Progress 05/01/24 to 04/30/25
Outputs
Target Audience:The target audience includes the plant science community at large, plant biologists, and wheat scientists and wheat growers. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project trained four research scientists, a graduate student, an undergraduate student, and a technician in genome editing, wheat genetics, and molecular biology. At SDSU, post-doctoral scientist Dr. Ming Ma, previously trained in rice biology, continued working on the project, focusing on the Objectives 2 and 3. He conducted biotin labelling, protein extraction, protein detection by Western Blot, protein purification, and protoplast isolation and transformation, RNA extraction, GreenCUT&RUN and phenotyping the transgenic plants. Postdoctoral scientist Dr. Peng Cheng, previously trained in developmental biology, joined the team in July 2024 and worked on the Objective 1 of the project. He genotyped and phenotyped the S-D1b/S-D1a NIL, S-D1/bs-b1 and S-D1b/s-ad3 populations. He also performed mutagenesis S-d1b and identified a new allele. MS graduate student Brandon Larson and undergraduate student William Hummel were trained in DNA extraction and PCR genotyping. William also worked on the analysis of RNA-seq and GreenCUT&RUN data more recently. Technician Yanhang Zhang worked on wheat transformation. 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). Conference presentation. The results from the project were presented at the Midwest ASPB conference held in March at University of Nebraska-Lincoln (product table). What do you plan to do during the next reporting period to accomplish the goals?Objective 1. Fine-tune S1 expression dosage. Complete phenotyping of S1 and S3 KO mutations in the S-D1b background and the new missense mutation. Objective 2. Identify S1-interacting proteins. Compare the phosphoproteome between Fielder, S-A1bS-D1b double mutant, and YFP-TbID- S-D1b transgenic plants. Identify S1-interacting proteins (SIPs) from primary roots and coleoptiles by LC-MS/MS profiling YFP-YFP-TbID and YFP-TbID- S-D1b. Validate the top SIP candidates by Y2H, BIFC, and phosphorylation assay. Objective 3. Identify TaBZR partners and targets. Identify BZR1-interacting proteins (BIPs) from young spikes by LC-MS/MS profiling YFP-YFP-TbID and BZR1- YFP-TbID. Validate the top BIP candidates by Y2H, BIFC, and phosphorylation assay. Genomic profiling BZR1-binding sites from young spike tissue by GreenCUT&RUN.
Impacts
What was accomplished under these goals?
Goal One: (70% Accomplished) Dosage effect of S-d1b on agronomic traits. We phenotyped the wild-type (S-D1a), homozygous S-D1b, and their heterozygous plants in the Fielder wheat background for 11 agronomic traits. Compared to the wild type, homozygous S-D1b mutants exhibited significant reductions in leaf angle, plant height, main spike weight, thousand-grain weight, and coleoptile length, while spikelet density and grain width increased (p < 0.0167), but no significant differences were observed between heterozygous S-D1b and wild-type plants (p > 0.0932). However, a dosage effect was detected in spike length and grain length, as heterozygous plants showed significant improvement compared to both homozygous S-D1b mutant and wild-type plants (p < 0.0377). The spikelet number per spike remained unaffected by the S-D1b. Effect of S1 homolog knockouts on the S-d1b background. We generated S-D1b NILs in s-b1, and s-ad3 mutant backgrounds. Compared to the Fielder, s-ad3 flowered early (p = 0.0004) as expected, but reduced plant height, spike length, and tiller number (p < 0.0377). No significant change was detected between them on leaf angle, spikelet number per spike, and spikelet density (p > 0.2023). While S-D1b/s-ad3, as compared to s-ad3, showed significant early flowering, reduced leaf angle, plant height, and spike length, and increased tiller number and spikelet density, it showed no difference in this trait as compared to S-D1b plants (p > 0.3195). We also evaluated the S-D1b effect on agronomic traits in the s-b1 background, where S-D1b significantly reduced plant height, spike length, spikelet number, grain size, and grain weight but increased spikelet density (p < 0.0003), but not flowering date, leaf angle, and tiller number (p > 0.11748). Collectively, s1 and s3 mutants improved flowering earliness but not plant height, spike length, and grain size. Identification of new S1 alleles. We developed a CRISPR-mutagenesis population via RNP bombardment targeting TaHLH489 in the S-D1b background, from which we selected 52 plants showing reduced density. A T1 family segregated for the non-dwarf traits. Genotyping the non-dwarf plants did not detect mutations in TaHLH489 but a missense mutation in position 173 of S-D1b, which overwrites the S-D1b effect. This mutation significantly increased plant height, spike length, grain number per spike (per spikelet), grain length, grain width, and grain weight (p < 0.0215). Compared to wild type Fielder, the new mutant showed reduction in spike length, spikelet number per spike, and grain length and increased grain number per spikelet (p < 0.0184) Impact. These findings demonstrate that the agronomical traits respond to S-D1b and its homolog dosage differentially, implicating potential applications in wheat breeding and yield improvement. Discovery of the missense mutation opens a new approach for engineering S1 protein for improvement of wheat. Goal two: (15% Accomplished) Developing YFP-TbID constructs and transgenic plants. We developed constructs S1-YFP-TbID and S-D1b-YFP-TbID, in which the transgene cassettes are driven by maize ubiquitin promoter (ZmUbi) with the S1 at the N terminus. We obtained 11 T0 transgenic plants for the former and 26 T0 plants for the latter. All the transgenic plants were confirmed by PCR genotyping. Of the 37 transgenic plants, two S-D1b-YFP-TbID T1 plants showed medium dwarf and increased spike density. Although YFP signals were detected in these plants under a fluorescent microscope and by western blot, biotin labelling signal is very weak. To improve the efficiency, we made a YFP-TbID- S-D1b construct, in which the S-D1b is located at the C terminus of the fusion protein. We have transformed it into Fielder and obtained 12 hygromycin-resistant regeneration seedlings from the first batch transformation. Development of double sphaerococcum mutant line for phosphoproteomics. We combined S-A1b and S-D1b by crossing their NILs and obtained the double sphaerococcum mutant line based on the super dwarf phenotype and genotyping. This double mutant can be used for identification of the S1-mediated phosphoproteomics as a backup. Impact. Development of the YFP-TbID- S-D1b transgenic plants and the double sphaerococcum mutant line laid a foundation for identifying S-interacting proteins. Phenotyping of the S1-YFP-TbID and S-D1b-YFP-TbID transgenic plants and immune-assay of the fusion proteins enriched our experience with the TbID technology. Goal three: (65% Accomplished) Phenotyping BZR1-YFP-TbID and BZR-1d-YFP-TbID transgenic plants. Compared to negative control (FYP-YFP-TbID) plants, BZR1-YFP-TD plants have lax plant type (increased tiller angle and leaf angle), elongated nodes, slender spikes, slender seeds (increased grain length but reduced grain width). The lax plant type is also observed in BZR-1d-YFP-TbID plants. Identification BZR1-interacting proteins. While YFP fluorescence signals in control plants are in cytoplasm and nuclei in guard and mesophyll cells, strong fluorescence signals in the BZR1-YFP-TD plants were localized in nuclei of the mesophyll cells. We performed TbID-mediated proximity labeling (PL) using one-month-old leaves of BZR1-YFP-TbID and negative control YFP-YFP-TbID transgenic plants. The streptavidin-enriched biotinylated protein samples were profiled by LC-MS/MS, which identified 151 enriched proteins in the TaBZR1-YFP-TbID transgenic plants. The bait BZR1 is significantly enriched, suggesting the PL enrichment is successful. The remaining 150 proteins include positive control proteins known to interact physically with BZR1, such as TOPLESS (TPL) and a TPL-related protein, and several interesting proteins, two ABC1-like kinases, a mitogen-activated protein kinase (MAPK), a protein-serine/threonine phosphatase, and a SET domain-containing protein (histone methylase). The BZR1-MAPK interaction has been confirmed by yeast 2 hybrid assay. Transcriptomic analysis of BZR1-YFP-TD transgenic plants. BZR1 is a key transcription factor (TF) transducing the BR signal by modulating thousands of genes in the model plant Arabidopsis. Driven by ZmUbi promoter, BZR1-YFP-TbID is overexpressed in the transgenic plants. Taking the advantage of overexpression, we are identifying the BZR1-mediated transcriptome by RNA-seq profiling the transcriptomes of young spikes of the YFP-YFP-TbID (negative control) and BZR1-YFP-TbID transgenic plants. A total of ~ 120 million PE150 reads were generated for six samples (three biological replicates of each genotype). A total of 4311 DEGs, 2215 upregulated and 2096 downregulated, were identified. The annotation of the DEGs is underway. Identification of BZR1 binding site by GreenCUT&RUN. We adopted GreenCUT&RUN, a new method for the genome-wide detection of native protein-DNA interactions using use of a GFP nanobody-MNase fusion protein (enh-MNase), to avoid the important drawbacks of ChIP. We prepared the enh-MNase and established a protocol in wheat (see other products). We isolated nuclei from the young spikes of YFP-YFP-TbID (negative control) and BZR1-YFP-TbID transgenic plants, performed GreenCUT&RUN, constructed six GreenCUT&RUN-seq libraries, three libraries for each genotype, which were pooled and submitted to a sequencing server. Sequencing the pooled libraries produced ~90 Gb of raw data, ~15 Gb per library/biological replicate. Mapping the reads to the wheat genome and quantification of signals and prediction of the BZR1-binding are underway. Impact. The results facilitate dissecting the BZR-mediated regulation network in wheat, and the validated transgenic plants are valuable resources for the investigation of BR signal in other biologic processes. Purification of the enh-MNase and establishment of GreenCUT&RUN protocol pave the way for efficient genomic profiling of TF binding sites in wheat.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2025
Citation:
Ming Ma, Andres Reyes, Shouling Xu, Wanlong Li. Establishing the TurboID-based proximity labeling system for studying BZR1 signaling in wheat. Poster: 601 (2025 Midwest ASPB Abstract Book.docx - Google Docs; https://docs.google.com/document/d/1lL5c8ekwHpRNz09J0QjAp5Ve1VxLWQkp/edit?tab=t.0)
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2025
Citation:
Peng Cheng, Yanhang Zhang, Wanlong,Li. Dosage-dependent effect of s1-d mutation on agronomical traits in wheat.
Poster: 700 (2025 Midwest ASPB Abstract Book.docx - Google Docs; https://docs.google.com/document/d/1lL5c8ekwHpRNz09J0QjAp5Ve1VxLWQkp/edit?tab=t.0)
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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
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