Study On The Function And Mechanism Of MiR56/SPL9 In Arabidopsis Immunity

The functions of microRNA156（miR156）and its targeted SQUAMOSA PROMOTER BINDING PROTEIN-LIKE（SPL）transcription factor genes in plant development have been widely investigated.Here,three Arabidopsis transgenic plants,Pro35S:MIR156（miR156 overexpression mutant）,Pro35S:MIM156（miR156repression mutant）and ProSPL9:rSPL9（one of the SPLs,SPL9,overexpression mutant）were used to investigate the immunity function of miR156/SPLs network during[Pseudomonas syringae pv.tomato（Pst）DC3000]infection.The main results are as follows:1.miR156/SPL9 regulates the accumulation of Reactive Oxygen Species（ROS）in Arabidopsis.In both one-week-old seedlings and five-weeks-old juvenile leaves,The concentration of H₂O₂ and O^2.-,NADPH oxidase activity and the expression levels of antioxidant-related genes（GR、CAT、MADR3 and MADR6）and the activity of antioxidant-related enzymes（SOD,CAT,GR,POD,MADR and DHAR）significantly decreased in Pro35S:MIR156 mutant,but dramatically increased in ProSPL9:rSPL9 and Pro35S:MIM156 mutant compared with those in wild-type Col-0plants.2.miR156/SPL9 regulates salicylic acid（SA）signaling pathway.RT-qPCR data showed that the mRNA expression levels of SA-signaling genes PR（PR1,PR2,PR4and PR5）and NPR（NPR1,NPR3 and NPR5）were significantly down-regulated in Pro35S:MIR156 but up-regulated in ProSPL9:rSPL9 and Pro35S:MIM156 relative to those in Col-0 plants.3.miR156/SPL9 modulates disease response.When inoculated with Pst DC3000by syringe infiltration,Pro35S:MIR156 is more susceptible to Pst DC3000,while Pro35S:MIM156 and ProSPL9:rSPL9 are more resistant to Pst DC3000 than WT.After bacterial challenge,the PR1 and NPR1 transcript levels were up-regulated.However,the increased fold were lower in Pro35S:MIR156 but higher in Pro35S:MIM156 and ProSPL9:rSPL9 than that in Col-0 plants.Consequently,the shift in resistance ability of the indicated mutants to bacterial challenge is likely due to suppression or activation of the SA signaling pathway.4.Ablation of miR156-mediated bacterial susceptibility by H₂O₂.We observed milder leaf symptoms and detected lower levels of bacterial population in Col-0 and Pro35S:MIR156 plants treated with H₂O₂ compared with the mock control.However,Pro35S:MIR156 plants still exhibited significantly higher levels of bacterial growth compared with WT.In addition,the expression of SA signaling genes was significantly increased in H₂O₂ treated plants compared with the mock controls.In Pro35S:MIR156plants,the transcript levels of SA responsive genes were less affected than that in WT after H₂O₂ treatmentThese results suggest that the ablation of miR156-induced susceptibility to Pst DC3000 infection by H₂O₂ was due to activation of the SA signaling pathway.In conclusion,we have demonstrated that ROS accumulation and SA signaling gene expression were suppressed in miR156 overexpression mutants but elevated in miR156 suppression mutants and SPL9 overexpression mutants.As a result,miR156induced susceptibility to Pst DC3000 infection,while SPL9 played an active role in antibacterial immunity in Arabidopsis.Furthermore,foliar H₂O₂ treatment triggered activation of the SA signaling pathway,leading to mask the miR156-mediated Pst DC3000 susceptibility.Taken together,our results provide new insights into the immune functions of the miR156/SPLs network,which appear to involve regulation of ROS accumulation and activation of the SA signaling pathway.