High Throughput Isolation And Functional Characterization Of Rice Root Bacteria

Rice is one of the most important food crops in the world.Stable rice production is important for food security.Plant roots contain a large number of microorganisms which play important roles for plant.Many microbes can promote rice growth,increase nutrient uptake efficiency,induce systemic resistance,and improve abiotic stress tolerance.A deep understanding of the mechanisms of such beneficial interactions between rice and bacteria will illuminate new strategies for sustainable rice production.The rapid development of plant microbiomics,which analyze microbes by high throughput sequencing,provide unprecedented view of plantmicrobe interactions.However,full elucidation of plant-microbe interactions and utilization of beneficial bacterial resources in agriculture require a comprehensive collection of bacterial strains.This study aims to optimize high throughput technologies for isolation and functional characterization of endophytic bacteria from rice root,which provide useful bacterial resources for rice microbiome and rice disease control.We tested a variety of different culture techniques and methods,and collected bacterial strains that covers the core bacterial microbiota of rice.Meanwhile,we took the advantages of bioluminescence-based Firefly luciferase(Fluc)and Nanoluciferase(Nluc)reporting systems to screen beneficial bacteria,which are widely used in gene expression analysis.We tested the feasibility of dual-reporter rice lines for rapid screening of beneficial bacteria.The main results of this study are summarized as follows.1.To isolate and collect rice root bacteria,high throughput culturomics and classical spreading methods were used in this study.(1)For high throughput culturation,bacterial suspension from rice root were dilute to appropriate concentration and then added to 96-well plates containing liquid media.A total of 122 plates were used in this study.Averagely,40%(38/96)wells per each plate contained bacterial isolate(s),resulting approximate 4500 bacterial isolates from rice root.We amplified and sequenced the 16 S r RNA genes of bacterial isolates in the first 14 plates(ZH1 ～ ZH14).The results recovered the taxa information of bacterial taxa from 397 wells in 96-well plates.(2)For classic spreading approach,different nutrient mediums were tested.After spreading root suspension and incubate for 4-7 days in agar plates,R2 A medium generated more colonies with diverse shapes and colors than other nutrient medium.The feasibility of isolating and cultivating anaerobic bacteria from rice roots was also tested with an anaerobic workstation.These bacterial colonies were individually picked up and grown in liquid medium.A total of 1816 bacterial isolates have been collected and characterized by sequencing 16 S r RNA genes.Together,bacterial isolates collected by these two approaches generated bacterial libraries that covered 50% of rice root bacterial microbiota(relative abundance greater than 0.1%).These bacterial isolates belong to four phyla: Proteobacteria,Actinobacteria,Bacteroidetes,and Firmicutes.2.To rapidly screen bacteria stains that potentially affected rice basal defense and nutrient uptake,a dual reporter rice genetic lines were used to monitor the expression of marker genes after inoculating different bacteria.In this reporter line,PR5 promoter,a defense marker genes,drived Nanoluciferase(Nano Luc)and UBI10 promoter expressed Firefly luciferase(Fluc).After inoculating four different bacteria with PR5::Nano Luc rice,the Nluc/Fluc ratio was used to examine the expression of PR5 genes.The results of this reporter assay showed that four bacterial isolated affected the expression of PR5,which was confirmed using quantitative RT-PCR.Using the same strategy,bacterial isolates OTU54 was identified for its ability to affect NRT1.1B expression.The above results demonstrate the feasibility of rice dualreporter transgenic rice plants to quickly screen beneficial bacteria.3.In order to verify the function and colonization of isolated bacteria,we further analyzed the auxin degrading probiotic OTU54.We labeled this strain by introducing a plasmid expressing green fluorescent protein(GFP).After inoculated OTU54(GFP)strains with rice root,GFP signals were observed in root tips,confirming that OTU54 is a colonized in endophytic sphere.GFP-tagged OTU54 does not affect its ability to degrade auxin.Confrontation culture of OTU54 with other auxin-producing rhizobia strains M15 and B27 showed no interaction between OTU54 and M15 or B27.In summary,in this study,a large number of rice root bacteria were cultured through various cultivation methods,which provided useful resources for ricemicrobe interaction studies.We also shown that dual-reporter rice lines(PR5::Nano Luc)can be used for rapid bacterial functional screening,which provides a new method to isolate beneficial bacteria that elicit defense.Using these resources and reporter lines,we identified and characterized a beneficial strain OTU54.Together,this thesis generated a pipeline from bacterial isolating,functional screen,and characterization,which may help to studying the mechanism of rice-microbe interaction in future.