| Ralstonia solanacearum is one of the ten most devastating plant pathogenic bacteria in the world,and bacterial wilt of plants caused by Ralstonia solanacearum is a major worldwide bacterial disease.There is still lack of a rapid and convenient R.solanacearum inoculation method,which severely limits disease resistance breeding.R.solanacearum secretes 60 to 90 effectors into the plant cells by type III secretion system during plant-R.solanacearum interactions.These effectors suppress plant defense and facilitate bacterial infection by interfering with plant physiology and biochemistry in susceptible host plants.In contrast,these effectors trigger plant defense in host plant containing the corresponding disease resistant genes.Rip21 induces hypersensitive response in Nicotiana benthamiana,and is recognized by N.benthamiana.However,the underlying recognition mechanism is still unclear.In this study,we used Rip21 as a model to explore the molecular mechanism of N.benthamiana-R.solanacearum recognition,providing a scientific basis for the development of novel prevention and control strategies of bacterial wilt.The main findings of this study are as follows:1.Establishment of a rapid R.solanacearum inoculation method,which provides technical support for the evaluation of plant resistance to bacterial wilt and speeding up crop resistant breeding.MS medium was used to prepare plant materials,and 6-day-age plant seedlings were inoculated with R.solanacearum.The plant disease resistance was evaluated by measuring the bacterial titers of R.solanacearum in plants after inoculation.N.benthamiana was used as plant material.Based on the infection characteristics of R.solanacearum,the inoculation method was developed by inoculating R.solanacearum in a petri dish,and the colonization of the N.benthamiana after inoculation at different concentrations and different time points was evaluated.The result showed that the rapid inoculation method can reflect the dynamic changes of bacterial colonization in N.benthamiana at different inoculation concentratuions and different inoculation time points,and the appropriate concentration of R.solanacearum and the sampling time were finally determined.The colonization of R.solanacearum GMI1000 and R.solanacearum CQPS-1 in N.benthamiana was then analyzed,and the result showed that this method can effectively reflect the difference in N.benthamiana resistance to two different R.solanacearum strains.Finally,the established R.solanacearum inoculation method was also used to evaluate the resistance of four common cultivated tobacco varieties to R.solanacearum.The results showed that Honghuadajinyuan showed obvious susceptibility to R.solanacearum,whereas DB101 showed resistance to R.solanacearum CQPS-1.2.Indication that Ralstonia solanacearum Rip21 can be recognize in N.benthamiana,and identification of the potential regulatory proteins involved in Rip21 recognition in N.benthamiana.Rip21 can induce cell death in N.benthamiana.In order to further clarify whether Rip21 is recognized by N.benthamiana,a homology-mediated gene knockout method was used to generate Rip21 knockout mutants,and analyzed their cell death induction ability in N.benthamiana.The result showed that the cell death induction ability of Rip21 knockout mutants was significantly reduced compared to wild-type strain,indicating that this effector could be recognized by the disease resistance genes in N.benthamiana.Then Rip21 was used as bait to identify its interaction proteins in N.benthamiana by the yeast two-hybrid method.A total of 181 potential Rip21-interacting proteins in N.benthamiana were identified in the preliminary screening.It was further showed that Rip21 could interact with Nb DAG1,Nb DAG2 and Nb NPR1.In order to clarify the role of these three proteins in Rip21 recognition in N.benthamiana,we first tested the effects of transient expression of these three N.benthamiana genes on Rip21-induced cell death.Transient expression of these three genes suppresses Rip21-induced cell death.Nb DAG1,Nb DAG2 and Nb NPR1 are all involved in the recognition of Rip21 effectors in N.benthamiana.We further silenced Nb DAG1,Nb DAG2 or Nb NPR1 in N.benthamiana by virus-mediated gene silencing approach,and analyzed Rip21-induced cell death in these gene-silenced plants.The results showed that the cell death induced by Rip21 was significantly enhanced in Nb NPR1-silenced plants compared to that in the control.The above results indicate that Nb NPR1 plays a key role in the recognition of Rip21 in N.bentamiana,providing a basis for further research on the recognition mechanism of Rip21. |
