A user-friendly platform for yeast two-hybrid library screening using next generation sequencing

Abstract

Yeast two-hybrid (Y2H) is a well-established genetics-based system that uses yeast to selectively display binary protein-protein interactions (PPIs). To meet the current need to unravel complex PPI networks, several adaptations have been made to establish medium- to high-throughput Y2H screening platforms, with several having successfully incorporated the use of the next-generation sequencing (NGS) technology to increase the scale and sensitivity of the method. However, these have been to date mainly restricted to the use of fully annotated custom-made open reading frame (ORF) libraries and subject to complex downstream data processing. Here, a streamlined Y2H library screening strategy, based on integration of Y2H with NGS, called Y2H-seq, was developed, which allows efficient and reliable screening of Y2H cDNA libraries. To generate proof of concept, the method was applied to screen for interaction partners of two key components of the jasmonate signaling machinery in the model plant Arabidopsis thaliana, resulting in the identification of several previously reported as well as hitherto unknown interactors. Our Y2H-seq method offers a user-friendly, specific and sensitive screening method that allows identification of PPIs without prior knowledge of the organism's ORFs, thereby extending the method to organisms of which the genome has not entirely been annotated yet. The quantitative NGS readout allows to increase genome coverage, thereby overcoming some of the bottlenecks of current Y2H technologies, which will further strengthen the value of the Y2H technology as a discovery platform.

Fig 1. Function of TOPLESS and NINJA in JA signaling in A. thaliana.

(A) In the absence of JAs, bHLH-type MYC TFs interact with the Jas domain of JAZ proteins that in turn interact with NINJA via their ZIM domain. The EAR motif of NINJA is essential for recruitment of the TPL co-repressors through the TPL domain (TPD). (B) In the presence of JA-Ile, JAZ proteins interact with the ubiquitin E3 ligase SCF^COI1 complex, leading to the proteasomal degradation of JAZs and consequent release of the NINJA–TPL complex from the MYC TFs, which leads to the transcriptional activation of JA-responsive genes by de-repressed MYC TFs.

Fig 2. Y2H-seq workflow.

The first checkpoint involves analysis of bait functionality through a Y2H assay with known interactors (preys). Alternatively, immunoblot analysis can be carried out in checkpoint 1 to validate protein expression of the fusion proteins in case no interactors are known of a particular bait prior to the screen. Subsequently, the actual Y2H screening with the functional bait is carried out by supertransformation of the bait yeast strain with the prey cDNA library. In checkpoint 2, the plasmids are extracted and Sanger sequenced for some of the colonies obtained on the selective plates. Next, pooling of all colonies on the plates is carried out, all plasmids from the pool are isolated in a single extraction and the inserts of the plasmids are amplified by PCR. In checkpoint 3, qPCR is performed to verify for enrichment of expected preys with a particular bait Y2H-seq PCR mix. For that, semi-quantitative qPCR analysis is carried out relative to the PCR product of a screen with an empty bait vector or the cDNA library itself. Finally, NGS and data analysis is performed to obtain a final list of putative interactors of the interested bait proteins.

Fig 3. Y2H of the NINJA and TPL-N bait proteins with positive and negative control prey proteins.

Y2H analysis of NINJA and TPL-N baits, fused to the DBD, and preys, fused to the AD, grown for 2 days on selective medium Synthetic Defined (SD)-Leu-Trp-His (-3). Transformed PJ69-4α yeast strains were also grown for 2 days on SD-Leu-Trp (-2) medium to confirm growth capacity. Direct interaction was confirmed between (A) NINJA and PPD1, JAZ1, JAZ2 and JAZ4, and (B) TPL-N and auxin/indole-3-acetic acid 17 (IAA17) and NINJA.

Fig 4. Y2H-seq selective growth.

(A-C) The EMPTY (A), NINJA (B), and TPL-N (C) Y2H-seq screenings were performed on selective SD-Leu-Trp-His + 5mM 3-AT plates. Per bait, a single transformation reaction is carried out, after which each transformation mix is plated on 3 plates for selection. A representative plate for each bait is shown.

Fig 5. qPCR assessment of the NINJA Y2H-seq screen.

JAZ1, JAZ2, JAZ12, TIFY8 and PPD1 were overrepresented in the PCR products of the NINJA screening compared to the PCR products of the A. thaliana cDNA library (Library). Statistical significance was determined by a Student’s t-test (***P<0.001).

Fig 6. qPCR assessment of the TPL-N Y2H-seq screening.

Only IAA30 was overrepresented in the PCR products of the TPL-N screening compared to the A. thaliana cDNA library (Library) cDNA insert amplicons. Statistical significance was determined by a Student’s t-test (***P<0.001).

Fig 7. NGS coverage of the N-TPL interactors using cutoff of SNR_N-TPL/EMPTY>6 and FPKM_N-TPL>100.

The depth of the NGS coverage for each gene, visualized by the coverage track, is aligned to the gene model. Coding sequences are represented by thick black boxes, 5’ and 3’ untranslated regions by thin black boxes and introns by thin black lines, respectively. The grey boxes in the gene model correspond to the EAR motif.

Fig 8. NGS coverage of the N-TPL interactors using cutoff of SNR_N-TPL/EMPTY>6 and FPKM_N-TPL>100.

The depth of the NGS coverage for each gene, visualized by the coverage track, is aligned to the gene model. Coding sequences are represented by thick black boxes, 5’ and 3’ untranslated regions by thin black boxes and introns by thin black lines, respectively. The grey boxes in the gene model correspond to the EAR motif.

Fig 9. Y2H analysis of potential interaction partners of NINJA.

Y2H analysis of NINJA, fused to the DBD, and potential interaction partners, fused to the AD of the GAL4 TF (in the pDEST22 vector), grown on selective medium SD-Leu-Trp-His (-3). Transformed PJ69-4α yeast strains were also grown on SD-Leu-Trp (-2) medium confirm growth capacity. No direct interactions could be observed for potential preys identified in the Y2H-seq with scoring values below the threshold of SNR_NINJA/EMPTY>7.2 and FPKM_NINJA values>100.

Fig 10. Binary Y2H validation of potential interaction partners of N-TPL.

Y2H analysis of N-TPL, fused to the DBD, and potential interaction partners, fused to the AD of the GAL4 TF (in the pDEST22 vector), grown on selective medium SD-Leu-Trp-His (-3). Co-transformed PJ69-4α yeast strains were also grown on SD-Leu-Trp (-2) medium to confirm growth capacity. No direct interaction was confirmed between ATCKA2 encoded by AT3G5000 and N-TPL, in contrast to the interactions with all other potential interactors selected from the list with a threshold of SNR_NINJA/EMPTY>7.2 and FPKM_NINJA<100 values. * indicates a truncated version of the protein, as it was present in the Y2H cDNA library.