Mechaniam Of Disease Resistance Of Populus Davidiana×p.Alba Var.pyramidlis Under Eliciting Plant Response Protein TatEpl1 Of Trichoderma Atrovirite Inducing

Trichoderma spp.,which is widespread,and effective biocontrol agent,has huge potential and widely used in agriculture and forestry,for example,inhibiting various plant pathogenic fungi,inducing plant immune resposes,promoting plant growth and degradating the pesticide residue and heavy metals in soil.Under the bio-control mechanism studying,some small molecular proteins secreted by Trichoderma were found that they could stimulate the host plant defense response on pathogenic fungi.For example,Eliciting Plant Response Protein 1(Epll)belongs to the cerato-platanin protein family and characterized by low molecular weight,so it has a higher study value and broad development prospect as the new plant inducer-fungi activated protein.In this study,the 417 bp TatEpll gene predicted molecular weight 14.3 kDa and an isoelectric point of 5.57 was cloned from T.atroviride ACCC30153 and code 138 aa,which has high hydrophobicity,and the DNA sequence of TatEpll was 487 bp in length.The BlastP results showed TatEpll protein belongs to the cerato-platanin protein family,and TatEpll amino acid sequence shared 88%identity with reported eliciting plant response-like proteins from T virens Gv29-8.TatEpll transcription of T.atroviride ACCC30153 was differentially up-regulated under nine inductive culture conditions(containing MM,carbon starvation,nitrogen starvation,1%powdered stems or leaves from Shanxin poplar seedlings,1%powdered cell walls of A.alternata or C.chrysosperma and 5%(v/v)fermentation supernatant of A.alternata or C.chrysosperma)by RT-qPCR analysis;The obviously higher expression level of TatEpll gene was shown under carbon starvation,cell walls of pathogenic fungus and powdered stems or leaves from Shanxin poplar seedlings,and the expression level of TatEpll gene was the highest under powdered stems or leaves from Shanxin poplar seedlings,up to the 216 and 217.8 fold of the control at 16 and 4 h,respectively.The results shown the transcription level changes of TatEpll gene was caused by carbon starvation,pathogenic fungus and woody plant,and the the expression level of TatEpll gene was the highest under powdered stems or leaves from Shanxin poplar seedlings.To study the function of TatEpll protein,the recombinant prokaryotic expression vector pGEX-TatEpl1 was successful structured and the recombinant strain BL21-TatEpll was obtained.The recombinant E.coli protein E-rTatEpll was obtained under the IPTG inducing,and a clear protein band was produced with a molecular weight of approximately 40.37 kDa(14.37 kDa of target protein+26 kDa of GST label)on the SDS-PAGE gel.The protein was inclusion body protein.Meanwhile,the recombinant eukaryotic expression vector pPIC9K-TatEpll was also successful structured and the recombinant strain GS115-TatEpll was obtained.The yeast recombinant protein Y-rTatEpl1 was obtained under the methanol inducing,and a clear protein band was produced with a molecular weight of approximately 14.37 kDa on the Tricine-SDS-PAGE gel.The results indicated that TatEpllgene could largly express in E.coli and P.pastor is,respectively.Compared with the control,(1)the growth of Shanxin poplar under inducing was obviously increasing.The average growth of Shanxin poplar was 10.3%of the control under E.coli recombinant protein E-rTatEpll,and that was 12.5%of the control under yeast recombinant protein Y-rTatEpll,and the growth of root was very strong.(2)The biological enzyme activityof Shanxin poplar under inducing strongly changed.Under E.coli recombinant protein E-rTatEpll,all of enzymes activities compared with controls had strong changes,and PPO,PAL,SOD,POD and CAT had the largest growth in 5d,5d,2d,5d and 12h,4.0,6.0,0.9,2.2 and 5.6 time of the control,respectively;Under yeast recombinant protein Y-rTatEpl1,all of the enzymes activities compared with controls had also strong changes,and PPO,PAL,SOD,POD and CAT had the largest growth in 3d,3d,3d,12h and 5d,3.3,3.1,3.3,1.6 and 7.7 time of the control,respectively.(3)Under E.coli recombinant protein E-rTatEpll or yeast recombinant protein Y-rTatEpl1,the expression of signal-related genes in the salicylic acid,jasmonic acid,and auxin signaling pathways in Shanxin poplar had the distinct transcription pattern changes.(4)Under E.coli recombinant protein E-rTatEpll or yeast recombinant protein Y-rTatEpl1,the Shanxin poplar importantly produced the disease resistance to A.alternate.These results showed that TatEpll protein,as an elicitor,is required for activation of systemic acquired resistance and induced systemic resistance in Shanxin poplar,and also stimulates Shanxin poplar growth.To sum up,cloning,bioinformatics analysis and transcription pattern research of TatEpll gene provide basic data for eliciting plant response protein studying;heterologous expression and characteristic research of TatEpll protein provide theoretical basis and technical support for development and utilization of the new plant inducer,meanwhile,establish the good foundation for production and application of new pesticides.