Molecular Mechanism Underlying Regulation Of Wheat Root Colonization By The Catabolite Repressor/activator Cra In Pantoea Alhagi

Drought is one of the major environmental pressures that affect global crop growth and productivity.Global warming and water shortages accelerate the degree of environmental degradation.Therefore,there is an urgent need to enhance the resistance of crops and improve their ability to fight drought.Plant endophytes can help plants deal with stress from outside and enhance their ability to resist pressure.The precondition for plant-microbe interaction is that endophytes can effectively colonize plants.In our previous study,we discovered Pantoea alhagi LTYR-11Z,an endogenous bacterium that promotes plant growth.The strain can effectively colonize the root tissue of wheat and improve the drought resistance of the host.In the previous stage,by constructing the transposon insertion mutant library of this strain,a globally regulated gene cra related to colonization was screened.Using transcriptome data analysis（RNA-seq）,the transcript levels of 284 genes in cra knockout mutants were found to be significantly different from those in the wild type,of which 180 genes were positively regulated by Cra proteins and 104 were negative by them.Regulation.By digging deeper into the transcriptome data,it was found that the eps operon（B1H58₁4485-B1H58₁4530）was significantly down-regulated in?cra expression.Through real-time PCR,β-galactosidase activity assay,and gel blocking migration（EMSA）,we determined that Cra directly regulated the transcription of the eps operon,and then predicted and passed the point spiking.The needle verified the binding site of the promoter of Cra and eps operons.This study also examined the production of exopolysaccharide（EPS）in wild-type P.alhagi LTYR-11Z,?cra mutants and complementary strains,and confirmed the positive regulation of extracellular polysaccharide biosynthesis by Cra.Subsequently,crystal violet staining was used to detect the biofilm production of wild-type,?cra,and?tuaG mutants.It was found that Cra promotes the formation of biofilm by promoting the expression of the eps operon.In the transcriptome data,the anti-FlhD₄C₂ factor homologue ydiV（B1H58₁8220）was down-regulated more than 2.2-fold in?cra,indicating that Cra may regulate flagella biosynthesis.In this study,we found that the mutant cra gene had increased locomotor ability through the swimming experiment.Compared with the single knockout cra gene,the locomotor ability of the strain was further increased after knocking out cra and ydiV genes,and we failed to recover cra in the double mutant.The recovery of exercise capacity to the wild-type level indicates that Cra inhibits the exercise capacity of P.alhagi LTYR-11Z by modulating the expression of the ydiV gene.To demonstrate that YdiV acts as an anti-Flh D₄C₂ factor,we used EMSA to examine the effect of His6-YdiV on the interaction between His6-FlhD₄C₂ and the fli A promoter.The results indicate that ydiV encodes FlhD₄C₂ and fliA in P.alhagi LTYR-11Z.The promoter-bound anti-FlhD₄C₂ factor;similarly we determined that Cra directly and positively regulates the ydiV gene and predicts and validates the binding site for the promoter of Cra and ydiV.In this study,in vitro enzyme activity assay was used to detect YedQ protein in P.alhagi LTYR-11Z with dual guanylate cyclase activity,and it was determined that Cra directly positively regulates the expression of yedQ gene.By measuring intracellular c-di-GMP content,the intracellular c-di-GMP level of mutants lacking the cra gene was found to decrease to 48%of the normal level,indicating that Cra regulates intracellular c-di-GMP levels.The phenotypic test found that compared with the wild-type P.alhagi LTYR-11Z,?yedQ showed increased exercise capacity,decreased EPS production,and decreased biofilm formation capacity.Based on the above results,we conclude that Cra directly binds to the eps operon,ydiV,and yedQ gene promoters and activates transcription,promotes extracellular polysaccharide production and anti-FlhD₄C₂ synthesis,and increases intracellular c-di-GMP level,leading to increased biofilm formation capacity of bacteria,and decreased ability to exercise,thereby enhancing its colonization capacity.Previous studies have shown that Cra is a regulatory protein that responds to carbon metabolism in bacteria and is controlled by the level of sugar metabolism.Under gluconeogenic conditions,Cra exerts its transcriptional regulatory function in the form of a tetramer;in the process of glycolysis,the regulation of Cra can be catabolized by the glycolytic intermediates fructose-1-phosphate（F1P）and fructose-1,6-Diphosphate（FBP）inhibition.In this regard,by changing the carbon source in the medium,we found that the transcriptional activation of the eps operon,ydiV,and yedQ genes was more pronounced in the gluconeogenic environment than in the glycolytic environment.The addition of FBP in the EMSA experiment reduced the formation of the corresponding Cra-DNA complex,demonstrating that the binding of the promoters of Pra with eps,ydiV and yedQ in P.alhagi LTYR-11Z can be inhibited by the glycolytic metabolite FBP.The pfkA gene mutation that encodes 6-phosphofructokinase can contribute to the transient accumulation of intracellular F6P,but the concentration of metabolic intermediates such as FBP decreases.After the pfkA gene was knocked out in this study,the promoter activities of eps,ydiV,and yedQ were compared with wild-type strains.Significantly enhanced,this also proves this view.In addition,plant root exudates play an important role in plant-microbe interactions and may regulate the expression of colonization-associated genes in plant-promoting bacteria.This study found that water-cultured rhizosphere secretions from wheat for 14 days can be enhanced.Cra activation of its target gene expression.In summary,P.alhagi LTYR-11Z identified the role of different response to carbon sources in the regulation of extracellular polysaccharide synthesis,flagella biosynthesis,and c-di-GMP synthesis,and then regulate the mechanism of its colonization behavior.A model has been established to explain the mechanism of CRA-mediated colonization regulation of bacterial carbon metabolism regulators.This study proposes a new strategy for regulating the colonization behavior of plant endophytes in response to different carbon sources in the environment.The research results obtained have laid a foundation for the further in-depth study of bacterial carbon-dependent colonization and have important theoretical values.Potential application prospects.