The Transcription Factor APIP5-mediated Programmed Cell Death And Immune Responses In Rice

Rice is one of the most important food crops in the world,and rice blast,caused by the fungal pathogen Magnaporthe oryzae,is the most destructive rice fungal diseases.The disease routinely causes10–30%yield losses.Understanding how to effectively suppress M.oryzae infection,and enhance innate immunity in rice plants is important for the control of the disease.We previously cloned the broad-spectrum resistance（R）gene Piz-t in rice and its cognate Avr gene AvrPiz-t in M.oryzae.In this project,we analyzed the function and regulatory mechanism of the transcription factor APIP5,a target of the effector AvrPiz-t.We uncovered a mechanism how M.oryzae triggers host necrosis and how plants inhibit the fungal transition from the biotrophic to the necrotrophic stage.We found that AvrPiz-t interacts with the bZIP-type transcription factor APIP5 in vitro and in vivo using yeast two-hybrid,GST-Pull-down,Co-IP assays,and the N terminus of APIP5 is required for the interaction.Co-transformation assay in yeast indicated that APIP5 forms homodimers.The transactivation activity assay in yeast showed that full-length APIP5 and APIP5N display a strong autoactivation.Furthermore,APIP5N has strong transcriptional activity in protoplasts and this transcriptional activity can be specifically suppressed by AvrPiz-t.Subcellular localization assay using confocal microscopy indicated that full-length APIP5,which contains both functional NLS and NES motifs,mainly localizes in the cytoplasm and membrane in N.benthamiana cells and rice protoplasts.In addition,APIP5 co-localizes with AvrPiz-t in the cytoplasm.We also found that silencing of APIP5 in rice plants induces cell death phenotypes,and enhances resistance against M.oryzae and ROS production after PAMPs treatment.The qPCR analysis showed that,relative to their expression in segregating wild-type（WT）plants,the expression of negative regulators of cell death is significantly downregulated,while the expression of defense marker genes is upregulated in APIP5 RNAi lines.Ectopic expression of AvrPiz-t in rice plants promotes the cell death phenotypes of APIP5-silenced plants,and more H₂O₂ accumulation in the APIP5 RNAi*AvrPiz-t plants.In addition,much lower transcript and protein levels of APIP5,and lower expression levels of negative regulators of cell death in the APIP5 RNAi*AvrPiz-t plants than in the APIP5 RNAi plants,indicating AvrPiz-t enhances APIP5-mediated cell death.The Piz-t,the cognate NLR protein for AvrPiz-t,partially suppresses APIP5-mediated cell death in the APIP5 RNAi*Piz-t-HA plants,these plants show reduced H₂O₂ accumulation and more transcript and protein levels of APIP5,and higher expression levels of negative regulators of cell death.Interestingly,the protein level of Piz-t is largely reduced in the APIP5RNAi*Piz-t-HA rice plants,indicating APIP5 is required for the accumulation of Piz-t.Furthermore,GST pull-down and LCI assays showed that Piz-t can interact with APIP5 directly.Inoculated non-Piz-t plants with an isogenic M.oryzae strain with AvrPiz-t,the APIP5 level is gradually declined.However,when Piz-t-HA plants were inoculated with the same isolate,Piz-t-HA and APIP5 protein levels are gradually increased,suggesting that AvrPiz-t strongly suppresses the APIP5 level to promote necrosis when Piz-t is not present,but Piz-t stabilizes APIP5 to prevent necrosis at the necrotrophic stage.In turn,APIP5 positively regulates the accumulation of Piz-t to enhance resistance against M.oryzae infection.These results demonstrate a novel mechanism for the suppression of effector-triggered necrosis at the necrotrophic stage by a pair of transcription factor and NLR receptor in plants.