Functional Characterizations Of Cell Death-inducing Effectors In Phytophthora Capsici

Species of the oomycete genus Phytophthora are possibly the greatest damaging microbes of dicotyledonous plants and comprise notorious microbes,such as Phytophthora infestans and P.capsici.P.capsici attacks over 45 cultivated plants and weeds.There is no effective chemical or cultural approach existing that limits disease colonization when the environment become warm and wet.In addition,there are no resistant varieties of crops identified up to now for domestication.The microbe produces huge amounts of zoospores to aid in colonization besides secreting different types of effectors that manipulates the plant immune system.P.capsici effectors are of two types;apoplastic and cytoplasmic effectors.Apoplastic effectors localize to the plant extracellular space while cytoplasmic effectors are intracellular effectors that enter the host plant cell.P.capsici RxLR and Crinkler（CRN）effectors are cytoplasmic effectors that have conserved motifs to enable them translocate into the plant cell.They enter the host cell and manipulate host immunity thereby enhancing pathogenicity.However,the mechanisms by which these effectors enhance pathogenicity are unclear hence need to be characterized.Here,we screened RxLR and CRN effectors that can induce cell death in planta and selected two effectors for further characterizations of their roles in manipulation of plant immune responses.First,we screened 47 CRN effectors by overexpression in Nicotiana benthamiana and N.tabacum.Three genes（PcCRN4,PcCRN23 and PcCRN42）exhibit cell death-inducing activities in N.benthamiana.PcCRN4 triggered strong cell death in both plants.Furthermore,PcCRN4 triggered cell death in Solanum lycopersicum which is a host of P.capsici.The gene expression results indicated that PcCRN4 is highly induced at the infection stages and required nuclear localization signal（NLS）signal to induce cell death as fusion to nuclear exclusion signal（NES）eliminated the cell death induction.Overexpression of PcCRN4 proteins in N.benthamiana enhanced susceptibility to P.capsici.Subcellular localization results showed that PcCRN4 localized to the plant nucleus,and the localization was required for its virulence function.Silencing PcCRN4 in P.capsici significantly reduced pathogen virulence.The expression of the pathogenesis-related gene PR1b in N.benthamiana was significantly induced when plants were inoculated with PcCRN4-silenced P.capsici transformant compared to the non-silenced lines.Callose deposits were more abundant at sites inoculated with PcCRN4-silenced transformant compared to those inoculated with controls,indicating that silencing of PcCRN4 in P.capsici reduced the ability of the pathogen to suppress plant defenses.The transcript levels of AtMC1 and LSD1 which are PCD gene promoters were up-regulated in wild-type while transcript levels BI-1 which is a PCD suppressor/inhibitor were down-regulated in wild-type and up-regulated in PcCRN4-stable silenced line.The transcript levels in PAD4 were unaffected in wild-type and PcCRN4-silenced line.This suggests that PcCRN4 induce cell death by manipulating expression of cell death-related genes.Overall,our results demonstrate that PcCRN4 is a virulence essential effector and it needs target to the plant nucleus to suppress plant immune responses,such as reactive oxygen species（ROS）and callose deposits,therefore promotes pathogen infection.Secondly,we screened 68 RxLR effectors from P.capsici by overexpression in Nicotiana benthamiana and N.tabacum.Three genes（PcRxLR242,PcRxLR207 and PcRxLR214）were obtained for their cell death-inducing activities in N.benthamiana;PcRxLR242 and PcRxLR207 induced cell death in both plants in which PcRxLR242 had stronger activities.Moreover,PcRxLR242 triggered cell death in S.lycopersicum.Subcellular localization results revealed that PcRxLR242 was a cytosolic effector and transient expression in N.benthamiana reduced susceptibility to P.capsici.Silenced transformants from P.capsici significantly increased microbe pathogenicity in N.benthamiana.The pathogenesis-related gene PR1b was down regulated by the silenced transformants compared to the unsilenced lines.Staining with diaminobenzidine（DAB）showed that silenced lines exhibited weak stain indicating that silencing PcRxLR242 increased the ability of the plant to produce H₂O₂ hence increased ROS.Trypan blue staining of the diseased part inoculated with PcRxLR242 proteins reduced hyphal intensity.Similarly,PcRxLR242-silenced transformant exhibited increased colony diameter as compared to the wild-type.Inoculation of the PcRxLR242-silenced transformants on non-host N.tabacum enhanced susceptibility to P.capsici colonization.Together,these findings show that PcRxLR242 may be an avirulence effector and enhances plant immune responses in both host and non-host plants.