Identification Of Field Host And Symptom-Determining Effector Of Jujube Witches'-Broom Associated Phytoplasma

Phytoplasma is a vital pathogen infecting crops and ornamental plants.Jujube witches' broom(JWB)disease is caused by phytoplasma,a member of subgroup 16 S rV-B,which can infect a variety of plants such as Zizyphus jujuba,Robinia pseudoacacia and Euonymus maackii.Jujube is an important cash crop in Shandong province.JWB seriously affects the safety of the jujube industry.At present,the potential hosts and mechanisms of symptom formation of JWB are unclear.This study analyzed the potential host of JWB phytoplasma,predicted and analyzed the potential effectors involved in the formation of witches' broom and leaf-like flowers symptoms,and identified host proteins interacted with the two possible effectors.The main results are as follows:(1)Screened the hosts of JWB phytoplasma in the field.15 phytoplasma-like samples from eight different host species collected from Yantai and Tai'an,Shandong province,were analyzed based on 16 S rRNA sequences.It showed that 11 samples from Ziziphus jujube,Cerasus pseudocerasus,Ailanthus altissima,Diospyros kaki,Vitex negundo L.var.heterophylla,Conyza Canadensis and Ulmus pumila host species contain phytoplasma.Sequences similarity between Jujube Witches' Broom strain nky in GenBank is higher,99.07%.The phylogenetic analysis and iphyclassifier analysis showed that all the Phytoplasma isolates obtained in this study belong to subgroup 16 S rV-B.(2)Effector JWB406 is a determinant of witches' broom symptoms.In total,57 proteins were predicted to be possible secreted proteins based on SignalP v3.0 and TMHMM v2.0 with default parameters.Twenty-eight were potential effectors.The heterologous expression of JWB406 and JWB406 transgenic Arabidopsis caused witches' broom symptoms,indicating that JWB406 is related to the formation of witches' broom symptoms.The presence of JWB406 in different parts of N.benthamiana plants was confirmed by immunocolloidal gold technology.Confocal microscopy showed that JWB406 is located in the cytoplasm and nucleus.JWB406 interacting host proteins were screened using Arabidopsis cDNA library.In total,53 host interacting proteins were obtained.It showed that JWB406 interacts with SPL11 and GATA3,but does not interact with TCP13 protein in class II CIN-TCPs,indicating that there is another mechanism of witches' broom symptom formation.(3)Effector JWB157 is a determinant of leaf-like flowers symptoms.The heterologous expression of N.benthamiana and the transgenic expression of JWB157 in Arabidopsis thaliana all caused the symptoms of leaf-like flowers symptoms.Effector JWB157 is located in the cytoplasm and nucleus.A total of 92 proteins interacting with JWB157 were obtained.