Functional Study Of The Phytophthora Infestans Effector PITG＿06087 And Its Target StPUB33 In Potato Late Blight Resistance By Molecular Marker-assisted Selection

Late blight,caused by Phytophthora infestans,is destructive disease of potato.Pathogens infection can trigger plant innate immunity which sequentially halts the attack of pathogens.According to the different mechanism,plant innate immunity can be divided into PTI/MTI(PAMPs/MAMPs-triggered immunity)and ETI(effector-triggered immunity).Plant pathogens deliver effector proteins into host cells to suppress or manipulate plant innate immunity for their colonization.Previous researches show that transient expression of effector Pi06087 or silencing its target gene Nb PUB33 in N.benthamiana reduces late blight resistance significantly.In order to further explore the function of effector Pi06087 and its target St PUB33 in potato late blight resistance,Pi06087 overexpression and St PUB33 RNAi transgenic lines were produced.The late blight resistance of these transgenic lines was further evaluated.The main results obtained are as follows:1.Bioinformatics analysis shows that St PUB33 contains a U-box domain and an S＿TKc domain,the typical domains of E3 ubiquitin ligase enzyme.The potato gene expression data collected from PGSC database shows that St PUB33 expression has no significant changes after treatment with salt,ABA,IAA,GA3,wounding and P.infestans inoculation.No significant diffrences of St PUB33 expression were observed in different potato materials indicating that St PUB33 is expressed constitutively with low level in potato.2.Over-expression and RNAi vector of St PUB33 were constructed and introduced into E-3 potato.29 St PUB33 RNAi transgenic lines,2 over-expression transgenic lines were obtained.Gene expression levels of St PUB33 were tested by q RT-PCR.6 St PUB33 RNAi transgeniclines were further selected to identify the late blight resistance.The date of lesion diameter,sporangia productivity,mycelium growth rate and histochemical staining(reactive oxygen and callose accumulation)showed that,compared to the control E3,the RNAi plants show increased susceptible to P.infestans.The results indicate that St PUB33 contributes to potato late blight resistance.3.17 transgenic lines stablely express P.infestans effector Pi06087 were obtained.Comprehensive analysis including lesion diameter,sporangia productivity and mycelium growth rate showed that the late blight resistance of transgenic lines is reduced significantly.It demonstrates that effector Pi06087 can suppress the potato immune response and decreased the resistance of potato agianst P.infestans.4.Expression of St PUB33 and some PTI marker genes were analysised by q RT-PCR before and after treatment with flg22 in St PUB33 RNAi and Pi06087 stable expression transgenic lines.The results showed that St PUB33 can be induced in ‘E-3 potato’ after treatement with flg22 indicating St PUB33 paticipates in basic immune response of potato.The expression of PTI marker genes(including St WRKY7,St WRKY8,Pti5 and St Acre31)are suppressed in St PUB33 RNAi lines when they were treated with flg22.The expression of 3 PTI marker genes(St WRKY7,St WRKY8 and St Acre31)are suppressed significantly in Pi06087 transgenic lines,indicating the effector Pi06087 of P.infestans can inhibit the basic immune response of potato.This study provided useful information for further investegating the molecular mechanisms of how the P.infestans effector Pi06087 manipulates potato immune response throught interacting with its host’s target St PUB33.