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 SCFCOI1 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.
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.
(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.
Table 1.
Transformation efficiency of Y2H screenings using EMPTY, TPL-N and NINJA as baits.
To ensure a full screening coverage of the A. thaliana Y2H cDNA library, screening of at least 1 x 106 yeast colonies is advised [43]. Titers are based on the numbers of colony forming units on control plates as illustrated for the TPL-N screen in S1 Fig.
Table 2.
Sanger sequencing of isolated NINJA and TPL-N preys.
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).
Table 3.
Signal-to-noise ratio of the FPKM values of NINJA and EMPTY Y2H-seq screenings.
Genes with SNRNINJA/EMPTY>7.2 were retained, listed and ranked from high to low SNR. Flagged genes are italicized. Previously reported interactors of NINJA are indicated in bold. Potential interactors that were tested for binary interaction in further validation assays are underlined.
Table 4.
Signal–to-noise ratio of the FPKM values of TPL-N and EMPTY Y2H-seq screenings.
Genes with SNRTPL-N/EMPTY>6 were retained, listed and ranked from high to low SNR. Flagged genes are italicized. Previously reported interactors of TPL are indicated in bold. Potential interactors that were tested for binary interaction in further validation assays are underlined. A ‘Y’ in bold font indicates the presence of an EAR domain in the wrong frame or in an untranslated region of the gene.
Fig 7.
NGS coverage of the N-TPL interactors using cutoff of SNRN-TPL/EMPTY>6 and FPKMN-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 SNRN-TPL/EMPTY>6 and FPKMN-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 SNRNINJA/EMPTY>7.2 and FPKMNINJA 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 SNRNINJA/EMPTY>7.2 and FPKMNINJA<100 values. * indicates a truncated version of the protein, as it was present in the Y2H cDNA library.