Dataset Information

Rapid validation of transcriptional enhancers using agrobacterium-mediated transient assay.

ABSTRACT: Background:Enhancers are one of the most important classes of cis-regulatory elements (CREs) and play key roles in regulation of transcription in higher eukaryotes. Enhancers are difficult to identify because they lack positional constraints relative to their cognate genes. Excitingly, several recent studies showed that plant enhancers can be predicted based on their distinct features associated with open chromatin. However, experimental validation is necessary to confirm the predicted enhancer function. Results:We developed a rapid enhancer validation system based on Nicotiana benthamiana. A set of 12 intergenic and intronic enhancers, identified in Arabidopsis thaliana, were cloned into a vector containing a minimal 35S promoter and a luciferase reporter gene, and were then infiltrated into N. benthamiana leaves mediated by agrobacterium. The enhancer activity of each candidate was quantitatively assayed based on bioluminescence measurement. The data from this luciferase-based validation was correlated with previous data derived from transgenic assays in A. thaliana. In addition, the relative strength of different enhancers for driving the reporter gene can be quantitatively compared. We demonstrate that this system can also be used to map the functional activity of a candidate enhancer under different environmental conditions. Conclusions:In summary, we developed a rapid and efficient plant enhancer validation system based on a luciferase reporter and N. benthamiana-based leaf agroinfiltration. This system can be used for initial screening of leaf-specific enhancers and for validating candidate leaf enhancers from dicot species. It can potentially be used to examine the activity of candidate enhancers under different environmental conditions.

SUBMITTER: Lin Y

PROVIDER: S-EPMC6402126 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Rapid validation of transcriptional enhancers using agrobacterium-mediated transient assay.

Lin Yuan Y Meng Fanli F Fang Chao C Zhu Bo B Jiang Jiming J

Plant methods 20190306

<h4>Background</h4>Enhancers are one of the most important classes of <i>cis</i>-regulatory elements (CREs) and play key roles in regulation of transcription in higher eukaryotes. Enhancers are difficult to identify because they lack positional constraints relative to their cognate genes. Excitingly, several recent studies showed that plant enhancers can be predicted based on their distinct features associated with open chromatin. However, experimental validation is necessary to confirm the pred ...[more]

PMID: 30873216

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Project description:Agrobacterium-mediated transient expression (AMTE) has been widely used for high-throughput assays of gene function in diverse plant species. However, its application in monocots is still limited due to low expression efficiency. Here, by using histochemical staining and a quantitative fluorescence assay of β-glucuronidase (GUS) gene expression, we investigated factors affecting the efficiency of AMTE on intact barley plants. We found prominent variation in GUS expression levels across diverse vectors commonly used for stable transformation and that the vector pCBEP produced the highest expression. Additionally, concurrent treatments of plants with one day of high humidity and two days of darkness following agro-infiltration also significantly increased GUS expression efficiency. We thus established an optimized method for efficient AMTE on barley and further demonstrated its efficiency on wheat and rice plants. We showed that this approach could produce enough proteins suitable for split-luciferase assays of protein-protein interactions on barley leaves. Moreover, we incorporated the AMTE protocol into the functional dissection of a complex biological process such as plant disease. Based on our previous research, we used the pCBEP vector to construct a full-length cDNA library of genes upregulated during the early stage of rice blast disease. A subsequent screen of the library by AMTE identified 15 candidate genes (out of ~2000 clones) promoting blast disease on barley plants. Four identified genes encode chloroplast-related proteins: OsNYC3, OsNUDX21, OsMRS2-9, and OsAk2. These genes were induced during rice blast disease; however, constitutive overexpression of these genes conferred enhanced disease susceptibility to Colletotrichum higginsianum in Arabidopsis. These observations highlight the power of the optimized AMTE approach on monocots as an effective tool for facilitating functional assays of genes mediating complex processes such as plant-microbe interactions.

Project description:We present here a modified, sonication-assisted transient transformation assay for rapid analysis of cis-regulatory elements. We tested promoter elements from MIR159B locus of Brassica juncea by generating stable transgenic lines and compared the transcriptional activity of GUS reporter with that of the transient assay method. To obtain reliable and repeatable results, and to omit false-positive data, we optimized several parameters including sonication duration and cycle and concentration of Agrobacterium tumefaciens measured as optical density (O.D.) at 600 nm. To the best of our knowledge, this is the first report of promoter characterization of MIR159B from Brassica juncea, and comparative analysis of stable and transient lines. Our analysis shows that the protocol described herein allows understanding promoter activity/transcriptional control in tissues other than leaf or protoplast which have remained the mainstay for transient analysis thus far. We tested reporter gene GUS under the control of constitutive promoter, CaMV 35S, and MIR159b from Brassica juncea. We optimized the duration of sonication (5-, 10- and 15-min cycle), bacterial density (measured as O.D at 600 nm?=?0.6/0.8/1.0) and Agro-infection time (5, 10, 15 min), and co-cultivation (12-, and 24-h). Sonication cycle of 10-min, followed by Agro-infection and co-cultivation with Agrobacterium tumefaciens with O.D. 600 nm?=?0.8 and for 12 h was found to be optimum. We could successfully express reporter genes in deep-seated tissues such as floral organs and pollen grains where it was previously not possible to perform transient assay. Constitutive GUS activity was observed when reporter was placed under control of the constitutive promoter of CaMV 35S. Reporter GUS when placed under transcriptional control of MIR159b promoter from Brassica juncea showed reporter activity in floral tissues, in mature pollen grains. Comparative analysis of reporter activity from stable transgenic lines at T2 generation with that of transient assay system reveals identical to near-identical reporter activity. Transient assay could be successfully performed in tissues collected not only from Arabidopsis thaliana, but also from Brassica juncea and Brassica nigra to demonstrate its wide applicability. Our modified method thus has the potential of quick and rapid analysis of promoter activity and allows us to record the developmental dynamics and spatio-temporal expression pattern driven by specific promoters. Suitable modification and controls should also allow analysis of hormonal regulation and identification of trans-factors via DNA-protein interactions. Furthermore, this method can also be extended to study promoters under various environmental conditions that otherwise do not allow growth and complete life cycle of healthy plants and can be modified to test reporter activity in other non-model plants or plants with long life cycle.

Dataset Information

Rapid validation of transcriptional enhancers using agrobacterium-mediated transient assay.

Publications

Rapid validation of transcriptional enhancers using agrobacterium-mediated transient assay.

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