| Facing the rapid growth of the world population in the 21 st century,it is urgent to improve the global agricultural production capacity.However,the hazards caused by excessive use of fertilizers seriously affect the human survival environment,resulting in soil degradation,desertification,water source pollution,and other problems.Microbial fertilizers produced by plant growth promoting rhizobacteria(PGPR)have great potential to be more friendly as substitutes for chemical fertilizers.However,there are problems with the instability of the field efficacy of microbiological fertilizers.It is very important to improve the stability of the application effect of microbial fertilizer.The rhizosphere chemotactic competence of PGPR is closely related to its growth promotion effect.In this study,we focused on improving the chemotactic rhizocompetence of PGPR.Using the typical PGPR strain Pseudomonas sp.UW4 as experimental material,we conducted research from three aspects,and the main results are as follows.1.Identification of the strong chemoattractant for rhizosphere chemotaxis colonization by Pseudomonas sp.UW4 using molecular docking technologyIdentification of the strong chemoattractant for PGPR chemotaxis colonization in the rhizosphere is essential for constructing efficient PGPR engineered strains.Conventional methods such as plate and capillary chemotaxis,as well as root colonization assays,are laborious and inaccurate.In this study,we used molecular docking to quickly and easily screen for the strong chemoattractants of UW4.Firstly,we conducted molecular docking simulations of 15 UW4 chemoreceptors(family I receptors)with 59 common plant root exudate molecules to calculate their binding free energies.Among the amino acid,organic acid,and sugar compounds,we selected 6 compounds from each class,totaling 18 compounds,and further measured their chemotaxis threshold concentrations for UW4 using capillary assays.We found a significant negative correlation(p < 0.0001)between the absolute value of the lowest binding free energy for the 18 compounds with UW4 family I chemoreceptors,and the logarithm of the chemotaxis threshold concentration for UW4.Among them,1-aminocyclopropane-1-carboxylic acid(ACC)had the lowest binding free energy with the UW4 wp116 receptor,compared to other family I receptors and the 59 plant root exudate molecules.ACC also had the lowest chemotaxis threshold concentration for UW4.These results suggest that molecular docking simulations can replace chemotaxis experiments to quickly identify strong chemoattractants for PGPR rhizosphere chemotaxis colonization,and the strong chemoattractant for UW4 rhizosphere chemotaxis colonization is ACC.2.Creation of highly efficient plant growth-promoting engineered strain of Pseudomonas sp.UW4 with a C-terminal pentapeptide grafted onto the receptor for the strong chemoattractant for rhizosphere chemotaxis colonizationMost of the PGPR strains have at least one chemotactic receptor with a pentapeptide which can bind to the chemotactic methyltransferase Che R and methylesterase Che B and mediated methylation of the receptors and then affect the chemotactic response of the bacteria.In order to improve the rhizosphere chemotactic competitiveness of Pseudomonas sp.UW4,we explored the grafting of pentapeptides onto the C-terminal of the UW4 ACC chemotactic receptor to improve its chemotactic response to ACC and the rhizosphere chemotactic competitiveness.Firstly,by comparing and analyzing the C-terminal amino acid sequences of28 chemotactic receptors annotated in the genome of Pseudomonas sp.UW4,it was predicted that there might be pentapeptides at the ends of the three chemotactic receptors.Then,employing molecular simulation docking and in vitro methylation reactions,we found that only the C-terminal pentapeptide(PEKPR)of chemotactic receptor wp502 was able to bind to the chemotactic Che R2 and mediate the methylation of wp502.This pentapeptide has no sequence conservation with other bacterial pentapeptides.The pentapeptide was grafted onto the C-terminal of the ACC chemotactic receptor wp116 of UW4 to construct an engineered strain UW4-1.The engineered strain UW4-2 was constructed by cutting off the C-terminal pentapeptide of the chemotactic receptor wp502 of UW4-1.The plate chemotactic response of UW4-1 to ACC was 36.0% and 17.2% higher than that of UW4 and UW4-2,respectively,but there was no difference in the chemotactic response to arginine and succinic acid among the three strains.Wheat incultived in sterile bottles and inoculated with these three strains,the colonization of UW4-1 in the rhizosphere of wheat was 93.2% and 37.1% higher than that of UW4 and UW4-2,respectively.Wheat incultived in pots and inoculated with these three strains,the colonization of UW4-1 in the rhizosphere of wheat was 62.0% and 14.3% higher than that of UW4 and UW4-2,respectively.Compared with UW4 and UW4-2,the root length of wheat inoculated with UW4-1 increased by 6.3% and 4.3%,the root dry weight increased by 10.3% and 5.5%,the aboveground height of wheat increased by 16.0% and 5.1%,and the aboveground dry weight of wheat increased by 16.5% and 13.2%,respectively.The above results indicate that grafting pentapeptides onto the C-terminal of the chemotactic receptor wp116 corresponding the strong chemoattractant for rhizosphere chemotaxis colonization by PGPR can significantly improve the chemotactic response of PGPR to this strong chemoattractant,the rhizosphere chemotactic competitiveness and the effect of promoting crop growth.3.Creation of an efficient gene editing system for PGPRCompared to other genetic engineering techniques,gene editing technology can more efficiently manipulate the genes of PGPR strains.However,the first challenge in applying gene editing technology in PGPR is to ensure the engineered cells survive CRISPR/Casinduced double-strand DNA breaks(DSBs).Our group discovered in the previous study that the single-stranded DNA-binding protein(SSB)of E.coli can catalyze DNA homologous recombination and non-homologous recombination,and the protein is relatively small and easy to manipulate.To explore its application in gene editing,in this study,we knocked out or replaced the λ-Red homologous recombination enzyme system in p Cas/p Target F with SSB and T4 DNA ligase,and constructed a p CasΔRed/p Target F,p Cas-SSB/p Target F,and p CasT4L/p Target F gene editing systems.To investigate whether SSB effectively mediates the HR repair of DSBs and improves the efficiency of CRISPR/Cas9 gene editing,p CasΔRed + p Target F-lac Z + donor DNA,p Cas/p Target F-lac Z + donor DNA,and p Cas-SSB + p Target F-lac Z + donor DNA were transformed into E.coli MG1655.No colonies grew in the plates transformed with p CasΔRed+ p Target F-lac Z + donor DNA.Plates transformed with p Cas + p Target F-lac Z + donor DNA and plates transformed with p Cas-SSB + p Target F-lac Z + donor DNA grew blue colonies and white colonies(mutations in the lac Z gene),the gene editing efficiency observed in the culture that was transformed with p Cas-SSB/p Target F-lac Z and donor DNA was 21.4% higher than that in the culture that was transformed with p Cas/p Target F-lac Z and donor DNA.Pseudomonas sp.UW4 was transformed with p Cas-SSB + p Target F-wp116 + donor DNA and the wp116 gene of UW4 was edited by homologous recombination with 100% gene editing efficiency,the mutants almost lost their chemotactic response to ACC.To determine whether SSB effectively mediates NHEJ to promote CRISPR/Cas9 gene editing,p Cas-SSB/p Target F-lac Z and p Cas-T4L/p Target F-lac Z were transformed into E.coli MG1655 and the gene editing efficiency observed in the culture that was transformed with p Cas-SSB/p Target F-lac Z was 33.2% higher than that in the culture that was transformed with p Cas/p Target F-lac Z.To validate whether SSB mediated DSB repair independently of Rec A and Rec BCD,p CasΔRed/p Target F-lac Z or p Cas-SSB/p Target F-lac Z as well as donor DNA as needed were transformed into E.coli MG1655-2(Δrec A,Δrec BCD,ΔSSB).The gene editing efficiency of the two transformed cultures transformed with p Cas-SSB/p Target F-lac Z and p CasSSB/p Target F-lac Z with donor DNA was no difference.Innovation points:(1)A molecular modeling docking method was established to efficiently identify the strong chemoattractants for PGPR rhizosphere chemotaxis colonization.(2)A new pentapeptide sequence from Pseudomonas sp.UW4 strain was discovered,and an engineered strain that improved the rhizosphere chemotactic competitiveness and growth promoting effect of PGPR strain was created by grafting the pentapeptide onto the Cterminal of the UW4 chemotactic receptor corresponding to the strong chemoattractant;(3)The newly created p Cas-SSB/p Target F gene editing system enables more efficient editing the genes of PGPR strains. |
