| Plant growth–promoting rhizobacteria(PGPR)have been widely used in agriculture for their significant plant growth–promoting and biocontrol functions.PGPR could be made into microbial fertilizer or bio-agent and applied to the plant rhizosphere.The key to exert the beneficial function of PGPR is that the microbes need to colonize the plant rhizosphere efficiently.They swim to the plant rhizosphere by sensing the chemical attraction of plant root exudates,and then attach,and form biofilm on the plant root surface.Among them,the biofilm formation ability of PGPR is a key factor affecting their rhizosphere colonization efficiency.Quorum sensing regulates a variety of microbial behaviors.Typical group sensing signals include AHLs in Gram-negative bacteria and autoinducer peptides in Gram-positive bacteria and autoinducer-2(AI-2),AI-2 is a quorum sensing signal shared by both Gramnegative and positive bacteria,however,there is currently lack of understanding for the regulatory function and regulatory mechanism of AI-2 in Gram-positive probiotic Bacillus.Bacillus velezensis SQR9 is a typical plant growth–promoting rhizobacterium,which can colonize the rhizosphere efficiently and promote plant growth.In this study,strain SQR9 was used to explore the regulating function of AI-2 on the colonization of Bacillus.The main results of this study are as follows:1.AI-2 promoted biofilm formation and motility of the strain SQR9 and promoted its colonization in plant rhizosphere.We identified the AI-2 synthetic gene luxS on the SQR9 genome and clarified that SQR9 can generate AI-2 from luxS gene.Comparing the biofilm formation,motility and extracellular matrix synthesis of SQR9 at different AI-2 concentrations,it was found that the addition of AI-2 could significantly promote biofilm formation,motility and extracellular matrix synthesis of wild-type SQR9,and restore the biofilm,motility and extracellular matrix synthesis defects caused by luxS deletion.Further studies have found that AI-2 can promote the colonization of the strain SQR9 in the rhizosphere of maize.2.AI-2 indirectly promoted biofilm formation of the strain SQR9 through inhibiting the spore formation.In order to explore the mechanism by which AI-2 promotes rhizosphere colonization of SQR9,we studied the transcriptome of SQR9 through RNA-seq,and combined with qPCR experiments,we found that the addition of AI-2 can greatly reduce the transcription of genes related to spore formation,without significantly affecting genes such as biofilm formation.In addition,the spore formation frequency of ΔluxS was higher than that of wild-type SQR9,and the spore formation frequency of SQR9 strain decreased along with the increase of exogenous AI-2 concentration.These above results show that AI-2 mainly indirectly improves its rhizosphere colonization capacity by inhibiting spore formation of strain SQR9.3.AI-2 inhibited the activity of ComA by binding RapC and promoted its interaction with ComA,therefore reduced phosphorylation of Spo0 A which led to the reduced sporulation.Spo0 A is a key factor to regulate biofilm formation and spore formation in Bacillus.Low phosphorylation levels of Spo0 A promotes cell differentiation to extracellular matrix producers to promote biofilm formation;high phosphorylation levels of Spo0 A promotes cell differentiation into dormant formed by spores.We found AI-2 decreased the expression of rapC.Further study showed that ΔrapC failed to reduce sporulation in response to AI-2.Moreover,mutant strain deficient in ComA,the regulator of another quorum sensing system,failed to respond to AI-2 as well.Molecular interaction experiments showed that AI-2 could bind RapC and promote its binding with ComA to inhibit the regulatory activity and reduce the phosphorylation of Spo0 A to inhibit spore formation.4.AI-2 mediates cross-species communication of strain SQR9 and relieves dormancy of Bacillus under soil slight stress conditions.Since AI-2 is a generic quorum sensing signal for both Gram-positive and Gram-negative bacteria,we speculate that strain SQR9 may sense AI-2 from other microbes in response to the population density of the target microbe.We found that the culture supernatant of wildtype Escherichia coli significantly inhibits SQR9 spore formation,while the E.coli mutant strain that lacks AI-2 synthesis gene cannot inhibit SQR9 spore formation.The above results suggest that strain SQR9 may have the potential function to sense other microbes and thus regulate it enters of dormancy.We further suggest that the inhibition of spore formation by AI-2 may allow it to function as a microbial fertilizer additive to assist Bacillus probiotics in the slight stress conditions of the soil,the principle of which is to prevent Bacillus from entering dormancy due to the stress.Experiments have shown that AI-2 is able to inhibit spore formation of strain SQR9 in soil environments and is able to alleviate the dormancy of SQR9 under mild drought conditions.Moreover,SQR9 could sense exogenous AI-2 to inhibit its own spore formation in soil environment,and could be affected by AI-2 under suitable water and mild drought conditions.These results suggested that AI-2 could promote SQR9 resistance to drought stress.In conclusion,our study revealed that the quorum sensing signal AI-2 in probiotic Bacillus SQR9 promotes RapC binding to the regulatory protein ComA by self-binding RapC,this process reduced the phosphorylation level of Spo0 A in cells in a quorum sensing interaction manner,thereby inhibiting spore formation and indirectly promote biofilm formation,further affect the rhizosphere colonization of SQR9.In addition,we found that AI-2 can mediate cross-species communication of strain SQR9 and alleviate the dormancy of SQR9 under soil slight stress conditions. |
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