Contribution Of Several Proteins On Virulence And Comparison Of Sugar Matabolism Pathways In Pectobacterium

Pectobacterium is one of the top 10 plant bacterial pathogens,which mainly causes blackleg and soft rot diseases of plants.The main virulence factors are a set of plant cell wall degrading enzymes,including pectinase,and cellulase,etc.Pectobacterium has a rich diversity of species and a wide range of host plants.Although selection of resistant plant varieties and improvement of field management can partly reduce the economic loss of the disease,there are no effective measures to prevent it.As the Chinese government has encouraged potato as a staple food over last year,the planting area for potato is constantly expanding in China.Black-leg and soft rot diseases will potentially be important inrestricting development of the potato industry in China.Therefore,research on the mechanisms of Pectobacterium infection is essential to develop effective disease control measures in the future.A previous study of our group conducted an analysis of P.carotovorum subsp.carotovorum strain PccS1 and its interaction with Zantedeschia elliotiana cultivar 'Black Magic'.Using 2-DE(two-dimensional electrophoresis)coupled with mass spectrometry,53 proteins were identified.It is showed that proteins PotF and KdgR are up-regulated in planta.The LysR family regulator KdgR plays a critical role in the regulation of plant cell wall degrading enzymes and subsequent pectin degradation inPectobacterium and Dickeya species,while PotF is asubstrate-binding protein of putrescine ABC transporter PotFGHI.To determine the roles of KdgR and PotFGHI during the infection,deletion mutant ApotFGHI and AkdgR were constructed by homologue recombination.Extracellular enzyme and pathogenicity assays showed that AkdgR had slightly enhance pectinase activity on plates,while both ApotFGHI and AkdgR had no effect on the virulence of PccS1.It was revealed that PotFGHI and KdgR appear not to be necessary for PccS1 virulence during infection.The proteomic results also revealed 2-keto-3-deoxy-phosphategluconate aldolase(Eda)is essential to the virulence of PccS1.Eda is a key enzyme of the Entener-Dordoroff pathway,which is a gluconate metabolism pathway in bacteria.The ED pathway has been shown to contribute to the colonization and virulence of the animal pathogens E.coli,Vibrio choleraeand Shigella flexneri.To better understand its role in Pectobacterium infection,we conducted both biological and phylogenetic analysis of the ED pathway.Result from a previous study of Pectobacterium interaction with the host plant Zantedischia elliotiana showed that a mutant of edain P.carotovorum subsp.carotovorum strain PccS1was significantly attenuated in virulence.Interestingly,analysis of the genomic sequence showed that PccSl has an uncompleted pathway as it lacks phosphogluconate dehydratase(Edd),while a related species in the same genus P.atrosepticum has a complete ED pathway.To further demonstrate the role of the ED pathway in infection of Pectobacterium,this study constructed mutants of two ED pathway key enzymes in Pba SCRI1039(Pba-Aeda and Pba-?edd)using homologous recombination.The slide assay showed that virulence of Pba-?eda was decreased by 50%on potato tubers,while Pba-Aedd has no effect on virulence.Pba-?eda had little effect on initial growthin LB and MM + 0.2%Glucose media.Results from extracellular enzyme assays showed that Pba-?eda was reduced in pectinase activity by 50%of wild type.Furthermore,Pba-?eda was not able to utilize pectin degrading products(polygalacturonic acid and galacturonic acid)as a sole carbon source to grow.All reduced phenotypes in ?edacould be restored by inducing an external vector expressing eda.On the other hand,Pba-?edd was unaffected in growth and pectinase activity.These results reveal that Eda is involved in the breakdown of pectin in the plant during infection and/or of the regulation of extracellular enzymes,but that this involvement is independent of the role of Eda in the ED metabolic pathway.To elucidate more details about how eda was involved in virulence,five regulator genes(kdgR,hexA,hexR,rsmA and rsmB)and 6 pectinase genes(pelA,pelB,pelC,pelW,pehA and pmeB)were analyzed at the transcriptional level.Results of qRT-PCR showed that although expression of regulator genes in Pba-?eda was unchanged compared with the wild type in the same condition,expression of pectinase genes pelC,pelW,pmeB were reduced 8,28 and 181 fold in Pba-?eda,respectively.In addition,an in planta assay on potato showed that the number of bacteria per gram of plant tissue in Pba-?eda treated plants reached its peak value of 105,which was only 1/1000 that of WT,indicating that ?eda hugely reduced the colonization of Pba on potato.In summary,the ED pathway is unnecessary for virulence in Pba.However,eda,which is involved in the ED pathway but also independently in the breakdown of pectins,is involved through the utilization and regulation,of pectinase enzymes,leading to reduced colonization and utilization of plant breakdown products during infection.The fact that the two bacterial species used in this study(P.atrosepticum and P.carotovorum)showed differences in the gene content of the ED pathway,an important pathway in bacterial metabolism,suggests that changes in metabolism may play an important role in the host range of these pathogens.In addition to the ED pathway,three other sugar metabolism pathways,namely Embden-Meyerhof-Pamas(EMP),Pentose Phosphate(PP)and Pectin degradation(PD),are the major pathways required to transfer sugar or sugar acid to pyruvate in Pectobacterium.In this study,we therefore wanted to look at possible gene gain and/or loss of within these pathways in Pectobacterium and related genera in order to assess how such changes might impact on host range and disease symptoms and development.We therefore analyzed the gene loss and horizontal gene transfer(HGT)events of the four metabolic pathways during the evolution of Pectobacterium and related species,and the effect of these events on pathway function by conducting multi-locus sequence analysis of 28 loci(10 housekeeping core genes and 18 accessory genes)from 30 isolates(including 11 Pectobacterium isolates,4 Dickeya isolates,6 Pantoea isolates and 9 Erwinia isolates).The phylogenetic tree estimated from the concatenated alignments of the 10 housekeeping genes(HK10)showed the expected four clades(Pectobacterium,Dickeya,Pantoea and Erwinia)but also the position of the 6 new ChinesePectobacterium isolates.Tanglegram analysis and Shimodaira-Hasegawa testswere conducted to compare the topology of each accessory gene tree with the core gene tree(HK10).The results revealed that there are 4 accessory gene trees that have significant differences in topology compared to the HK10 core tree.In the 11 Pectobacterium isolates in this study,there appear to have been 4 putative HGT events in three loci(glk,pehA and pemA),and the gene loss events have mainly occurred in the edd locus of the ED pathway of 8 of the Pectobacterium isolates.For isolates of Dickeya,Pantoea,and Erwinia,the gene loss events are mainly in genes of the PD pathway.Furthermore,one of the candidate horizontal gene transferevents involving the pemA locus involves the PC1 and the PccS110 isolates,with the PC1 locus showing evidence of intragenenic recombination.Our results suggest that 10 housekeeping genes are enough to build a well resolved phylogenetic tree;gene loss events are associated with the gene function,and HGT might have happened in the basic metabolism pathway genes.