Preliminary Functional Investigation Of 11 Phytophthora Infestans Effectors

Potato(Solanum tuberosum L.)suffered from various diseases in its growth process,which cause huge economic losses every year.One of the most devastating diseases is late blight which caused by the Irish potato famine agent Phytophthora infestants.Scientists have already realized that P.infestants secrets effectors into plant through forming haustoria to manipulate plant innate immunity.So,the key point to understand the competition between plant and pathogen is effector.The genome of the Phytophthora infestants contains over 500 genes encoding candidate effector proteins carrying the host cell entry motif RxLR-dEER.Our research based on the effectors screened by our British partner,11 effectors were picked up in present research;The preliminary functions of these effectors were identified by agrobacterium mediated transient expression system in N.benthamiana.Firstly,we identified the expression pattern of these effectors and protein expression in plant;then,the subcellular localization of these effectors were observed;we inoculated P.infestans on N.benthamiana after infiltrated with effectors to testing whether effectors affects the invasion and colonization of P.infestans;at last,we verified if these effectors could suppress plant innate immunity of several ETI and PTI pathways.The main results as follows:1.When the dominant P.infestans strain “14-2” was inoculated onto the N.benthamiana.The expression level of effector genes were measured at different time courses post inoculated infection(0,24,48 and 72 hpi),11 effector genes identified from the P.infestans strain “T30-4” were all up-regulated at early stage after inoculation.2.The expression of effectors were identified by Western-blot in N.benthamiana.Sampling after 1 day post-infiltration.Total protein was extracted and used in Western-blot.The results showed that 11 effectors were all expressed in the plant.3.To determine the subcellular localizations of effectors,we fused green fluorescent protein to 11 effectors on the upstream of their signal peptide cleavage site.Fusion plasmids were expressed transiently in N.benthamiana using agroinfiltration.The results showed that Pi08278,Pi15972,Pi19617 and Pi07555 strictly localized to the cytoplasmic,Pi16195,Pi17063 and Pi00582 localized to plasma membrane,Pi07550 and Pi21388 localized to both nuclear and nucleolar,Pi09732 localized to the plasma membrane and cytoskeleton-like strands.4.To evaluate whether these effectors could boost P.infestans colonization,P.infestans “88069” was inoculated in N.benthamiana leaves after 1 d infiltration,lesion size was measured 7 d post inoculation.The results showed that Pi15972,Pi19617,Pi16195 and Pi09732 can boost P.infestans colonization;Pi08278,Pi17063,Pi00582,Pi07550,Pi21388 and Pi01934 showed no effects on P.infestans colonization;Interestingly,Pi07555 can suppress P.infestans colonization.5.Effectors were co-infiltrated with INF1,XEG1,Avr3a/R3 a and Avr4/Cf4 which could induce HR reaction on N.benthamiana.The degree of HR reaction triggered by INF1,XEG1,Avr3a/R3 a and Avr4/Cf4 were measured.Of the 11 effectors screened,we found no effector could partially or comletly suppress HR induced by INF1,Avr3a/R3 a,Avr4/Cf4 and XEG1.