Enhanced Y1H assays for Arabidopsis

Gaudinier, Allison; Zhang, Lifang; Reece‐Hoyes, John S.; Taylor-Teeples, Mallorie; Pu, Li; Liu, Zhijie; Breton, Ghislaı̀n; Pruneda‐Paz, Jose L.; Kim, Dahae; Kay, Steve A.; Walhout, Albertha J.M.; Ware, Doreen; Brady, Siobhán M.

doi:10.1038/nmeth.1750

Cited by 115 publications

(151 citation statements)

References 13 publications

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“…The NIA1 109-bp DNA fragment (16) and the NRT2.1 150-bp DNA fragment (49) were used for screening the transcription factor library as described (59,72). Bait strains were obtained by homologous recombination of pLacZi (Clontech) bait vectors in the yeast YM4271 (Clontech).…”

Section: Methodsmentioning

confidence: 99%

Nitrate foraging by Arabidopsis roots is mediated by the transcription factor TCP20 through the systemic signaling pathway

Guan

Wang

Nacry

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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To compete for nutrients in diverse soil microenvironments, plants proliferate lateral roots preferentially in nutrient-rich zones. For nitrate, root foraging involves local and systemic signaling; however, little is known about the genes that function in the systemic signaling pathway. By using nitrate enhancer DNA to screen a library of Arabidopsis transcription factors in the yeast one-hybrid system, the transcription factor gene TEOSINTE BRANCHED1/CYCLO-IDEA/PROLIFERATING CELL FACTOR1-20 (TCP20) was identified. TCP20, which belongs to an ancient, plant-specific gene family that regulates shoot, flower, and embryo development, was implicated in nitrate signaling by its ability to bind DNA in more than 100 nitrate-regulated genes. Analysis of insertion mutants of TCP20 showed that they had normal primary and lateral root growth on homogenous nitrate media but were impaired in preferential lateral root growth (root foraging) on heterogeneous media in split-root plates. Inhibition of preferential lateral root growth was still evident in the mutants even when ammonium was uniformly present in the media, indicating that the TCP20 response was to nitrate. Comparison of tcp20 mutants with those of nlp7 mutants, which are defective in local control of root growth but not in the root-foraging response, indicated that TCP20 function is independent of and distinct from NLP7 function. Further analysis showed that tcp20 mutants lack systemic control of root growth regardless of the local nitrate concentrations. These results indicate that TCP20 plays a key role in the systemic signaling pathway that directs nitrate foraging by Arabidopsis roots. TCP20 | nitrate | root foraging | systemic signaling | Arabidopsis

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Section: Methodsmentioning

confidence: 99%

Nitrate foraging by Arabidopsis roots is mediated by the transcription factor TCP20 through the systemic signaling pathway

Guan

Wang

Nacry

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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208

188

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“…In the Y1H system, reporter gene expression is activated when a TF interacts with the DNA bait. A root TF collection (Gaudinier et al, 2011) containing >650 genes was fused to a transcription activation domain (AD) and used as a prey. A total of 17 root stele-expressed TFs were found to bind to the LAX3 promoter sequence (Table S1).…”

Section: Identification Of Putative Lax3 Transcriptional Regulatorsmentioning

confidence: 99%

“…The experiments were performed as described in Gaudinier et al (2011). Interactions were called for TFs that activated at least one reporter assay.…”

Section: Yeast One-hybrid Assaysmentioning

confidence: 99%

“…The correct clone was recombined with pMW2 (HIS3 reporter vector) and pMW3 (lacZ reporter vector) (Brady et al, 2011) using LR clonase II. Because the steleexpressed transcription factor collection (Gaudinier et al, 2011) did not contain LBD29 transcription factor, a construct was generated to clone the LBD29 coding sequence into the pDest-AD-2 µ plasmid. The cDNA sequence of LBD29 was amplified (5′-CACCATGACTAGTTCCAGCTC-3′ and 5′-CGAGAAGGAGATGTAGCCAAAATT-3′) and cloned into the pENTR-DTopo entry vector (Invitrogen).…”

Section: Cloning For Luciferase and Y1h Assaysmentioning

confidence: 99%

“…The cDNA sequence of LBD29 was amplified (5′-CACCATGACTAGTTCCAGCTC-3′ and 5′-CGAGAAGGAGATGTAGCCAAAATT-3′) and cloned into the pENTR-DTopo entry vector (Invitrogen). The entry vector was used in a gateway LR cloning reaction (LR clonase II; Invitrogen) with pDest-AD-2 µ (Gaudinier et al, 2011) to create a GAL4-activation domain fusion Y1H prey vector.…”

Section: Cloning For Luciferase and Y1h Assaysmentioning

confidence: 99%

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Lateral root emergence in Arabidopsis is dependent on transcription factor LBD29 regulating auxin influx carrier LAX3

et al. 2016

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Lateral root primordia (LRP) originate from pericycle stem cells located deep within parental root tissues. LRP emerge through overlying root tissues by inducing auxin-dependent cell separation and hydraulic changes in adjacent cells. The auxin-inducible auxin influx carrier LAX3 plays a key role concentrating this signal in cells overlying LRP. Delimiting LAX3 expression to two adjacent cell files overlying new LRP is crucial to ensure that auxin-regulated cell separation occurs solely along their shared walls. Multiscale modeling has predicted that this highly focused pattern of expression requires auxin to sequentially induce auxin efflux and influx carriers PIN3 and LAX3, respectively. Consistent with model predictions, we report that auxin-inducible LAX3 expression is regulated indirectly by AUXIN RESPONSE FACTOR 7 (ARF7). Yeast one-hybrid screens revealed that the LAX3 promoter is bound by the transcription factor LBD29, which is a direct target for regulation by ARF7. Disrupting auxin-inducible LBD29 expression or expressing an LBD29-SRDX transcriptional repressor phenocopied the lax3 mutant, resulting in delayed lateral root emergence. We conclude that sequential LBD29 and LAX3 induction by auxin is required to coordinate cell separation and organ emergence.

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High‐Throughput Yeast One‐Hybrid Screens Using a Cell Surface gLUC Reporter

Bonaldi

Kang

et al. 2019

CP Plant Biology

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Gene‐centered yeast one‐hybrid (Y1H) screens using arrayed genome‐wide transcription factor (TF) clone collections provide a simple and effective strategy to identify TF‐promoter interactions using a DNA fragment as bait. In an effort to improve the assay we recently developed a Y1H system that uses a cell surface Gaussia luciferase reporter (gLUC59). Compared to other available methods, this luciferase‐based strategy requires a shorter processing time, enhances the throughput and improves result analysis of gene‐centered Y1H screens. Here, we described the procedure to perform high‐throughput screens using this novel strategy, which involves a protocol for mating two haploid yeast strains carrying an arrayed TF clone collection and a promoter::gLUC59 reporter, respectively, and a protocol for analyzing gLUC59 activity in the resulting diploid cells. © 2019 by John Wiley & Sons, Inc.

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Enhanced Y1H assays for Arabidopsis

Cited by 115 publications

References 13 publications

Nitrate foraging by Arabidopsis roots is mediated by the transcription factor TCP20 through the systemic signaling pathway

Nitrate foraging by Arabidopsis roots is mediated by the transcription factor TCP20 through the systemic signaling pathway

Lateral root emergence in Arabidopsis is dependent on transcription factor LBD29 regulating auxin influx carrier LAX3

High‐Throughput Yeast One‐Hybrid Screens Using a Cell Surface gLUC Reporter

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