Genome Editing Of BdFLS2 In Brachypodium Distachyon And Phosphoproteomic Analysis Of Poaceae For Pathogen Resistance

The plant immune response against environmental pathogens starts with the sense of pathogen-associated molecular patterns(PAMPs)by cell surface receptors called PRRs(pattern recognition receptors).The following protein phosphorylation transduces the downstream signaling into the nucleus and activate expression of defense genes.Present knowledge on plant innate immunity was mainly gained from studies on the dicot plants such as Arabidopsis,while understanding of defense mechanism of the monocot poaceae is just beginning.Plant diseases have always been an important problem in agriculture.Poaceae contains the most important food,and feed crops in the world,thus research on the rice disease resistance is of great significance.In this study two kinds of classic mode poaceae plants Brachypodium distachyon and rice were selected to research the molecular mechanism of resistance.Important mutants were obtained through the CRISPR/Cas9 genome editing technology,and screening of important phosphorylation protein for disease resistance based on the analysis of phosphorylation proteomic.With further biochemical experiment,following results were obtained:(1)We successfully obtained the FLS2 knockout mutant(bdfls2)of the important receptor kinase associated with PTI through CRISPR/Cas9 system in Brachypodium distachyon.It was further verified that the receptor was indeed involved in the recognition of the corresponding PAMP flg22 through the assays of ROS burst and PR gene expression.(2)FLS2 can’t be fully used to analyze the mechanism of PTI,and there are few studies on the protein kinases related to disease resistance in Brachypodium distachyon.As the technique of protein phosphorylation in rice,another model plant of poaceae,is more mature compared to Brachypodium distachyon.This study started with screening of OGs-induced phosphorylated proteins in rice suspension cells with phosphoproteomic approaches,aiming to identify some key proteins involved in rice defense signaling and illustrate their roles in PAMPs perception and signal transduction.In all,a total of 1175 phosphosites were identified,including 449 phosphoproteins.(3)OsWAK1 was found as the potential receptor of OGs,and 9 potential key signaling components were screened.Meanwhile the genetic mutants of these key signaling components were also generated using the CRISPR-Cas9 systems for further study.Through biochemical experiments,it is preliminarily confirmed that OsWAK1 is induced by OGs and actives PTI.(4)We generated a simple method for examining PTI in rice seedlings based on lateral root growth inhibition upon PAMPs treatments.This simple and robust PTI assay provides an efficient approach to investigate defense mechanisms in rice.In conclusion,a series of target proteins related to disease resistance were screened based on phosphorylated proteomics in this study,which provided new phosphorylated proteomics support for the study of disease resistance of poaceae.A large number of potential mutants of disease-resistant related phosphorylated proteins were obtained,which laid a good material foundation.At the same time,a simple and efficient mutant screening system was established,which provided a complete method for large-scale screening of PTI mutants.This study may provide valuable insights into the molecular basis of the broadspectrum resistance of rice and may also provide new strategies and targets for the development of diseaseresistant rice varieties.