Yeast two hybrid screen of a pollen tube cDNA library

A screen for putative protein binding partners of AtARO1 was carried out with a cDNA library made from germinated pollen tubes (see section 2.20.1) using AtARO1 as bait. After amplification of the pollen tube cDNA and a human control cDNA by long distance PCR and subsequent purification, the integrity and average size of both probes was determined on an agarose gel. With 250 to 500 bp, the average size of cDNA fragments from pollen tubes was slightly lower than in human control cDNA, where most fragments ranged from 250 to 750 bp. The pollen tube cDNA and the vector pGADT7-Rec were co-transformed into yeast strain AH109. The transformation efficiency was 0.8 x 10⁶ transformants for 3 µg vector used and therefore slightly lower than the expected 1x 10⁶ transformants/3 µg vector. Accordingly, the library titer also was lower than the expected number of cells. Only 1.7 x 10⁷ colonies/ml instead of the expected ≥2.0 x 10⁷ colonies/ml were obtained. After mating the bait (pGBKT7-ARO1) with the host cDNA library (mating efficiency: 0,7%), diploids were plated onto SD/-Leu/-Trp/-His low stringency selection plates. 754 diploid clones able to activate the HIS3 selective marker and therefore surviving this low stringency selection, were picked and spotted again on SD/-Leu/-Trp/-His (Fig. 3.32 A, B) as well as two times on SD/-Leu/-Trp/-His/-Ade (4x drop out medium, 4xDO; Fig. 3.32 C, D) selective plates. Colonies surviving the high stringency selection

four replica clones four replica clones

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on 4xDO medium, able to activate the HIS3 and ADE2 reporter genes, were tested for activation of a third reporter, lacZ with an x-Gal assay (Fig. 3.32E, F).

Fig. 3.32. Yeast two hybrid screening using AtARO1 as bait and a cDNA library from germinated pollen tubes.

Diploids were tested for activation of reporter genes with low stringency (HIS3 reporter gene) and high stringency (HIS3 and ADE2 reporter genes) selection plates and with an x-Gal assay (HIS3, ADE2 and lacZ reporter genes).

Two representative plates each are shown in this figure. A1, A12, H1 and H12 represent positions of clones on each plate (compare to Table 3.7). (A, B) Selection for HIS3 on low stringency medium. (C, D) Selection for HIS3 and ADE2 on high stringency medium. (E, F) Selection for the third reporter, lacZ, using an x-Gal assay. Blue color indicates activation of lacZ.

As a result, 59 colonies showed a range of weak to strong activation of the lacZ reporter gene and were classified into 4 groups according to corresponding X-Gal activity: 0 = very weak staining, + = weak staining, ++ = intermediate staining, +++ = strong staining and - = negative control (Table 3.7).

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Table 3.7 Very weak to strong activation of lacZ on 4xDO plates after the x-Gal assay. Colonies growing on 4xDO medium additionally show activation of HIS3 and ADE2 reporter genes.

Plate (1) 1 2 3 4 5 6 7 8 9 10 11 12 Plate (5) 1 2 3 4 5 6 7 8 9 10 11 12

On all plates, the negative and positive controls were spotted on positions H11 and H12 respectively, except for plate (1), where they were spotted by hand next to the full plate replica print. As shown in Table 3.7, 17 colonies showed a very weak staining and 29 colonies a weak staining, while intermediate staining could be observed in 10 colonies. A strong activation of the lacZ reporter, similar to that observed in positive controls, was only visible in 3 out of 754 analyzed clones. In order to identify the putative interacting proteins/protein fragments responsible for the activation of the reporter genes, 42 cDNAs of clones displaying a weak, intermediate or strong staining were sequenced, either after isolating plasmids and subsequent transformation in E. coli cells, or after direct amplification of the cDNA from the pGADT7 prey vector via PCR.

After sequencing the inserts from all 42 clones, the identified cDNA sequences were used for BLASTN searches of the Arabidopsis thaliana genome in the non-redundant nucleotide collection (nr/nt) at NCBI. 30 sequences matched to PGA3 (At3g07820), a gene encoding polygalacturonase 3, which is highly expressed in germinating pollen and growing pollen tubes (Torki et al., 1999). The remaining cDNA fragments represented three more cDNAs for polygalacturonases (At5g48140 and At2g23900), each matching to two of the sequenced fragments, and two other genes. One of these genes encodes a SKS13 copper ion binding protein (4 clones, At3g13400), the other a member of the pectin esterase

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family (1 clone, At5g07430). Two cDNAs gave no significant hit in the database, due to their length (132 bp and 64 bp). All cDNA clones identified in this screen are listed in the appendix (Table 7.2).

Although polygalacturonases are known to be secreted (Niogret et al., 1991), and therefore an interaction with intracellular AtARO1 is highly unlikely, plasmids from one representative clone for each gene identified were retransformed into yeast strain AH109 and re-mated with the bait pGBKT7-ARO1, as well as with the empty pGBKT7 vector in order to examine possible auto-activation of the fragments. Positive and negative controls from the kit were included in the tests. All cDNA clones tested showed an activation of both reporter genes for nutritional selection ADE2 and HIS3 (Fig. 3.33).

As the cDNA encoded peptides were able to activate the reporter genes independently of the bait protein, all cDNA fragments must be considered as false positives.

Fig. 3.33. Verification of protein interactions found in the yeast two-hybrid screen using the pollen tube cDNA library. One representative cDNA clone of each gene identified as a putative positive was used to retest the activation of reporter genes on 4XDO medium. Each candidate was mated with the bait, AtARO1-BD, and with an empty pGBKT7 vector. Growth of diploids containing the empty pGBKT7 indicates auto-activation of candidate genes and thus false-positives. (A) All candidate genes showed activation of the reporter genes HIS3 and ADE2 with both, the AtARO1 bait and the empty vector control. (B) Table describing performed matings. Yeast cells were mated and diploids spotted onto 4xDO selective media as indicated in the table. A1, AtARO1; BD, binding domain; 0-BD, empty pGBKT7 vector; RecT, SV40 large T-antigen; 53, murine p53; lam, human lamin C.

A1-BD x 6-A-2 A1-BD x 6-A-3 A1-BD x 6-E-7 A1-BD x 8-A-4 A1-BD x 8-D-4 0-BD x 6-A-2 0-BD x 6-A-3 0-BD x 6-E-7 0-BD x 8-A-4 0-BD x 8-D-4 BD-lam x AD-RecT BD-53 x AD-RecT

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