The Virulence Of Effector RxLR 121 Of Phytophthora Capsici

Phytophythora capsica is a kind of broad range host pathogen that could infect papper,tomato,Nicotiana benthamiana,and Arabidopsis.It’s very difficult to deal with it.The first report about P.capsica is in New Mexico,America,1922.Effectors could help micropathogen to infect and expand between the host cells or hosts.Most effectors of P.capsici could regulate the host’s immunity and promote the infection of itself.Only a few effectors could be recognized by the host resistant protein and the hypersensitive reaction accure,that could make the programed cell death.As the largest family of the oomycete effectors,1121 RxLR effectors of P.capsici could be searched at the FungiDB webset,and reports about them emerge in endlessly.As the result of our experience before,effector RxLR 121 could cause the N.benthamiana leaves cell death and reduce the cell death caused by elicitor INF1.There is a hypothesis that it could promote the infection.In this experience,we want to make sure about it,and then the gene silencing and transgene come to be the method.We cut off the singnal peptide of RxLR 121 and construct the pBIN-eGFP vector then transfer the vector to agribacerium GV3101.It comes to be convininent for P.capsici research after the pubic of the LT1534 genome by Lamour K et al.,2012.Taking LT1534 as the object of the study,we want to know the contribution of RxLR 121 to its’ infection and virulence.We use the silence vector pTOR-eGFP to make the production of RxLR 121 down-regulate in LT1534,and infect the host’s leaves to make sure the hypothesis.Using pBIN-eGFP vector,we could know that after the random insertion,RxLR 121 could make the Col-0 ecotype Arabidopsis growth to be sick.We hope the result could help the study about RxLR effectors in the future.Results were as follows:1.We use RNAi method,pTOR-eGFP as the vector,transfer the vector into protoplasts by DNA-PEG-calcium mediated protoplast transformation.After the reverse insertion worked,the gene silencing succeed.As the result of qRT-PCR shows that two silenced lines comes out because the expression level of RxLR 121 is under 30 %.One of them named RxLR 121-S13,the other RxLR 121-S22.The hyphal plugs were used to infect the leaves of pepper and N.benthamiana,after 60 hours or 48 hours,the necrosis of the leaves were observed.We use handheld long-wavelength UV lamp to distinguish the cell death part and the living cell.After photos had been taken,we used trypan blue solution to stain the leaves,after 24 hours,chloral hydrate solution was used to fade them and took down.As the result shows that RxLR 121 could promote the infection of P.capsici.2.As the in vitro cover experiment shows,after the overexpress of the RxLR 121:GFP with the agrobacterium 24 hours,the zoospores of silenced lines or wild type was used to infect.The handheld long-wavelength UV lamp could detect the GFP fluorescence and the necrosis part.The conclusion is that the in vitro experiment could recover the function of RxLR 121 successfully.As a result,RxLR 121 could promote the infection and virulence up to a point.3.After the reverse transcript PCR and Western Blot experiment,demonstrated that the insertion of the RxLR 121 without the peptide into the Arabidopsis could decrease the height,the inflorescence and the seed foundation seriously.