| As a microsymbiont of soybean,Bradyrhizobium japonicum plays an important role in symbiotic nitrogen fixation and sustainable agriculture.However,the survival of B.japonicum cells under desiccation can be seriously affected when inoculated on the seeds surface and in soils,this is a major concern affecting their nitrogen-fixing ability by establishing the symbiotic relationship with the host,leading to the decreased nodulation,low levels of nitrogen fixation and thus reduced crop yield.In this study,we isolated a water stress tolerant rhizobium from soybean root nodules,Bradyrhizobium japonicum 5038,which would be potentially used in commercial inoculation.A comprehensive analysis of the characteristics of desiccation resistance of strain 5038 and its related molecular mechanisms in response to drought will enriches our understanding of a stress-tolerant rhizobia in response to water-limited condition and the factors that affect its desiccation survival.These will be helpful for development of commercial inoculants that aim at enhance the cell survival.This will also promote the development of a new microbial formulation and improve the rhizobia inoculants used for agricultural soybean production system.In this study,the stress response of strain 5038 was studied from the following aspects:1.The influence of the factors on the desiccation survival of strain5038 were investigated,such as to evaluate the effects of factors(growth medium,growth phase,matrices,metabolites,etc.)on the desiccation survival of B.japonicum 5038.The results showed that the viability of B.japonicum 5038 was significantly affected by different RH levels,matrices,and soybean seed variety.Addition of different sugar metabolites prior desiccation indeed enhanced the cell survival rate,particularly,when combination of trehalose and salt plus riboflavin in the media showed the highest survival rate of 12.96%,which is much higher than control group(0.03%).2.The physiological characteristics of B.japonicum 5038 in response to the whole drying and rehydration process were studied,including the change of cell viability,intracellular trehalose content,exopolysaccharide,cellular protein content.The plate counting method showed that the two step decreases of strain 5038 was found during drying processes.For subsequent rehydration,cell viable counts showed increased trend but there was no statistically difference among R0.5,R3 and R9 treatments.Interestingly,both plate counting and live/dead fluorescence staining assays indicate that a number of viable but non-culturable(VBNC)cells exist in the culture medium upon the rehydration process.3.The whole genome of the strain was obtained by Pacbio sequencing technology,which laid the foundation for the subsequent analysis of the gene expression pattern at the transcriptional level.The complete genome sequence of strain B.japonicum 5038 was sequenced by Pacbio Sequel sequencing platform.The results showed that the genome of strain 5038 consists only a chromosome with a size of 9,238,642 bp,and GC content is of 63.66 mol%,and the sequencing coverages was 460 x.The genome was predicted to contain 8968 CDSs,370 GIs,431 TRs,396 minisatellite DNA,2 microsatellite DNA,55 t RNA,6 r RNA as well as 7 s RNA were predicted.A large number of genes related to desiccation were found in strain 5038,mainly involving three trehalose synthesis pathways(ots AB,tre S,tre YZ),transcription regulators(rpo H,rpo N),and molecular chaperones(gro ES,gro EL,Hsp20).4.To examine differential gene expression when B.japonicum 5038 was exposed to desiccation and subsequent rehydration stresses,RNA-seq whole-genome transcriptional analysis of strain 5038 was performed under10% relative humidity(RH),and subsequent 100% RH conditions,respectively.A total of 462 differentially expressed genes(DEGs,> 2.0-fold)were identified during desiccation,while 3,776 genes showed differential expression during the subsequent rehydration process.Desiccation-responsive genes are mainly involved in signal transduction,metabolisms of carbohydrates,amino acids,and lipids,inorganic ion transport,and energy production.Genes in almost all of these categories were far more up-regulated than down-regulated in the desiccation condition.Notably,trehalose biosynthetic genes(ots AB,tre S,and tre YZ)and genes lig D,opr B and a sigma factor rpo H were significantly induced by desiccation.Interestingly,most of desiccation-inducible genes displayed rehydration-repressed,except three genes encoding heat shock(Hsp20)proteins.For subsequent rehydration,genes involved in translation,transcription,cell membrane regulation,replication and repair,and protein processing were highly up-regulated.5.The upregulation of genes involved in trehalose biosynthesis pathways during the desiccation based on the transcriptome data indicating that disaccharide trehalose may play an important role in resistance dehydration.Additionally,the new finding of the upregulation of Rpo H sigma regulator encoding gene was also found in the strain,thus,biparental mating procedure was used to construct the ots A deletion mutant(Δots A),ots B deletion mutant(Δots B)and rpo H deletion mutant(Δrpo H)to study their roles in the desiccation resistance of strain 5038.The results showed that the mutant strains Δots A,Δots B,Δrpo H were more sensitive to desiccation than the wild type strain 5038,and with an obviously decreased in viable counts,and cell motility assay also showed different result.The growth curves of those mutant strains except mutant strain Δrpo H,showed no significant differences with strain 5038.And there were no obvious differences between those three mutants and the wild type strain in osmotic stress experiments of KCl and PEG6000. |
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