Genome-wide Identification And Gene Expression Analysis Of The Sec-dependent Secreted Protease And Cellulase In Erwinia Amylovora And Prokaryotic Expression And Purified Of The E.amylovora-encoded EAMY₃046

Fire blight is a devastating disease of pome fruits worldwide and caused by the bacterium Erwinia amylovora,an important quarantine phytopathogen in China.Since the middle of last century,one to two countries were reported to have fre blight disease each year.The disease has recently reached Central and East Asia,posing an imminent threat to the healthy development of apple and pear industry in China.Therefore,it is necessary to establish accurate,fast and sensitive detection methods to effectively prevent the introduction of E.amylovora into China.To secrete proteins contributing to environment niche and disease pathogenesis,pathogens evolve a variety of secretory pathways,among which sec-dependent secretory mechanism is widely existed in bacteria as the main transmembrane transport pathway of proteins.However,so far,the secretory proteins identified from E.amylovora are almost limited to effector proteins released from type III secretion system?T3SS?,and sec-dependent secretory proteins have not yet been reported.In this study,the sec-dependent secretory protease and cellulase of E.amylovora were explored,Meanwhile,the sec-dependent secreted protein EAMY₃046 was selected for prokaryotic expression and purification.The results were as follows:1.Bioinformatics analysis revealed that E.amylovora contained all the core components of sec system,indicating its ability in Sec-dependent secretion.2.Using bioinformatics technology and Escherichia coli-based alkaline phosphatase?PhoA?fusion system,15 sec-dependent secreted proteases,namely EaPrt1-15,were identified at the genome-wide level of E.amylovora.Among them,EaPrt1?2?5?6?7?8?9?10?13 and 15 are of serine protease,EaPrt3 and 11 are of cysteine protease,and EaPrt4?12 and 14 are of metallo protease.The E.amylovora was inoculated into the young pear fruits,the pear tissues were collected at 0,8 and 24 hpi?hours post-inoculation?respectively.The extracted total RNA was subjected to transcriptional expression of 15 sec-dependent secretory protease-encoding genes by reverse transcription quantitative real-time PCR?RT-qPCR?.The results showed that EaPrt4,5,8,14 and 15 displayed a steadily increased expression pattern in the course of 24-hour infection,while expression of EaPrt6,11 and 13 were continuously decreased.In addition,mRNAs of EaPrt1,2,3,7,9,10 and 12 peaked at 8 hpi,but dropped at 24 hpi,showing an ‘up-down' expression pattern.It was worth noting that compared with its expression level at 0 hpi,EaPrt7 showed a ³7-fold increase in expression at 8 hpi,but only increase to a ⁶-fold increase at 24 hpi,exhibiting the most significantly altered expression pattern among all tested genes.This result suggested that E.amylovora might secrete a series of proteases by Sec system to promote pathogen infection of host plants.3.Using bioinformatics technology and PhoA gene fusion system,we identified two Sec-dependent secreted cellulases,EAMY₃602 and EAMY₁236,from the whole genome of E.amylovora.In particular,EAMY₃602 is an endoglucanase,and EAMY₁236 is a ?-glucosidase.The E.amylovora was inoculated into the young pear fruits,transcriptional expression of 2 sec-dependent secretory cellulases encoding genes was analyzed by RT-qPCR,which showed that the mRNA expression levels of Ea3602,the coding gene of EAMY₃602,were continuously decreased at 8 h and 24 h,with no significant difference when compared with that at 0 h.On the contrary,the transcript levels of Ea1236,the coding gene of EAMY₁236,at 8 h and 24 h were 2.43-and 3.26-fold higher than that at 0 h.These data implied the Sec-dependent secreted endoglucanase EAMY₃602 had no implication in plant infection,but ?-glucosidase EAMY₁236 probably acted as a virulence factor that plays an important role in the process of infecting host plants.4.Using PCR,Ea3046,the coding gene of EAMY₃046 was cloned from the E.amylovora strain DSM 17948.Using bioinformatics technology and PhoA gene fusion system,we demonstrated that EAMY 3046 is a Sec-dependent secretion protein.The E.amylovora was inoculated into the young pear fruits,transcriptional expression of Ea3046 was analyzed by RT-qPCR,the results showed that Ea3046 showed a steadily increased expression in the course of E.amylovora infects host plants.In addition,the coding sequence of mEa3046 was inserted into the pET30 a,resulting in pET30a-mEa3046,and subsequently transformed into Escherichia coli BL21.Afterinduction with IPTG for 16 hours,the recombinant expression of mEa3046 at 15? was higher than that at 37?,andpresent as inclusion bodies.The expressed mEa3046 was further purified by Ni-IDA affinity chromatography,and SDS-PAGE analysis showed that the target protein was successfully purified.In summary,the study selected the E.amylovora sec-dependent secreted proteases and cellulases,and analyzed their gene expression pattern at the transcriptional level.In addition,prokaryotic expression and purification of Sec-dependent secretion protein EAMY₃046 were preformed.The data laid a foundation to further reveal the new pathogenic mechanism of E.amylovora and prepare antibodies for quarantine detection of this bacterium.