Function And Mechanism Analysis Of Two RxLR Effectors From Phytophthora Capcisi

Phytophthora capsiei is one kind of pathogenic oomycetes which can cause destructive diseases in solanaceous(pepper,tomato),legume(lima and snap beans)and most cucurbit hosts.Currently,we haven't set out some effective measures to control the disease since some fungicides can't reach a good effect on the disease caused by P.capsici.Thus,it is significant for the disease control to research the effective targets which can interact with P.capsici effectors.P.capsici is a hemibiotrophic oomycete pathogen secreting a battery of effectors to promote infection through regulates plant immune defense responses in biotrophic phase.In the varied of the effector secretome of plant pathogenic Oomycetes,a kind of important cytoplasm effectors with conservative RxLR-dEER domain in N-terminal was reported.The N-terminal region of the RxLR effectors is involved in secretion and targeting while the C-terminal domain is consistent with a role in effector activities.To know the function and mechanism of the RxLR effectors can help us to uncover the infection mechanism and make the control measures by genetic engineering of plant disease resistance.Through RNA-Seq,bioinformatics predictive analytics and agrobacterium-mediated transient expression,we gain an effector PcRxLR207 which can induce programmed cell death(PCD)in ANicoliana bentha,iana and an effector PcRxLR103 that promoted pathogen infection after expressed recombinant vector in N.henzhamiana.On this basis,we do some research for the mechanism analysis in the following:PcRxLR207 can interact with RBPs and induce cell death in plants:we gained an effector PcRxLR207 which can induce programmed cell death in N.henthamiana.To know the site of action of PcRxLR207 in plants,we expressed recombinant vector pBinGFP2::RxLR207 in N.benthamiana and found PcRxLR207 only in cytoplasm.To know the targets of PcRxLR207 in plants,we screen the gene BPA11?RRMP1?RRMP2 and RRMP3 from Arabidopsis cDNA library through yeast two-hybrid system.Through NCBI data bases blasts and bioinformatics predictive analytics,we find that the targets gene has conservative RNA-binding motif and may expressed RNA-binding proteins(RBPs).Through the verifying PcRxLR207 interact with RNA-binding proteins by yeast two-hybrid system and bimolecular fluorescence complementation system,the results show that PcRxLR207 can interact with RBPs and the interaction sites may in mitochondrion.Others,through the subcellular localization experiment of RBPs,we found that the location of BPA11,RRMP2 and RRMP3 in cytoplasm and nucleus of N.benthamiana leaves while RRMP1 located in cytoplasm.Compared with the different results of PcRxLR207 and RBPs in cytoplasm after interaction and before,we guess that PcRxLR207 may reduce the activity of RBPs to promote infection through changed the functional site of RBPs.PcRxLR103 facilitated P.capsici infection in early and late infection stage:we successfully cloned some RxLR effectors from P.capsici and cloned them into the virus X(PVX)vector.N.benthamiana leaves was infiltrated with Agrobacterium cells containing each effector construct and then the infiltrated leaves were challenged with P.capsici zoospores,and one effector PcRxLR103 that promoted pathogen infection was identified.The expression pattern determined by real time RT-PCR showed that PcRxLR103 effector is up-regulated in early infection stage,suggesting that PcRxLR103 effector may play an important role during infection.Cell death induction assay showed that PcRxLR103 effector can trigger weak cell death in N.benthamiana,however,the relationship between its cell death-inducing activity and virulence function is still unknown.We thought this effector can inhibit plant defense response to cause diseases.On the one hand,we get seeds of Arabidopsis plants transformed with PcRxLR103 through the floral dip method and fluorescence-accumulating seed technology.Under the fluorescence microscope,we can pick up T0 seeds that giving up green fluorescence.Similarly,we will screen until T3 seeds to get the stable inheritance seeds.Through transgenic Arabidopsis plants,we can research the function and signal pathway of PcRxLR103 in plant.On the other hand,we can screen and identify it target in hosts by Y2H and BiFC so that promote the research process of pathogenesis.