Screening And Functional Study On The Interaction Protein Of RNA Silencing Suppressor SWP16 Of Wheat Blue Dwarf Phytoplasma

Phytoplasma is a single-cell prokaryote with polymorphism,irregularity and no cell wall.According to statistics,there are more than 800 kinds of plant diseases caused by phytoplasma in the world,and there are more than 100 kinds in China.The report of phytoplasma disease in China as early as the end of the Ming Dynasty in Shen’s agricultural book recorded that mulberry shrinkage "every mulberry has a rash,there is no medical method,and it can’t stay",which has caused serious economic losses to economic trees and crops.Wheat blue dwarf phytoplasma(WBD phytoplasma)is an important food crop disease,which causes plant dwarfing,increased clumps of branches and yellowing of leaf tips.Most of the susceptible plants do not bear fruit because they can not heading normally,resulting in yield reduction in arid wheat areas in Northwest China.The yield loss in wheat areas with severe disease can reach 60%～80%,or even no harvest.Therefore,it is very important to study the pathogenic mechanism,host interaction and control technology of WBD phytoplasma.The genome map of wheat blue dwarf phytoplasma was sequenced in our laboratory,and identified the predicted 37 secretory proteins through the RNA silencing inhibitor identification system.It was found that the effector protein SWP16 has the function of inhibiting systematic RNA silencing and significantly promoting PVX infection.Therefore,it was initially determined to be an active RNA silencing suppressor.At present,there are no relevant research reports on the identification of phytoplasma RNA silencing suppressor and its interaction protein with the host at home and abroad.Therefore,this experiment takes looking for the interaction protein with silencing suppressor SWP16 as the starting point,screens the candidate protein by yeast two hybrid method,determines the interaction by combining yeast one-to-one verification,Bi FC,and co-localization experiments,and then finds the key interaction protein by VIGS and overexpression technology.To reveal the mechanism of WBD phytoplasma silencing inhibitor,the main research results are as follows:(1)Screening and bioinformatics analysis of interacting proteins.Using the isolated ubiquitin yeast two hybrid membrane system and p BT3STE-SWP16 as bait,a total of 22 interacting proteins were obtained from wheat library.Double-screening andα-Galactosidase detection showed that all 22 candidate proteins interacted with SWP16.GO annotation analysis was performed on the candidate proteins,and combined with the transcriptome sequencing results,four target proteins that may be related to the silencing pathway,such as cytopigment B561 domain-containing protein,ribulose bisphosphate carboxylase small chain PW9,Delta tonoplast intrinsic protein TIP2 and S-phase kinase associated protein 1(SKP1),were preliminarily selected to further verify the interaction.(2)The interaction between four candidate proteins and silencing suppressor SWP16 was determined by yeast two-hybrid(Y2H),Bi FC and Co-localization(immunofluorescence)experiments.The CDS sequences of the four candidate proteins were cloned and the yeast two-hybrid experiment was carried out.The results showed that all the four candidate proteins interacted with the silencing suppressor SWP16.Bi FC experiment showed that SWP16 interacted with the four candidate proteins mainly on the membrane,and the Co-localization(immunofluorescence)results further verified the interaction results.SKP1,as the connector protein of SCF(Skp1-Cul1-F-box)ubiquitin ligase complex,may play an important role in the silencing pathway.Therefore,fluorescence intensity analysis further confirmed that SWP16 promoted the expression of SKP1 gene.(3)The effect of SKP1 on the function of SWP16 was investigated by VIGS and overexpression technique.A virus-induced gene silencing vector TRV2-Nb SKP1 was constructed.After silencing Nb SKP1 gene,p BI121-GFP and p BI121-SWP16 was overexpressed.After 25 days of silencing Nb SKP1 gene,the phenotype of N.benthamiana16 c was observed under ultraviolet light.The relative expression of GFP was detected by RT-q PCR and protein quantification,and the results were consistent with the phenotypic results,suggesting that SKP1 plays a key role in the systemic silencing of SWP16.In conclusion,a total of 22 interacting proteins were obtained from yeast two-hybrid screen library,among which 4 proteins may be related to systematic silencing.SKP1 was identified as the key interaction protein for the function of RNA silencing suppressor SWP16 of wheat blue dwarf phytophyte.Silencing of Nb SKP1 gene can directly lead to the SWP16 loss of the function of silencing suppressor.It is speculated that SWP16 needs SKP1 to avoid plant recognition and degradation to maintain its own stability,or indirectly affect other pathways to inhibit the formation of silencing complex.This study is the first to screen the interacting proteins of phytoplasma RNA silencing suppressor,which will help to further analyze the pathogenic mechanism of WBD phytoplasma and explore new prevention and control technologies for phytoplasma diseases.