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><tbody><tr><td>Cetyltrimethylammonium bromide (CTAB)</td><td>Applichem</td><td>A6284</td><td>for DNA preparation</td></tr><tr><td>Trishydroxymethylaminomethane (Tris)</td><td>Applichem</td><td>A2264</td><td>for media and buffer</td></tr><tr><td>Sodium chloride (NaCl)</td><td>Applichem</td><td>A2942</td><td>for media and buffer</td></tr><tr><td>Ethylenediaminetetraacetic acid Disodium salt (NaEDTA)</td><td>Applichem</td><td>A2937</td><td>for media and buffer</td></tr><tr><td>N-Lauroylsarcosin sodium salt</td><td>Applichem</td><td>A7402</td><td>for DNA preparation</td></tr><tr><td>ammonium acetate&amp;nbsp;</td><td>Sigma-Aldrich (Fluka)</td><td>9688</td><td>for DNA preparation</td></tr><tr><td>2-mercaptoethanol</td><td>Applichem</td><td>A1108</td><td>for DNA preparation</td></tr><tr><td>Isopropanol (2-Propanol)</td><td>Applichem</td><td>A3928</td><td>for DNA preparation</td></tr><tr><td>Ethanol</td><td>Sigma-Aldrich (Fluka)</td><td>51976</td><td>for DNA preparation</td></tr><tr><td>RNAse</td><td>Applichem</td><td>A2760</td><td>for DNA preparation</td></tr><tr><td>Chloroform:Isoamyl 24:1</td><td>Applichem</td><td>A1935</td><td>for DNA preparation</td></tr><tr><td>Proofreading Polymerase (&lt;em&gt;e.g.&lt;/em&gt; iProof)</td><td>Bio-Rad</td><td>203433</td><td>for PCR</td></tr><tr><td>EcoRI</td><td>Thermo Scientific</td><td>ER0271</td><td>for cloning</td></tr><tr><td>SalI</td><td>Thermo Scientific</td><td>ER0641</td><td>for cloning</td></tr><tr><td>Column-based DNA Purification Kit (&lt;em&gt;e.g.&lt;/em&gt; QIAquick)</td><td>Qiagen</td><td>28104</td><td>for cloning</td></tr><tr><td>Kanamycin Sulfate</td><td>Applichem</td><td>A1493</td><td>for microbiological selection</td></tr><tr><td>3-Amino-1,2,4-Triazole (3-AT)</td><td>Sigma-Aldrich</td><td>A8056</td><td>for self-activation assay</td></tr><tr><td>L-Arginine Monohydrochloride&amp;nbsp;</td><td>Applichem</td><td>A3680</td><td>for yeast culture</td></tr><tr><td>L-Isoleucine&amp;nbsp;</td><td>Applichem</td><td>A3642</td><td>for yeast culture</td></tr><tr><td>L-lysine Monohydrate&amp;nbsp;</td><td>Applichem</td><td>A3448</td><td>for yeast culture</td></tr><tr><td>L-Methionine&amp;nbsp;</td><td>Applichem</td><td>A3897</td><td>for yeast culture</td></tr><tr><td>L-Phenylalanine&amp;nbsp;</td><td>Applichem</td><td>A3464</td><td>for yeast culture</td></tr><tr><td>L-Threonine&amp;nbsp;</td><td>Applichem</td><td>A3946</td><td>for yeast culture</td></tr><tr><td>L-Tyrosine&amp;nbsp;</td><td>Applichem</td><td>A3401</td><td>for yeast culture</td></tr><tr><td>L-Uracile</td><td>Applichem</td><td>A0667</td><td>for yeast culture</td></tr><tr><td>L-Valine&amp;nbsp;</td><td>Applichem</td><td>A3406</td><td>for yeast culture</td></tr><tr><td>L-Adenine Hemisulfate Salt&amp;nbsp;</td><td>Applichem</td><td>A1596</td><td>for yeast culture</td></tr><tr><td>0.22 &amp;micro;m Pore Filter</td><td>Sartorius</td><td>16541</td><td>for sterile filtration</td></tr><tr><td>L-Histidine Monohydrochloride</td><td>Applichem</td><td>A3719</td><td>for yeast culture</td></tr><tr><td>L-Leucine&amp;nbsp;</td><td>Applichem</td><td>A3496</td><td>for yeast culture</td></tr><tr><td>L-Tryptophane&amp;nbsp;</td><td>Applichem</td><td>A3410</td><td>for yeast culture</td></tr><tr><td>Yeast Nitrogen Base&amp;nbsp;</td><td>Sigma-Aldrich</td><td>51483</td><td>for yeast culture</td></tr><tr><td>D-Glucose Monohydrate&amp;nbsp;</td><td>Applichem</td><td>A1349</td><td>for yeast culture</td></tr><tr><td>Agar</td><td>Fisher Scientific</td><td>BP2641-1</td><td>for yeast and bacteria culture</td></tr><tr><td>Peptone</td><td>Applichem</td><td>A2208</td><td>for yeast culture</td></tr><tr><td>Polyethylene Glycol 4000 (PEG)</td><td>Applichem</td><td>A1249</td><td>for yeast transformation</td></tr><tr><td>Lithium Acetate Dihydrate (LiOAc)</td><td>Applichem</td><td>A3478</td><td>for yeast transformation</td></tr><tr><td>Salmon Sperm DNA&amp;nbsp;</td><td>Applichem</td><td>A2159</td><td>for yeast transformation</td></tr><tr><td>Antibody against Lex-A tag (&lt;em&gt;e.g.&lt;/em&gt; Anti-LexA DNA binding region)</td><td>Millipore</td><td>06-719</td><td>for Western blot</td></tr><tr><td>Plasmid Miniprep kit (&lt;em&gt;e.g.&lt;/em&gt; GenElute Plasmid Miniprep Kit)</td><td>Sigma-Aldrich</td><td>PLN350</td><td>for plasmid purification from yeast and bacteria</td></tr><tr><td>Acid Washed Glass Beads</td><td>Sigma-Aldrich</td><td>G8772</td><td>for plasmid purification from yeast</td></tr><tr><td>Ampicillin Sodium Salt</td><td>Applichem</td><td>A0839</td><td>for microbiological selection</td></tr><tr><td>Yeast Extract</td><td>Applichem</td><td>A1552</td><td>for yeast culture</td></tr><tr><td>Vacuum concentrator centrifuge (&lt;em&gt;e.g.&lt;/em&gt; Concentrator 5301)</td><td>Eppendorf</td><td>Z368245 (Sigma)</td><td>for DNA preparation</td></tr><tr><td>Bait vector (&lt;em&gt;e.g.&lt;/em&gt; pLexA-N)</td><td>Dualsystems</td><td>P01004</td><td>for Y2H</td></tr><tr><td>Prey vector (&lt;em&gt;e.g.&lt;/em&gt; pGAD-HA)</td><td>Dualsystems</td><td>P01004</td><td>for Y2H</td></tr><tr><td>Control prey vector (&lt;em&gt;e.g.&lt;/em&gt; pLexA-p53)</td><td>Dualsystems</td><td>P01004</td><td>for Y2H</td></tr><tr><td>Control bait vector (&lt;em&gt;e.g.&lt;/em&gt; pACT-largeT)</td><td>Dualsystems</td><td>P01004</td><td>for Y2H</td></tr><tr><td>Electrocompetent &lt;em&gt;E. coli&lt;/em&gt; (&lt;em&gt;e.g.&lt;/em&gt; MegaX DH10B T1R Electrocomp Cells)</td><td>Invitrogen</td><td>C640003</td><td>for cloning</td></tr><tr><td>Yeast reporter strain (NMY51:MATahis3&amp;Delta;200 trp1-901 leu2-3,112 ade2 LYS2::(lexAop)4-HIS3 ura3::(lexAop)8-lacZ ade2::(lexAop)8-ADE2 GAL4, &lt;em&gt;e.g.&lt;/em&gt; NMY51)</td><td>Dualsystems</td><td>P01004</td><td>for Y2H</td></tr><tr><td>Plastic paraffin film (&lt;em&gt;e.g.&lt;/em&gt; Parafilm M)</td><td>Sigma-Aldrich</td><td>P7793-1EA</td><td>for yeast and bacteria culture</td></tr><tr><td>Gas permeable sealer (&lt;em&gt;e.g.&lt;/em&gt; BREATHseal)</td><td>Greiner</td><td>676 051</td><td>for yeast culture</td></tr><tr><td>PCR Cycler (&lt;em&gt;e.g.&lt;/em&gt; T100 Thermal Cycler)</td><td>Bio-Rad</td><td>1861096</td><td>for PCR</td></tr><tr><td>quantitative PCR Cycler (&lt;em&gt;e.g.&lt;/em&gt; CFX96 Touch Real-Time PCR Detection System)</td><td>Bio-Rad</td><td>1855195</td><td>for quantitative PCR</td></tr><tr><td>Microcentrifuge (&lt;em&gt;e.g.&lt;/em&gt; Centrifuge 5417 R)</td><td>Eppendorf</td><td>5417 R</td><td>general lab equipment</td></tr><tr><td>Centrifuge (&lt;em&gt;e.g.&lt;/em&gt; Centrifuge 5804 R)</td><td>Eppendorf</td><td>5804 R</td><td>general lab equipment</td></tr><tr><td>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><td>Scalpell</td><td>Swann-Morton</td><td>0301</td><td>for DNA preparation</td></tr><tr><td>Sterile filter (0.22 &amp;micro;m; Polyethersulfone)</td><td>VWR-International</td><td>514-0073 (European Catalogue number)</td><td>for yeast and bacteria culture</td></tr><tr><td>Petri dishes &amp;Oslash; 90 mm&amp;nbsp;</td><td>Greiner Bio-One</td><td>633180</td><td>for yeast and bacteria culture</td></tr><tr><td>Petri dishes &amp;Oslash; 150 mm&amp;nbsp;</td><td>Greiner Bio-One</td><td>639161</td><td>for yeast culture</td></tr><tr><td>Photometer (&lt;em&gt;e.g.&lt;/em&gt; BioPhotometer)</td><td>Eppendorf</td><td>550507804</td><td>for yeast culture</td></tr><tr><td>Photometer Cuvettes</td><td>Brand</td><td>759015</td><td>for yeast culture</td></tr><tr><td>Water Bath (&lt;em&gt;e.g.&lt;/em&gt; Water Bath Memmert WB7)</td><td>Memmert</td><td>WB7</td><td>for yeast transformation</td></tr><tr><td>Western Blot Equipment</td><td>Bio-Rad</td><td>diverse</td><td>for protein detection</td></tr><tr><td>dNTPs</td><td>5Prime</td><td>2201210</td><td>for cloning</td></tr><tr><td>Shaker (Erlenmeyer) Flasks (100 mL; 1,000 mL; 2,000 mL) and breathable cotton plugs (&lt;em&gt;e.g.&lt;/em&gt; Duran Erlenmeyer narrow-neck flasks)</td><td>Sigma Aldrich</td><td>Z232793, Z232858, Z232866</td><td>for yeast and bacteria culture</td></tr><tr><td>Shaking Incubator (&lt;em&gt;e.g.&lt;/em&gt; GFL 3031)</td><td>GFL</td><td>3031</td><td>for yeast and bacteria culture</td></tr><tr><td>Incubator</td><td>Binder</td><td>KB 53 (E3.1)</td><td>for yeast and bacteria culture</td></tr><tr><td>Ice Maker (&lt;em&gt;e.g.&lt;/em&gt; Ice Maker IF 825)</td><td>Omniwash</td><td>IF 825</td><td>general lab equipment</td></tr><tr><td>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><td>Pipettes and disposable pipette Tips (different volumina: 10 - 1,000 &amp;micro;L)</td><td>Eppendorf</td><td>diverse</td><td>general lab equipment</td></tr><tr><td>Spreader</td><td>Sigma-Aldrich</td><td>SPR-L-S01</td><td>for yeast and bacteria culture</td></tr><tr><td>Vortex shaker (&lt;em&gt;e.g.&lt;/em&gt; MS 3 Minishaker)</td><td>IKA</td><td>3617000</td><td>general lab equipment</td></tr><tr><td>T4-Ligase</td><td>Thermo Fisher</td><td>EL0011</td><td>for cloning</td></tr><tr><td>Fridge (4 &amp;deg;C) (&lt;em&gt;e.g.&lt;/em&gt; Liebherr Medline FKEX 5000)</td><td>Liebherr</td><td>81.767.580.4</td><td>general lab equipment</td></tr><tr><td>Freezer (-20 &amp;deg;C) (&lt;em&gt;e.g.&lt;/em&gt; Liebherr Comfort Professional G5216)</td><td>Liebherr</td><td>G5216</td><td>general lab equipment</td></tr><tr><td>Graduated Cylinder (&lt;em&gt;e.g.&lt;/em&gt; Brand)</td><td>Sigma Aldrich</td><td>Z327352; Z327417; Z327441&amp;nbsp;&amp;nbsp;</td><td>general lab equipment</td></tr><tr><td>Serological Pipettes (&lt;em&gt;e.g.&lt;/em&gt; Fisherbrand)</td><td>Thermo Fisher</td><td>S55701</td><td>for yeast and bacteria culture</td></tr><tr><td>Mechanical Pipettor (&lt;em&gt;e.g.&lt;/em&gt; Pipetboy acu 2)</td><td>Integra-Biosciences</td><td>155000</td><td>general lab equipment</td></tr><tr><td>Cuvettes for Electroporation (1 mm)</td><td>Molecular Bio Products</td><td>5510-11</td><td>for bacteria transformation</td></tr><tr><td>Electroporator (&lt;em&gt;e.g.&lt;/em&gt; Eporator)</td><td>Eppendorf</td><td>4309000019</td><td>for bacteria transformation</td></tr><tr><td>Gel and Blot imaging system (&lt;em&gt;e.g.&lt;/em&gt; ChemiDoc MP System)</td><td>Bio-Rad</td><td>1708280</td><td>general lab equipment</td></tr><tr><td>Conical centrifuge tubes (Polypropylene) (50 mL)</td><td>VWR-International&amp;nbsp;</td><td>525-0403 (European Catalogue number)</td><td>general lab equipment</td></tr><tr><td>Conical centrifuge tubes (Polypropylene) (13 mL)</td><td>BD Falcon</td><td>352096</td><td>general lab equipment</td></tr><tr><td>Dithiothreitol (DTT; 1 M)</td><td>Thermo Scientific</td><td>P2325</td><td>for ligation</td></tr><tr><td>adenosine triphosphate (ATP, &lt;em&gt;e.g.&lt;/em&gt; ATP Solution (10 mM))</td><td>Ambion&amp;nbsp;</td><td>AM8110G (distributed by Thermo Scientific)</td><td>for ligation</td></tr><tr><td>Dropout mix without histidine, leucine, tryptophan, and adenine (DO, &lt;em&gt;e.g.&lt;/em&gt; Yeast Synthetic Drop-out Medium Supplements)</td><td>Sigma-Aldrich</td><td>Y2021 Sigma&amp;nbsp;</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 ...

unravelling-function-bacterial-effector-from-non-cultivable-plant

Research

JoVE Journal

Immunology and Infection

11.5K Views.  Laimburg Research Centre. 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.

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

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

A Yeast 2-Hybrid Screen in Batch to Compare Protein Interactions

Screening for protein-protein interactions using the yeast 2-hybrid assay has long been an effective tool, but its use has largely been limited to the discovery of high-affinity interactors that are highly enriched in the library of interacting candidates. In a traditional format, the yeast 2-hybrid assay can yield too many colonies to analyze when conducted at low stringency where low affinity interactors might be found. Moreover, without a comprehensive and complete interrogation of the same library against different bait plasmids, a comparative analysis cannot be achieved. Although some of these problems can be addressed using arrayed prey libraries, the cost and infrastructure required to operate such screens can be prohibitive. As an alternative, we have adapted the yeast 2-hybrid assay to simultaneously uncover dozens of transient and static protein interactions within a single screen utilizing a strategy termed DEEPN (Dynamic Enrichment for Evaluation of Protein Networks), which incorporates high-throughput DNA sequencing and computation to follow the evolution of a population of plasmids that encode interacting partners. Here, we describe customized reagents and protocols that allow a DEEPN screen to be executed easily and cost-effectively.

Batch processing of yeast 2-hybrid screens allows for direct comparison of the interaction profiles of multiple bait proteins with a highly complex set of prey fusion proteins. Here, we describe refined methods, new reagents, and how to implement their use for such screens.

Screening for protein-protein interactions using the yeast 2-hybrid assay has long been an effective tool, but its use has largely been limited to the ...

Biochemistry

Identification of Host Pathways Targeted by Bacterial Effector Proteins using Yeast Toxicity and Suppressor Screens

Intracellular bacteria secrete virulence factors called effector proteins into the host cytosol that act to subvert host proteins and/or their associated biological pathways to the benefit of the bacterium. Identification of putative bacterial effector proteins has become more manageable due to advances in bacterial genome sequencing and the advent of algorithms that allow in silico identification of genes encoding secretion candidates and/or eukaryotic-like domains. However, identification of these important virulence factors is only an initial step. Naturally, the goal is to determine the molecular function of effector proteins and elucidate how they interact with the host. In recent years, techniques like the yeast two-hybrid screen and large-scale immunoprecipitations coupled with mass spectrometry have aided in the identification of protein-protein interactions. Although identification of a host binding partner is the crucial first step toward elucidating the molecular function of a bacterial effector protein, sometimes the host protein is found to have multiple biological functions (e.g., actin, clathrin, tubulin), or the bacterial protein may not physically bind host proteins, depriving the researcher of crucial information about the precise host pathway being manipulated. A modified yeast toxicity screen coupled with a suppressor screen has been adapted to identify host pathways impacted by bacterial effector proteins. The toxicity screen relies on a toxic effect in yeast caused by the effector protein interfering with the host biological pathways, which often manifests as a growth defect. Expression of a yeast genomic library is used to identify host factors that suppress the toxicity of the bacterial effector protein and thus identify proteins in the pathway that the effector protein targets. This protocol contains detailed instructions for both the toxicity and suppressor screens. These techniques can be performed in any lab capable of molecular cloning and cultivation of yeast and Escherichia coli.

Bacterial pathogens secrete proteins into the host that target crucial biological processes. Identifying the host pathways targeted by bacterial effector proteins is key to addressing molecular pathogenesis. Here, a method using a modified yeast suppressor and toxicity screen to elucidate host pathways targeted by toxic bacterial effector proteins is described.

Intracellular bacteria secrete virulence factors called effector proteins into the host cytosol that act to subvert host proteins and/or their associated ...

Genetics

A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions

Significant efforts were gathered to generate large-scale comprehensive protein-protein interaction network maps. This is instrumental to understand the pathogen-host relationships and was essentially performed by genetic screenings in yeast two-hybrid systems. The recent improvement of protein-protein interaction detection by a Gaussia luciferase-based fragment complementation assay now offers the opportunity to develop integrative comparative interactomic approaches necessary to rigorously compare interaction profiles of proteins from different pathogen strain variants against a common set of cellular factors.
This paper specifically focuses on the utility of combining two orthogonal methods to generate protein-protein interaction datasets: yeast two-hybrid (Y2H) and a new assay, high-throughput Gaussia princeps protein complementation assay (HT-GPCA) performed in mammalian cells.
A large-scale identification of cellular partners of a pathogen protein is performed by mating-based yeast two-hybrid screenings of cDNA libraries using multiple pathogen strain variants. A subset of interacting partners selected on a high-confidence statistical scoring is further validated in mammalian cells for pair-wise interactions with the whole set of pathogen variants proteins using HT-GPCA. This combination of two complementary methods improves the robustness of the interaction dataset, and allows the performance of a stringent comparative interaction analysis. Such comparative interactomics constitute a reliable and powerful strategy to decipher any pathogen-host interplays.

This article focuses on the identification of high-confident interaction datasets between host and pathogen proteins using a combination of two orthogonal methods: yeast two-hybrid followed by a high-throughput interaction assay in mammalian cells called HT-GPCA.

Significant efforts were gathered to generate  large-scale comprehensive protein-protein interaction network maps. This is  instrumental to understand the ...

Screening and Identification of RNA Silencing Suppressors from Secreted Effectors of Plant Pathogens

RNA silencing is an evolutionarily conserved, sequence-specific gene regulation mechanism in eukaryotes. Several plant pathogens have evolved proteins with the ability to inhibit the host plant RNA silencing pathway. Unlike virus effector proteins, only several secreted effector proteins have showed the ability to suppress RNA silencing in bacterial, oomycete, and fungal pathogens, and the molecular functions of most effectors remain largely unknown. Here, we describe in detail a slightly modified version of the co-infiltration assay that could serve as a general method for observing RNA silencing and for characterizing effector proteins secreted by plant pathogens. The key steps of the approach are choosing the healthy and fully developed leaves, adjusting the bacteria culture to the appropriate optical density (OD) at 600 nm, and observing green fluorescent protein (GFP) fluorescence at the optimum time on the infiltrated leaves in order to avoid omitting effectors with weak suppression activity. This improved protocol will contribute to rapid, accurate, and extensive screening of RNA silencing suppressors and serve as an excellent starting point for investigating the molecular functions of these proteins.

Here, we present a modified screening method that can be extensively used to quickly screen RNA silencing suppressors in plant pathogens.

RNA silencing is an evolutionarily conserved, sequence-specific gene regulation mechanism in eukaryotes. Several plant pathogens have evolved proteins ...

Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput identification of protein-protein interactions (PPI) for full-length transmembrane proteins. To this end, our lab developed the split-ubiquitin based Membrane Yeast Two-Hybrid (MYTH) system. This technology allows for the sensitive detection of transient and stable protein interactions using Saccharomyces cerevisiae as a host organism. MYTH takes advantage of the observation that ubiquitin can be separated into two stable moieties: the C-terminal half of yeast ubiquitin (Cub) and the N-terminal half of the ubiquitin moiety (Nub). In MYTH, this principle is adapted for use as a 'sensor' of protein-protein interactions. Briefly, the integral membrane bait protein is fused to Cub which is linked to an artificial transcription factor. Prey proteins, either in individual or library format, are fused to the Nub moiety. Protein interaction between the bait and prey leads to reconstitution of the ubiquitin moieties, forming a full-length 'pseudo-ubiquitin' molecule. This molecule is in turn recognized by cytosolic deubiquitinating enzymes, resulting in cleavage of the transcription factor, and subsequent induction of reporter gene expression. The system is highly adaptable, and is particularly well-suited to high-throughput screening. It has been successfully employed to investigate interactions using integral membrane proteins from both yeast and other organisms.

Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions

MYTH allows the sensitive detection of transient and stable interactions between proteins that are expressed in the model organism Saccharomyces cerevisiae. It has been successfully applied to study exogenous and yeast integral membrane proteins in order to identify their interacting partners in a high throughput manner.

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput ...

Biology

Identification of Growth Inhibition Phenotypes Induced by Expression of Bacterial Type III Effectors in Yeast

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within the cell, type III effectors subvert host cellular processes to suppress immune responses and promote pathogen growth. Numerous type III effectors of plant and animal bacterial pathogens have been identified to date, yet only a few of them are well characterized. Understanding the functions of these effectors has been undermined by a combination of functional redundancy in the effector repertoire of a given bacterial strain, the subtle effects that they may exert to increase virulence, roles that are possibly specific to certain infection stages, and difficulties in genetically manipulating certain pathogens. Expression of type III effectors in the budding yeast Saccharomyces cerevisiae may allow circumventing these limitations and aid to the functional characterization of effector proteins. Because type III effectors often target cellular processes that are conserved between yeast and other eukaryotes, their expression in yeast may result in growth inhibition phenotypes that can be exploited to elucidate effector functions and targets. Additional advantages to using yeast for functional studies of bacterial effectors include their genetic tractability, information on predicted functions of the vast majority of their ORFs, and availability of numerous tools and resources for both genome-wide and small-scale experiments. Here we discuss critical factors for designing a yeast system for the expression of bacterial type III effector proteins. These include an appropriate promoter for driving expression of the effector gene(s) of interest, the copy number of the effector gene, the epitope tag used to verify protein expression, and the yeast strain. We present procedures to induce expression of effectors in yeast and to verify their expression by immunoblotting. In addition, we describe a spotting assay on agar plates for the identification of effector-induced growth inhibition phenotypes. The use of this protocol may be extended to the study of pathogenicity factors delivered into the host cell by any pathogen and translocation mechanism.

In this video, we describe a procedure for the expression of bacterial type III effectors in yeast and the identification of effector-induced growth inhibition phenotypes. Such phenotypes can be subsequently exploited to elucidate effector functions and targets.

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within ...

Split Green Fluorescent Protein System to Visualize Effectors Delivered from Bacteria During Infection

Bacteria, one of the most important causative agents of various plant diseases, secrete a set of effector proteins into the host plant cell to subvert the plant immune system. During infection cytoplasmic effectors are delivered to the host cytosol via a type III secretion system (T3SS). After delivery into the plant cell, the effector(s) targets the specific compartment(s) to modulate host cell processes for survival and replication of the pathogen. Although there has been some research on the subcellular localization of effector proteins in the host cells to understand their function in pathogenicity by using fluorescent proteins, investigation of the dynamics of effectors directly injected from bacteria has been challenging due to the incompatibility between the T3SS and fluorescent proteins.
Here, we describe our recent method of an optimized split superfolder green fluorescent protein system (sfGFPOPT) to visualize the localization of effectors delivered via the bacterial T3SS in the host cell. The sfGFP11 (11th &#946;-strand of sfGFP)-tagged effector secreted through the T3SS can be assembled with a specific organelle targeted sfGFP1-10OPT (1-10th &#946;-strand of sfGFP) leading to fluorescence emission at the site. This protocol provides a procedure to visualize the reconstituted sfGFP fluorescence signal with an effector protein from Pseudomonas syringae in a particular organelle in the Arabidopsis and Nicotiana benthamiana plants.

Fluorescent protein-based approaches to monitor effectors secreted by bacteria into host cells are challenging. This is due to the incompatibility between fluorescent proteins and the type-III secretion system. Here, an optimized split superfolder GFP system is used for visualization of effectors secreted by bacteria into the host plant cell.

Bacteria, one of the most important causative agents of various plant diseases, secrete a set of effector proteins into the host plant cell to subvert the ...

Biomolecular Interaction Studies Using Yeast Two-Hybrid Technique 

Isolating Interaction-Null&#47;Impaired Mutants Using the Yeast Two-Hybrid Assay

Protein-protein interactions are one of the most basic processes that underlie biological phenomena. One of the simplest and best ways to understand the role(s) and function(s) of a specific protein-protein interaction is to compare the phenotype of the wild-type (with the relevant protein-protein interaction) and those of mutants that lack the relevant interaction. Therefore, if such mutants can be isolated, they will help to elucidate the related biological processes. The yeast two-hybrid (Y2H) procedure is a powerful approach not only to detect protein-protein interactions but also to isolate interaction-null/impaired mutants. In this article, a protocol is presented to isolate interaction-null/impaired mutants using Y2H technology. First, a mutation library is constructed by combining the polymerase chain reaction and efficient seamless cloning technology, which efficiently excludes the empty vector from the library. Second, interaction-null/impaired mutants are screened by the Y2H assay. Because of a trick in the Y2H vector, undesired mutants, such as those with frameshift and nonsense mutations, are efficiently eliminated from the screening process. This strategy is simple and can, therefore, be applied to any combination of proteins whose interaction can be detected by the two-hybrid system.

Author Spotlight: Enhanced Isolation of Interaction-Null Mutants in Yeast

The interactions of biomolecules, such as protein-protein interactions, are the molecular basis of biological functions. If interaction-null/impaired mutants that specifically lack the relevant interaction can be isolated, they will greatly help to understand the function(s) of this interaction. This article presents an efficient way to isolate interaction-null/impaired mutants.

Protein-protein interactions are one of the most basic processes that underlie biological phenomena. One of the simplest and best ways to understand the ...

Identifying Drugs that Affect Polymerization of Bacterial Cytoskeletal Proteins

Fission Yeast as a Platform for Antibacterial Drug Screens Targeting Bacterial Cytoskeleton Proteins

Bacterial cytoskeletal proteins such as FtsZ and MreB perform essential functions such as cell division and cell shape maintenance. Further, FtsZ and MreB have emerged as important targets for novel antimicrobial discovery. Several assays have been developed to identify compounds targeting nucleotide binding and polymerization of these cytoskeletal proteins, primarily focused on FtsZ. Moreover, many of the assays are either laborious or cost-intensive, and ascertaining whether these proteins are the cellular target of the drug often requires multiple methods. Finally, the toxicity of the drugs to eukaryotic cells also poses a problem. Here, we describe a single-step cell-based assay to discover novel molecules targeting bacterial cytoskeleton and minimize hits that might be potentially toxic to eukaryotic cells. Fission yeast is amenable to high-throughput screens based on microscopy, and a visual screen can easily identify any molecule that alters the polymerization of FtsZ or MreB. Our assay utilizes the standard 96-well plate and relies on the ability of the bacterial cytoskeletal proteins to polymerize in a eukaryotic cell such as the fission yeast. While the protocols described here are for fission yeast and utilize FtsZ from Staphylococcus aureus and MreB from Escherichia coli, they are easily adaptable to other bacterial cytoskeletal proteins that readily assemble into polymers in any eukaryotic expression hosts. The method described here should help facilitate further discovery of novel antimicrobials targeting bacterial cytoskeletal proteins.

Author Spotlight: Exploring Cytoskeletal Dynamics to Unveil Novel Antibiotics Through Innovative Cell-Based Assays

Fission yeast is used here as a heterologous host to express bacterial cytoskeletal proteins such as FtsZ and MreB as translational fusion proteins with GFP to visualize their polymerization. Also, compounds that affect polymerization are identified by imaging using a fluorescence microscope.

Bacterial cytoskeletal proteins such as FtsZ and MreB perform essential functions such as cell division and cell shape maintenance. Further, FtsZ and MreB ...