Screening And Disease Resistant Role Of Avirulent Effector Proteins From Ralstonia Solanacearum CQPS-1

As a model pathogenic bacterium,Ralstonia solanacearum has a complex pathogenic mechanism.The typeⅢsecretion system（T3SS）is one of the main virulence determinants of Ralstonia solanacearum.The typeⅢeffectors（T3Es）secreted by T3SS play an important role in either pathogenicity in the susceptible host plants or induction of immune responses in the resistant host plants.When the effectors were recognized by the host cells,it will trigger a hypersensitive response（HR）and stimulate the host’s defense system.Although the number of the T3Es is large,there are few studies on their relationship with resistance of host plant.Tabacco,as one of the hosts of Ralstonia solanacearum,is deeply damaged by the bacterial wilt in the southern region of China.Breeding cultivars that are reistant to tobacco bacterial wilt is considered to be the effective method of disease control.However,at present,there is no systematic evaluation of the resistant tobacco cultivars to bacterial wilt in China to screen out the cultivars with strong resistance.Therefore,the existing resistant tobacco cultivars to bacterial wilt in our country were collected and the more resistant cultivars were screened.Using the strain with complete genome information as the material and screening the avirulent effectors in the resistant tobacco by the transient expression system,it will help to analyze the pathogenic mechanism of the pathogen,provide a theoretical basis for breeding lasting resistant cultivars to tobacco bacterial wilt and preventing and controlling it.The main research results that we obtained in this study are as follows.1.The tobacco materials GDSY,G3 and 2015-614 with strong resistance to the strongly pathogenic strain CQPS-1 were screened from 18 tobacco materials that were resistant to bacterial wilt by artificial inoculation at the seeding stage.Eighteen resistant tobacco materials to the bacterial wilt cultivated in eight different regions of the country were selected.At the seeding stage,the tobacco resistance was evaluated at 10⁷ cfu/m L or10⁸ cfu/m L inoculation concentration in the greenhouse using a non-invasive root irrigation treatment.The results showed that the resistance of tobacco to the strain decreased as the inoculation concetration increased.The disease index of tobacco material GDSY was 3.33 and 6.25 at 10⁷ cfu/m L and 10⁸cfu/m L,respectively,showing high resistance to strain CQPS-1.In addition,tobacco materials G3 and2015-614 were also relatively stable in resistance,and the disease index was less than 40 under both inoculation conditions,showing disease resistance.2.Three effectors Rip26,Rip28 and Rip61 that produce a necrotic phenotype on high resistant tobacco material GDSY leaves were screened by Agrobacterium-mediated transient expression system.Fourty effector proteins were cloned from R.solanacearum CQPS-1,and the transient expression vector of 39 effector protein genes were successfully constructed using the plant expression vector p GWB505.Using Agrobacterium-mediated transient expression system to identify effectors on highly resistant tobacco GDSY leaves,it was revealed that the effector protein Rip28 induced strong necrosis in leaf tissue,which was basically consistent with the phenotype produced by the positive control Rip AA_GMI1000.And the effectors Rip26 and Rip61 induced slight yellowing necrosis in leaf tissue nine days after injection.In addition,the effector protein Rip28 induced tissue necrosis in the leaves of other resistant tobacco cultivars,while the effector proteins Rip26 and Rip61 had no phenotype when injected into the leaves of other resistant tobacco cultivars.3.By gene knockout technology,we clarified the role of three effector proteins in physiological and biochemical functions and virulent properties of Ralstonia solanacearum.It was confirmed that the effector proteins played important roles in resistance of GDSY to bacterial strain CQPS-1.The effector protein gene of wild-type strain CQPS-1 was knocked out by homologous recombination and mutant strains?rip26,?rip28 and?rip61 were obtained.The results showed that compared with the wild-type strain,the deletion of the effector protein gene had no effect on the colony morphology and normal growth,which had increased biofilm formation,enhanced the metabolic ability of nitrogen source and relatively weakens the metabolic ability of carbon source,whlie the motility of mutant strains?rip28 and?rip61 were decresed.When the effector protein genes rip26,rip28 or rip61 of the strain was missing respectively,the resistant material GDSY reduced resistance to strain CQPS-1 and no longer showed high resistance,the disease was severe and disease index was62.5,70 and 80,respectively.The mutant strains?rip26 and?rip28 became more proliferative in GDSY leaves and sterile seedlings in dishes.The proliferation ability of mutant strains?rip26 and?rip28 was 100 times as fast as the wild-type strain in sterile seedlings in dishes,and 30 times and 15times as fast as the wild-type strain in leaves,respectively.4.Bioinformatics analysis clarified the structural characteristic of effector protein Rip28,and constructed its prokaryotic expression vector Rip28-p GEX-6p-1,which realized the large expression of protein Rip28 in vitro.Spraying protein Rip28 on tobacco leaves reduced the occurrence of tobacco bacterial wilt under indoor pot condition.The full-length of effector protein Rip28 gene was 2799 bp and encoded 932 amino acids,with a predicted theoretical molecular weight of 101.64 k Da and an isoelectric point of 6.83,and it was a hydrophilic protein.No transmembrane domain and N-terminal signal peptide were found in effector proteins Rip28,while two predicted functional structural domains existed in Rip28,a FAD dependent oxidoreductase domain of 98-477 amino acid intervals and a protein kinase domain of 614-914 amino acid intervals.The prokaryotic expression vector Rip28-p GEX-6p-1 was constructed and a large amount of expression of protein Rip28 was realized in vitro.Under the condition of indoor pot,spraying protein on leaves twice at an interval of 2 days before inoculation could reduce the occurrence of tobacco bacterial wilt,with reduced disease index.The concentration of 200μg/m L had the best control effect.