| Bacterial fruit blotch(BFB)is a devastating seed-borne bacterial disease that threatens the melon and watermelon industry worldwide.It rapidly spreads on melon and watermelon and causes serious losses,as well as other cucurbits including cucumber,pumpkin,etc.BFB is listed as a quarantine pathogen in China due to its severe damage,wide host range and rapid spreading.The causal agent of BFB is a gram-negative bacterium named Acidovorax citrulli,which has a polar flagella and transmits via attaching on seed surface.Previous study found that a Lux R family regulator,AcrR,contributed to multiple biological traits of A.citrulli,inculding the virulence and motility,as well as the flagellar assembly.The gene acr R encoding AcrR showed potential structural character of two-component regulation system(TCS)with its upstream gene acr K.To investigate the AcrK and AcrR form a TCS and the regulatory mechanism of AcrR on the vrulence and motility of A.citrulli,we carried out bioinfermatics analysis on AcrK and AcrR,and constructed acr K and acr Kacr R mutant strains based on the wild-type strain Aac-5 of A.citrulli,consequentially showed the regulatory function of AcrK/AcrR on virulence and motility of A.citrulli.We also verified the interaction between AcrK and AcrR through bacterial two hybrid and GST pull-down assay.The results provided solid evidence of the interaction between AcrK and AcrR.To further investigate the regulatory mechanism of AcrR on virulence and motility of A.citrulli,we constructed AcrR-tagged strains for Chromatin Immunoprecipitation(Ch IP)assay,and determined the biological traits of these strains.The results showed that:(1)AcrK/AcrR played critical role in regulation of virulence and motility of A.citrulli;The acr K,acr Kacr R mutant strains ?acr K,?acr K?acr R were constructed,and in turn the complementation of the mutant strains were also generated.The virulence,motility and flagellum formation were determined and observed.The results showed that,the ?acr K and ?acr K?acr R attenuated in virulence,lost motility and were unable to form flagellum,while the complementation strains restored the virulence,motility and flagella as the wild type strain Aac-5,indicating the regulatory role that AcrK/AcrR plays in the virulence and motility of A.citrulli.(2)There exits interaction between AcrK and AcrR;Bioinformatic analysis showed that AcrK is a signal transduction of histidinekinase,and the AcrR is a Lux R family transcriptional regulator.AcrK and AcrR potentially form a two-component regulatory system.The interaction between AcrK and AcrR was verified by using Bacterial Two Hybrid assay and GST pull-down assay.(3)AcrK and AcrR forms two-component regulation system;The AcrK and AcrR were expressed by using heterologous expression,and then purified for further use.The phosphorylation of AcrR by AcrK were observed in phos-tag assay,where the migration of AcrR was changed by the pre-phosphorylated AcrK on SDS-PAGE gel,indicating that AcrK and AcrR form a two-component system via phosphorylation of AcrR by AcrK.(4)The Ch IP stains showed similar biological traits with wild-type strain Aac-5,could be used in further studies on regulatory mechanism.Three antibiotic-labeled vectors were used in the Ch IP strain construction,and strain p BBRTet-acr RHA-?acr R,p BBRKam-acr RHA-?acr R and p BBRAmp-acr RHA-?acr R were eventually constructed.Biological assays showed that the virulence,motility,flagellum formation,growth rate in planta,and hypersensitive respones on tobacco of the three Ch IP strains were same as those of the wild-type strain Aac-5,indicating that the AcrR was expressed in these strains as same as in the Aac-5.The 3 strains could be used to capture the binding DNA of AcrR,and provide a foundation of disclosure the regulatory role of AcrK/AcrR on the virulence and motility of A.citrulli. |
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