Experiment Summary

CytoTrap two-hybrid system provides an alternate strategy to detect protein-protein interactions in yeast. In this system, bait protein is fused with human son of sevenless (hSos) protein, and a cDNA library or prey protein is expressed by fusion with myristoylation signal which anchors the prey fusion protein to yeast cell membrane. Protein interaction between bait and prey proteins recruits the hSos protein to the cell membrane, where hSos activates the Ras signaling pathway, leading to the survival of temperature-sensitive Saccharomyces cerevisiae (S. cerevisiae) strain cdc25H at 36 °C. In the CytoTrap two-hybrid system, detection of protein interaction occurs in the cytoplasm near cell membrane and is not dependent on transcription activation of reporter genes. Hence, the system is particularly useful for identifying interaction partners of transcription factors and proteins that need post-translational modification in the cytoplasm, which could not be used as bait proteins in conventional transactivation-based yeast two-hybrid systems. Here we describe the construction of a cDNA library from the model plant Arabidopsis and a procedure for screening interaction proteins of AtSR1/CAMTA3, a Ca2+/CaM-regulated transcription factor from this library. This procedure could be adapted to identify interacting partners of interested proteins from other organisms.

Materials and Methods

1. XL10-Gold Kanr Ultracompetent cells
2. RNeasy plant mini kit
3. Oligotex mRNA mini kit
4. CytoTrap two-hybrid system
5. YeastmakerTM yeast transformation system 2
6. AccuScript
7. RNase H
8. DNA polymerase I
9. UltraPureTM phenol: chloroform: isoamyl alcohol (25:24:1, v/v)
10. T4 ligase
11. T4 polynucleotide kinase
12. Xho I
13. Acid-washed glass beads
14. TritonTM X-100
15. Sodium dodecyl sulfate (SDS)
16. Sodium chloride (NaCl)
17. Tris
18. EDTA
19. Yeast extract
20. Peptone
21. Dextrose
22. Adenine sulfate
23. Yeast nitrogen base without amino acids
24. DO supplement -His/-Leu/-Trp/-Ura
25. Histidine
26. Tryptophan
27. Galactose
28. Raffinose
29. Yeast lysis solution
30. YPDA/YPAD
31. SC/-LU (glucose)
32. SC/-LU (galactose)
33. 10x STE buffer

Equipment

1. Replica plating mold
2. RNase-free microcentrifuge tube
3. Microcentrifuge
4. Shaker
5. Incubator
6. 150-mm plate
7. PCR thermocycler

Procedure

1. Constructing and testing bait-hSos fusion protein

1. DNA encoding bait protein is cloned into the pSos vector (plasmid map: http://www.chem-agilent.com/pdf/strata/217438.pdf) by the conventional enzyme-ligation method. The plasmid construct is confirmed by DNA sequencing to ensure the correct reading frame between hSos and bait.

2. hSos-bait should be tested for auto-activation or interact with the myristylation signal prior to the CytoTrap two-hybrid screen.

a) The pSos bait plasmid needs to be co-transformed into yeast host cell cdc25H with negative control either pMyr or pMyr-Lamin C using YeastmakerTM Yeast Transformation System 2.

b) All the yeast culture procedures need to be conducted at room temperature (22-25 °C). After co-transformation, yeast cells are spread on synthetic media plate without leucine and uracil (SC/-LU) containing glucose, and incubate at room temperature for 4-5 days to allow yeast colonies to grow.

c) Six independent colonies are picked and placed on SC/-LU medium plate containing galactose, and the plates are kept at room temperature for 1 h, and then transferred to 36 °C for 3 to 6 days for yeast growth.

2. Generating a CytoTrap two-hybrid cDNA library

CytoTrap two-hybrid cDNA libraries can be constructed using mRNA from various organisms. Here we use the mRNA from the model plant Arabidopsis as an example. Briefly, mRNA is extracted and purified from leaf tissue of wild-type Arabidopsis plant (Col-0), then first-strand cDNA is synthesized using a hybrid oligo (dT) linker-primer that contains an XhoI restriction site and a high-fidelity reverse transcriptase. 5-methyl dCTP is used in place of regular dCTP during the synthesis of first strand cDNA, and the methylated XhoI sites in the resulting cDNA will be resilient to XhoI digestion. The second-strand cDNA is synthesized using DNA polymerase I. An adapter containing a cohesive EcoRI end and a blunt end is linked to the polished double stranded cDNA, and then the cDNA was digested with XhoI. The resulting cDNA containing EcoRI at 5’ end and XhoI at 3’ end is ligated into pMyr vector pre-cut by EcoRI and XhoI, so the cDNA is inserted in a sense expressional orientation driven by the Gal1 promoter (PGAL1). The cDNA library carried in pMyr plasmid (plasmid map: http://www.chem-agilent.com/pdf/strata/217438.pdf) is then transformed into high efficiency E. coli XL10-Gold Kan cells for propagation and storage. The detailed procedure is described below.

cDNA synthesis:

1. mRNA purification. Leaf samples (~2 g) from 4-week-old Arabidopsis Col-0 plants are harvested, and total RNA is extracted from the leaf tissues using RNeasy Plant Mini Kit. Poly (A) mRNA is then purified from 1 mg of total RNA using Oligotex mRNA Mini Kit for cDNA synthesis (Zhang et al., 2014).

2. First-strand cDNA synthesis is carried out using high fidelity reverse transcriptase AccuScript.

a) Add the following reagents to an RNase-free microcentrifuge tube:

10x first-strand buffer: 5 μl

First-strand methyl nucleotide mixture: 3 μl

Linker-primer (1.4 μg/μl): 2 μl

RNase Block Ribonuclease Inhibitor (40 U/μl): 1 μl

mRNA: Equal to 5 μg

DEPC-treated water: Bring volume to 50 μl

b) Incubate the reaction at room temperature for 10 min to allow primer to anneal to template.

c) Add 3 μl of AccuScript reverse transcriptase to the reaction and incubate the reaction at 42 °C for 2 h.

3. Second-strand cDNA synthesis

a) Put the first-strand cDNA reaction tube on ice, and add the following reagents to the tube:

10x second-strand buffer: 20 μl

Second-strand dNTP mixture: 6 μl

Sterile ddH₂O: 139 μl

RNase H (5 U/μl): 1 μl

DNA polymerase I (9.0 U/μl): 11 μl

b) Mix the reaction gently and spin down the reaction.

c) Incubate at 16 °C for 3 h, and then put the tube on ice. The resulting double-strand cDNA (ds cDNA) should be ready for adapter ligation.

4. Blunting cDNA termini and ligating the EcoR I adapter

5. Phosphorylating the EcoRI end

6. Purify ds cDNA with CHROMA SPINTM+TE-400 columns. (Procedures are performed at room temperature if not specified.)

7. Ligating cDNA into pMyr vector and transform into E. coli.

C. Screening a CytoTrap two-hybrid cDNA library

The bait and cDNA library plasmids are co-transformed into temperature sensitive host strain cdc25H. The transformation reaction is spread on SC/-LU plates containing glucose at room temperature to allow positive transformants to grow, and then the transformants are replica plated to SC/-LU plates containing galactose and incubated at 36 °C to select clones which expressing the bait and a potential interaction partner genes. The positive clones are further analyzed to confirm the bait-specific protein interaction. The flowchart of library screening is shown in Figure 1.

1. Co-transformation of bait and cDNA library plasmids into host strain cdc25H. This step is conducted as suggested by YeastmakerTM Yeast Transformation System 2 manual except that all the yeast culture steps must be conducted at room temperature unless otherwise specified to reduce rate of revert mutation of cdc25H. The transformation reactions are spread on SC/-LU plates (150-mm) containing glucose and kept at room temperature for 3 to 4 days until colonies grow to 2-3 mm in diameter. It is ideal to have ~3,000 colonies on each plate, and it is necessary to screen at least 1 million colonies.

2. Replica plate the colonies grown up to SC/-LU plates (150-mm) containing galactose, and leave the plates at room temperature for one hour before moving to the selective temperature of 36 °C for incubation of 3 to 6 days. Colonies containing putative interacting partners and reverting mutants will grow up and it is necessary to pick up the colonies when they are 2-3 mm in diameter during selection, and not wait to pick up at the end of selection.

3. Pick up colonies grown up on SC/-LU plates containing galactose at 36 °C, and dissolve the colonies in 25 μl sterile dH₂O. Dot 5 μl of yeast suspension to four plates: Two SC/-LU containing glucose and two SC/-LU containing galactose. Incubate one SC/-LU plate containing glucose and one SC/LU plate containing galactose at room temperature, and the other two plates at 36 °C.

Fig. 1 CytoTrap two-hybrid library screening procedure.

D. Analyze positive interaction candidates

The primary positive candidates from library screen need to be further tested to see whether the interaction is bait-specific and nature of the prey genes need to be identified for functional analysis.

1. Isolate prey plasmids from positive candidates.
2. Yeast co-transformation to test bait-specific protein-protein interaction.