"Candidatus Liberibacter asiaticus" Secretes Nonclassically Secreted Proteins That Suppress Host Hypersensitive Cell Death and Induce Expression of Plant Pathogenesis-Related Proteins

Although emerging evidence indicates that bacteria extracellularly export many cytoplasmic proteins referred to as nonclassically secreted proteins (ncSecPs) for their own benefit, the mechanisms and functional significance of the ncSecPs in the extracellular milieu remain elusive. "Candidatus Liberibacter asiaticus" (CLas) is a fastidious Gram-negative bacterium that causes Huanglongbing (HLB), the most devastating citrus disease globally. In this study, using the SecretomeP program coupled with an Escherichia coli alkaline phosphatase assay, we identified 27 ncSecPs from the CLas genome. Further, we demonstrated that 10 of these exhibited significantly higher levels of gene expression in citrus than in psyllid hosts and particularly suppressed hypersensitive response (I-IR)-based cell death and H2O2 overaccumulation in Nicotiana benthamiana, indicating their opposing effects on early plant defenses. However, these proteins also dramatically enhanced the gene expression of pathogenesis-related 1 protein (PR-1), PR2, and PR-5, essential components of plant defense mechanisms. Additional experiments disclosed that the increased expression of these PR genes, in particular PR-1 and PR-5, could negatively regulate HR-based cell death development and H2O2 accumulation. Remarkably, CLas infection clearly induced gene expression of PR-1, PR-2, and PR-5 in both HLB-tolerant and HLB-susceptible species of citrus plants. Taking together these data, we hypothesized that CLas has evolved an arsenal of ncSecPs that function cooperatively to overwhelm the early plant defenses by inducing host PR genes. IMPORTANCE In this study, we present a combined computational and experimental methodology that allows a rapid and efficient identification of the ncSecPs from bacteria, in particular unculturable bacteria like CLas. Meanwhile, the study determined that a number of CLas ncSecPs suppressed HR-based cell death and thus indicated a novel role for the bacterial ncSecPs in the extracellular milieu. More importantly, these ncSecPs were found to suppress cell death presumably by utilizing host PR proteins. The data overall provide a novel clue to understand the CLas pathogenesis and also suggest a new way by which phytopathogens manipulate host cellular machinery to establish infection.