Abstract
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.
Highlights
Gram-negative bacteria are the causal agents of numerous diseases in plants and animals
Our objective was to construct a small array of deletion strains covering the yeast synthetic lethal (SL) interaction network and use this array to identify cellular processes affected by the expression of bacterial effectors (Fig. 1)
We describe a simple strategy that employs an array of yeast deletion strains to identify cellular processes targeted by virulence proteins
Summary
Gram-negative bacteria are the causal agents of numerous diseases in plants and animals. Many of these bacteria encode a syringe-like structure termed the type III secretion system, which delivers effector proteins into the host cell during infection [1]. The use of yeast in the study of bacterial effectors is based on the observation that these proteins often target fundamental cellular processes that are conserved among all eukaryotes. In agreement with this premise, the expression of many T3Es from plant and animal pathogens inhibits yeast growth [6,9]. Toxic phenotypes induced by bacterial effectors in yeast were used in suppressor screens for the identification of eukaryotic targets of the effectors [10,11]
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