Functional Study On The Periplasmic Protein CtvP Of Xanthomonas Oryzae Pv. Oryzae To Overcome Copper Nutritional Immunity

Host metal homeostasis changes dramatically in response to bacterial infections,including production of metal sequestering proteins and the bombardment of bacteria with toxic levels of metals.This physiological behavior is defined as host nutritional immunity.After inoculation with Xanthomonas oryzae pv.oryzae（Xoo）,the concentration of copper ions in the stems increased markedly,which indicated that Xoo had encountered the host copper ion nutrition immunity when infecting rice,that is,the host was gathering poisonous copper ions in the infected site to poison the invading pathogen.So it’s necessary to overcome the host copper ions immune for the pathogen,however,the research on the antagonistic mode of copper ion nutrition immunity is focus on the animal pathogens,and the role of copper ion tolerance for plant pathogen during infects host is known very limited.Xanthomonas are good models to study the battle for copper metals at the host pathogen interface.Xanthomonas oryzae pv.oryzae as an important plant pathogen can cause bacterial blight.The disease causes seriously reduction of rice production and threatens food security.Our group focus on Xanthomonas oryzae pv.oryzae PXO99^A.The gene knockout technique was used to screen the genes related to virulence in PXO99^A,and it was found that mutant of a unknown functional protein could cause the virulence rapid decreased,and the virulence of mutant could be partially recovered by gene complement,this protein was named CtvP（Copper tolerance and virulence related protein）in this study.Through evolutionary analysis,it was found that the CtvP protein was conserved in the genus of Xanthomonas.Further informatics analysis found that CtvP protein contained a copper-ion-binding domain NosD（periplasmic copper-binding protein）.Due to the CtvP protein contains a NosD domain,it is speculated that this protein may be the copper ion tolerance.Protein localization is often closely related to function,and bioinformatics predicts that CtvP proteins may be located in periplasm.firstly,the subcellular localization of CtvP protein is validated.The expression of CtvP fusion protein with eGFP label was detected by western blot after separation of PXO99^Acells,confirming that CtvP protein was located in the cell periplasm.Further research on the function of CtvP protein was confirmed,by adding the high concentration of copper ions in vitro to detect ctvP deletion mutant and wild type growth inhibition differences,found that ctvP gene deletion caused PXO99^Aresistance to copper ions decreased.In order to further explore the mechanism of copper ion tolerance mediated by CtvP protein,we studied the relationship between CtvP and classical copper tolerance related proteins.Through the bacterial two-hybrid system,it was found that CtvP can interact both with the copper ion tolerance-related homologous protein CopA and CopB.The copper ion tolerance function of CopA and CopB protein in PXO99^Awas validated,found the copper tolerance of copA and copB deficient mutants decreased significantly,Based on the above results,it is speculated that CtvP and CopA and CopB constitute the copper ion transfer chain,which is used to pump the copper ions out of the cell or to fix the copper ions in the periplasm.The results of real-time fluorescence quantitative PCR and Gus enzyme activity assay showed that the ctvP gene expression was raised under high concentration of copper pressure.Similarly,the gene expression of copA and copB was increased under the stimulation of copper ions.Although excess copper ions are toxic to cells,the right amount of copper ions is essential for the normal life of cells.we speculate that the expression of ctvP,copA,copB copper tolerance genes in PXO99^Awere accurately regulated.Sensing and transducing extracellular signals are usually mediated by a two-component signal transducing system,and the two-component signal transducing system ColS/ColR in Xanthomonas citri subsp.citri is related to copper tolerance.The Copper tolerance related function of homologous protein ColS/ColR in PXO99^Awas verified,found the copper tolerance of colS deficient mutant was significantly decreased,and the two-component signal transducing system responded to Cu²⁺stimulation.The results of real-time fluorescence quantitative PCR showed that the expression of ctvP was positively regulated by ColS/ColR,and CtvP in turn positively regulated colS expression,but there was no direct interaction between CtvP and ColS/ColR.Therefore,we screened the transcriptional regulatory factors that may regulate ctvP expression in the downstream of the two-component signal transducing system ColS/ColR.Through the bacterial single hybridization system,it was found that the ctvP promoter had interaction with transcription regulatory factor Zur.By adding the high concentration of copper ions in vitro to detect zur deletion mutant and wild type growth inhibition differences,found the transcription regulatory factor Zur was also involved in copper resistance.The results of gene expression detection showed that Zur negatively regulated ctvP expression,and zur gene was positive regulated by ColS/ColR.It is noteworthy that the expression of hrp gene associated with virulence in ctvP deficient mutant was significantly reduced.In the PXO99^A,the expression of hrp gene was increased in the copper stimulation.Based on the above results,we preliminarily speculate on the copper balance regulation network in PXO99^A.In low copper,the transcription regulation factor Zur suppresses the expression of ctvP,at this time does not need the CtvP-mediated copper efflux;When the copper ion pressure increases,the periplasmic CtvP senses high concentrations of copper ions in the extracellular surroundings and transmits the signal to the two-component signal system ColS/ColR,and then ColS/ColR activates the expression of zur.Upon Cu binding,there is a conformational change in the dimer of Zur and the target gene ctvP transcript is de-repressed,simultaneously activate the expression of virulence hrp gene and pth XOI to promote Xoo overcoming the copper ions nutritional immunity.In summary,we found that virulence-related protein CtvP not only mediate copper ions detoxification,but also as sensor involved in copper ion balance and virulence regulation,and plays an important role in Xanthomonas oryzae pv.oryzae to overcome the host copper ions nutritional immunity.Further study of CtvP will help us to understand how plant pathogens overcome host nutritional immunity.