Pseudomonas syringae utilizes the type III secretion system to translocate effector proteins into plant cells, where they can contribute to the pathogen's ability to infect and cause disease. Recognition of these effectors by resistance proteins induces defense responses that typically include a programmed cell death reaction called the hypersensitive response. The YopJ/HopZ family of type III effector proteins is a common family of effector proteins found in animal-and plant-pathogenic bacteria. The HopZ family in P. syringae includes HopZ1a PsyA2 , HopZ1b PgyUnB647 , HopZ1c PmaE54326 , HopZ2 Ppi895A and HopZ3 PsyB728a . HopZ1a is predicted to be most similar to the ancestral hopZ allele and causes a hypersensitive response in multiple plant species, including Arabidopsis thaliana. Therefore, it has been proposed that host defense responses have driven the diversification of this effector family. In this study, we further characterized the hypersensitive response induced by HopZ1a and demonstrated that it is not dependent on known resistance genes. Further, we identified a novel virulence function for HopZ2 that requires the catalytic cysteine demonstrated to be required for protease activity. Sequence analysis of the HopZ family revealed the presence of a predicted myristoylation sequence in all members except HopZ3. We demonstrated that the myristoylation site is required for membrane localization of this effector family and contributes to the virulence and avirulence activities of HopZ2 and HopZ1a, respectively. This paper provides insight into the selective pressures driving virulence protein evolution by describing a detailed functional characterization of the diverse HopZ family of type III effectors with the model plant Arabidopsis.The intimate interaction between plant bacterial pathogens and their hosts is believed to have driven the stepwise evolution of plant resistance mechanisms which successful pathogens have evolved to suppress (11,18,28). The gram-negative bacterial phytopathogen Pseudomonas syringae uses the type III secretion system to translocate virulence proteins (also known as type III effectors) directly into host cells, where they can suppress host defense responses (1, 9). However, plants have evolved resistance genes that can recognize effectors or the cellular perturbations that they cause and can induce defense responses that can thwart the infection process (15, 40). In fact, certain type III effector genes were originally called avirulence (avr) genes due to their ability to compromise the virulence of bacteria that express them. Often associated with the recognition of specific type III effectors by plant resistance proteins is the rapid cell death known as the hypersensitive response (HR), which is localized to the site of infection (23). Recent genome-wide screens have identified hundreds of type III effector proteins in P. syringae pathovars that can be subdivided into approximately 50 families (35). Although individual effectors have been studied to determine their virulence a...