Molecular Mechanism Of Rhizosphere Chemotaxis By The Plant Growth-promoting Rhizobacterium Bacillus Amyloliquefaciens SQR9

Plant growth-promoting rhizobacterias?PGPRs?are a group of bacteria that can promote plant growth and suppress soil-borne diseases.Application of PGPRs as microbial fertilizers is a promising approach for reducing chemical fertilizer input and promoting the sustainable development of agriculture in China.Efficient colonization of the applied PGPRs in rhizosphere is prerequisite for exerting the roles of plant growth promotion and suppression of soil-borne diseases,which is based on the chemotaxis reaction of PGPRs towards plant roots induced by root exudates.Bacillus amyloliquefaciens is a relatively important PGPR in rhizosphere,but little research of its molecular mechanism of rhizosphere chemotaxis has been studied.This research will focus on the PGPR strain Bacillus amyloliquefaciens SQR9,which was isolated from cucumber rhizosphere by this laboratory and have been widely applied in agricultural production as the microbial fertilizer.Previous studies showed that SQR9 revealed positive chemotactic response to several small molecular weight organic acids in cucumber root exudates?CREs?.This study is mainly focused on the methyl-accepting chemotaxis proteins?MCPs?in SQR9 and chemoattractants in CREs.Firstly,the potential mcp genes were predicated according to the standard of the existence of a methy-accepting?MA?domain.Secondly,the B.amyloliquefaciens SQR9 mutant deficient in all mcp genes was constructed using an unmarked genetic manipulation based on multiple gene deletions that rely on a counter-selectable marker,pheS.Single-mcp-gene complementary strains were obtained by intergrating each gene into the chromosomal amyE locus of the all mcp genes deficient mutant.Thirdly,the chemotaxis assay was performed by using a microfluidic SlipChip device to identify the chemoattractants in CREs and their corresponding chemoreceptors in SQR9.Finally,the concentration of the key chemoattractants in CREs was studied by Ultra Performance Liquid Chromatography-Mass spectrum/Mass spectrum?UHPLC-MS/MS?,and we conducted chemotaxis assays using different CREs concentrations,individual exudate components as well as recomposed exudates,taking into account their concentrations in CREs.The main results of this study are summarized as follows.1.SQR9 has eight MCPs with McpA and McpC play key roles in root colonization.Based on the whole genomic sequence of B.amyloliquefaciens SQR9?NCBI accession no.CP006890?,the eight potential genes,namely mcpA?mcpB?mcpC?v529₁0510?mcpR??tlpA?tlpB?hemATandyfmS,encoding for MCPs were predicated according to the standard of the existence of a MA domain.The B.amyloliquefaciens SQR9 mutant deficient in all eight predicated mcp genes was constructed using an unmarked genetic manipulation based on multiple gene deletions that rely on a counter-selectable marker,pheS.The eight mcp genes were separately integrated into the chromosomal amyE locus of the chemoreceptor-deficient SQR9?8mcp to obtain eight single-mcp-gene complementary strains.To assess the roles of rhizosphere chemotaxis in root colonization by SQR9,we compared the colonization ability of wild-type SQR9 and SQR9?8mcp on cucumber roots,and found that the colonized population of the mutant was significantly impaired by two orders of magnitude as compared with the wild-type?2.6× 105 CFU per g of root vs.2.5×107 CFU per g of root?.Since the growth patterns of the wild-type and SQR9?8mcp were similar,the deficient colonization by SQR9?8mcp suggests that the MCPs-mediated chemotaxis to plant-secreted molecules is necessary for root colonization by SQR9.To clarify the significant MCPs,we monitored the root colonization of eight individual mcp gene-complementary strains on cucumber seedlings with fully developed cotyledons.It was found that complementation of McpA or McpC partially restored the colonized population to approximately?8?9?× 106 CFU g-1 root,which was significant higher than that of SQR9?8mcp.While complementation of other six MCPs didn't influence the bacterial root colonization when comparing with SQR9?8mcp.Subsequently,the double mcp gene-complementary strain were constructed and it was found to completely restored the colonization ability of SQR9?8mcp as the wild-type?SQR9?8mcp/mcpAmcpC,2.4× 107 CFU per g of root?.2.Forty-four chemotactic compounds from cucumber root exudates,including 20 amino acids,14 organic acids and 10 others,were identified for SQR9.In this study,39 chemoattractants and 5 chemorepellents,including amino acids,organic acids and sugars,were identified from 98 tested components of root exudates for SQR9 by using the microfluidic SlipChip device.To determine the chemoreceptor responsible for sensing these root-secreted chemoattractants and chemorepellents,each of the eight MCP encoding genes was individually complemented in SQR9?8mcp?and the chemotaxis of the complementary strains to these 44 compounds were tested.Firstly,expression of McpA?SQR9?8mcp/mcpA?significantly restored chemotaxis to 20 compounds,including five amino acids?aspartic acid,glutamic acid,isoleucine,lysine,and tyrosine?,ten organic acids?citric acid,malic acid,oxalic acid,fumaric acid,succinic acid,phthalic acid,adipic acid,dehydroascorbic acid,glyceric acid,and 3-hydroxypropionic acid?,and five other compounds?hydroxycarbamate,mannose,ribose,fucose,and ribitol?.Secondly,McpB was only responsible for sensing four amino acid attractants?glycine,tryptophan,asparagine,and glutamine?,as well as two chemorepellents,salicylic acid and sodium decanoate.Thirdly,McpC was mostly responsible for sensing amino acids,since nine of the chemoattractants were amino acids?valine,alanine,threonine,proline,serine,leucine,cystine,methionine,and histidine?,and one organic acid?gluconic acid?.Fourthly,complementation of McpR?SQR9?8mcp/mcpR?fully restored the chemotactic response to arginine.Fifthly,SQR9A8mcp/tlpA only showed a response to a chemorepellent,DL-dithiothreitol.Sixthly,SQR9?8mcp/llpB responded to five chemoattractants?phenylalanine,maltose,fructose,dulcitol and inosine?,mostly sugars,and a potential chemorepellent of pentadecanoic acid.Finally,complementation of hemAT and yfmS did not have any chemotactic effect regarding to these tested compounds.3.The concentration of 20 chemoattractants in root exudates that directly sensed by McpA or McpC that were quantified by using an UHPLC-MS/MS approach.To understand the contribution of the individual chemtoattractants to root colonization related chemotaxis,it is indispensable to obtain quantitative measurements of the abundance of compounds in exudates.Using an UHPLC-MS/MS approach,attempts have been made to quantify the 33 attractants for McpA and McpC in exudates at cotyledon stage?5?10 days after transplantation?.Totally 20 compounds were successfully quantified,including citric acid,malic acid,lysine,succinic acid,glyceric acid,glutamic acid,gluconic acid,valine,fumaric acid,mannose,aspartic acid,isoleucine,serine,leucine,proline,threonine,adipic acids,tyrosine,alanine,and methionine,ranging from high concentration to low.4.The rhizospheric chemotaxis to cre by SQR9 is dominantly driven by a handful of strong attractants.SQR9 failed to reveal chemotaxis to neither 1×nor 10×cre?1×cre,concentration in the cucu,ber seedling containing recipient used to generate root exudates?,or 1 ×nor 10×Mix-20?1×Mix-20,namely a mixture of the twenty chemoattractants at a concentration that corresponded to 1×cre?.At 100× cre,malic acid was the strongest attractant,followed by Lys,Ser,and fumaric acid that triggering moderate attraction of SQR9;whereas mannose,glyceric acid,Ile,gluconic acid,and Glu induced only weak chemotaxis of SQR9;the remaining 11 individual components caused only very minor responses.The general pattern at 1000× cre was similar to that at 100× cre,but additional compounds caused attraction of SQR9.In detail,malic and gluconic acid triggered strongest chemotaxis of SQR9;fumaric acid,Ala,mannose,and glyceric acid were found to be moderate attractants;and Lys,Thr,Val,succinic acid,and Glu induced weak response of the bacteria;finally,the other 9 chemicals didn't attract SQR9 cells.Subsequently we ordered these 20 attractants according to their magnitude of chemotaxis index as determined for 100× and 1000× cre?I30,chemotactic index,I30>0.6 or<0.4 indicate attractant and repellence responses,respectively,whereas 0.4?I30?<0.6 means no taxis?.Starting with the compound that caused least chemotaxis,i.e.,Try in 100× and Leu in 1000× concentrations,respectively,we then generated two sets of 19 different compound mixtures,by adding consecutively individual compounds according to the sequence of increasing chemotaxis.Chemotaxis assays were then conducted with these mixtures.At 100× cre,such increases were noted following the addition of Leu to Tyr?Mix-2?,while the addition of a series of weak and moderate chemoattractants?from Mix-3 to Mix-19?didn't significantly alter the chemotactic response and the corresponding 130 values ranged from 0.65?0.80.Importantly,supplement of the strong attractant malic acid led to a significant increase in the value from 0.80 to 0.90?Mix-20?.With regard to 1000× conditions,the significant increase was observed only following the inclusion of Pro?Mix-2?and Thr?Mix-13?;Supplying most of other chemicals led to increase in the I30 value,but didn't significantly alter the I30.In conclusion,McpA and McpC play key roles in mediating chemotaxis to root exudates by SQR9.The rhizospheric chemotaxis to root exudtaes by SQR9 is dominantly driven by a handful of strong attractants.This study contributes to understanding of molecular interaction mechanisms between plants and PGPRs,and to provide theoretical instructions for development and application of relevant microbial fertilizer products.