A High-Throughput Screening System for Arabidopsis Transcription Factors and Its Application to Med25-Dependent Transcriptional Regulation

Abstract

The activities of transcription factors (TFs) require interactions with specific DNA sequences and other regulatory proteins. To detect such interactions in Arabidopsis, we developed a high-throughput screening system with a Gateway-compatible Gal4-AD-TF library of 1589 Arabidopsis TFs, which can be easily screened by mating-based yeast-one-hybrid (Y1H) and yeast-two-hybrid (Y2H) methods. The efficiency of the system was validated by examining two well-characterized TF-DNA and TF-protein interactions: the CHE-CCA1 promoter interaction by Y1H and NPR1-TGAs interactions by Y2H. We used this system to identify eight TFs that interact with a Mediator subunit, Med25, a key regulator in JA signaling. We identified five TFs that interacted with the GCC-box cis-element in the promoter of PDF1.2, a downstream gene of Med25. We found that three of these TFs, all from the AP2-EREBP family, interact directly both with Med25 and the GCC-box of PDF1.2, suggesting that Med25 regulates PDF1.2 expression through these three TFs. These results demonstrate that this high-throughput Y1H/Y2H screening system is an efficient tool for studying transcriptional regulation networks in Arabidopsis. This system will be available for other Arabidopsis researchers, and thus it provides a vital resource for the Arabidopsis community.

The Arabidopsis Mediator subunit MED16 regulates iron homeostasis by associating with EIN3/EIL1 through subunit MED25

“…B€ ackstr€ om et al (2007) proposed that Mediator recruits and works with species-specific transcription factors through either newly evolved subunits or subunits with one conserved Mediator-interacting domain and one species-specific regulator-interacting domain. This proposal is supported by recent research that the ACID domain of MED25 interacts with many transcription factors in Arabidopsis (Elfving et al, 2011;Ou et al, 2011;Zhu et al, 2011;C ßevik et al, 2012) and our results that MED25 interacts with YID1/MED16 through its conserved N-terminal vWF-A domain. We noticed, however, that neither YID1/MED16 nor MED25 was induced by iron deficiency ( Figure S6), which raised the question of how the Mediator complex was assembled under iron-deficient conditions.…”

“…That MED25 interacts with YID1/MED16 in vivo suggests that MED25 is probably a tail subunit. The interaction of MED25 with different transcription factors regulates multiple physiological processes (Elfving et al, 2011;Ou et al, 2011;Chen et al, 2012), because tail subunits usually interact with transcription factors through conformational changes (Casamassimi and Napoli, 2007;Toth-Petroczy et al, 2008;Meyer and Fu, 2012). The N-terminal vWF-A domain of MED25 was conserved among metazoans, Arabidopsis and other plant species.…”

“…MED25 interacts with transcription factors in the JA signaling pathway (Ou et al, 2011;C ßevik et al, 2012;Chen et al, 2012), with med25 mutants showing JA-mediated disease resistance phenotypes (Kidd et al, 2009). Therefore, the interaction between MED25 and the two crucial components (EIN3/EIL1) in the ethylene signaling pathway may affect ethylene-mediated plant growth (Alonso et al, 2003;Guo and Ecker, 2003;Potuschak et al, 2003).…”

“…However, we did not find any transcription factor that interacted with YID1/MED16 in a yeasttwo-hybrid assay using the Arabidopsis transcription factor collection (Ou et al, 2011). To test if YID1/MED16 interacted indirectly with transcription factors through other Mediator subunits, we examined the interaction between YID1/MED16 and other subunits in the tail module and the nearby region.…”

“…MED25 is an important Mediator subunit having multiple functions in the defense response and organ development (Kidd et al, 2009;Xu and Li, 2011;Chen et al, 2012). Several transcription factors in various pathways have been reported to interact with MED25 (Ou et al, 2011;C ßevik et al, 2012). MED25 contains three domains: a von Willebrand type A (vWF-A) domain crucial for Mediator binding; an ACID domain interacting with transcription factors; and a Q-rich domain (Cerdan and Chory, 2003;Mittler et al, 2003;B€ ackstr€ om et al, 2007).…”