Crystallographic Analysis Of IMPDH And GMPS From Candidatus Liberibacter Asiaticus And Mechanisms Of Induced Resistance In Cirtus Against HLB By Bacillus Amyloliquefaciens GJ1

Citrus production is facing an unprecedented crisis due to Huanglongbing(HLB)disease.HLB is associated with Candidatus Liberibacter asiaticus(Las),Ca.L.americanus(Lam),and Ca.L.africanus.Currently,no effective HLB management approach exists once the trees become infected.Consequently,new strategies and methods are urgently needed to prevent and control citrus HLB.Structure-based drug screening is a fast and effective method for screening specific inhibitors.Inosine 5’-monophosphate dehydrogenase(IMPDH)and GMP synthetase(GMPS),two key enzymes in the purine biosynthetic pathway.The inhibition of IMPDH and GMPS activity can reduce the ability of pathogen infection.More and more researches revealed that IMPDH and GMPS can be used as the potential antibacterial target.Furthermore,plant growth-promoting rhizobacteria(PGPR)providing a safe and effective method to prevent plant diseases.Previous studies in our lab have founded that the biocontrol agent Bacillus amyloliquefaciens GJ1 can reduce the infection rate of HLB.In this study,we carried out two aspects of research,one is to target CLas IMPDH and CLas GMPS for protein expression,purification,crystallization screening and structure-based inhibitor screening,the other is to explore the mechanism of B.amyloliquefaciens GJ 1 prevention and control HLB.The main results are as follows:1.Crystal structure of CLas IMPDH and evaluation of bronopol and disulfiram as potential CLas IMPDH inhibitors by using enzyme kinetic.First,a CLas IMPDH variant without the Cystathionine beta synthase(CBS)domain(CLas IMPDHΔS)was designed.The pure and uniform states protein was obtained by protein purification.After the initial crystallization conditions were tested and further optimization.The resolution of the best diffracting crystals was 2.55(?).The space group of CLas IMPDHΔS was C121(PDB ID:6KCF).Eight IMPDH inhibitors were selected to perform molecular docking,and the-CDOCKER＿ENERGY of bronopol and disulfiram was 11.19 and 25.03,respectively.Bronopol and disulfiram showed a typical Lineweaver Burk plot for non-competitive inhibition.The inhibition assay suggested that the K_i values of bronopol and disulfiram were 234 and 616 n M,respectively.2.The resolution of the best diffracting CLas GMPS crystals was 4(?)and ZINC192586 was the potential inhibitor for CLas GMPS using structure based high-throughput screening.First,CLas GMPS protein was purified using the Ni-NTA resin affinity chromatograph,followed by high-resolution gel filtration column.Cells carrying pET28a-CLas GMPS plasmid were used to perform the expression and purification.The best crystals were obtained and the resolution of the best diffracting crystals was 4(?).Cells carrying pET28atplus-CLas GMPS plasmid using for expression and purification.The crystal of CLas GMPS was obtained by the sitting-drop technique.For the apo CLas GMPS variant,the resolution of the best diffracting crystals was 8(?).For the CLas GMPS and IMP complex crystal,the resolution of the best diffracting crystals was 5(?).A high-throughput virtual screening and molecular docking were performed,ZINC192586、ZINC117323、ZINC084693 and ZINC072272 were found to have a higher binding affinity with CLas GMPS.3.B.amyloliquefaciens strain GJ1 could improve the photosynthesis ability of citrus plants and induce the expression of defense genes to enhance the ability of plants to resist Huanglong disease.Citrus were irrigated with B.amyloliquefaciens strain GJ1served as the experimental group,the untreated plants served as the control group.B.amyloliquefaciens strain GJ1 treated and untreated leaves were collected and used for transcriptome analyses.Differential gene expression and metabolic pathway analysis,we focus on photosynthesis pathway.The PRM results showed that the abundance of Cs2g07330,Cs5g31180,and Cs5g34450 in photosynthesis pathway were upregulated 1.84-,2.01-,and 1.56-fold,respectively.Under the treatment of Bacillus amyloliquefaciens GJ1,the photosynthetic parameters,chlorophyll content,resistance-associated enzyme content and the expression of defense realated genes were significantly higher than the control treatment.Compared to the control treatment,the content of starch content and soluble sugar were significantly lower.The marker genes,WRKY22 and GST1 were upregulated7.5-,4.5-fold,respectively.The accumulation of ROS in citrus with treatment of B.amyloliquefaciens strain GJ1 flag22 were significantly higher than untreated plants,and induced the defense related gene expression in citrus.Surfactin was identified from the fermentation broth of B.amylolyticus strain GJ1 by HPLC(high performance liquid chromatography).These results indicated that B.amylolyticus strain GJ1 might improved the immunity of citrus by enhancing the expression of the resistance-related genes,and resulted in changes of starch,sugars,chlorophylls,and resistance-associated enzyme in citrus.