| Bacterial blight of soybean is the most common bacterial disease of soybean,which can cause production drawdown.P.savastanoi pv.glycinea is the pathogen of soybean bacterial blight.Our lab isolated two pathogens of P.savastanoi pv.glycinea from diseased leaves of soybean that from the northeast of China.We named them as A1 and SI.The type? secretion system and type ? effectors are playing important roles in pathogenesis.Type? effectors consist of Avr proteins,DSP family proteins and harpins.To study function of type ? effectors from P.savastanoi pv.glycinea in the pathogenesis.This research mainly aspects as follows:whole genome sequencing of A1 strain,comparative analysis of amino sequences between HrpZpsgs1 and HrpZpsgAl,function of type ? effectors from A1 strain.1.To analyze the characteristics of A1 strain and understand the whole genome sequencing,we got the sequences information and features of genome by the high-thoughput sequencing technology.The whole genome of A1 strain,which consists of a circular chromosome sized 5755153 bp and a plasmid sized 71352 bp The chromosome genome has 5001 protein-coding genes and the plasmid has 85 coding genes.The genome contains 14 sRNA genes,sixteen 5s-16s-23s ribosomal rRNA operon and 65 tRNA genes.The chromosome genome GC content is 59.41%,and the plasmid genome GC content is 54.05%.The function of 4004 protein-coding genes from chromosome had been known.We analysed the chromosome genome by bioinfoamatics technology,and the results as follows:(1)A1 strain has 33 metabolic pathways.(2)There are 134 proteins of Al strain which can interact with proteins in the PHI database.And 87 of those proteins play an important role in pathogen virulence.(3)This strain has a complete secretion system.The type III secretion system was encoded by 55 genes,and can secrete 17 type ? effectors.(4)The numbers of pathogenic virulence factors about 230,including effectors,extracellular polysaccharide,lipid polysaccharide,and so on.This work for studying Al genome sequencing which made a contribution to reveal the molecular pathogenesis of P.savastanoi pv.glycinea.2.Comparative analysis of their amino sequences showed that three different regions mainly existed in C-terminal.In order to clarify the relationship of these differential regions of HrpZ proteins and the ability in eliciting the HR and disease resistance,we constructed 6 recombinant proteins by respectively exchanging three differential regions between HrpZPsgS1 and HrpZpsgAl.After expression and purification,6 recombinant proteins about 41×103 were got,HR elicition ability and TMV disease resistance ability were detected.HrpZs(s2?A2)and HrpZA(A3,s3)could induce stronger HR in tobacco compared with the wild-type proteins,furthermore,all of 6 recombinant proteins could reduce the phenotype caused by inoculation with Tobacco Mosaic Virus(TMV)than wild-type proteins,among them HrpZA(A3?S3)and HrpZs(s3?A3)showed the best effects.These results showed that the 7,8,9 a-helices were the main regulatory region of HR,the 8,9 a-helices were also related to the disease resistance.This study provides the theoretical basis for molecular modification of HrpZ harpin protein in the future.3.To study function of type ? effectors from P.savastanoi pv.glycinea in the pathogenesis.After analysing the whole genome sequence,we got three effectors sequence and cloned them by PCR.The full length of hopAAl gene contained a 1452bp open reading frame,encoding 484 amino acid residues.The full length of hopAEl gene contained a 2739bp open reading frame,encoding 913 amino acid residues.The full length of hopAH2 gene contained a 1248bp open reading frame,encoding 416 amino acid residues.There were not any signal peptide when analyzed and predicted by the tools on the NCBI.Three effectors were inserted in the binary PVX vector,and the constructs were transformed into Agrobacterium tumefaciens GV3101.To analyzed the biological function of three effectors by Agrobacterium-mediated transient expression technology.The results showed that three effectors can promote Phylophthora nicotianae infection Nicotiana benthamiana after injected with effectors 24 h.Real-time PCR results showed that HopAA1 could inhibit the expression of transcriptional factors WRKY,HopAH2 could inhibit expression of the ethylene signal regulatory transcription factors EIN2 and the SAR positive regulatory factor NPR1,and HopAEl could inhibit expression of EIN2.Therefore,type ? effectors of P.savastanoi pv.glycinea Al could suppress the plant innate immunity during pathogenesis.Our work proved a basis to study the interaction between type ? effectors and target protein in plants. |
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