Synergism Of Protein YukE Secreted By Type Ⅶ Secretion System Of Bacillus Velezensis SQR9 In Plant Growth Promotion

Long-term excessive application of chemical fertilizers has led soil acidification,compaction and reduced nutrient utilization.Rational application of biofertilizer can reduce the same of chemical fertilizer.Bio-fertilizer is environment friendly and can improve the soil quality and reduce environment pollution.Efficient colonization of beneficial bacteria in rhizosphere is a prerequisite for microbial fertilizer to exert its beneficial functions.Type Ⅶ secretion system is a unique secretion system that only occur in Gram-positive bacteria.It generally interacts with the host through secreting proteins and therefore enhances the colonization of the bacteria.Bacillus is the main group probiotics and has been widely used in bio-fertilizers.Bacillus velezensis SQR9 is a probiotics isolated and identified from healthy cucumber rhizosphere,which effective on growth-promoting and control of soil-borne diseases.In this study,we found that genome of Bacillus velezensis SQR9 encodes an functional type Ⅶ secretion system and its secreted protein Yuk E.However,the function of this system and its secreted protein in root colonization of Bacillus velezensis remains unclear.We explored the function of type Ⅶ secretion system and the secreted protein Yuk E in root colonization of Bacillus velezensis SQR9,and further explored its synergist of bio-fertilizer and demonstrated its significance in fertilizer development.1.We identified the gene cluster of type Ⅶ secretion system in Bacillus velezensis SQR9 by genomic comparison,which containing yue D,yue C,yue B,yuk AB,yuk C,yuk D and yukE,and then generated a series of mutants that lack the coding genes of type Ⅶ secretion system by gene knockout.Subsequently,we found that the colonization efficiency of SQR9 in rhizosphere of cucumber and Arabidopsis decreased along with the deficiency of type Ⅶ secretion system or the Yuk E.2.The pET system was used to construct an engineered strain expressing and purifying the protein Yuk E.Rhizosphere colonization experiments experiments showed that exogenous addition of 5 μM purified Yuk E significantly improved the colonization and enhanced the growth-promoting effect of SQR9 in the rhizosphere.3.Cucumber pot experiments demonstrated that T3 treatment(Yuk E and strain SQR9 were added together)showed the best growth promotion among all treatments after 16 and20 days of transplanting,and the concentration for Yuk E synergistic on SQR9 was 5 μM.4.We constructed the strain SQR9(yukE :: yukE-gfp)with Yuk E gene in situ-labeled with GFP fluorescent protein by molecular biology methods,and the secretion of Yuk E in strain SQR9 was calibrated.The Yuk E content of SQR9 cultured in LB medium was thick,up to 0.14 μg/m L,which is ergastic to be used as bio-fertilizer synergist in agricultural production.In conclusion,the protein Yuk E secreted by type Ⅶ secretion system of SQR9 significantly improve the colonization ability and has a potential synergistic effect on the biofertilizer,which provide a basis for the development of synergist of bio-fertilizer made by Bacillus spp.