| The rapid development of intensive farming and increasing application of agricultural chemicals in modern agriculture have caused negative effects on crop production,such as decrease of soil fertility and occurrence of soil-borne diseases.Plant growth-promoting rhizobacteria(PGPR)is defined as a heterogeneous group of rhizosphere colonizing bacteria,which can stimulate plant growth and/or suppress soil-borne pathogens.The effective colonization of applied PGPRs in the rhizosphere is prerequisite for exerting the roles of plant growth promotion and suppression of soil-borne diseases.Therefore,deep understanding the mechanisms of plant-PGPRs interaction is of great significance for guiding the rational application of PGPRs.As a PGPR strain,Paenibacillus polymyxa SQR-21(SQR-21)was isolated and indentified from a healthy watermelon rhizosphere in a severely diseased watermelon field.In the previous studies,SQR-21 strain showed a tremendous inhibition of pathogen in vitro and significant growth promoting effects on watermelon plants in hydroponic system.SQR-21 strain also revealed outstanding biocontrol and growth promotion performance under both greenhouse and field conditions.SQR-21 strain can establish a cooperative and friendly relationship with the watermelon root system,but the detailed interaction mechanism remains to be defined.Firstly,the effects of amino acids in root exudates of watermelon on the chemotactic reaction and root colonization of SQR-21 were investigated.Subsequently,RNA-Seq methodology was carried out to evaluate(1)the effects of concentrated watermelon root exudates on gene transcription of SQR-21 in vitro.(2)the dynamic transcriptome of SQR-21 that co-cultured with watermelon root system for different times in situ.(3)the transcriptional profiling among planktonic,rhizospheric,and root-colonized SQR-21 cells after co-cultured with the watermelon root system in situ for 3 days.The main results obtained were summarized as follows:1.Thirteen amino acids were identified from watermelon root exudates collected in hydroponic system by automatic amino-acid analyzer.Drop assay was performed to investigate the qualitative chemotactic reaction of SQR-21 towards these amino acids.Results indicated that SQR-21 strain showed positive chemotactic reactions to 11 amino acids,especially to threonine,phenylalanine,arginine,and histidine,but not to methionine and tyrosine.The capillary tube test was performed to evaluate the quantitative chemotactic reaction of SQR-21 towards these amino acids,and the results suggested that the SQR-21 strain showed intense chemotactic reaction towards the amino acids within the concentration of 40-80 μM,especially arginine,histidine,and phenylalanine.Finally,on the basis of synthetically considering of drop assay and capillary tube test results,phenylalanine,arginine,and histidine were then chosen to study the effects of exogenous amino acids on the colonized population of SQR-21 recruited by watermelon roots,and the results confirmed that adding exogenous arginine,histidine,and phenylalanine of 60 μM in the rhizosphere could promote recruitment to SQR-21,as revealed by the significantly increased population on watermelon roots.2.The concentrated root exudates of the watermelon could significantly stimulate the growth of SQR-21 in 1/20 TSB medium containing 10%soil extracts.The transcriptional profiling of SQR-21 towards the root exudates under shaken condition was analyzed by Illumina high-throughput transcriptome sequencing.Results indicated that the significantly regulated genes covered 12.4%(625 genes)of the whole genome of SQR-21 and 446 genes of which were up-regulated.The main regulated genes belonged to metabolism(including metabolism and transport of carbohydrate and amino acids,and energy production and conversion),secondary metabolites biosynthesis,transport and catabolism(including antibiotics biosynthesis),defense mechanisms(including multidrug transporter),transcription(including two-component system)and cell motility.Overall,root exudates of watermelon promoted the growth and metabolism of SQR-21 and induced its antibiotics production.3.The dynamic transcriptome of SQR-21 co-cultured with watermelon root under different timepoint(0 h,1 h,3 h,and 9 h)were analyzed by Illumina high-throughput transcriptome sequencing.Results showed that the transcriptional profiling of SQR-21 co-cultured with watermelon for a short time(1 h and 3 h recorded as S1 and S3,respectively)was close to its initial status(time zero recorded as SO),as the numbers of significantly regulated genes were 9 and 21 compared to S0,respectively;while after co-cultured for 9 h(recorded as S9),SQR-21 cells revealed a considerable transcriptome difference with the nonexposed status(SO),and the number of significantly regulated genes was 1008.In order to avoid the additional influence of the environmental changes on the transcriptome of SQR-21 cells at 0 h,S1 was chosen as the control treatment and the initial state for comparison with S9.Results suggested that 870 genes were significantly regulated between these two treatments,covering 17.2%(870 genes)of the whole genome of SQR-21 and 275 of which were up-regulated.The main regulated genes belonged to carbohydrate transport and metabolism,energy production and conversion(involved in electron transfer chain and oxidation-reduction reaction)and transcription.Overall,after co-cultured with watermelon root in situ for 9 h,SQR-21 cells revealed more positive metabolism activity,but the transcriptional profiling of genes relevant to secondary metabolites biosynthesis and defense mechanisms showed less significant changes.4.The transcriptional profiling of planktonic(P),rhizospheric(R)and root-attached(colonized,C)SQR-21 cells after in situ co-cultured with watermelon root for 3 days were analyzed by Illumina high-throughput transcriptome sequencing.Results indicated that the transcriptional profiling of planktonic cells and rhizospheric cells were similar,while a considerable difference was obtained between root-colonized cells and rhizospheric cells,revealing 199 significantly regulated genes,which covered 3.9%of the whole genome of SQR-21 and 88 of which were up-regulated.The main regulated genes belonged to metabolism(including carbohydrate transport and metabolism and energy production and conversion),cell wall/membrane/envelope biogenesis(including glycosyltransferase)and transcription(including biofilm formation).Moreover,as compared with planktonic cells,abrB(negative regulator of biofilm formation)in root-colonized cells was significantly down-regulated and several genes encoding glycosyltransferase and endoglucanase in root-colonized cells were significantly up-regulated.Overall,comparing to planktonic and rhizospheric cells,root-colonized SQR-21 was more active in metabolism and more resistant to environmental stresses,which showd harboring better abilities of rhizosphere adaptation. |
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