Identification Of Functional Fragments Of HrpZ_psgs1in Inducing Disease Resistance And Enhancing Growth And RNA-Sep Analysis Of Rice Treated With HrpZ_psgs1from Pseudomonas Syringae Pv.Glycinea

Harpin HrpZ of plant-pathogenic bacteria Pseudomonas syringae elicits hypersensitive response(HR)and pathogen defense as well as enhances growth in plants.Recently,studies of harpin protein of Pseudomonas.syringae pv.glycinea were rare,and studies of HR,disease resistance and growth promotion were less.Our lab have cloned and expressed the HrpZpsgs1 protein from Pseudomonas.syringae pv.glycinea S1 strain,study showed HrpZpsgS1 protein has the similar biological effects with other harpin proteins,could induce hypersensitive response(HR),enhances the resistance of tobacco to TMV,promotes the growth of tobacco and so on.In order to study the functional domain related to HR,disease resistance and growth promotion of HrpZpsgs1,this study tested the bioactivities of HrpZpsgS1 and 11 recombinant proteins on plants,to provide the theoretical basis for the crystal structure of the harpin protein.We predicted the second structure of HrpZpsgs1,it contains 1 ?-sheet and 9 a-helixes,these 9 a-helixes were crucial for the structure of protein.Based on this prediction,11 mutants which deleted different parts of HrpZpsgS1 were obtained by PCR or inverse-PCR.After cloned,expressed and purified,we got the recombinant proteins.The sizes of purified HrpZpsgs1 and 11 mutants were tested by SDS-PAGE and were identical to the calculated value.After salt removal using the ultrafiltration centrifugal tubes,protein concentrations were measured by BCA method,they were about 1.5mg/mL-5mg/mL.HrpZpsgsi protein has the similar biological effects with other harpin proteins,could induce hypersensitive response(HR),enhances the resistance of tobacco to TMV,promotes the growth of tobacco and so on.In order to study the functional domain of HrpZpsgs1,this study tested the bioactivities of HrpZpsgs,and 11 recombinant proteins on plants,they contained HR eliciting ability,tobacco resistance to TMV,rice resistance to Xanthomonas oryzae pv.oryzae and growth promotion of rice seeds.The HR testing results showed that C-terminal is indispensable for HR elicitation,and some parts in the N-terminal acted as the regulatory role.Then among 11 recombinant proteins,we found HrpZ?89-124 and HrpZ?254-298 all had better effects in inducing the resistance of tobacco to Tobacco Mosaic Virus(TMV)and rice to Xanthomonas oryzae pv.oryzae as well as the growth promotion of rice seeds.The results of the Real-time PCR indicated that HrpZ?89-124 and HrpZ?254-298 resulted in greater levels of expression of genes related to HR,pathogen defense and growth process.This study indicated that the genetic modified protein HrpZ?89-124 and HrpZ?254-298 had potential in development as protein-type of biocontrol agents.Harpin protein can induce the plant to start multiple signaling pathways,and activate a variety of resistance mechanism,improves different agronomic performance.Previous experiments have proved HrpZPsgS1 protein and recombinant proteins could elicit the expression of genes related to HR,pathogen defense and growth process in tobacco or rice plants.In order to understand the molecular mechanisms mediated by harpins in plant,our study use the transcriptome sequencing technology to analyze the differentially expressed genes of rice(Oryza saliva L.ssp.japonica)leaves after dealed with HrpZpsgsi protein for 12 h,with water treatment as control.Finally,the analysis results showed that 500 genes of HrpZPsgs1 protein treatment present different expression,among them,316 genes were up-regulate expressed and 184 genes were down-regulate expressed.GO annotation and KEGG pathway analysis revealed that these differentially expressed genes mainly involve in energy metabolism,amino acid metabolism,plant hormone,G protein signaling,protein kinase,Ca2+ signaling,phenylpropanoid metabolism,transcription factor and so on.In summary,under normal physiological conditions,transcripts related to the plant growth and development and response to biotic and abiotic stress were differentially expressed after HrpZPsgS1 protein treatment.Our work proved the previous plantphenotype results in rice,and provided a theoretical basis to explain the biological effects mediated by harpins in plants.