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Gram-negative bacterial pathogens inject type III secreted effectors (T3SEs) directly into host cells to promote pathogen fitness by manipulating host cellular processes. Despite their crucial role in promoting virulence, relatively few T3SEs have well-characterized enzymatic activities or host targets. This is in part due to functional redundancy within pathogen T3SE repertoires as well as promiscuous individual T3SEs that can have multiple host targets. To overcome these challenges, we conducted heterologous genetic screens in yeast, a non-host organism, to identify T3SEs that target conserved eukaryotic processes. We screened 75 T3SEs from the plant pathogen Pseudomonas syringae and identified 16 that inhibited yeast growth on rich media and eight that inhibited growth on stress-inducing media, including the acetyltransferase HopZ1a. We focused our further analysis on HopZ1a, which interacts with plant tubulin and alters microtubule networks. We first performed a Pathogenic Genetic Array (PGA) screen of HopZ1a against ~4400 yeast carrying non-essential mutations and found 95 and 10 deletion mutants which reduced or enhanced HopZ1a toxicity, respectively. To uncover putative HopZ1a host targets, we interrogated both the genetic- and physical-interaction profiles of HopZ1a by identifying yeast genes with PGA profiles most similar (i.e. congruent) to that of HopZ1a, performing a functional enrichment analysis of these HopZ1a-congruent genes, and by analyzing previously described HopZ physical interaction datasets. Finally, we demonstrated that HopZ1a can target kinesins by acetylating the plant kinesins HINKEL and MKRP1.ARTICLE SUMMARYBacterial pathogens utilize secretion systems to directly deliver effector proteins into host cells, with the ultimate goal of promoting pathogen fitness. Despite the central role that effectors play in infection, the molecular function and host targets of most effectors remain uncharacterized. We used yeast genomics and protein interaction data to identify putative virulence targets of the effector HopZ1a from the plant pathogen Pseudomonas syringae. HopZ1a is an acetyltransferase that induces plant microtubule destruction. We showed that HopZ1a acetylated plant kinesin proteins known to regulate microtubule networks. Our study emphasizes the power of yeast functional genomic screens to characterize effector functions.