The Study Of The NBS-LRR Protein Pik-H4 Mediates Molecular Mechanism In Rice Blast Resistance

Rice as the one of most important crop in the world,which supplies the carbohydrate energy for more than half of the world's population.But the ascomycetous fungus Magnaporthe oryzae caused rice blast and Xanthomonas oryzae pv oryzae caused rice blight are maximally threaten the rice safely production.Major resistance genes encoding the NBS-LRR protein that play the critical intermediate receptor of signaling transduction in the immunity response under the pathogen attack,which directly recognize the pathogen secretory Avirulence effector and activate the downstream defense responses.Rice disease resistance genes regulated complicated network involve multiple physiological and biochemical reaction,to deep elaborate the detailed molecular immunity pathway induced by the resistance genes not only required for revealing the plant-pathogen interaction mechanism,but also providing the theoretical guidance for pyramiding multi-genes in rice resistance breeding.This study aims to comprehensive illustrate the mechanism of Pik-H4 induced blast and blight resistance in broad-spectrum resistance line H4,Pik-H4 encoded NBS-LRR protein directly recognize the pathogen avirulence effector AvrPik and activate the ETI immunity responses.Previously using the yeast-two hybrid screening,the transcriptome sequencing and ITRAQ have identified the Pik-H4 induced potential disease resistance pathway.Here,we found that the Pik-H4 interacte with transcription factors OsBIHD1 and OsCOL9 to regulate the immunity and plant growth by the hormone crosstalk pathway at transcription level.Furthermore,Pik-H4 repressed the lignin deficient caused rice blast resistance reducing and growth development abnormal via down-regulating the mRNA level of cytoplasmic protwin OsAAE3.In addition,Pik-H4 activated immunity signaling pathway also contained rice blight resistance,OsPrx30 depended on ROS signaling pathway negatively regulated the bacterial blight,which induced by the M.oryzae infected in Pik-H4 NIL.The main conclusions are as follows:1.Rice blast resistance gene Pik-H4 is consists of two adjacent genes Pik₁-H4 and Pik₂-H4,the coil-coil domain of the Pik₁-H4 encoded NBS-LRR protein interacts with the HD domain of the homeodomain transcription factor OsBIHD1 in the nucleus.The knockout of OsBIHD1 in rice lines carrying Pik-H4 largely compromised the resistance of the rice lines to M.oryzae,While overexpression of OsBIHD1 resulted in enhanced expression of the pathogenesis-related?PR?gene PR1b and ethylene?ET?synthesis gene OsACO3,and the OsBIHD1 expression was able to induce by the ET synthesis precursor ACC,OsBIHD1 was also found to directly bind to the cis-element TGTCA in the promoter region of OsACO3,OsBIHD1 enhanced blast resistance depended on ethylene-synthesis pathway.In addition,OsBIHD1 overexpression provoked dwarfism and reduced brassinosteroid?BR?insensitivity through repressing the expression of several critical genes involved in BR biosynthesis and BR signaling,and BR catabolic genes?CYP734A2,CYP734A4,CYP734A6?were significantly up-regulated,OsBIHD1 repressed the plant growth via directly binding the cis-element in the CYP734A2 promoter region to cause the BR deactivation.Our results collectively suggest a model that OsBIHD1 is required for Pik-H4-mediated blast resistance through modulating the trade-off between resistance and growth by coordinating brassinosteroid-ethylene pathway.2.Previous analysis indicate the Pik-H4 interacted with CONSTANS-like 9?OsCOL9?in yeast cells.OsCOL9 belonged to group II of the COL protein family,OsCOL9 encoded transcription factor and contained transcriptional activation activitiy through its middle region?MR?.Magnaporthe oryzae infection induced OsCOL9 expression,and the highest expression level of OsCOL9 in leaves,and transgenic OsCOL9 knock-out rice plants showed increased pathogen susceptibility.OsCOL9 was a critical regulator of pathogen-related genes,which were also activated by exogenous salicylic acid?SA?and ACC,the precursor of ethylene?ET?.Further analysis indicated that OsCOL9over-expression increased the expressions of phytohormone biosynthetic genes,NPR1,WRKY45,OsACO1 and OsACS1,which were related to SA and ET biosynthesis.Interestingly,we found that OsCOL9 physically interacted with the scaffold protein OsRACK1 through its CCT domain,and the OsRACK1 expression was induced in response to exogenous SA and ACC as well as M.oryzae infection.Rhythmic pattern analysis suggested that OsCOL9 and OsRACK1 responded to the change of daylight,which was regulated by the circadian clock.3.We previously identified an AMPBP OsAAE3 interacted with Pik-H4 in yeast cells.A phylogenetic analysis showed that OsAAE3 was most likely 4CL-like 10 in an independent group,and localized to cytoplasm,and it could be expressed in various tissues.Histochemical staining of transgenic plants carrying OsAAE3 promoter-driven GUS??-glucuronidase?reporter gene suggested that OsAAE3 was expressed in all tissues of rice.Furthermore,OsAAE3-OX plants showed increased susceptibility to M.Oryzae,but knock-out OsAAE3 not affected the phenotype of plant disease resistance,and this finding was attributable to decreased expression of pathogen-related 1a?PR1a?,POD-related genes,and low level of peroxidase?POD?activity.Moreover,OsAAE3 over-expression resulted in increased content of H₂O₂,leading to programmed cell-death induced by reactive oxygen species?ROS?.In addition,OsAAE3 over-expression repressed the floret development,exhibiting dramatically twisted glume and decreased fertility rate of anther.Meanwhile,the expressions of lignin biosynthesis genes were significantly decreased in Os AAE3-OX plants,thereby leading to reduced lignin content,and plant dwarfism.Taken together,OsAAE3functioned as a negative regulator in rice blast resistance,floret development and lignin biosynthesis.4.In a previous transcriptome and proteome analysis of early response genes in rice during Magnaporthe oryzae infection,we identified the difference expression of a Class III PRXs precursor gene OsPrx30 in resistance and susceptible lines.Meanwhile,we found the OsPrx30 expression also induced by the Xanthomonas oryzae in rice resistance line H4.GUS staining and tissue specific assay indicate the majority of OsPrx30,which primarily expresses in leaves,roots,and stems of the rice,localize at the endoplasmic reticulum,peroxidase and plasma membrane.Moreover,OsPrx30-OX increased the susceptibility to X.oryzae through maintaining a high level of peroxidase activity and reducing the H₂O₂content,while knock-out of OsPrx30 showed the opposing results,OsPrx30 depended on the POD-induced ROS pathway regulated the rice blight resistance.Furthermore,we identified OsATH1,an AT-hook domain containing protein,as a transcription factor that specifically binds to AT-rich region in the OsPrx30 promoter.Knockout of OsATH1increase the resistance to X.oryzae,which is similar to OsPrx30 overexpression,RNA-seq indicated the OsPrx30 up-regulated in osath1-ko.Finally,we demonstrated that Os ATH1suppressed OsPrx30 by inhibiting histone acetylation,especially H3 acetylation,at the OsPrx30 promoter.Together,our study comprehensive investigated the Pik-H4 mediated disease resistance pathway,we found the Pik-H4 regulated the trade-off between the host ETI immunity and plant growth,and associated with AMP-binding proteins to modulate the cell wall component lignin biosynthesis,and used the AT-hook and C?PRX to mediate the ROS signaling pathway that co-exist in fungal-bactrium defense response.Our results lay the theoretical foundation for understanding the NBS-LRR disease resistance protein induced molecular mechanism,as well as provide the theoretical guidance for pyramiding multi-genes in rice resistance breeding program.