LysR-type Transcriptional Regulator LrhA Regulate The Synthesis Of Menaquinone And Its Antioxidant Function Study In Pantoea Alhagi LTYR-11Z

Drought problems caused by water shortages have a huge impact on crop production.Therefore,improving the drought resistance of crops has become t he focus of attention and research hotspots.Numerous studies have shown that endophytes can help host plants resist abiotic stress such as drought and salinity.However,the mechanism of endophytic bacteria promote crops'drought resistance is not very clear,which hinders the identification and practical application of endophytic bacteria that promote plant resist drought stress.In the early stage,Pantoea alhagi LTYR-11Z,isolated from the surface-sterilized leaves of Alhagi sparsifolia Shap.,can effectively colonize the rhizoplane of wheat to promote its growth and enhance its resistance to drought stress.1.Starting from the global transcriptional regulator LrhA,the survival rate of WT(p B BR1MCS-1),?lrhA(p BBR1MCS-1)mutant and?lrhA(p BBR1MCS1-lrhA)treated with CHP and H₂O₂was measured.We discovered the?lrhA mutant showed a lower survival rate and the sensitivity of the?lrhA was almost completely alleviated by complementation of the plasmid p BBRMCS1-lrhA.The result showed that the LrhA play a vital role in P.alhagi LTYR-11Z response to oxidative stress.2.The transcriptomes of the?lrhA mutant and the WT strain were analyzed using RNA sequencing(RNA-seq).Compared to the WT results,men D,men H,men B,men C,men E genes involve in menaquinone biosynthesis were downregulated over 1.5 fold(P<0.05)in?lrhA.These genes belong to men FDHBCE operon.q RT-PCR found that the expression of men operon gene,such as men B,men C,men D and men F was significantly lower than that in wild type.By using p K18mobsac B-P_{men FDHBCE}::lac Z,we found men operon activity was much lower in the?lrhA strain and men operon activity was fully restored by introducing p BBR1-lrhA plasmid expressing.We examined the interaction between LrhA and the men operon promoter using electrophoretic mobility shift assay(EMSA).We found LrhA and the men operon promoter conformed DNA–protein complexes.DNase I footprinting assay were conducted with the fluorescence(5'-FAM)labeled to further determined precisely the binding site of LrhA.The result showed the DNA sequence containing 5'-CAGCCTCTGACCAACCGGGAAGAGGCTG-3'was clearly protected against DNase I by LrhA if adding increasing amounts of the LrhA protein(0–4?g)into the reactions.Taken together,our findings indicate that LrhA regulated men operon expression by directly binding to the men operon promoter.3.In addition to the men FDHBCE operon,the men A also involved in the biosynthesis of menaquinone in Pantoea alhagi LTYR-11Z.The?men A,?men D mutants and?men A(men A),?men D(men D)were constructed.The survival rate of these strains treated with CHP and H₂O₂ were measured.?men D and?men A mutants were significantly more sensitive to the oxidant than wild-type strains and the sensitivity of the?men D and?men A mutants to CHP and H₂O₂ was almost completely alleviated by complementation of the p KT100-men D and p K T100-men A plasmids,respectively.These dates showed that deletion of men D or men A genes involved in the biosynthesis of menaquinone reduced resistance to oxidative stress.The intracellular ROS levels in P.alhagi LTYR-11Z wild-type and?men A,?men D mutants and?men A(men A),?men D(men D)challenged with CHP and H₂O₂ was measured by using the fluorogenic probe 2?,7?-dichlorodihydr of luorescein diacetate(DCFHDA).?men A,?men D mutants had significantly higher ROS levels after exposure to CHP and H₂O₂compared with wild-type,indicating that menaquinone synthesized in P.alhagi LTYR-11Z can significantly reduce intracellular ROS levels.4.The pot experiment in wheat revealed that the shoot length,root length,fresh weight and dry weight of plants inoculated with?men A and?men D were lower than the plants inoculated with P.alhagi LTYR-11Z.It indicates that the menaquinone synthesis pathway in P.alhagi LTYR-11Z has the effect of assisting the strains to promote drought resistance.In summary,this study found that LrhA plays a vital role in P.alhagi LTYR-11Z resist drought stress.LrhA directly positively regulates men FDHBCE operon expression,which removes excessive intracellular ROS to maintain the stability of intracellular ROS.Menaquinone also enhance P.alhagi LTYR-11Z's ability to promote drought resistance in wheat.This result is of great significance for studying the mechanism of P.alhagi LTYR-11Z promoting wheat to resist drought stress.