Identification Of The Hypersensitive Response-suppressing Effectors From Candidatus Phytoplasma Ziziphi And Primary Investigation Of Their Function Mechanism

Candidatus phytoplasma ziziphi,belonging to the bacterial kingdom,phylum Tenericutes,order Nonsteroidal Mycoplasma,and family Nonsteroidal Mycoplasma,is the causative agent of Jujube witches’room（JWB）,a devastating disease of jujube trees.The bacterium resides in the sieve tubes of plant phloem,and leads to typical symptoms of phyllody,the proliferation of shoots（Witches’broom）,stunting,and even death.So far,Candidatus phytoplasma ziziphi remain unculturable in artificial media,which seriously restricts the research on its pathogenic biology.When interacting with the hosts,the plant pathogens,usually secrete proteins（also called effectors）through various mechanisms to facilitate their infection and colonization.Non-classical secretion is a mechanism that widely exists in bacteria,and the resulting proteins,termed non-classically secreted proteins（nc Sec Ps）,play important roles in the bacterial life.However,this kind of secreted proteins have not yet been fully explored in phytoplasmas.In this study,on the basis of Candidatus phytoplasma ziziphi,we identified the phytoplasma encoded non-classically secreted proteins and further explored their biological functions and molecular mechanisms.The main experiments and results are as follows:（1）A total of 694 annotated proteins from Candidatus phytoplasma ziziphi were screened by using the Secretome P 2.0 coupled with an Escherichia coli-based alkaline phosphatase（pho A）assay,and leading to identification of 25 nc Sec Ps.（2）By using agroinfiltration,six of the 25 Candidatus phytoplasma ziziphi-encoded nc Sec Ps（nc Sec P3,nc Sec P9,nc Sec P14,nc Sec P16,and nc Sec P22）were defined as hypersensitive response（HR）suppressor that blocked HR and H₂O₂ accumulation triggered by both the pro-apoptotic mouse protein Bax and the Phytophthora infestans elicitin INF1 in Nicotiana benthamiana.（3）Subcellular localizations of the six HR suppressors were observed in the epidermal cells of N.benthamiana by using green fluorescence protein（GFP）as a reporter.The results showed that nc Sec P3were predominately localized in the nuclear,nc Sec P14 was generally localized in both the cytoplasm and the nuclear membrane,nc Sec P22 was inclined to target the nucleolus,and nc Sec P9,nc Sec P12 and nc Sec P16 were almost evenly distributed in the cells.（4）RT-q PCR analysis revealed that the six HR suppressors significantly enhanced the transcriptional expression of the programmed cell death（PCD）inhibitors in plant,including three pathogenesis-related proteins（PR）,PR-1,PR-2,and PR-5,and the Bax inhibitors,BI-1and BI-2.The data suggested that these six HR suppressors might inhibit HR by enhancing the expression of plant PCD inhibitors.（5）The plant expression vector p35S-nc Sec P14 harboring the nc Secp14 gene was constructed,and further introduced into Agrobacterium tumefaciens.By using the floral-dip method,the transgenic Arabidopsis thaliana plants expressing nc Sec P14 were generated,thus providing the plant materials to further investigate the molecular mechanism of HR-suppressing factors encoded by Candidatus phytoplasma ziziphi.Phytoplasmas are the important plant pathogenic prokaryotes,and the previous studies on their secreted proteins are confined to the Sec-dependent secreted proteins.Here,the study validated that Candidatus phytoplasma ziziphi encodes an arsenal of nc Sec Ps,and six of them function as HR suppressors.In addition,the study explored the molecular mechanism that Candidatus phytoplasma ziziphi suppresses HR of host plants using nc Sec Ps.The data expanded the research scope of the phytoplasmal secreted proteins,enriched the knowledge on the phytoplasmal pathogenic biology,and also might provide new theoretical basis for the scientific control of Jujube witches’-broom.