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0 Q&A 395 Views Jul 20, 2023

Virus-mediated transient gene overexpression and gene expression silencing can be used to screen gene functions in plants. Sugarcane mosaic virus (SCMV) is a positive strand RNA virus in the Potyviridae family that has been modified to be used as vector to infect monocots, including maize (Zea mays), for transient gene overexpression and gene expression silencing. Relative to stable transformation, SCMV-mediated transient expression in maize has the advantages of being faster and less expensive. Here, we describe a protocol for cloning constructs into the plasmid vector pSCMV-CS3. After maize seedlings are transformed with pSCMV-CS3 constructs by particle bombardment, the virus replicates and spreads systemically in the plants. Subsequent infections of maize seedlings can be accomplished by rub inoculation with sap from SCMV-infested plants. As an example of a practical application of the method, we also describe virus-induced gene silencing (VIGS) of fall armyworm (Spodoptera frugiperda) gene expression. Transgenic viruses are created by cloning a segment of the fall armyworm target gene into pSCMV-CS3 prior to maize transformation. Caterpillars are fed on the virus-infected maize plants, which make dsRNA to silence the expression of the fall armyworm target gene after ingestion. This use of SCMV for plant-mediated VIGS in insects allows rapid screening of gene functions when caterpillars are feeding on their host plants.


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0 Q&A 7901 Views Feb 5, 2019
Identification of specific DNA binding sites of transcription factors is important in understanding their functions. Recent techniques allow us to investigate genome-wide in vivo binding positions by chromatin immunoprecipitation combined with high-throughput sequencing. However, to further explore the binding motifs of transcription factors, in-depth biochemical analysis is required. Here, we describe an efficient protocol of protein-DNA interactions based on a combination of our in vitro transcription/translation system and AlphaScreen® technology. The in vitro transcription/translation system supports an efficient and quick way of protein synthesis by alleviating cumbersome cloning steps. In addition, AlphaScreen® system provides a highly sensitive, quick, and easy handling platform to investigate the protein-DNA interactions in vitro. Thus, our method largely contributes to comprehensive analysis of the biochemical properties of transcription factors.
0 Q&A 16581 Views Jul 20, 2017
Determination of a gene expression in guard cells is essential for studying stomatal movements. GUS staining is one means of detecting the localization of a gene expression in guard cells. If a gene is specially expressed in guard cells, the whole cotyledons or rosette leaf can be used for GUS staining. However, if a gene is expressed in both mesophyll and guard cells, it is hard to exhibit a clear expression of the gene in guard cells by a GUS staining image from leaf. To gain a clear guard cell GUS image of small G protein ROP7, a gene expressed in both mesophyll and guard cells, we peeled the epidermal strips from the leaf of 3-4 week-old plants. After removing the mesophyll cells, the epidermal strips were used for GUS staining. We compared the GUS staining images from epidermal strips or leaf of small G protein ROP7 and RopGEF4, a gene specifically expressed in guard cells, and found that GUS staining of epidermal strips provided a good method to show the guard cell expression of a gene expressed in both mesophyll and guard cells. This protocol is applicable for any genes that are expressed in guard cells of Arabidopsis, or other plants that epidermal strips can be easily peeled from the leaf.
0 Q&A 19705 Views Oct 5, 2015
Production of functional eukaryotic RNA is a very elaborate process that involves a complex interplay between transcription and various RNA processing activities, including splicing, 5’ capping, and 3’ cleavage and polyadenylation (Bentley, 2014). Accurate mapping of RNA ends provides a valuable tool to assess transcriptional and post-transcriptional events giving rise to different gene transcripts. The abundance of such transcripts most likely depends on exogenous and developmental cues, or mutations. In the reference plant Arabidopsis, perturbation of the HUA-PEP post-transcriptional regulatory factors (Rodríguez-Cazorla et al., 2015) leads to the accumulation of aberrant transcripts of the key floral homeotic gene AGAMOUS (AG) (Yanofsky et al., 1990) that retain intronic sequence. It was determined by 3’ RACE reactions that such erroneous transcripts correspond to premature processing and polyadenylation events taking place at the AG intron region. Here we describe a protocol that is suitable for analysis of relatively abundant transcripts and also for detecting aberrant RNA species that are likely prone to rapid turnover. Likewise, the method, here adapted to Arabidopsis reproductive tissues, can be applied to characterize RNA species from other organs (leaf, root) and/or other plant species. We provide a detailed protocol of our 3’ RACE procedure comprising four major parts: Total RNA extraction, RNA amount determination and quality control, the RACE procedure itself, and isolation of the resulting RACE products for cloning and sequencing.
0 Q&A 11753 Views Sep 20, 2015
The T-DNA (transferred-DNA) region of virulent Agrobacterium tumefaciens (A. tumefaciens) strain is transferred and integrated into the plant genome, and thereby the T-DNA genes are expressed in transformed plant cells. This protocol was used to analyze the transcription start sites (TSSs) of agrobacterial T-DNA genes within plant crown gall tumor. Firstly, the stems of Arabidopsis thaliana were inoculated by A. tumefaciens strain C58 and developed crown gall tumor. Subsequently, the mRNA was extracted from the crown gall tumor and then used for amplification of 5’ cDNA ends by 5’ Rapid Amplification of cDNA Ends (5’ RACE) assay. The full-length cDNAs were generated in reverse transcription reactions and used to analyze TSSs. Here, TSSs of three oncogenes, IaaH, IaaM and Ipt were analyzed as examples. This protocol also allows for identification of TSSs of the other agrobacterial T-DNA genes that expressed in plant cells.
0 Q&A 10108 Views Aug 20, 2014
We developed an in vivo method to assay plant transcription factor (TF)–promoter interactions using the transient expression system in Nicotiana benthamiana (N. benthamiana) plants. The system uses the Arabidopsis stay green (SGR) gene as a reporter. Induction of SGR expression in N. benthamiana causes chlorophyll degradation and causes leaves to turn yellow.
0 Q&A 12265 Views Jun 20, 2014
There are different direct and indirect methods available to study gene expression in plant systems. In this protocol we describe a modified expression assay using transgenic plants expressing the luciferase gene under the control of phosphate transporter PHT1;1 promoter. This assay was originally optimized for analyzing the repression of PHT1;1 promoter in response to arsenate As (V) which can be adapted to study the kinetics of transcriptional regulation of any gene in response to biotic or environmental stimuli measuring Luciferase activity in Arabidopsis thaliana.
0 Q&A 9589 Views May 5, 2013
Transient gene expression via biolistic particle delivery is a widely used technique for gene functional analysis in plants. In this protocol we describe a modified single-cell transient expression assay through transformation with a particle inflow gun of the model PDS-1000/He system (Bio-Rad). This assay was originally optimized for analyzing cell death activity and disease resistance function of the barley MLA (mildew locus A) disease resistance proteins against the powdery mildew fungus, which can be further adopted for other purposes for other types of plant proteins and in some other plant species, including Arabidopsis thaliana.



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