Construction Of Isogenic Tagged System In Ralstonia Solanacearum And Its Application To Evaluate Bottleneck Effect Against Host Infection

Ralstonia solanacearum(R.solanacearum)is one of the ten most difficult plant bacterial diseases(R.solanacearum ranks second)because of its wide host range,serious damage and difficult prevention.In southern provinces and cities of China,especially in tropical and subtropical regions,R.solanacearum can infect a variety of important economic crops such as tobacco,tomato,potato,peanut,etc.,and lead to large-scale production reduction or total crop failure in some fileds.To date,there is no generally effective ways for prevention and control.As a soil-borne pathogenic bacteria,R.solanacearum infection undergoes the main infection link of root surface attachment,infection,colonization and proliferation,and produces a large amount of viscous extracellular polysaccharides,which seriously blocks the water and nutrient transport in the vascular bundle catheter,resulting in rapid wilting and death of plants.Infection process of R.solanacearum is constrained by many links,including rhizosphere environment,root surface barrier,root immunity,vascular bundle catheter pressure,and plant immunity.This constraint phenomenon is also called’bottleneck effect’.The bottleneck effect value(called N_bvalue)can be quantified by evaluating abundance changes of the’founding population’that successfully invaded host compared to the original population.In this study,we used the’isogenic tagged system’to mark the wild-type strain(OE1-1)with different DNA barcodes and contruced the original population.After treatment with different bottleneck effects,each barcoding strain in the founding population could be detected with high-throughput DNA sequencing approcach and then N_bvalues were calculated by evaluated abundance changes of each barcoding strain.Mains results in this study were presented as following:1.Establishment of the isogenic tagged system and detection of each barcoding strain with high-throughput DNA sequencing approcach(1)Establishment of the isogenic tagged system and construction of the original populationBased on the Tn7-mediated genomic site-specific insertion system,90R.solanacearum strains with different barcode markers were constructed in this study.Each barcode markered R.solanacearum strain exhibited identical growth characteristics(in vitro growth and in planta growth)and infection profiles as the wild-type strain OE1-1.These 90 barcode markered were mixed with equal proportions to construct the original population.(2)Abundance detdction of of the original population with High-throughput DNA sequencing approachThese barcode markered strains were also mixed weith different ratio and sampled at different growth phases of Lag Phase,Log Phase and Stationary Phase in broth medium.Abundance of these barcode markered strains was identical to these mixed proportions and was stabilized at different growth phases,confirming that the isogenic tagged system was appropriate to evaluate abundance variation of each barcode markered strain in the founding population compared to the original population and detect their relative abundance changes.(3)Feasibility of the generated original population for host infection toward tomato plantsThe original population with 90 barcode-markered strains was subjected to invade tomto plants and the founding population in tomato stems was recovered for abundance analysis.Tomato plants were inoculated with methods of soil-soaking,root-cutting and petiole cutting,which mimic natural invasion through root crotex,directly invading root tissues and directly invading xylems vessels,respectiley and refter to different barrier of bottleneck.A total of about 75 barcode-markered strains were detected that form the founding population even through the loosest bottleneck of the petiole cutting on susceptible tomato plants(Ailsa Criga,AC),indcaitng that riginal population with 90 barcode-markered strains was appropriate to evaluate different bottlenecks blocking infection of R.solanacearum toward host plants.2.Quantification of different bottleneck barriers blocking infection of R.solanacearum toward tomato plantsThe soil-soaking inoculation refers to the strongest bottleneck barrier of physical barrier of roots,and that of the petiole cutting refers to the loosest bottleneck barrier of immune barrier inside xylems vessels.The root-cutting inoculation refers to moderate bottleneck barrier that blocks colonization inside roots and migration to the overground part.The original population was subjected to invade two tomto varities of susceptible(AC)and resistant tomato(Hawaii 7996,H)with three inoculation methods to quantify bottleneck values(N_b)that block R.solanacearum infection.(1)N_bvalues of different bottleneck barriers against R.solanacearum infectionThe N_bvalues of different bottleneck barriers from susceptible tomato(AC)with inoculation methods of soil-soaking,root-cutting and petiole cutting were approximate15,182 and 222,respectively,which met with barrier strength from these inoculation methods.The N_bvalues from resistant tomato(H)with three inoculation methods were10,16 and 95,respectively.There was no significant difference among N_bvalues from H inoculated with method of the soil-soaking and root-cutting.Noet that N_bvalues from H inoculated with method of the soil-soaking was just slightly lower than that from AC,while N_bvalues from H with root-cutting and petiole-cutting were substantially lower than that from AC,indicating that physical barrier of roots was strong in both susceptible and resistant tomato plants,while the immune barrier inside resistant tomato plants was much stronger than than inside susceptible tomato plants.In addition,the study found that the’bottleneck effect’of R.solanacearum migration to the stem of the two tomatoes was weak.(2)Effects of different bottleneck effects on the abundance of R.solanacearum progenyAccording to the statistics of the founding population with abundance greater than10%in each plant,it was found that the detection rates of highly resistant(H)and susceptible(AC)tomato plants were 94%and 6%respectively when tomato seedlings were infected by’root-cutting inoculation’.When the two tomato seedlings were infected by’petiole-cutting inoculation’,The detection rate of resistant tomato was 16%,and the founder population with abundance greater than 10%was not detected in susceptible(AC)tomato.When the two tomato seedlings were infected by’soil-soaking inoculation’,the detection rates in the two tomato plants were 94%and85%,respectively.Obviously,the higher abundance of R.solanacearum invaded the host plant earlier,and then occupied the niche and proliferated.The strong’infection bottleneck’can seriously restrict the invasion of R.solanacearum to host plants.Only a few’original populations’of R.solanacearum can successfully invade in the early stage,so the abundance of’founding populations’of offspring varies greatly.The weaker’infection bottleneck’had weaker constraints on R.solanacearum infection,allowing most of the’original population’to invade successfully in the early stage,so the’founding population’had relatively uniform abundance.(3)Correlation analysis between bottleneck value(N_b)and population abundanceThe linear correlation analysis between N_bvalue and population abundance showed that’genetic bottleneck’and’population bottleneck’were significantly co-variant.Under the treatment of resistant and susceptible plants and different infection methods,the population abundance increased rapidly with the increase of N_bvalue.Under the treatments of’soil-soaking inoculation’,’root-cutting inoculation’and’petiole-cutting inoculation’of resistant and susceptible plants,the N_bvalue was negatively linearly correlated with population abundance.