Identification Of Jujube Witches' Broom Phytoplasma Pathogenic Effector And Mechanism Of Shoot Proliferation Induced By Zaofeng6

Ziziphus jujuba belongs to Rhamnaceae,which is the most economic specie among the rhamniaceae.Jujube witches' broom(JWB)is a kind of fatal disease in the cultivation of jujube and bring serious economic losses to the jujube industry in China.The pathogen of jujube witches'broom is caused by candidatus phytoplasma ziziphi.To the date,the pathogenic mechanism of candidatus phytoplasma ziziphi has still not been clarified due to absence of culture medium for culturing phytoplasma.In recent years,a large number of researches have proved that effector was responsible for the phytoplasma pathogenesis.Phytoplasmas employ effectors to disturb plant defense and ensures its aggressiveness to host plant.Effectors can disturb plant development and morphogensis by interacting with the target proteins.The key to clarify the pathogenic mechanism of JWB phytoplasma is to screen the pathogenic effectors and find out the target proteins.In this study,bioinformatics methods were used to screen the candidate effectors from the genome of candidatus phytoplasma ziziphi.The pathogenic function of phytoplasma effector of Jujube Witches' broom was determined by constructing effector-expressing transgenic Arabidopsis seedlings and Z.jujuba transformants.The targets directly interacting with pathogenic effector in jujube were screened through the Yeast two-hybrid screening and protein-protein interaction analysis.The interaction network of target proteins was analyzed to clarify the life process of pathogenic effectors involved in.The function of target of pathogenic effectors was analyzed for clarifying the pathogenic mechanism of JWB phytoplasma.The main results are listed below:1.Eight candidate JWB phytoplasma effectors were identified by bioinformatics methods.A total of 8 candidate effectors located in potential mobile unit with N-terminal signal peptide and without transmembrane region were screened according to the information of JWB genome and named Zaofengl to Zaofeng8.Among them,Zaofeng3 belongs to SAP54 group and Zaofeng6 was SAP 11-like effector.By analyzing physical and chemical properties of the effector proteins,we found that the candidate effectors were all short peptides,which was consistent with the characteristics of phytoplasma effectors reported previously.2.Zaofeng6 were identified as pathogenic effectors of JWB phytoplasma.Eight JWB phytoplasma candidate effectors were linked to the plant over-expression vector pSAK277,and then transferred into Arabidopsis by Agrobacterium mediated transformation system for function identification in Arabidopsis and jujube.Meanwhile,the candidate effectors were transiently expressed in tobacco leaves to explore their effects on plant defense.Finally,Zaofeng6,which can induce plant diseases symptom and significantly stimulate plant defense response was screened as the JWB phytoplasma pathogenic effector.Zaofeng6 mainly induced the phenotype of witches' broom in jujube,which was consistent with the phenotype of plants infected with JWB phytoplasma.3 Screening of target proteins of Zaofeng6 in jujubeIn this study,a yeast two hybrid library of pGADT7-cDNA of Ziziphus jujuba was constructed for screening the target proteins of Zaofeng6.A total of 11 target proteins of Zaofeng6 including ZjTCP7 and ZjTCP16 were screened,which were mainly involved in the growth and development,morphogenesis and plant defense.The interaction between Zaofeng6 and ZjTCP7 in the nucleus was confirmed by yeast two hybrid and BiFC assay.The first two a helices of Zaofeng6 was the key regions for interaction with ZjTCP7.Zaofeng6 was proved to degrade ZjTCP7 after targeting it by qRT-PCR and Western blot assay.4 Functional verification of ZjTCP7 in Ziziphus jujubaWe speculated that Zaofeng6 induced the symptom of witches' broom by degrading ZjTCP7.To prove that,zjtcp7 mutant jujube seedlings were obtained by CRISPR technique.It was found that zjtcp7 mutant jujube seedlings exhibited typical phenotype of witches'broom.5.The mechanism of ZjTCP7 regulated phenotype of witches'broom in jujube seedlings was elucidated.In this study,we demonstrated that ZjTCP7 regulated the transcription of key genes(ZjCCD 7,ZjCCD8 and ZjCYP711A1)involved in the synthesis of strigolactone by Yeast One Hybrid assay.qRT-PCR detection showed that ZjTCP7 positively regulated the expression level of ZjCCD7,ZjCCD8 and ZjCYP711A1 genes.The results showed that ZjTCP7 regulated the branching of jujube seedlings through the strigolactone pathway,while Zaofeng6 reduced the level of strigolactone by degradation ZjTCP7.