Performing Department
(N/A)
Non Technical Summary
Long-lived perennial plants like trees are important in providing food, fiber and ecosystem services. Nitrogen is an essential nutrient that impacts plant growth and productivity. Annual plants must acquire and assimilate nitrogen from soil to support growth and development. In contrast, nitrogen acquisition and use in perennial plants and trees is more complex than annual plants since a major portion of nitrogen demand is met by internal seasonal cycling that reuses assimilated and reduced nitrogen. Seasonal nitrogen cycling is an annual recursive process that increasingly contributes to the nitrogen budget as trees age. Consequently, seasonal nitrogen cycling affords a competitive advantage in nitrogen limited environments and is important to the ecology of nitrogen use in trees and is a major factor contributing to Nitrogen Use Efficiency.The contribution of stored nitrogen to forest tree nutrient budgets varies among species. And for poplars (Populus) spring growth relies almost exclusively on nitrogen from storage reserves. Besides being important to forest trees, seasonal nitrogen cycling and nitrogen reserve mobilization has long been recognized as being important in fruit trees. In this project, state-of-the-art CRISPR gene editing methods will be used in combination with molecular biology research approaches to understand the molecular and genetic basis of how seasonal nitrogen cycling is controlled and regulated in poplar. This builds upon the discovery that the plant hormone ethylene represses the expression of the poplar bark storage protein genes, which play a central role in season nitrogen cycling. This will be accomplished through (1) CRISPR gene editing of genes that appear to regulate this process, (2) biochemical approaches to test if the proteins encoded by these gene interact, (3) molecular biology approaches to determine if these protein factors bind to DNA regulatory sequences in the bark storage protein genes, and (4) discover if other regulatory factors regulate bark storage protein gene expression and seasonal nitrogen cycling using CRISPR-based screens.This research will advance understanding of seasonal nitrogen cycling and directly addresses the research priorities of the Physiology of Agricultural Plants program (A1152). Specifically, this research falls within the research priority of "Nutrient uptake, assimilation, accumulation, and/or utilization, particularly increase efficiency in using nitrogen, or phosphorus or other supplemental nutrients". In addition, this project will also advance knowledge concerning regulatory factors involved in the establishment and regulation of N source-sink relationships.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
0%
Developmental
0%
Goals / Objectives
The overall goal of this project is to identify the molecular factors and determine their regulatory role in mediating BSP gene expression and seasonal N cycling. This will be accomplished through 4 specific objectives.Objective 1: Functionally determine in planta the role and interactions of the ethylene regulated ERF12 and ERF41 transcription factors in SD-induced BSP expression using novel CRISPR gene editing approaches. Preliminary results indicate that ERF41, and to a lesser extent ERF12, are involved in regulating BSP expression, yet further research is required to definitively establish this function. Additionally, results from this objective will also determine if these factors act independently to regulate BSP expression or are cooperative interactions involved in this regulation.Objective 2: Genetically ascertain if ERF12 and ERF41 physically interact with each other or with other factors. Gene regulation involving AP2/ERF transcription factors involves homo- and heterodimerization and differential binding of dimer combinations to cis-DNA sequences. Therefore, it is possible that ERF12/ERF41 homo- and/or heterodimers are involved in regulating SD-induced BSP expression. It is also conceivable that these factors also interact with other SD-induced or repressed ERFs. This objective will use yeast two-hybrid genetic screens to determine if these factors can physically interact and if they interact with other SD-induced or repressed ERFs.Objective 3: Determine if ERF12 and ERF41 bind to the DRE ethylene response element consensus sequence CCGAC present in the BSP gene promoters. Transcriptional regulation involves the binding of transcription factors to specific cis-DNA sequences that mediates temporal and spatial gene expression. If ERF12 and ERF41 are transcriptional activators of BSP expression, then it is likely that they bind to the DRE ethylene response element consensus sequence CCGAC present in the promoter regions of the poplar BSP genes. In this objective we will determine if these factors bind to the CCGAC consensus sequence found in the promoters of BSP.Objective 4: Identify if other SD-associated transcription factors regulate BSP expression using CRISPR genomic screens. We have identified additional transcription factors whose expression is either induced or repressed in poplar bark during SD, yet it is not known if they have a role if mediating BSP expression and seasonal N cycling. In this objective we will use powerful CRISPR genetic screens to determine if other SD-associated transcription factors also regulate BSP expression.
Project Methods
Objective 1. Functionally determine in planta the role and interactions of the ethylene regulated ERF12 and ERF41 transcription factors in SD-induced BSP expression using novel CRISPR gene editing approaches. Two CRISPR gene editing approaches will be used to decipher the in vivo functional role and interactions of ERF12 and ERF41 in regulating BSP expression. The first approach will create indel mutants targeting the ERF domains to generate combinations of ERF12 and ERF41 gene knockouts using the multiplexed CRISPR-LbCas12a-RRVQ system developed by Co-PD Qi Transgenic poplars will be produced for ERF12, ERF41 and ERF12 combined with ERF41 using methods routinely used in our labs. Transgenic poplars will be genotyped by DNA sequencing to verify the nature and location of mutations and identify biallelic mutations. The goal is to produce the following genotypes: (1) erf12 (knockout)/ERF41(wildtype); (2) ERF12/erf41; (3) erf12/erf41 and ERF12/ERF41 (wildtype control). A minimum of 3 independent lines that harbor the respective biallelic mutations for each of the three genotypes will be used to determine the relationship between genotype and BSP expression under SD coupled BSP expression and under conditions uncoupled from SD photoperiod. Expression of BSPA, BSPB, BSPC will then be determined by RT-qPCR. The second approach will use the novel CRISPR-Combo method for the simultaneous activation of one gene while mutating or knocking out a second gene. To accomplish this, either ERF12 or ERF41 expression will be activated while at the same time the other ERF gene will be targeted for editing. CRISPR edited lines will be selected that show elevated levels of expression for either ERF12 or ERF41 combined with the corresponding biallelic mutation in the other ERF gene. The selected lines (at least 3 independent lines) will then be treated with LD and SD a and the bark expression of BSPA, BSPB, BSPC, along with ERF12 and ERF41 will be determined by RT-qPCR.Objective 2: Genetically ascertain if ERF12 and ERF41 physically interact with each other or with other factors.To determine if poplar ERF12 and ERF41 can interact we will use yeast two-hybrid screens. Because efficient and effective commercial kits exist for conducting yeast two-hybrid screens and have been previously used by the PDs then we intend to use the Matchmakerä Gold Yeast Two-Hybrid system. First, each ERF will be cloned and tested for autoactivation and toxicity. Bait constructs will be made using the pGBKT7 vector which contains the Gal4 DNA-binding domain (BD) while prey constructs will be made in the pGADT7 vector containing the Gal4 transcriptional activation domain (AD). The CDS of each ERF will be cloned in each vector allowing for reciprocal interaction studies. Bait constructs are grown in yeast strain Y2HGold while prey constructs are grown in yeast strain Y187. Following mating to create diploids, interactions will be detected using 4 reporter genes AUR1-C, HIS3, ADE2, and MEL1, which combines drug and nutritional reporters resulting in lower background activity. If we detect positive interactions between ERF12 and ERF41 we will use BiFC assays in transfected Nicotiana benthamiana leaves to further confirm in vivo interactions.Objective 3: Determine if ERF12 and ERF41 bind to the DRE ethylene response element consensus sequence CCGAC present in the BSP gene promoters. To test if ERF12 or ERF41 bind to the ERF GCC boxes present in the BSPA, BSPB and BSPC promoters, we will amplify two DNA fragments of 150bp from each of the BSP promoters, one fragment with the putative binding sites and the other without the binding sites. The fragments will be purified prior to use in Electrophoresis Mobility Shift Assays (EMSAs). The CDS of both ERF12, ERF41 along with variants where the ERF domain is mutated through changes in the ERF domain amino acid sequence will be cloned into the pET32a(+) vector to produce His-tagged proteins grown in E. coli strain BL21(DE3). ERF12-His, ERF41-His and the ERF-His domain mutant proteins will be affinity purified with Na2+-NTA agarose resin and used for EMSA binding assays. After confirming whether the ERF12, ERF41 or their ERF variants bind to the respective 150 bp BSP promoter fragment, smaller 27 bp fragments along with mutated versions of the GCC box will be synthesized and further used for EMSA. EMSA using GCC containing fragments and mutated versions will be performed using the EMSA Kit (Invitrogen) with SYBR Green and SYPRO Ruby stains according to product instructions using non-denaturing polyacrylamide gels. Further validation of binding will use in vivo chromatin immunoprecipitation (ChIP) assays. C terminal GFP tagged versions (ERF12-GFP and ERF41-GFP) of the ERFs and GFP tagged ERF domain mutants will be constructed and transfected into poplar leaf protoplasts. After an appropriate incubation period, successful transfection will be verified by examining protoplasts for GFP fluorescence. Following transfection and validation of GFP fluorescence, protoplasts will be harvested by centrifugation followed by protein to DNA crosslinking using formaldehyde. The crosslinking reaction will be stopped with glycine and chromatin will be isolated using the EpiQuick ChIP Kit. Anti-GFP and IgG (negative control) antibodies will be used for chromatin immunoprecipitation. DNA recovered from the anti-GFP and IgG ChIPs will be assayed for enrichment of the ERF GCC sequences by qRT-PCR and normalized against 5% input DNA.Objective 4: Identify if other SD-associated transcription factors regulate BSP expression using CRISPR genomic screens. We will generate a gRNA library for additional SD-associated transcription factors that will be used to produce stable poplar transgenics that can then be screened and phenotyped. Furthermore, this approach will also provide a benchmark for the poplar research community for the use of this powerful screening method. We will use a modified sgRNA2.0 scaffold with 3¢ barcodes that do not interfere with CRISPR activation. Based on the promoter sequence similarity of P. tremula and P. alba in the poplar hybrid INRA 717-1B4, four sgRNAs will be designed to activate both alleles of each selected TF, by following our recently published design guideline. Approximately 200 oligos (50 genes x4 sgRNAs) will be synthesized and cloned into the CRISPR-Act3.0 following our established pipeline. After NGS-based validation of high coverage (>95%), the plasmid library will be used for activation. The pooled CRISPR-Act3.0 vectors will be transformed into Agrobacterium tumefaciens (C58pmp90), and the pooled library will be used to generate stable poplar transgenics using Populus tremula x alba 717-1B4 that already contains a BSPA promoter-GUS construct.