Biological Mechanism Involved In Enhancement Of Rhizosphere Colonization Of Beneficial Rhizobacterium SQR9 Mediated By An Indole-3-acetic Acid-synthesis Key Gene YsnE

Plant growth-promoting rhizobacteria(PGPRs)are a type of beneficial microorganisms that can colonize in rhizo sphere and have the functions of plant growth-promotion or biocontrol.Based on the outstanding plant growthpromotion/biocontrol effects and environmentally friendly characteristics,PGPRs are being more and more widely used as inoculants or bio-fertilizers in agricultural production,which is thought to be important for Green Agriculture.Therefore,in-depth understanding of the rhizosphere interaction mechanism between PGPRs and plants is of great important for improving the application effects of beneficial microbes and the relevant bio-fertilizers.Bacillus amyloliquefaciens SQR9 is a commercialized PGPR strain that isolated from the rhizosphere of healthy cucumber plants.Synthesis of indole-3-acetic acid(IAA)is the main mechanism contributed to plant growth-promotion by strain SQR9;ysnE is a key gene that involved in the IAA synthesis pathway in SQR9.This study was performed focusing on the ysnE-mediated rhizosphere interaction between strain SQR9 and cucumber plants.First,the effects of gene ysnE on colonization of strain SQR9 on cucumber roots in different systems were evaluated;then,the biological manner that mediates the rhizosphere colonization of SQR9 by gene ysnE,was investigated;finally,metabolomics and transcriptomics methods were used to identify the key compounds that influence the bacterial colonization,from the different components in local root exudates response to wild-type SQR9 and mutant ?ysnE inoculation.The main results are as follows:1.Rhizosphere colonization of mutant ?ysnE that deficient in IAA synthesis,was significantly impaired as compared with wild-type SQR9,in single-inoculation,competitive colonization,and split-root system.In single-inoculation(wild-type and the mutant ?ysnE were inoculated in cucumber rhizosphere as the same concentration),mixed inoculation(wild-type and the mutant were mixed in equal concentration and inoculated in the same plant),and split-root inoculation(wild-type and the mutant were equally inoculated in the left and right side of the same cucumber plant in split-root equipment,respectively)systems,colonization of mutant ?ysnE on cucumber roots(0.86 X 105 CFU g-1 root weight)was all much lower than that of the wide type(1.0 ×106 CFU g-1 root weight).Moreover,the growth in root exudates medium,cell motility(swimming and swarming),chemotaxis to cucumber root exudates,and biofilm formation between wild-type SQR9 and the mutant ?ysnE,were all found to be similar.2.With regards to the left and right side of the same cucumber roots(in splitroot system)inoculating wild-type SQR9 and mutant ?ysnE,there was no difference in plant immunity response and root oxygen secretion,but the root exudates of the left side(receiving wile-type)was more favorable for inducing bacterial growth and chemotaxis,when comparing with the right side(receiving?ysnE).In the split-root system,the same cucumber plant roots were inoculated with the wild-type SQR9 in the left side while ?ysnE in the right side;the expression pattern of the defense genes,as well as the root oxygen-secretion levels(0.027?mol O2 h-1 g-1 and 0.028 ?mol O2 h-1 g-1),were found to be similar in both sides.The root exudates in left side(receiving wild-type)and right side(receiving ?ysnE),were also collected as RE-W and RE-M,respectively.In the growth assay,the wild-type and mutant both reached the maximum growth amount after 8 hours in the RE-W medium(OD600=0.3),while the maximum growth amount was only 0.2 in the RE-M medium.The chemotaxis of ?ysnE to the two kinds of exudates under different concentrations(0.1,1.0,10,100,and 1000 mg L-1)was further investigated.It was found that 10 mg L-1 of RE-W can attract cells of 1.0×106 CFU mL-1,while that for RE-M of the same concentration was only 4.7×105 CFU mL-1;the recruited cells reached 1.0×106 CFU mL-1 only until the concentration of RE-M increased to 100 mg L-1.These results indicate that the IAA produced by wild-type SQR9 enhances its local rhizosphere interaction with cucumber,which affects the root exudates composition and therefore promotes its own rhizosphere colonization.3.The IAA produced by strain SQR9 induced the local secretion of carbohydrates(D-galactose,glucose,etc)and malic acid,thereby enhancing the bacterial rhizosphere colonization.The root exudates composition and gene expression profiles of cucumber roots inoculated with wild-type SQR9 and mutant?ysnE were determined by metabolomics and transcriptome methods,respectively.It was found that in the split-root system,inoculation of SQR9 induced more root secretion of carbohydrates(D-galactose,glucose,etc)and organic acids(malic acid)than that inoculating the mutant ?ysnE,and partially genes encoding sugar and malic acid transporters were also up-regulated by wild-type SQR9.Moreover,it was found that adding malic acid at concentrations of 10'5 and 10-4 M can significantly increase the population of root-colonized ?ysnE from 2.2×104 CFU g-1 root weight to 7×104 CFU g-1 root weight,suggesting that malic acid can enhance the root colonization of mutant ?ysnE within a certain concentration.In summary,the IAA produced by PGPR SQR9 can induce the local root secretion of sugars and malic acid,which recruits strain SQR9 by stimulating its growth and chemotaxis,and finally enhance its root colonization.This study expands the understanding of rhizosphere colonization mechanism of beneficial bacteria from the perspective of bacteria-medicated rhizosphere interaction,and can provide a theoretical basis for guiding the development and application of related bio-fertilizer products.