In this work, we describe a simple strategy that employs an array of yeast deletion strains to identify cellular processes targeted by virulence proteins. Our strategy is based on the observation that maximal coverage of the yeast SL interaction network does not require the entire collection of null deletion strains. The major advantage of this strategy is that it uses a single 96-well plate instead of over fifty 96-well plates that are used when the entire yeast deletion strain collection is screened. As a proof of concept, we showed that the array of the deletion strains was sufficient to accurately predict a previously identified cellular process targeted by the Shigella
T3E OspF 
. Next, we employed the array of the deletion strains to investigate the Xanthomonas campestris
T3E XopE2 for which no cellular target was described. We found that XopE2 was congruent to genes that were all involved in cell wall biogenesis and organization, implying that XopE2 affected these processes. Indeed, we showed that XopE2 caused sensitivity to the cell wall stressing agents caffeine and SDS. Subsequently, we found that XopE2 affected the ER stress response, which is tightly linked to cell wall organization and biogenesis 
. Thus, we demonstrated the applicability of our approach for studying the functions and targets of bacterial T3Es.
Our approach has several advantages over screening the yeast null deletion strain collection. First, it is simple, convenient and economical, requiring less than 10 days to complete a full screen with relatively few plates. Second, working with a single 96-well plate simplifies the analysis of the results and allows for more repetitions to be made. Importantly, in contrast to previous approaches, our approach does not necessitate the use of a robot, lowering the initial investment required for performing the screen and making it accessible to any laboratory studying virulence proteins that function inside the host cell. Nevertheless, using robotic plating, it is possible to simultaneously screen a large repertoire of effectors, an intriguing possibility in light of the growing number of bacterial proteins identified as effectors.
It should be noted that our approach is suitable for studying bacterial T3Es that target conserved eukaryotic processes found in yeast. It is not expected to yield significant results for T3Es that affect specific processes that cannot be found in yeast.
The hypersensitive deletion strains identified in the screen can be used in additional ways. First, the hypersensitive deletion strains can be used to screen for genes, which upon over-expression, suppress the growth inhibition phenotype caused by the T3Es. Finding such suppressors can assist in identifying the cellular processes that are targeted by the T3Es. Second, the hypersensitive deletion strains can be used to classify T3Es of various pathogens into functional groups, laying the foundation for future study of “functional effector families”.
Several factors affected our selection of the expression vector. Our system employs the GAL1/10 promoter, a strong promoter whose activity is regulated by the carbon source in the medium. An important feature of the GAL1/10 promoter is that it does not require the use of modified yeast strains, which simplified the construction of the array. The use of an inducible expression vector enabled us to perform the transformation step under conditions in which the expression of the bacterial effector is repressed, grow the transformed cells to saturation and only then spot them on inducing and repressing plates. In this way, we eliminated the effect of variations in transformation efficiency between deletion strains. Another important factor that influenced our selection of the expression vector was the number of copies of the effector gene in the cell. It was previously suggested that high-level expression of the bacterial effector (when using a 2 micron vector) might result in non-specific activity of the effector 
. Our system uses a centromere-containing vector to obtain low-level expression of the bacterial effector and thus to increase the specificity of the assay. The expression vector that we use also contains a single myc tag, which allows to monitor the expression of the effector in the cell. The tag is fused to the C-terminal tail of the effector and owing to its short size it is not likely to affect the expression or the function of the effector.
Our approach requires the transformation of the array of deletion strains with the vector encoding the bacterial effector. One way to avoid this step is to transform a single yeast strain with the vector encoding the bacterial effector, and by mating and meiosis to transfer the vector to the deletion strains. However, this approach, known as the synthetic genetic array (SGA) methodology 
, is much slower, requiring at least two weeks, not including the time required for the transformation of the starting strain 
. Nevertheless, the SGA methodology should be considered when a large number of bacterial T3Es are screened simultaneously, ideally with the aid of a robot. Another mating-based approach, which is expected to be much faster than the SGA methodology, is called selective ploidy ablation (SPA) 
. This approach employs a universal plasmid donor strain that contains conditional centromeres on every chromosome. The plasmid-bearing donor strain is mated to a recipient, followed by removal of all donor-strain chromosomes, producing a haploid strain containing the transferred plasmid. One limitation of the SPA approach is that chromosomes destabilization requires growth on galactose, which induces the expression of the bacterial effector in our system.
Finally, although we concentrated our work on bacterial T3Es, our approach can be easily employed to study other types of virulence proteins that function inside host cells, such as bacterial type IV and type VI secreted effectors, fungal effectors and viral proteins. In conclusion, the approach presented in this work provides an excellent platform for studying the functions and cellular targets of bacterial effectors and other virulence proteins.