Analysis Of Molecular Mechanism On Salicylic Acid Induced Gene NbPR-R Promoting RNA Silencing

RNA silencing is a defense mechanism against virus and foreign nucleic acids in eukaryotes and plays an important role in defence of plants.The plant hormone salicylic acid is an immune signaling molecule that required for systemic acquired resistance(SAR).More and more studies showed that salicylic acid may play a role in enhance RNA silencing,such as salicylic acid treatment affecting the expression of genes related to RNA silencing,enhancing plant resistance to viruses.But its mechanism is still unknown.Therefore,in-depth study on the molecular mechanism of interaction between the two is helpful to reveal the regulation mode of RNA silencing.The results could provide theoretical basis for plant virus disease control and antiviral breeding.This study combined salicylic acid treatment with the transcriptome sequencing technology to reveal the molecular mechanism of salicylic acid enhancing RNA silencing.The main results were as follows:(1)Transcriptome sequencing analysis was performed to screen RNA silencing related genes induced by salicylic acid(SA).Nicotiana benthamiana leaves were infiltrated with Agrobacterium carrying 35S-GFP after 1 day of treatment with 2mM salicylic acid.GFP fluorescence was observed under a handheld long wave ultraviolet lamp.It was found that the GFP fluorescence of SA treated leaves was weaker than that of water treated leaves.Northern blot assays showed that the lower level of GFP m RNA and the higher level of GFP siRNA in the SA infiltrated leaves than that of the water treated leaves.These results indicate that SA can enhance RNA silencing.Total RNA that extracted from the above treatments was used for transcriptome sequencing analysis and five differentially expressed genes were analyzed using RNAi technology.GFP fluorescence observation showed that salicylic acid induced gene NbPR-R enhanced RNA silencing mediated by transient infiltration.The CDS sequence of NbPR-R contains 681 nucleotides and encods 226 amino acids.Sequence analysis showed that NbPR-R was highly homologous with NtTLP1 of N.tabacum and contained 16 cysteine residues and 3 characteristic domains which were conservative in thaumatin-like proteins.(2)NbPR-R affected the expression of RNA silencing related genes.RNA silencing enhancement of NbPR-R was further verified with Northern blot assay.qRT-PCR detection showed that the transient overexpression of NbPR-R enhanced the expression levels of RNA silencing related genes generally,and NbDCL3,NbAGO6,NbAGO7,NbAGO10 expression level decreased after down-regulate NbPR-R.Thus,we speculate that NbPR-R may affect upstream steps of RNA silencing.(3)NbPR-R regulated the expression level of NbCAT and affected the accumulation of hydrogen peroxide in leaves.The chemical staining of DAB and NBT showed that NbPR-R promoted the accumulation of hydrogen peroxide,but not affect the superoxide anion level in the infiltrated zone.qRT-PCR showed that NbPR-R reduced the expression level of NbCAT.NbCAT was the enzyme responsible for the decomposition of hydrogen peroxide,and down-regulation of NbCAT increased the accumulation of hydrogen peroxide in the leaves.TRV:NbCAT vector was used to reduce the expression of NbCAT.After 7 days post infiltrated with TRV:NbCAT,35S-GFP was transiently expressed in the upper leaves of the plant.And the GFP fluorescence in TRV:NbCAT infiltrated plants was weaker than that of TRV empty vector.This indicates that NbPR-R may increase the accumulation of hydrogen peroxide by down-regulating the expression of NbCAT,thereby enhancs the transient infiltration mediated RNA silencing.(4)Hydrogen peroxide enhanced the transient infiltration mediated RNA silencing.In order to verify the above hypothesis,N.benthamiana leaves were infiltrate with Agrobacterium carrying 35S-GFP after 1 day of treatment with 1.5 mM hydrogen peroxide.And GFP fluorescence of hydrogen peroxide treated leaves was weaker than that of water treatment.Northern blot assay showed that the lower level of GFP mRNA and higher level of GFP siRNA was detected comparing with that of the water treatment.These results showed that hydrogen peroxide is indeed enhance RNA silencing.qRT-PCR detection showed that the expression levels of RNA silencing related genes were up-regulated significantly,and the expression levels of NbAGO6,NbAGO7 and NbAGO10 were significantly increased in the N.benthamiana.In summary,salicylic acid accumulation induces the expression of NbPR-R,NbPR-R inhibits the expression of NbCAT,and the hydrogen peroxide accumulation enhances RNA silencing mediated by transient infiltration in N.benthamiana.