Translate this page to:
Choose Language…
In JoVE (1)
Other Publications (6)
Articles by Dor Salomon in JoVE
JoVE General
Identification of Growth Inhibition Phenotypes Induced by Expression of Bacterial Type III Effectors in Yeast
Department of Plant Sciences, Tel Aviv University
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.
Other articles by Dor Salomon on PubMed
Bypassing Kinase Activity of the Tomato Pto Resistance Protein with Small Molecule Ligands
The tomato (Solanum lycopersicum) protein kinase Pto confers resistance to Pseudomonas syringae pv. tomato bacteria expressing the AvrPto and AvrPtoB effector proteins. Pto specifically recognizes both effectors by direct physical interactions triggering activation of immune responses. Here, we used a chemical-genetic approach to sensitize Pto to analogs of PP1, an ATP-competitive small molecule inhibitor. By using PP1 analogs in combination with the sensitized Pto (Pto(as)), we examined the role of Pto kinase activity in effector recognition and signal transduction. Strikingly, while PP1 analogs efficiently inhibited kinase activity of Pto(as) in vitro, they enhanced interactions of Pto(as) with AvrPto and AvrPtoB in a yeast two-hybrid system. In addition, in the presence of PP1 analogs, Pto(as) bypassed mutations either at an autophosphorylation site critical for the Pto-AvrPto interaction or at catalytically essential residues and interacted with both effectors. Moreover, in the presence of the PP1 analog 3MB-PP1, a kinase-deficient form of Pto(as) triggered an AvrPto-dependent hypersensitive response in planta. These findings suggest that, rather than phosphorylation per se, a conformational change likely triggered by autophosphorylation in Pto and mimicked by ligand binding in Pto(as) is a prerequisite for recognition of bacterial effectors. Following recognition, kinase activity appears to be dispensable for Pto signaling in planta. The chemical-genetic strategy applied here to develop specific small molecule inhibitors of Pto represents an invaluable tool for the study of biological functions of other plant protein kinases in vivo.
A Chemical-genetic Approach for Functional Analysis of Plant Protein Kinases
Plant genomes encode hundreds of protein kinases, yet only for a small fraction of them precise functions and phosphorylation targets have been identified. Recently, we applied a chemical-genetic approach to sensitize the tomato serine/threonine kinase Pto to analogs of PP1, an ATP-competitive and cell-permeable small-molecule inhibitor. The Pto kinase confers resistance to Pst bacteria by activating immune responses upon specific recognition of bacterial effectors. By using PP1 analogs in combination with the analog-sensitive Pto, we shed new light on the role of Pto kinase activity in effector recognition and signal transduction. Here we broaden the use of this chemical-genetic approach to another defense-related plant protein kinase, the MAP kinase LeMPK3. In addition, we show that analog-sensitive but not wild-type kinases are able to use unnatural N(6)-modified ATP analogs as phosphodonors that can be exploited for tagging direct phosphorylation targets of the kinase of interest. Thus, sensitization of kinases to analogs of the small-molecule inhibitor PP1 and ATP can be an effective tool for the discovery of cellular functions and phosphorylation substrates of plant protein kinases.
Ssz1 Restores Endoplasmic Reticulum-associated Protein Degradation in Cells Expressing Defective Cdc48-ufd1-npl4 Complex by Upregulating Cdc48
The endoplasmic reticulum (ER)-associated protein degradation (ERAD) pathway eliminates aberrant proteins from the ER. The key role of Cdc48p-Ufd1p-Npl4p is indicated by impaired ERAD in Saccharomyces cerevisiae with mutations in any of this complex's genes. We identified SSZ1 in genetic screens for cdc48-10 suppressors and show that it upregulates Cdc48p via the pleiotropic drug resistance (PDR) network. A pSSZ1 plasmid restored impaired ERAD-M of 6myc-Hmg2 in cdc48-10, ufd1-2, and npl4-1, while SSZ1 deletion had no effect. Ssz1p activates Pdr1p, the PDR master regulator. Indeed, plasmids of PDR1 or its target gene RPN4 increased cdc48-10p levels and restored ERAD-M in cdc48-10. Rpn4p regulates transcription of proteasome subunits and CDC48, thus RPN4 deletion abolished ERAD. However, the diminished proteasome level in Deltarpn4 was sufficient for degrading a cytosolic substrate, whereas the impaired ERAD-M was the result of diminished Cdc48p and was restored by expression of pCDC48. The corrected ERAD-M in the hypomorphic strains of the Cdc48 partners ufd1-2 and npl4-1 by the pCDC48 plasmid, and in cdc48-10 cells by the pcdc48-10 plasmid, combined with the finding that neither pSSZ1 nor pcdc48-10 restored ERAD-L of CPY*-HA, support our conclusion that Ssz1p suppressing effects is brought about by upregulating Cdc48p.
Expression of Xanthomonas Campestris Pv. Vesicatoria Type III Effectors in Yeast Affects Cell Growth and Viability
The gram-negative bacterium Xanthomonas campestris pv. vesicatoria is the causal agent of spot disease in tomato and pepper. X. campestris pv. vesicatoria pathogenicity depends on a type III secretion system delivering effector proteins into the host cells. We hypothesized that some X. campestris pv. vesicatoria effectors target conserved eukaryotic cellular processes and examined phenotypes induced by their expression in yeast. Out of 21 effectors tested, 14 inhibited yeast growth in normal or stress conditions. Viability assay revealed that XopB and XopF2 attenuated cell proliferation, while AvrRxo1, XopX, and XopE1 were cytotoxic. Inspection of morphological features and DNA content of yeast cells indicated that cytotoxicity caused by XopX and AvrRxo1 was associated with cell-cycle arrest at G0/1. Interestingly, XopB, XopE1, XopF2, XopX, and AvrRxo1 that inhibited growth in yeast also caused phenotypes, such as chlorosis and cell death, when expressed in either host or nonhost plants. Finally, the ability of several effectors to cause phenotypes in yeast and plants was dependent on their putative catalytic residues or localization motifs. This study supports the use of yeast as a heterologous system for functional analysis of X. campestris pv. vesicatoria type III effectors, and sets the stage for identification of their eukaryotic molecular targets and modes of action.
Sensitizing Plant Protein Kinases to Specific Inhibition by ATP-competitive Molecules
The highly conserved nature of the protein kinase catalytic domain and the low permeability of plant cell membranes pose a challenge to the development of specific inhibitors that target individual protein kinases in vivo. Here, we describe a chemical-genetic approach to specifically sensitize individual plant kinases to cell-permeable small molecules that do not inhibit wild-type kinases. In this approach, a single amino-acid substitution is introduced in the ATP-binding site of the enzyme enabling specific binding of ATP-competitive molecules. Cell-permeable molecules can then be used to specifically target the sensitized allele in transgenic Arabidopsis thaliana plants that do not express the wild-type form of the kinase. This strategy provides a useful tool for the functional characterization of protein kinases in planta and for the dissection of the signaling pathways in which they are involved.
A Simple Yeast-based Strategy to Identify Host Cellular Processes Targeted by Bacterial Effector Proteins
Bacterial effector proteins, which are delivered into the host cell via the type III secretion system, play a key role in the pathogenicity of gram-negative bacteria by modulating various host cellular processes to the benefit of the pathogen. To identify cellular processes targeted by bacterial effectors, we developed a simple strategy that uses an array of yeast deletion strains fitted into a single 96-well plate. The array is unique in that it was optimized computationally such that despite the small number of deletion strains, it covers the majority of genes in the yeast synthetic lethal interaction network. The deletion strains in the array are screened for hypersensitivity to the expression of a bacterial effector of interest. The hypersensitive deletion strains are then analyzed for their synthetic lethal interactions to identify potential targets of the bacterial effector. We describe the identification, using this approach, of a cellular process targeted by the Xanthomonas campestris type III effector XopE2. Interestingly, we discover that XopE2 affects the yeast cell wall and the endoplasmic reticulum stress response. More generally, the use of a single 96-well plate makes the screening process accessible to any laboratory and facilitates the analysis of a large number of bacterial effectors in a short period of time. It therefore provides a promising platform for studying the functions and cellular targets of bacterial effectors and other virulence proteins.
