Candidatus Phytoplasma ziziphi encodes non-classically secreted proteins that suppress hypersensitive cell death response in Nicotiana benthamiana

Increasing evidence is proving the biological significance of the phytoplasma-secreted proteins. However, besides a few Sec-dependent secretory proteins, no other phytoplasma-secreted proteins have been reported yet. Candidatus Phytoplasma ziziphi is a phytoplasma that causes witches’-broom, a devastating jujube disease prevalent in east Asia. In this study, using the SecretomeP server coupled with an Escherichia coli -based alkaline phosphatase assay, we identified 25 non-classically secreted proteins (ncSecPs) from Ca . P. ziziphi, a novel type of secreted protein associated with phytoplasmas. Among them, six were characterized as hypersensitive cell death response (HR) suppressors that significantly attenuated both Bax- and INF1-triggered HR and H 2 O 2 accumulation in Nicotiana benthamiana , indicating a so-far unknown role of the phytoplasma-secreted proteins. Further, we demonstrated that despite the diverse subcellular localizations in the N. benthamiana cells, the six HR-suppressing ncSecPs enhanced the gene expression of several known cell death inhibitors, including pathogenesis-related proteins (NbPR-1, NbPR-2, and NbPR-5) and Bax inhibitor-1 (NbBI-1 and NbBI-2). Together, our data indicated that Ca . P. ziziphi has evolved an arsenal of ncSecPs that jointly circumvent HR by activating the plant cell death inhibitors, thus providing new insight into understanding the pathogenesis of phytoplasmas.