The Effect Of Hydroxycoumarins On Regulating Virulence Characteristics Of Ralstonia Solanacearum

During the interaction of pathogen and host plants,plant secondary metabolites are enriched reponsed to pathogen infection,resulting improve bacteria growth.Meanwhile,host plants produce specie small molecular metabolites that has antibacterial activity,through regulating plant defense system during pathogen invade.Therefore,plant secondary metabolites play an important role in the interaction between pathogen and host plants.Coumarins,naturally derived compounds composed of fused benzene and?-pyrone rings,are widely present in plant species and shown to possess a variety of biological activities.Coumarins secreted by plant roots shown changing rhizosphere microbiota and promoting plants health.Furthermore,coumarin are accumulated in infected tissues reponsed to pathogen infection and shown antibacterial or antifungal activity.Coumarins are proven play an important role in regulation of virulence characteristics of pathogen,plant health.Therefore,it is interesting to study regulation of certain coumarins on virulence characteristics of plant pathogen,and understand the role of coumarins in the relationship between pathogen,rhizosphere microbiota and host plant.Bacterial wilt is a soil-borne bacterial disease caused by Ralstonia solanacearum?R.solanacearum?,one of the world's most important bacterial plant pathogens.R.solanacearum heavily infecting more than 250 plant species in 54 families,including many economic and vegetable crops such as tobacco,tomato,eggplant and causing serious economic losses.Tranditional chemical methods have proven shown few control effenciey in the field conditions.Meanwhile,beneficial microbial agents display poor stability and low control effenciey.Recently,many studies focued on the antibacterial activity of coumarins in medicine,and key role in the interaction of pathogen and host plants.Meanwhile,coumarins are represent safety,effective and environmentally friendly activity.However,few studies focued on biological activity virulence characteristics regulation of coumarins against R.solanacearum.Therefore,in this study we measured the antibacterial activity of coumarins against R.solanacearum.Then,three hydroxycoumarins?umbelliferone,esculetin and daphnetin?were choosed to evaluate biological activity and cell structure,virulenve characteristics of R.solanacearum.Furthermore,using RNA-Seq to explore the mechinism of hydroxycoumarins on the transcription level of R.solanacearum.Then,selected lpxB as target gene and used homologous recombination to construct lpxB mutant to evaluated the regulation of lipopolysaccharide in R.solanacearum.In addition,evaluated the mechanism of umbellifone on suppressing expression of type III secretion system,and daphnetin as extracellular polysaccharides inhibitor reduced virulence of R.solanacearum on tobacco.Finally,we evaluated the effect of hydroxycoumarins on the control effenciency of tobacco bacterial wilt in greenhouse and field condition.The main research results that we obtained in this research are as follows.1 Effect of antibacterial activity of coumarin against R.solanacearum and2D-QSAR analysisIn order to evaluate the effects of 29 coumarins on the antibacterial activity against R.solanacearum.The results shown that the antibacterial activity of 16 coumarins against R.solanacearum are better than the positive control?thiediazole copper?.Among the coumarins,daphnetin shown best antibacterial activity with IC₅₀ is 16.13 mg L^-1.The position and number of hydroxyl substituents determine antibacterial activity of coumarins against R.solanacearum.Hydroxyl substitutions at C-6,C-7 and C-8 sites enhanced antibacterial activity of coumarins.Compared with single hydroxy substituent,coumarins with two hydroxy substituents displayed better antibacterial activity against R.solanacearum.Then,we constructed a quantitative structure-activity relationship?QSAR?model of antibacterial activity of coumarins using Pearson analysis,which included Mor23u,DISPv,R3u and HATS8v as descriptors and R² value achieved0.861.This model can explain 86.1%of antibacterial activity of target compounds.2 Biological effects of hydroxycoumarin?Hycs?on R.solanacearum2.1 Effect of hydroxycoumarins on antibacterial activity and cell construct on R.solanacearumIn this study,antibacterial activity of three hydroxycoumarins on R.solanacearum were mearsured by growth curve and minimal inhibit concentration.The results shown that 50-100 mg L^-11 of hydroxycoumarins inhibited growth of R.solanacearum.The minimum inhibitory concentrations of daphnetin,esculetin and umbelliferone against R.solanacearum were 64 mg L^-1,192 mg L^-1,and 256 mg L^-1.Furthermore,compared with control treatment,the survival rates of daphnetin,esculetin and umbelliferone were significantly lower with 7.23%,17.81%and 63.61%,respectively.Transmission electron microscope shown that esculetin and daphnetin destroyed cell membrane structure of R.solanacearum,caused leakage of cytoplasm and cell death.2.2 Effect of hydroxycoumarins on the infection characteristics of R.solanacearumDaphnetin,esculetin and umbelliferone significantly inhibit the biofilm formation of R.solanacearum.The inhibitory rates of 10-50 mg L^-1 daphnetin and esculetin were 44.98%-98.59%and 13.85%-71.76%.The effects of hydroxycoumarins on the motility activity of R.solanacearum were measured by plate swimming method.The results shown that 5-50 mg L^-1 hydroxycoumarins inhibits the motility activity of R.solanacearum,and daphnetin displayed best inhibition activity with swimming diameter decreasing 0.76-fold,0.69-fold,0.65-fold and 0.29-fold.Furthermore,10mg L^-1 and 25 mg L^-1 daphnetin significantly reduced extracellular polysaccharides secretion with1.38-fold and 2.63-fold.In addition,compared with control,umbelliferone,esculetin and daphnetin significantly suppressed the transcriptional expression of flagella motility-related genes fliA and flhC with 1.57,2.40.2.35-fold and 1.46,1.60 and 1.52-fold.Otherwise,esculetin and daphentin significantly reduce the expression of biofilm formation-related genes lecM with1.88-fold and 2.90-fold.Interesting,daphnetin specifically inhibit the expression of transcription regulator xpsR and epsE with 1.70-fold and 1.28-fold compared to control.3 Molecular mechanisms of R.solanacearum in response to hydroxycoumarins3.1 Transcriptome analysis of R.solanacearum in response to hydroxycoumarinsThe prokaryotic chain-specific transcriptome sequencing was used to analyze the transcriptional level of R.solanacearum in response to hydroxycoumarins?umbelliferone,esculetin and daphnetin?.Compared with the control,significantly differently expressed genes of umbelliferone,esculetin and daphnetin treatment were 344,698 and 922,respectively.In the differential expressed genes,there were 191 genes in common among three hydroxycoumarins treatment.Based on gene function annotation,it was found that hydroxycoumarins significantly suppressed expression of lipopolysaccharide synthesis,fatty acid synthesis,ribosomal subunit coding and oxidative phosphorylation.Also induced genes expression of oxidative stress-related and drug efflux pumps.According to GO enrichment and KEGG pathway analysis,the differential expressed genes of R.solanacearum in response to hydroxycoumarins were mainly enriched in the cell membrane,plasma membrane,ribosome,transporter activity and translation.KEGG pathway shown that focused on secondary metabolite biosynthesis,ribosome,ABC transporter,oxidative phosphorylation,fatty acid metabolism pathways.The results shown that hycs can inhibit the transcriptional expression of flagella-related genes and type III secretion system-related genes.Meanwhile,daphnetin specifically inhibited extracellular polysaccharides synthetic gene cluster?Rsp1010-Rsp1020?.3.2 The mechanism of hydroxycoumarins control expression of lipopolysaccharide synthesis gene lpxBBased on destroy of hydroxycoumarins on bacterial cell membrane structure of R.solanacearum,we analysis transcriptional expression of membrane related genes supplemented with Hycs.The results displayed that daphnetin and esculetin specifically inhibited gene expression of lipopolysaccharide biosynthesis in R.solanacearum,such as Rsc0135,Rsc0136,lpxB,lpxA,fabZ and lpxD were significantly suppressed with 4.45-9.58 fold and 5.15-10.83 fold.Among them,lpxB was down-regulated 10.83-and 9.58-fold by esculetin and daphnetin treatment.According to gene function,lpxB is involved in a lipid A disaccharide synthase and plays an important role in the lipopolysaccharide synthesis pathway.In order to evaluate effect of lpxB on bacterial growth and virulence of R.solanaceaum,we used homologous recombination method to construct lpxB mutant.The growth of the lpxB deletion mutant was significantly lower than that of the wild-type R.solanacearum CQPS-1.Meanwhile,the mobility activity of lpxB mutant was decreased 2.67-fold,3.18-fold and 4.97-fold after inoculating for 36 h,48 h and 60 h.Meanwhile,biofilm formation were significantly lower with 2.57-fold compared with control.The disease index result found that lpxB mutant can delay the occurrence of tobacco bacterial wilt.The results indicated that hydroxycoumarin suppressed biosynthesis of lipopolysaccharide,destroyed bacterial membrane and inhibited growth of R.solanacearum,causing reduce of virulence on tobacco.4 Mechanism of umbelliferone and daphnetin regulate virulence factor of R.solanacearum4.1 Umbelliferone suppresses transcriptional expression of type III secretion system through hrpG-hrpB and prhG-hrpB pathwayTo determine how umbelliferone?UM?affects the regulatory components of T3SS regulators,mRNA levels of T3SS regulators were measured by RT-PCR.The results showed that expression of some T3SS upstream regulators was signidicantly reduced,such as hrpG,prhG and hrpB,compared to the DMSO control.However,UM had no effect on expression of other T3SS upstream regulators?prhA,prhR,prhI,and prhJ?.The results indicated that UM mediated regulation of R.solanacearum T3SS regulators mainly through hrpG-hrpB and prhG-hrpB pathways.UM significantly suppressed gene expression of type III effectors?ripX,ripD,ripP1,ripR,RipTAL,and ripW?with 3-4 fold.UM significantly suppressed the wilting disease proves by reducing colonization and prolideration in tobacco roots and stems.4.2 The mechanism of daphnetin on the regulation of extracellular polysaccharide secretion of R.solanacearumIn order to determine daphnetin?DA?on suppression of extracellular polysaccharide associated genes,we used RNA-Seq and RT-PCR to evaluate gene expression of R.solanacearum supplemented with DA.The results showed that DA suppressed transcriptional expression of the extracellular polysaccharide synthesis gene cluster?Rsp1010-Rsp1020?via transcription regulator xpsR but did not affect other transcriptiona regulator.Among the EPS synthesis genes,epsB,epsC and wecC were down-regulated 9.38,9.32,8.75 times,respectivitly.Furthermore,we used homologous recombination assay to constructe the mutants in R.solanacearum.The results indicated that epsB,epsC and wecC deletion mutants did not affect growth of R.solanacearum in liquid medium.Compared with the control,three mutants significantly reduced extracellular polysaccharide and biofilm formation,but induced the bacterial mobility activity of R.solanacearum.Compared with control,biofilm formation was suppressed by 2.17-fold,2.11-fold and 2.35-fold.Furthermore,epsB,epsC and wecC mutant showed delay the occurrence of tobacco bacterial wilt.epsB and wecC mutants completely lose pathogenicity,and epsC deletion mutants have significantly reduced pathogenicity on tobacco.Further,the biocontrol effect of epsB as a non-pathogenic bacterial agent on tobacco bacterial wilt was evaluated.The results display that early application of epsB-deficient mutants effectively delayed the occurrence of tobacco bacterial wilt with relatively control efficiency of 53.25%.5 Effect of hydroxycoumarins on control efficiency of tobacco bacterial wiltIn order to evaluate the control efficiency of hydroxycoumarins on tobacco bacterial wilt,we used greenhouse assay and field experiments.The results showed that control efficiency of root application in advance was better than root application after inoculation,foliar spray application had less effect.Hydroxycoumarins significantly reduced the bacterial colonization in roots and bases stem of tobacco.Compared to control,bacterial population of tobacco roots supplemented with 50 mg L^-1 umbelliferone,esculetin and daphnetin was suppressed with 1.58-fold,11.03-fold and 22.54-fold.Meanwhile,tobacco stem bacterial density was reduced by 4.19-22.27 fold,5.99-37.04 fold and 11.12-61.67fold.Furthermore,Hycs delayed the incidence of tobacco bacterial wilt.Daphnetin shown best controlling effenciency against tobacco bacterial wilt,the control efficiency is 38.27%-80.03%.The control effect of daphnetin was maintained at 40.52%⁵9.47%under field condition,which is siminal with control agent thiopenthium copper?37.98%⁶2.26%?.The control efficiency of esculetin was 24.21%⁶9.42%,36.43%⁵3.96%in the greenhouse and field condition.In summary,this study focued on plant secondary metabolites-coumarins,firstly analyzed antibacterial activity of coumarins against R.solanacearum.Then study molecular mechanism of R.solanacearum reponse to hydroxycoumarins treatment using transcriptome sequencing.The results shown hydroxycoumarins inhibit growth of bacteria,suppress genes expression of lipopolysaccharide synthesis,resulting destroy bacterial cell membranes structural stability.Then,we evaluated regulation of hydroxycoumarins on virulence characteristics of R.solanacearum.The results shown hydroxycoumarins can reducebiofilm formation of R.solanacearum.Umbelliferones displayed suppress T3ss regulator gene expression through hrpG-hrpB and prhG-hrpB pathways.Daphnetininhibited expression of the extracellular polysaccharide synthesis genes cluster and reduced the extracellular polysaccharide via controlling transcriptional regulatory factor xpsR.The control effect of hydroxycoumarins on tobacco bacterial wilt was evaluated.Application of esculetin and daphnetin in advance to root can effectively control the occurrence of tobacco bacterial wilt.This study understand mechanism of hydroxycoumarins on regulating virulence characteristics of R.solanacearum.Our study provides a new idea for green ecological control development for bacterial wilt.