TARGET

All about the experimental technique called TARGET (Transient Assay Reporting Genome wide Effect of Transcription Factors)

co-developed with Bastiaan Bargmann at NYU-Biology.

You can order the TARGET plasmid pBeaconRFP-GR --> [here]

Vector NTI file here

Plant protoplasts are transfected with a plasmid (pBeaconRFP_GR) that expresses the TF-of-interest fused to GR, which allows the controlled entry of the chimeric GR–TF into the nucleus by addition of the GR–ligand dexamethasone (DEX; Schena and Yamamoto,1988). In addition, the vector contains a separate expression cassette with a positive fluorescent selection marker (red fluorescent protein; RFP) which enables fluorescence-activated cell sorting (FACS) of successfully transformed protoplasts (Bargmann and Birnbaum, 2009). This purification step allows reliable qPCR or transcriptomic analysis of multiple independent transfections, which would otherwise be hampered by the presence of a population of untransformed cells that varies from experiment to experiment. Lastly, the effect of target gene induction by DEX treatment is measured in the presence or absence of the translation inhibitor cycloheximide (CHX), allowing for the distinction of direct (Blue in Figure below) and indirect (Red in Figure below) target genes of the TF under study.

Design Principles (Probing Gene Regulatory Networks as in "electronics")

Technically

References using the system:

**** First paper describing the technique applied on ABI3 as a proof of concept.

Bargmann BO, Marshall-Colon A, Efroni I, Ruffel S, Birnbaum KD, Coruzzi GM, Krouk G

TARGET: A Transient Transformation System for Genome-Wide Transcription Factor Target Discovery.

Mol Plant. 2013 May;6(3):978-80.

paper Studying HRS1 a highly Nitrate responsive GARP TF.

Medici A, Marshall-Colon A, Ronzier E, Szponarski W, Wang R, Gojon A, Crawford NM, Ruffel S, Coruzzi GM, Krouk G

AtNIGT1/HRS1 integrates nitrate and phosphate signals at the Arabidopsis root tip.

Nat Commun. 2015 Feb 27;6:6274.

paper Studying bZIP1 a Nitrogen related TF controlling N metabolism.

Para A, Li Y, Marshall-Colón A, Varala K, Francoeur NJ, Moran TM, Edwards MB, Hackley C, Bargmann BOR, Birnbaum KD, McCombie WR, Krouk G, Coruzzi G

"Hit-and-run" transcriptional control by bZIP1 mediates rapid nutrient signaling in Arabidopsis.

Proc Natl Acad Sci U S A. (2014) June 23, doi:10.1073/pnas.1404657111