Name: unravelling the function of a bacterial effector from a non-cultivable plant pathogen using a yeast two-hybrid screen
Uploaded: 2017-01-20T13:00:00
Duration: 11 min 30 s
Description: Watch this Scientific Journal Video about Unravelling the Function of a Bacterial Effector from a Non-cultivable Plant Pathogen Using a Yeast Two-hybrid Screen at JoVE.com

We thank Christine Kerschbamer, Thomas Letschka, Sabine Oettl, Margot Raffeiner, and Florian Senoner from the Laimburg Research Centre and Mirelle Borges Dias Schnetzer from Dualsystems Biotech AG for technical support and Julia Strobl for proofreading the manuscript. This work was performed as part of the APPL2.0 project and was partially funded by the Autonomous Province of Bozen/Bolzano, Italy and the South Tyrolean Apple Consortium.

The authors declare that they have no competing financial interests.

In the Y2H screen, the potential effector protein is co-expressed with different hypothetical interacting proteins (step 7). Each growing yeast clone contains the bait but a (potentially) different interactor. The interacting proteins are encoded in a cDNA library that is cloned into prey plasmids. The bait and the prey plasmids each co-cistronically code for a part of a yeast transcription factor. In case of a physical interaction between the bait (effector) and a prey plasmid-encoded interactor, the two transcription factor parts (the DNA binding domain and the activation domain) are united, and the reporter gene expression is induced. The yeast is able to grow on histidine- and adenine-depleted SD selection plates. The kind of prey cDNA library is dependent upon the screened effector and must be chosen accordingly. The prey and bait vector, as well as the yeast strain, must be compatible. In this screen, a LexA DNA binding domain and the Gal4 activation domain were used as the compatible yeast transcription factor units. The cDNA library can be individually constructed, custom made, or commercially obtained. The preparation of the cDNA prey library is not part of this protocol. In this protocol, a self-constructed, normalized cDNA library from the RNA of Malus x domestica leaves was used and cloned into pGAD-HA41. The effector (bait)-expressing yeast strain was transformed with the prey library, and clones were selected on histidine- and adenine-depleted SD selection plates. To extract plasmid DNA from the yeast colonies, a column-based DNA plasmid mini kit for bacteria is recommended in combination with a mechanical disruption step using glass beads to improve yeast lysis (step 8). In this plasmid purification, the bait and the prey vector will be purified simultaneously in relatively low concentrations. However, the amount of plasmid DNA is enough to identify the interaction partner through sequencing without prior plasmid propagation (step 8.4). As an alternative, the DNA purified in step 8.4 can be transformed into competent E. coli and selected with prey vector-mediated antibiotic resistance (e.g., ampicillin the in case of pGAD-HA). The selected E. coli colonies carry only the pGAD-HA library plasmid, and high-yield plasmid purification can be performed with these clones.
The successful transformation of pLexA-N and pGAD-HA constructs complements the tryptophan and leucine auxotrophy of NMY51. An interaction between the effector and a protein (encoded on pGAD-HA) leads to the activation of the reporter system in NMY51 strains. In the case of self-activation, NMY51 transformed with the effector expressing pLexA-N in combination with the empty library vector pGAD-HA grows on selective plates lacking trp-leu-his-ade, due to the unwanted activation of the NMY51 reporter system40. The self-activation can be weak and can occur in the absence of trp-leu-his, or the activation can be strong and characterized by growth on trp-leu-his-ade plates41. Self-activation can cause a massive background of false-positive clones in the actual Y2H screen. To avoid this, a self-activation test with the bait must be conducted, in which the effector-expressing vector is co-transformed with the empty library vector. In this experimental setting, reporter gene expression must not be induced. If self-activation (i.e., growth on selective plates) is visible in the test, the medium can be supplemented with different concentrations of 3-amino-1,2,4-triazole45 (3-AT). 3-AT is an inhibitor of imidazoleglycerol-phosphate dehydratase (HIS3), an enzyme important during histidine biosynthesis46. Supplementation with 3-AT can reduce the weak effects of self-activation during Y2H screens47,48. Different concentrations of 3-AT should be tested. In this protocol, 1 - 40 mM 3-AT were used. The lowest concentration that leads to the repression of self-activation should subsequently be used in the Y2H screen. The selective plates of the self-activation assay can only be evaluated if the SD-trp-leu plates of the co-transformation contain a sufficient number of clones. As an indication &#8805; 500 colonies per 90-mm (diameter) petri dish are sufficient. To determine the exact transfection efficiency, it is recommended to prepare serial dilutions of the co-transformed yeast and to spread them on SD-trp-leu plates.
To reduce self-activation, the effector can be cloned into pLexA-C, a bait expression vector that fuses the LexA tag to the C-terminus of the protein. The orientation of the Lex-A tag can attenuate self-activation24. However, it is not possible in every case to reduce or abolish self-activation. The formation of red or reddish colonies indicates a weak or false-positive interaction. The ade2 reporter gene of NMY51 is only activated when it comes to a protein-protein interaction, which in turn blocks the accumulation of a red dye in this yeast strain40. In the absence of an interaction, NMY51 are reddish, while colonies carrying strong interactors are white. The observation of the colony color on the selection plates thus provides a further important hint to judge if the respective colonies are true- or false-positive interactors.
It is eventually necessary to adapt or change certain assay settings with respect to the nature of the bait and the interaction characteristics. By now, a number of improvements and derivative techniques of the common Y2H have been established to allow for the analysis of rather difficult protein interactions in different host systems. A review by Stynen et al.49 addresses and describes different aspects of Y2H, its improvements, and adaptations, and thus provides useful information about how to choose the appropriate interaction assay.
Y2H screens and derived techniques have become widely used in different research fields, wherever identification of binary protein interactions is required. Even if critical controls are performed, such as self-activation tests of the bait and one-to-one re-transformations of the identified interacting proteins, the Y2H is prone to produce false results26,50,51. The yeast as a model is not suitable for all interaction studies. Yeast does not necessarily constitute a cellular environment that supports the appropriate post-translational modification and folding for every protein24. Furthermore, in the Y2H setting, the proteins are over-expressed, and their expression is not controlled by their natural promoter. The Y2H forces the proteinaceous interaction partners to the nucleus, which is not necessarily their natural subcellular destination. The native cellular circumstances of the interaction thus may not be reflected by the yeast and may lead to false-negative or false-positive results. Most Y2H are based on yeast auxotrophy complementation as a selective principle. This nutritional selection is characterized by high sensitivity, but at the cost of decreased selectivity compared to other (e.g., chromogenic reporter) assays49. If unreducible self-activation (see above) or other limitations occur for a certain effector, the Y2H is not an appropriate assay25. In Table 1 and Figure 1, the expected results of the self-activation test are given. The absence of real interactions (i.e., false negatives) can be caused by protein toxicity, incorrect translational protein processing, steric hindrance of the interaction, underrepresented interaction partners in the prey library, membrane localization of the bait, or missing components necessary for the interaction24.
It is recommended to de novo amplify the full-length gene of the identified interacting protein from cDNA, subclone it into pGAD-HA, and perform a one-to-one interaction assay by transforming the generated pGAD-HA construct into the bait-expressing NMY51 strain. This is necessary since the observed interactor present in the prey library might only be a fragment of a bigger protein. However, the information for the respective full-length gene is only accessible if considerable genomic and transcriptomic sequence data is available. The transformation protocol for the self-activation assay described here can be applied for such a one-to-one assay, and the interaction between the bait and the interactor must be reproducible.
Interactions identified in a Y2H screen must always be confirmed by another independent technique. This independent technique must be closer to the natural setting of the actual protein-protein interaction. In the case of the phytoplasmal effector ATP_00189, the interaction with the TCP transcription factors of Malus x domestica was verified in planta with bimolecular fluorescent complementation (BiFC) in Nicotiana benthamiana protoplasts28 (not part of this protocol). The effector protein ATP_00189 was derived from the plant pathogen P. mali. In planta BiFC was thus chosen to verify ATP_00189 interactions with the Malus x domestica transcription factors previously identified in the Y2H28. BiFC is a protein-protein interaction assay that does not require the subcellular translocation of the interacting proteins to the nucleus in order to activate the reporter system52. Furthermore, the in planta expression and modification machinery mimics the environment of the natural interaction between the plant bacterial effector in its plant host. However, a global screen using BiFC is not feasible.
There are several steps in the protocol that must be carefully addressed. When amplifying the gene of interest (step 4), it is important to rule out that primers bind to plant DNA. Thus, DNA from non-infected plants must be included as a negative control. The sequence of the gene of interest must not contain the EcoRI and SalI restriction sites that are used for the directional cloning of the insert. If the sequence does contain these sites, different restriction enzymes for cloning must be chosen. Yeast transformation is a central method during this protocol. Transfection efficiency depends on the competency of the yeast cells, their viability, the growth state, and the quality of the transfection reagent53,54. For the proposed protocol, it is highly recommended to use yeast from fresh plates not older than two weeks (kept at 4 &#176;C) when performing the transformations. Often, the growth of transformed yeast is delayed when selective liquid cultures are inoculated with colonies from old agar plates. It is also helpful to use a liquid starter culture as an inoculum for the actual experiments and not to use the yeast directly from the plate. Low efficiency when transfecting the prey into the bait-expressing yeast can lead to the underrepresentation of certain prey proteins (potential interactors) and can thus skew the whole screen. In this protocol, a yeast transfection efficiency of ~150,000 cfu/&#181;g transfected DNA in the Y2H worked well.
Knowing the flaws and drawbacks of the Y2H technique and interpreting the results in a critical and appropriate manner is indispensable to drawing the correct conclusions. Y2H assays and the derivatives have been used for many years and have undergone many improvements and adaptations with respect to the different bait characteristics, the subcellular localization of the interaction, the expected binding partners, and other factors that might be required for the interaction (see Y3H55,56,57). Recently, array-based Y2H screens have been developed that allow for automated, high-throughput analysis of numerous baits against millions of preys58. The future most likely lies in automation and high-throughput approaches to this assay to allow for the elucidation of complex signaling pathways and interactomes in different research fields.

Yeast two-hybrid screens (Y2H) were developed about 27 years ago1 and have since been widely used in various fields to determine specific protein-protein interactions2,3,4,5,6,7,8,9,10,11. The physical interaction of a bacterial effector with a host target protein is often the precondition for the functional manipulation of this target protein. Analyzing these interactions has moved to the focus of many different sectors of infection biology12,13,14,15,16,17,18. The principle of the Y2H screen is that the interaction of two proteins leads to the reconstitution of a functional transcription factor that drives the expression of reporter genes. The known effector (the &#34;bait&#34;) is translationally fused to the DNA binding domain (DBD) of the transcription factor, and the potential interaction partners (the &#34;prey&#34;) are fused to the activation domain (AD) of the respective transcription factor. Since the interacting partner is unknown, a library of potential interactors is cloned into the so called &#34;prey-library.&#34; Normally, this library is made by cloning cDNAs into an appropriate prey vector expression plasmid encoding the AD that is compatible with the bait-vector encoded DBD. In case of an interaction, the reconstituted transcription factors induce the expression of reporter genes, which generally enable the growth selection of yeast. The identification of interactors is achieved by sequencing the prey plasmid with plasmid-specific primers.
Bacteria have developed various strategies to manipulate and exploit their host&#39;s metabolism or to evade defense mechanisms and secrete effector molecules via different bacterial secretion systems19,20,21. Determining the binding partners of these bacterial effectors is thus the first step in identifying the manipulated pathway in the host. This leads to an improved understanding of the specific pathogenicity mechanisms.
Y2H screens are performed to identify bacterial effector interactions during phytoplasmoses, and often, Y2H is a crucial initial experiment for the further characterization of molecular pathogenicity mechanisms in phytoplasma research22,23. However, the method description in most publications is rather scarce, and these techniques are often outsourced to biotech companies. To draw attention to the feasibility of this method, this protocol provides step-by-step information to identify interaction partners of a bacterial effector molecule in its natural host.
Despite the wide use and the fast forward approach of Y2H screens, it must not be forgotten that certain interactions might not occur in the yeast system. This is due to the fact that the yeast cell is used as a kind of &#34;in vivo reaction vessel&#34; with certain biological limitations. Several authors have addressed the advantages and disadvantages of Y2H and its derivatives11,24,25,26,27. General considerations are, for example, that the yeast cell might not provide the appropriate gene expression, (post-)translational, or translocational conditions for the respective proteins being studied. This can lead to false-negative results in the screen. Positive interactions can in turn be artefacts and might not occur in the natural situation (i.e., in case of effectors in the appropriate host). It is thus indispensable to confirm the interactions from the heterologous yeast expression system with an independent interaction test in a more closely related biological system.
In this study, the binding partners of the effector ATP_00189 from the non-cultivable plant pathogen Candidatus Phytoplasma mali (P. mali) were identified. The results provide important insights into the molecular mechanisms underlying the symptom development of apple proliferation28, a disease that causes high economic losses in affected apple-growing regions in Europe29.

<table border="0" cellpadding="0" cellspacing="0" width="1514">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:565px;">Cetyltrimethylammonium bromide (CTAB)</td>
			<td style="width:211px;">Applichem</td>
			<td style="width:324px;">A6284</td>
			<td style="width:415px;">for DNA preparation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Trishydroxymethylaminomethane (Tris)</td>
			<td>Applichem</td>
			<td>A2264</td>
			<td>for media and buffer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sodiumchloride (NaCl)</td>
			<td>Applichem</td>
			<td>A2942</td>
			<td>for media and buffer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Ethylenediaminetetraacetic acid Disodium salt (NaEDTA)</td>
			<td>Applichem</td>
			<td>A2937</td>
			<td>for media and buffer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">N-Lauroylsarcosin sodium salt</td>
			<td>Applichem</td>
			<td>A7402</td>
			<td>for DNA preparation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">ammonium acetate&#160;</td>
			<td>Sigma-Aldrich (Fluka)</td>
			<td>9688</td>
			<td>for DNA preparation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">2-mercaptoethanol</td>
			<td>Applichem</td>
			<td>A1108</td>
			<td>for DNA preparation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Isopropanol (2-Propanol)</td>
			<td>Applichem</td>
			<td>A3928</td>
			<td>for DNA preparation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Ethanol</td>
			<td>Sigma-Aldrich (Fluka)</td>
			<td>51976</td>
			<td>for DNA preparation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">RNAse</td>
			<td>Applichem</td>
			<td>A2760</td>
			<td>for DNA preparation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Chloroform:Isoamyl 24:1</td>
			<td>Applichem</td>
			<td>A1935</td>
			<td>for DNA preparation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Proofreading Polymerase (e.g. iProof)</td>
			<td>Bio-Rad</td>
			<td>203433</td>
			<td>for PCR</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">EcoRI</td>
			<td>Thermo Scientific</td>
			<td>ER0271</td>
			<td>for cloning</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">SalI</td>
			<td>Thermo Scientific</td>
			<td>ER0641</td>
			<td>for cloning</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Column-based DNA Purification Kit (e.g. QIAquick)</td>
			<td>Qiagen</td>
			<td>28104</td>
			<td>for cloning</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Kanamycin Sulfate</td>
			<td>Applichem</td>
			<td>A1493</td>
			<td>for microbiological selection</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">3-Amino-1,2,4-Triazole (3-AT)</td>
			<td>Sigma-Aldrich</td>
			<td>A8056</td>
			<td>for self-activation assay</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">L-Arginine Monohydrochloride&#160;</td>
			<td>Applichem</td>
			<td>A3680</td>
			<td>for yeast culture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">L-Isoleucine&#160;</td>
			<td>Applichem</td>
			<td>A3642</td>
			<td>for yeast culture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">L-lysine Monohydrate&#160;</td>
			<td>Applichem</td>
			<td>A3448</td>
			<td>for yeast culture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">L-Methionine&#160;</td>
			<td>Applichem</td>
			<td>A3897</td>
			<td>for yeast culture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">L-Phenylalanine&#160;</td>
			<td>Applichem</td>
			<td>A3464</td>
			<td>for yeast culture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">L-Threonine&#160;</td>
			<td>Applichem</td>
			<td>A3946</td>
			<td>for yeast culture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">L-Tyrosine&#160;</td>
			<td>Applichem</td>
			<td>A3401</td>
			<td>for yeast culture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">L-Uracile</td>
			<td>Applichem</td>
			<td>A0667</td>
			<td>for yeast culture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">L-Valine&#160;</td>
			<td>Applichem</td>
			<td>A3406</td>
			<td>for yeast culture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">L-Adenine Hemisulfate Salt&#160;</td>
			<td>Applichem</td>
			<td>A1596</td>
			<td>for yeast culture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">0.22 &#181;m Pore Filter</td>
			<td>Sartorius</td>
			<td>16541</td>
			<td>for sterile filtration</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">L-Histidine Monohydrochloride</td>
			<td>Applichem</td>
			<td>A3719</td>
			<td>for yeast culture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">L-Leucine&#160;</td>
			<td>Applichem</td>
			<td>A3496</td>
			<td>for yeast culture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">L-Tryptophane&#160;</td>
			<td>Applichem</td>
			<td>A3410</td>
			<td>for yeast culture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Yeast Nitrogen Base&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>51483</td>
			<td>for yeast culture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">D-Glucose Monohydrate&#160;</td>
			<td>Applichem</td>
			<td>A1349</td>
			<td>for yeast culture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Agar</td>
			<td>Fisher Scientific</td>
			<td>BP2641-1</td>
			<td>for yeast and bacteria culture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Peptone</td>
			<td>Applichem</td>
			<td>A2208</td>
			<td>for yeast culture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Polyethylene Glycol 4000 (PEG)</td>
			<td>Applichem</td>
			<td>A1249</td>
			<td>for yeast transformation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Lithium Acetate Dihydrate (LiOAc)</td>
			<td>Applichem</td>
			<td>A3478</td>
			<td>for yeast transformation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Salmon Sperm DNA&#160;</td>
			<td>Applichem</td>
			<td>A2159</td>
			<td>for yeast transformation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Antibody against Lex-A tag (e.g. Anti-LexA DNA binding region)</td>
			<td>Millipore</td>
			<td>06-719</td>
			<td>for Western blot</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Plasmid Miniprep kit (e.g. GenElute Plasmid Miniprep Kit)</td>
			<td>Sigma-Aldrich</td>
			<td>PLN350</td>
			<td>for plasmid purification from yeast and bacteria</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Acid Washed Glass Beads</td>
			<td>Sigma-Aldrich</td>
			<td>G8772</td>
			<td>for plasmid purification from yeast</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Ampicillin Sodium Salt</td>
			<td>Applichem</td>
			<td>A0839</td>
			<td>for microbiological selection</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Yeast Extract</td>
			<td>Applichem</td>
			<td>A1552</td>
			<td>for yeast culture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Vacuum concentrator centrifuge (e.g. Concentrator 5301)</td>
			<td>Eppendorf</td>
			<td>Z368245 (Sigma)</td>
			<td>for DNA preparation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Bait vector (e.g. pLexA-N)</td>
			<td>Dualsystems</td>
			<td>P01004</td>
			<td>for Y2H</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Prey vector (e.g. pGAD-HA)</td>
			<td>Dualsystems</td>
			<td>P01004</td>
			<td>for Y2H</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Control prey vector (e.g. pLexA-p53)</td>
			<td>Dualsystems</td>
			<td>P01004</td>
			<td>for Y2H</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Control bait vector (e.g. pACT-largeT)</td>
			<td>Dualsystems</td>
			<td>P01004</td>
			<td>for Y2H</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Electrocompetent E. coli (e.g. MegaX DH10B T1R Electrocomp Cells)</td>
			<td>Invitrogen</td>
			<td>C640003</td>
			<td>for cloning</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Yeast reporter strain (NMY51:MATahis3&#916;200 trp1-901 leu2-3,112 ade2 LYS2::(lexAop)4-HIS3 ura3::(lexAop)8-lacZ ade2::(lexAop)8-ADE2 GAL4, e.g. NMY51)</td>
			<td>Dualsystems</td>
			<td>P01004</td>
			<td>for Y2H</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Plastic paraffin film (e.g. Parafilm M)</td>
			<td>Sigma-Aldrich</td>
			<td>P7793-1EA</td>
			<td>for yeast and bacteria culture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Gas permeable sealer (e.g. BREATHseal)</td>
			<td>Greiner</td>
			<td>676 051</td>
			<td>for yeast culture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">PCR Cycler (e.g. T100 Thermal Cycler)</td>
			<td>Bio-Rad</td>
			<td>1861096</td>
			<td>for PCR</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">quantitative PCR Cycler (e.g. CFX96 Touch Real-Time PCR Detection System)</td>
			<td>Bio-Rad</td>
			<td>1855195</td>
			<td>for quantitative PCR</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Microcentrifuge (e.g. Centrifuge 5417 R)</td>
			<td>Eppendorf</td>
			<td>5417 R</td>
			<td>general lab equipment</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Centrifuge (e.g. Centrifuge 5804 R)</td>
			<td>Eppendorf</td>
			<td>5804 R</td>
			<td>general lab equipment</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Nuclease-free water (DNAse-free, RNAse-free, Protease-free)</td>
			<td>5Prime</td>
			<td>2500010</td>
			<td>for PCR and DNA works</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Scalpell</td>
			<td>Swann-Morton</td>
			<td>0301</td>
			<td>for DNA preparation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sterile filter (0.22 &#181;m; Polyethersulfone))</td>
			<td>VWR-International</td>
			<td>514-0073 (European Catalogue number)</td>
			<td>for yeast and bacteria culture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Petri dishes &#216; 90 mm&#160;</td>
			<td>Greiner Bio-One</td>
			<td>633180</td>
			<td>for yeast and bacteria culture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Petri dishes &#216; 150 mm&#160;</td>
			<td>Greiner Bio-One</td>
			<td>639161</td>
			<td>for yeast culture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Photometer (e.g. BioPhotometer)</td>
			<td>Eppendorf</td>
			<td>550507804</td>
			<td>for yeast culture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Photometer Cuvettes</td>
			<td>Brand</td>
			<td>759015</td>
			<td>for yeast culture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Water Bath (e.g. Water Bath Memmert WB7)</td>
			<td>Memmert</td>
			<td>WB7</td>
			<td>for yeast transformation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Western Blot Equipment</td>
			<td>Bio-Rad</td>
			<td>diverse</td>
			<td>for protein detection</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">dNTPs</td>
			<td>5Prime</td>
			<td>2201210</td>
			<td>for cloning</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Shaker (Erlenmeyer) Flasks (100 ml; 1000 ml; 2000 ml) and breathable cotton plugs (e.g. Duran Erlenmeyer narrow-neck flasks)</td>
			<td>Sigma Aldrich</td>
			<td>Z232793, Z232858, Z232866</td>
			<td>for yeast and bacteria culture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Shaking Incubator (e.g. GFL 3031)</td>
			<td>GFL</td>
			<td>3031</td>
			<td>for yeast and bacteria culture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Incubator</td>
			<td>Binder</td>
			<td>KB 53 (E3.1)</td>
			<td>for yeast and bacteria culture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Ice Maker (e.g. Ice Maker IF 825)</td>
			<td>Omniwash</td>
			<td>IF 825</td>
			<td>general lab equipment</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">0.2 ml, 1.5 ml and 2.0 ml Reaction Vials (Polypropylene)</td>
			<td>Eppendorf</td>
			<td>0030.124.332 (0.2 ml); 0030.123.328 (1.5 ml); 0030.123.344 (2.0 ml)</td>
			<td>general lab equipment</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Pipettes and disposable pipette Tips (different volumina: 10-1000 &#181;l)</td>
			<td>Eppendorf</td>
			<td>diverse</td>
			<td>general lab equipment</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Spreader</td>
			<td>Sigma-Aldrich</td>
			<td>SPR-L-S01</td>
			<td>for yeast and bacteria culture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Vortex shaker (e.g. MS 3 Minishaker)</td>
			<td>IKA</td>
			<td>3617000</td>
			<td>general lab equipment</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">T4-Ligase</td>
			<td>Thermo Fisher</td>
			<td>EL0011</td>
			<td>for cloning</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Fridge (4&#176;C) (e.g. Liebherr Medline FKEX 5000)</td>
			<td>Liebherr</td>
			<td>81.767.580.4</td>
			<td>general lab equipment</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Freezer (-20&#176;C) (e.g. Liebherr Comfort Professional G5216)</td>
			<td>Liebherr</td>
			<td>G5216</td>
			<td>general lab equipment</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Graduated Cylinder (e.g. Brand)</td>
			<td>Sigma Aldrich</td>
			<td>Z327352; Z327417; Z327441&#160;&#160;</td>
			<td>general lab equipment</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Serological Pipettes (e.g. Fisherbrand)</td>
			<td>Thermo Fisher</td>
			<td>S55701</td>
			<td>for yeast and bacteria culture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Mechanical Pipettor (e.g. Pipetboy acu 2)</td>
			<td>Integra-Biosciences</td>
			<td>155000</td>
			<td>general lab equipment</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Cuvettes for Electroporation (1 mm)</td>
			<td>Molecular Bio Products</td>
			<td>5510-11</td>
			<td>for bacteria transformation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Electroporator (e.g. Eporator)</td>
			<td>Eppendorf</td>
			<td>4309000019</td>
			<td>for bacteria transformation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Gel and Blot imaging system (e.g. ChemiDoc MP System)</td>
			<td>Bio-Rad</td>
			<td>1708280</td>
			<td>general lab equipment</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Conical centrifuge tubes (Polypropylene) (50 ml)</td>
			<td>VWR-International&#160;</td>
			<td>525-0403 (European Catalogue number)</td>
			<td>general lab equipment</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Conical centrifuge tubes (Polypropylene) (13 ml)</td>
			<td>BD Falcon</td>
			<td>352096</td>
			<td>general lab equipment</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dithiothreitol (DTT, e.g. DTT, 1M)</td>
			<td>Thermo Scientific</td>
			<td>P2325</td>
			<td>for ligation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">adenosine triphosphate (ATP, e.g. ATP Solution (10 mM))</td>
			<td>Ambion&#160;</td>
			<td>AM8110G (distributed by Thermo Scientific)</td>
			<td>for ligation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dropout mix without histidine, leucine, tryptophan, and adenine (DO, e.g. Yeast Synthetic Drop-out Medium Supplements)</td>
			<td>Sigma-Aldrich</td>
			<td>Y2021 Sigma&#160;</td>
			<td>for yeast culture (ready-made mix)</td>
		</tr>
	</tbody>
</table>

unravelling the function of a bacterial effector from a non-cultivable plant pathogen using a yeast two-hybrid screen

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria have evolved different strategies to manipulate their host metabolism for their own benefit. This bacterial manipulation is often coupled with severe symptom development or the death of the affected plants. Determining the specific bacterial molecules responsible for the host manipulation has become an important field in microbiological research. After the identification of these bacterial molecules, called &#34;effectors,&#34; it is important to elucidate their function. A straightforward approach to determine the function of an effector is to identify its proteinaceous binding partner in its natural host via a yeast two-hybrid (Y2H) screen. Normally the host harbors numerous potential binding partners that cannot be predicted sufficiently by any in silico algorithm. It is thus the best choice to perform a screen with the hypothetical effector against a whole library of expressed host proteins. It is especially challenging if the causative agent is uncultivable like phytoplasma. This protocol provides step-by-step instructions for DNA purification from a phytoplasma-infected woody host plant, the amplification of the potential effector, and the subsequent identification of the plant&#39;s molecular interaction partner with a Y2H screen. Even though Y2H screens are commonly used, there is a trend to outsource this technique to biotech companies that offer the Y2H service at a cost. This protocol provides instructions on how to perform a Y2H in any decently equipped molecular biology laboratory using standard lab techniques.

Before the actual Y2H screen can be performed the bait must be tested for self-activation. This is achieved by transforming the bait expression vector together with the empty prey library vector and checking growth on selective plates.
To analyze whether the phytoplasmal protein ATP_00189 is self-activating, the self-activation test was performed as described in section 5. The bait plasmid is complementing the trp and the prey plasmid the leu auxotrophy of S. cerevisiae NMY5140. A successful co-transformation is thus characterized by growth on selective plates lacking trp and leu. Interaction of the bait and a prey protein leads to a complementation of the his and ade auxotrophy of NMY51. If self-activation by the bait in the absence of an interaction appears, the yeast grows on selective plates lacking his and ade. Strong and weak self-activation can occur. Strong self-activation of the bait is characterized by growth of the co-transformed yeast on trp-leu-his-ade depleted selection plates. Weak self-activation leads to growth on trp-leu-his but not on trp-leu-his-ade depleted selection plates. Proper positive controls are indispensable for interpreting results of the self-activation assay. A summary of expected results of the self-activation assay and their interpretation is provided in Table 1 and visualized in Figure 1.
<img alt="Figure 1" src="/files/ftp_upload/55150/55150fig1.jpg" /> 
Figure 1: Example of Bait Self-activation Tests of a Bait Before Performing a Y2H Screen. S. cerevisiae NMY51 was co-transformed with interacting bait (pLEX-p53) and prey (pACT-largeT) as a positive control (left panel: a-c), a weakly self-activating plus an empty prey library vector (middle panel: d-f) and a not self-activating bait plus an empty prey library vector (right panel: g-i). The co-transformed yeast were cultured on SD plates lacking trp and leu (upper panel: a, d, g), trp, leu and his (middle panel: b, e, h) and trp, leu, his and ade (lower panel: c, f, i). Selection on medium lacking trp and leu is a positive control for successful co-transformation, as the bait vector complements the trp auxotrophy and the prey vector the leu auxotrophy of NMY51 (growth on SD-trp-leu). In case of an interaction between bait and prey or a self-activation of the bait, the reporter expression of NMY51 is turned on and complements the his and ade auxotrophy (growth on SD-trp-leu-his-ade). A weak self-activation is characterized by growth on SD-trp-leu-his plates (middle panel, d-f). Weak self-activation of a bait must be diminished prior for analyzing the bait in a Y2H screen, e.g. by adding 3-AT to the selective media. Amino acid depletions are indicated with &#34;-&#34; in the respective media name. <a href="http://cloudflare.jove.com/files/ftp_upload/55150/55150fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>pLexA-N-atp00189</td>
			<td>none</td>
			<td>colonies</td>
			<td>no growth</td>
			<td>no growth</td>
			<td>no growth</td>
			<td>no growth</td>
		</tr>
		<tr>
			<td>none</td>
			<td>pGAD-HA</td>
			<td>no growth</td>
			<td>colonies</td>
			<td>no growth</td>
			<td>no growth</td>
			<td>no growth</td>
		</tr>
		<tr>
			<td>pLexA-N</td>
			<td>none</td>
			<td>colonies</td>
			<td>no growth</td>
			<td>no growth</td>
			<td>no growth</td>
			<td>no growth</td>
		</tr>
		<tr>
			<td>pLexA-N</td>
			<td>pGAD-HA</td>
			<td>colonies</td>
			<td>colonies</td>
			<td>colonies</td>
			<td>no growth</td>
			<td>no growth</td>
		</tr>
		<tr>
			<td>pLexA-p53</td>
			<td>pACT-largeT</td>
			<td>colonies</td>
			<td>colonies</td>
			<td>colonies</td>
			<td>colonies</td>
			<td>colonies</td>
		</tr>
		<tr>
			<td>pLexA-N-atp00189</td>
			<td>pGAD-HA</td>
			<td>colonies</td>
			<td>colonies</td>
			<td>colonies</td>
			<td>no growth</td>
			<td>no growth</td>
		</tr>
	</tbody>
</table>
Table 1: Expected Results in a Bait Self-activation Assay. The transformation of S. cerevisiae NMY51 yields in differential growth on selective media based on the characteristics of co-transformed bait and prey. Growth on selective plates was evaluated upon transformation of different vector combinations (a-f) after 72 h incubation at 30 &#176;C. Weak self-activation is characterized by yeast growth on SD-trp-leu-his and strong self-activation by growth on SD-trp-leu-his-ade selection plates in the absence of an interaction partner. The pLexA-N encoded phytoplasmal effector ATP_00189 does not exhibit self-activation, which is characterized by the inability of the bait vector transformed NMY51 to grow in the absence of his and ade.
Depending on the bait, a Y2H screen can gain numerous yeast clones growing on selective plates. All clones must be analyzed and checked for possible redundancies. Even if a normalized cDNA library has been used, it is very likely that an interactor is represented in many different clones. Depending on the library cloning technique, it is also possible that only fragments of the full gene are inserted in some prey vectors. It is thus advisable to de novo amplify (from cDNA) and subclone the full length gene of the interactor and to test the interaction in a one-to-one Y2H analysis (Figure 2).
<img alt="Figure 2" src="/files/ftp_upload/55150/55150fig2.jpg" /> 
Figure 2: Example of an Interaction between Bait and Prey in a Y2H Experiment. A Yeast two-hybrid (Y2H) screen was performed and plasmids from positive interactor clones were purified. The prey vector was sequenced and the Malus x domestica host interaction partners MdTCP24 and MdTCP25 were identified. As a negative control MdTCP34 (for which no interactor was identified in the Y2H library screen) was subcloned in parallel. The full length genes were amplified from apple cDNA, subcloned into the prey vector (co-cistronically expressing the activation domain AD) and de novo co-transformed with the bait vector expressing the bacterial effector ATP_00189 coupled to the DNA binding domain (BD). This figure is taken from 28. <a href="http://cloudflare.jove.com/files/ftp_upload/55150/55150fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Unravelling the Function of a Bacterial Effector from a Non-cultivable Plant Pathogen Using a Yeast Two-hybrid Screen at JoVE.com

Unravelling the Function of a Bacterial Effector from a Non-cultivable Plant Pathogen Using a Yeast Two-hybrid Screen

Bacterial effector proteins are important for establishing successful infections. This protocol describes the experimental identification of proteinaceous binding partners of a bacterial effector protein in its natural plant host. Identifying these effector interactions via yeast two-hybrid screens has become an important tool in unravelling molecular pathogenicity strategies.

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria ...

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