Studies On The Mechanism Of PUB13 Regulating The Hypersensitive Response Of Arabidopsis Thaliana-Pseudomonas Syringae

Ubiquitination is a system that specifically modifies target proteins in cells and plays an important regulatory role in a variety of signaling pathways.A series of physiological and biochemical reactions such as cell death,innate immune response,and hormone synthesis are regulated by the ubiquitination system.Previous studies in this study showed that Arabidopsis PUB13 protein is a U-box protein with E3 ubiquitin ligase activity,which is dependent on salicylic acid(SA)for flowering time,cell death and innate immune response in Arabidopsis thaliana.Comprehensive regulation is carried out,but its mechanism of action in plant ETI responses has not been clarified.ETI can cause plants to produce severe disease resistance in a short period of time,which is characterized by Hypersensitive Response(HR)at the site of infection of the disease to inhibit the proliferation and infestation of pathogenic microorganisms.In order to explore the mechanism of PUB13 in Arabidopsis HR,this study used pub13 mutant and RIN4 overexpression material,yeast two-hybrid and Real-time PCR to analyze the fuction of PUB13 in HR at Arabidopsis thaliana.The main results are as follows:1.PUB13 can affect RPS2 mediated HR.After pub13 and Col-0 were inoculated with Pseudomonas syringae pv.tomato DC3000::AvrRpt2,Pseudomonas syringae pv.tomato DC3000::AvrRPM1 and other bacterial solutions,the HR reaction number of pub13 leaves was significantly less than Col-0.At the same time,after trypan blue staining,no cell death was detected in the pub13 leaf inoculation site.2.PUB13 interact with RIN4 to affect Arabidopsis HR.Yeast two-hybrid experiments were carried out with PUB13 as bait,RPS2,AVRRPT2 and RIN4 as target proteins(prey).The results showed that there was an interaction between PUB13 and RIN4.In addition,this study found that the transcription level of RIN4 and RPS2 in pub13 was inhibited.3.RIN4 was constructed into the binary expression vector pCAMBIA1300.It was transferred to Arabidopsis Col-0 plants by Agrobacterium-mediated genetic transformation.A RIN4-OX transgenic line capable of stable inheritance was identified and obtained.4.RIN4 inhibits RPS2-mediated HR.After RIN4 overexpressing plants and Col-0 were inoculated with Pst DC3000::AvrRpt2,Pst DC3000::AvrRPM1 and other bacterial liquids,the HR reaction number of RIN4 overexpressing plants was significantly less than that of Col-0.At the same time,after trypan blue staining,no cell death was detected in the RIN4 overexpressing leaf inoculation site.In addition,the number of statistical colonies found that the number of bacteria on the leaves of RIN4 overexpressing plants was higher than that of Col-0.5.RIN4 induces expression of PUB13.The transcription level of PUB13 in Col-0 and RIN4 overexpressing plants was detected by Real-Time PCR.It was found that the expression level of PUB13 in RIN4 overexpressing plants was higher than that in Col-0.