Mechanism Of NtRNF217 Gene Regulating Tobacco Resistance To Ralstonia Solanacearum And Inductive Effect Of The Agents To It

Bacterial wilt caused by Ralstonia solanacearum,is a serious threat to the development of crop industry all over the world.As a species complex,R.solanacearum has a wide range of distribution,a variety of host and complex genetic diversity,which makes the control of bacterial wilt particularly difficult.Facing the complex living environment,plants gradually formed a set of mature response mechanisms to overcome various kinds of stresses during long-term evolution.Each cell in a plant has an innate immune system which produces a hypersensitive response at the site of infection by pathogen and then transmits the signal to the entire plant,giving rise to SAR or ISR.Therefore,it is necessary to explore the mechanism of plant-pathogen interaction and find the key of gene that plays an important regulatory role in plant resistance.The E3 ubiquitin ligase family,an important class of proteins involved in ubiquitin modification of target protein,has been shown to be involved in plant resistance to various stresses.At present,there are few studies on regulating the resistance of plants to bacterial wilt by E3 ubiquitin ligase at home and abroad.Our laboratory screened some genes from Nicotiana benthamiana by transcriptome sequencing.These genes may be involved in tobacco resistance to bacterial wilt,and Nt RNF217 was selected for further exploring its functionally characteristics in resistance to bacterial wilt.Ulteriorly we explored the relationship between the transcription level of Nt RNF217 and the resistance of tobacco induced by exogenous substances.The main results of this study are as follows:1.Nt RNF217 was induced by R.solanacearum and exogenous hormones.Comparing the transcript levels of genes in the resistant cultivar PVH2254 and the susceptible cultivar Yunyan87 after R.solanacearum inoculation,the differentially expressed gene Nt RNF217 was screened by the transcriptome sequencing.The qRT-PCR detection further confirmed that Nt RNF217 was involved in the resistance regulation of tobacco to bacterial wilt.At the same time,SA,Me JA and ethephon were also found to induce the transcription of Nt RNF217,and the effect of SA treatment was the best.Sequence analysis revealed that Nt RNF217 encodes E3 ubiquitin ligase protein,which contains 239 amino acids and a RING domain.2.Overexpression of Nt RNF217 enhanced tobacco resistance to bacterial wilt.Nt RNF217 overexpression tobaccao were constructed successfully.The results showed that overexpression of Nt RNF217 significantly reduced the disease index of bacterial wilt and suppressed the colonization of R.solanacearum in tobacco roots.The leaves of Nt RNF217-OE tobacco also had stronger resistance to bacterial wilt than wild type tobacco.3.Overexpression of Nt RNF217 enhanced the hypersensitive reaction of tobacco,regulated the accumulation of ROS and the transcription of defence-related genes.Tissue staining results showed that overexpression of Nt RNF217 gene enhanced the hypersensitive reactions of tobacco leaves to R.solanacearum infection,and promoted the explosion of active oxygen at the pathogen infection point.QRT-PCR results confirmed that the relative transcript levels of the hypersensitive reactions maker genes Nt HIN1 and Nt HSR201 were up-regulated in Nt RNF217-OE tobacco after R.solanacearum infection.In addition,overexpression of Nt RNF217 enhanced the activity of antioxidant enzymes such as SOD,APX and CAT in tobacco leaves,and the relative expression levels of antioxidant enzymes genes were also activated.Afterwards,it is found that overexpression of Nt RNF217 increased the SA-,JA-and ET-responsive genes expression in tobacco,such as Nt PR2,Nt PDF1.2,Nt EFE26 and Nt ACC Oxidase.4.INA effectively activated the transcription of Nt RNF217 and enhanced tobacco resistance to R.solanacearum infection.BTH,INA,scopoletin,Bacillus subtilis and Bacillus polymyxa were selected to induce Nt RNF217 transcription in the experiment.In the non-infection and early infection stage of R.solanacearum,50 mg/L INA treatment had the best inducing effect for the transcription of Nt RNF217,and 125 mg/L scopolatin followed.In the middle term of bacterial wilt,INA treatment still had a certain effect on inducing Nt RNF217 transcription.Meanwhile,Pot experiments showed that INA treatment also significantly enhanced the resistance of tobacco to bacterial wilt.Scopoletin treatment induced tobacco resistance to bacterial wilt in the early stage but had no significant difference with the control in the late stage.There was no obvious effect on inducing the relative expression of Nt RNF217 and resistance bacterial wilt by other material treatments.