Identification Of RNA Silecing Suppressor Of Wheat Blue Dwarf Phytoplasma

RNA silencing suppressors increase the efficiency of pathogen infestation by inhibiting or interfering with the host RNA silencing pathway.To date,researches have shown that viruses,bacteria,and fungi can encode RNA silencing suppressors.The study of silencing suppressors is significant in revealing the interaction between pathogens and hosts,better analysising the inhibition mechanism of pathogens to host RNA silencing,and the prevention and control of diseases.Phytoplasma may also encode RNA silencing suppressors to increase the efficiency of self-infection.However,up to now,there have been no studies related to RNA silencing suppressors on phytoplasma.With the completion of the sequencing of the wheat blue dwarf genome,it is possible to identify RNA silencing suppressors.Therefore,we identified the silencing activity of the secreted proteins encoded by the wheat blue dwarf phytoplasma.The main results are as follows:（1）37 secreted proteins DNA gene were cloned and successfully constructed into the vector BI121,and successfully transformed into Agrobacterium GV3101.（2）Plant expression vectors of 37 secreted proteins were separately co-infiltrated with PBI121-GFP in leaves of 16c at the 4-5 leaf stage,while plants infiltrated by PBI121 empty vector as negative controls,PBI121-P19 and PBI121-GFP as positive controls.Results of phenotypic screening under UV light showed that SWP16 can inhibit GFP-induced systemic silencing.In order to verify the phenotypic results,we used real-time RT-PCR and western blot to verify the RNA level and protein level respectively.The validation results were consistent with the phenotypic results.（3）In order to determine the inhibitory activity of SWP16,the SWP16 gene was constructed into PVX vector,and Agrobacterium containing the PVX-SWP16 vector was infiltrated Nicotiana benthamtana to detect the effect of SWP16 on the pathogenicity of virus.Severe symptoms such as mosaic and curl appeared on the upper leaves and the system leaves of N.benthamiana infiltrated by PVX-SWP16 at the 10th day,while the upper leaves of the negative control had curly mild symptoms.Afterwards,we conducted quantitative analysis of the PVX virus coat protein（CP）gene and found that the quantity of PVX-CP in leaves infiltrated by PVX-SWP16 was significantly higher than that infiltrated by PVX empty vector,approximately 1600 times.This result showed that SWP16 not only aggravates the pathogenicity of PVX but also increases the accumulation of PVX virus,which indicated that SWP16 is a pathogenic factor that aggravates plant diseases.（4）At mRNA level,the expression levels of related genes in the RNA silencing pathway of SWP16 during host silencing inhibition were measured,including Nb DCL1-4,Nb AGO1,and Nb AGO2.On the first day,DCL1,DCL2,DCL3,DCL4,and AGO2 were down-regulated in the RNA silencing pathway.Therefore,it is speculated that SWP16 may act on the DCL,AGO synthesis pathway,and suppress gene expression.On the second day after injection,DCL1,DCL2,DCL3,and AGO2 were still down.On the fourth day after injection,all genes were significantly up-regulated.This may be due to the fact that SWP16 mainly does not exert its inhibitory activity by inhibiting the expression of DCL and AGO genes,but mainly exerts inhibitory activity by inhibiting the conduction of silencing signals.By constructing subcellular localization expression vector p CAMBAI1302-SWP16^△SP,the PSWP16^△SPwas located.The SWP16^△SPmainly distributed in the cytoplasm,especially concentrated on the cell membrane.In addition,a small amount of SWP16^△SPis located on the nucleus.SWP16 is mainly located in the nucleus and cell membrane.Subcellular localization also demonstrated that SWP16 may exert inhibitory activity through systemic transmission that suppresses silencing signals.