Identification of Target Proteins Involved in Cell Death Signaling Induced by a Phytophthora infestans Core Effector in Nicotiana benthamiana

Abstract

In nature, plants have developed a multi-layered defense system, since they are exposed to various environmental challenges such as pathogen attacks. A programmed cell death is induced to the formation of necrotic lesions that prevents further spread of the pathogens from the infection site. Recent studies showed that diverse pathogen effectors, pathogen-specific secreted molecules, interact with plant proteins to induce the cell death. In this study, I examine of plant target proteins of a Phytophthora infestans core effector 82 (CE82) which induces cell death in Nicotiana benthamiana and pepper to investigate how the pathogen effector triggers cell death in plant cells. The candidates of target protein interacting with CE82 were identified by co-immunoprecipitation (co-IP) and LC-MS/MS. Among the target protein candidates, four candidates are related to the cellular vesicle trafficking. In order to verify the function of four target protein candidates in CE82-induced cell death responses, virus-induced gene silencing (VIGS) was performed with the candidates. Although the gene expressions of the candidate genes were suppressed in N. benthamiana, there were no significant changes in cell death. Furthermore, I performed agro-infiltrated transient overexpression of the target genes for the gain-of-function study. Interestingly, CE 82-induced cell death was accelerated when CE82 was coexpressed with MON2 which is one of the target proteins related to vesicle trafficking. The cellular localization study revealed the localization of CE82 in vesicle-like or Golgi-like membrane. However, localization of CE82 was changed when it was coexpressed with MON2. I performed interaction studies using yeast-two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) between CE82 and MON2. Although direct interaction was not evident in Y2H, BiFC result indicated that CE82 interacts with MON2 in planta. Study between effectors of P. infestans and target proteins provides important information contributing to plant immunity.