout mutations with the S-D1b allele. To fine-tune the expression dosage of wheat BIN2 homologs, we are integrating S1 and S3 KO mutations with the S-D1b allele by genetic crossing. Two large populations were constructed from crosses between Fielder near isogenic line FDR-S1b and the s3 triple mutant s-3abd and between FDR-S1b and the s1 double mutant s-1ad. The F2 population was genotyped for KO mutations and S-D1b allele. Five homozygous combinations were identified, i.e., S-D1b/s-3a, S-D1b/s-b3, S-D1b/s-d3, and S-D1b/s-1a. Developing knockout TaHLH489 mutants in the S-D1b background. While S-D1b has a positive effect on the improvement of wheat plant architecture, grain quality, drought tolerance, NUE, and PUE but a negative effect on grain size and flowering time. To break the trade-offs, we need to improve grain size and flowering time. A recent report showed that TaHLH489 functions downstream S1 and reduces grain length (Lyu, et al. 2024. Plant Biotechnology Journal. https://doi.org/10.1111/pbi.14319). With this, we initiated an effort to develop KO mutation for TaHLH489 in the S-D1b background by RNP bombardment. Two sgRNAs were designed to target the conserved regions of among the A-, B-, and D-genome homoeologs of TaHLH489. The sgRNAs were incubated with the Cas9 protein to form the RNP, which were coated to gold particles. More than 500 immature embryos of FDR-S1b were bombarded in two batches with RNP-coated gold particles, from which more than 400 calluses were induced. Calluses of the first batch start to regenerate 41 plantlets. Impact. Cleaning the KO mutants and combining them with the S-D1b allele paved the way to determine the dosage effect of the BIN2 homologs on agronomic traits. Developing the TaHLH489 KO mutants would provide a path to break the S-D1b mediated trade-offs. Goal two: Identify S1-interacting proteins (10% Accomplished) Developing YFP-TbID constructs and transgenic plants. Previously, we developed GFP-TbID transgenic wheat for the S1 and BZR1 together with cognitive negative control. One inconvenience using GFP is its fluorescent signal is interfered by the autofluorescence from the non-transgenic Filder plant using the fluorescent microscopes in the SDSU Core Facility, and transgene expression must be detected by Western blot, which is not feasible for high-throughput assay. To overcome this problem and for rapid screen of transgene expression, we developed YFP-YFP-TbID as negative control and S1-YFP-TbID transgenic plants, in which the transgene cassettes are driven by maize ubiquitin promoter (ZmUbi). In addition, we also developed transgenic plants expressing S-D1b-YFP-TbID driven by S-D1 native promoter. Transgenic plants of these constructs can be used in a complementary manner. The S-D1b-YFP-TbID transgenic plants will not only be used for identifying S1-interacting protein but also for determining S-D1 expression pattern and profiling the S-mediated phosphoproteome. The expression of transgenes was validated by PCR sequencing and Western blot in addition to fluorescent microscopy. Detailed protocols for extracting and Western blotting of wheat proteins are posted on the lab webpage (https://www.sdstate.edu/li-lab). Optimizing proximity labeling protocols in wheat. Compared to the dicot model Arabidopsis, the monocot grasses like wheat have heavy wax covered on the surface of plant tissues, which would affect the penetration of biotin into cells thus labeling efficiency. To enhance the biotin labeling efficiency, we optimized the proximity labeling (PL) procedure by testing experimental parameters including pretreatment, vacuum, and biotin concentration. We found that vacuum for 5 to 10 minutes and/or brief pretreatment of 0.01% Silwet-77 can increase labeling efficiency, and 300µM is the optimal concentration of biotin. A detailed protocol is posted on the lab webpage (https://www.sdstate.edu/li-lab). Identifying S1-interacting proteins. Using the optimized protocol in a pilot experiment, protein samples were purified from one-week old seedlings of YFP-YFP-TbID and S1-YFP-TbID transgenic plants. LC-MS/MS profiling identified over 3,000 peptides. In addition to the bait protein S1, BZR1 is also detected, indicating that the PL protocol works in wheat. We are doing functional annotation of the peptides and their coding genes. Impact. Development of the S1-YFP-TbID and S-D1b-YFP-TbID transgenic plants and optimization of the LP protocol laid a foundation for identifying S-interacting proteins by LC-MS/MS approach. Goal three: Identify TaBZR partners and targets (1% Accomplished) Developing BZR1-YFP-TbID and BZR-1d-YFP-TbID transgenic plants. We also developed transgenic plants expressing BZR1-YFP-TbID and BZR-1d-YFP-TbID fusion proteins. The BZR1-YFP-TbID transgene is driven by ZmUbi promoter, and the BZR-1d-YFP-TbID transgene by BZR1 native promoter. The transgenic plants were validated by PCR, YFP, and Western blot assays. Impact. Development of the BZR1-YFP-TbID and BZR-1d-YFP-TbID transgenic plants laid a foundation for Identifying TaBZR partners and targets.
Publications
|