| Jujube（Ziziphus jujuba Mill.）is a one of the fruit trees with important economic value in China,and has a long cultivation history.Fruit of jujube is rich in biologically active ingredients such as vitamin C,polysaccharides,phenol and triterpene acid,which has high edible and medicinal value.Jujube witches’broom（JWB）,induced by phytoplasma infection,is a devastating disease in jujube production.Plants infected by phytoplasma show typical symptoms,such as witches’broom and phyllody（leaf-like floral organs）,which died in 2-3 years later,causing significant economic losses.However,the molecular mechanism of phytoplasma-induced leaf-like floral organs in jujube plants is not clear.Based on the identification of the SAP11-type effector SJP1/2,the effect of SJP1/2 on the development of jujube floral organs has not been reported.Recent studies have shown that auxin plays a key role in the formation of floral primordia and the development of floral organs in the plants.In this study,the healthy and JWB-infected’Fangchang Changzao’was used as the material.Preliminary analysis of the molecular mechanism of the phytoplasma effector SJP1/2 mediated IAA pathway interferes with the abnormal development of jujube flower by hormone detection,bimolecular fluorescent complimentary,bioinformatics analysis,real-time fluorescence quantitative analysis and other technologies.This provides a theoretical basis for biological control JWB and disease-resistant molecular breeding.The main results are as follows:1.Observe the phenotype of the’Fanchang Changzao’disease plant infected with JWB and collect samples of the flower development process in 3 periods（Stage 1:young bud stage,Stage 2:large bud stage,Stage 3:flowering stage）.The q RT-PCR technology was used to detect the change trend of SJP1/2 expression activity during the development of the diseased jujube flower.From young bud to flowering,the results showed that the expression of SJP1 was significantly decreased and SJP2 showed a significant upward trend in the JWB plants.2.Observe the phenotype of the healthy plant’Fanchang Changzao’and collect the samples of the development period of flower in three periods（Stage 1:young bud stage,Stage 2:large bud stage,Stage 3:flowering stage）.LC-ESI-MS/MS technique was used to detect the endogenous auxin content in the flower development of healthy jujube and JWB plants.The results showed that the IAA content showed a downward trend in healthy jujube plants and the IAA content showed an upward trend and then decreased of in JWB plants during the development of flowering.Comparing the endogenous IAA content between healthy jujube plants and JWB plants during the same period of flower development,the IAA content in healthy jujube plants was significantly higher than that of JWB plants.3.QRT-PCR was used to analyze the expression patterns of CIN-like TCP transcription factors during the flower development of JWB infected plants.Compared with healthy plants,the results showed that the expression of ZjTCP2/4/5/13-like gene was disturbed during the flower development,which might be caused by the infection of jujube diseased phytoplasma.4.The interaction between SJP1/2 and ZjTCP2 in plants was verified by bimolecular fluorescence complementary technology.The results showed that SJP1ΔNand SJP2ΔN,deleted the N-terminal and retained the C-terminal TCP-binding domain,were interacted with ZjTCP2 and the interaction signals were located in the nucleus.ZjTCP2 interacts with SJP2,the interaction signal is located in the nucleus.However,ZjTCP2 does not interact with SJP1.Co-expression of the jujube phytoplasma effector SJP1/2 and transcription factor ZjTCP2 in nicotiana tabacum,observation results show that SJP1/2and ZjTCP2 co-localize in the nucleus.The above results indicate that SJP2 interacts with ZjTCP2 in plants.The N-terminus of SJP1 affects the interaction between SJP1 and ZjTCP2 in plants.5.Bimolecular fluorescent complimentary was used to detect the interaction between ZjTCP2 and other TCP members.The results show that ZjTCP2 interacts with ZjTCP7（belonging to Class I-TCP）and also interact with ZjTCP1,ZjTCP12,and ZjTCP18（belonging to CYC/TB1）.The interaction signals are all located in the nucleus.It indicates that the member of CIN-TCP,Class I-TCP,and CYC-TB1 in jujube can form multiple compounds.6.Ten key genes for auxin synthesis,ZjYUCCAs,were identified and their promoter elements were predicted by bioinformatics.The results showed that promoter regions of ZjYUCCA4/5/6/7/10-2/10-4/11 contained TCP transcription factor binding sites.The expression patterns of ZjYUCCAs and ZjTCP2 genes in the three stages of flower development of healthy jujube and JWB plants were analyzed by q RT-PCR.The results showed that,compared with healthy plants,ZjTCP2 and ZjYUCCA2/4/5/6/7/10-3/10-4/11gene expression was disordered during the flower development of JWB plants.Among them,the similar expression trend of ZjYUCCA2/4/11 and ZjTCP2 may be involved in the metamorphosis development process of jujube flowers under phytoplasma infection.7.Through bioinformatics analysis,16 members of the auxin response factor ZjARFs were initially identified.QRT-PCR was used to analyze the differences in the expression of ZjARFs in the three stages of flower development of the healthy and JWB plants.The results showed that the expression of ZjARF1/2/3/4/8 was significantly different in the development process of healthy jujube and JWB plants,which may be related to the abnormal development of diseased jujube flowers.In summary,the SJP1/2 phytoplasma effector SJP1/2 interacts with the CIN-TCP transcription factor ZjTCP2 to disrupt the auxin signaling pathway and cause abnormal floral organ development. |
