Mechanism Of A Novel Deubiquitination Enzyme Regulating Arabidopsis Immunity

Plants are vulnerable to pathogens throughout their life cycle,and metabolites such as plant hormones play a vital role in resisting the invasion of pathogens.Hence,it is almost significant that to investigate the production and regulation mechanism.The research undoubtedly shed light on plant-pathogen interaction,immune signaling pathways in plants and new immune regulatory targets.Although accumulated researcsh reported that many secondary catabolites can participate in the regulation of plant immunity.Nevertheless,the function of plant primary catabolites in plant immunity is remains to be clarified.At present,considerable progress has been made in dicots represented by the Arabidopsis,whereas many crops are monocotyledonous plants,their genetic structure and regulation mechanism have undergone significant changes different from dicotyledonous plants,which results into that many research results in dicotyledonous plants cannot be effectively applied to food crops.Thus,the comparative analysis between monocots and dicots in the innate immune signal network,especially the functional difference analysis on the basic amino catabolic pathway,is of great value to the application of agricultural production.In the early-stage work in our lab,we found a novel protein called Pigm R,which can confers broad-spectrum disease resistance in rice.For sake of investigating its mechanism of broad-spectrum resistance of disease,we screened an interesting protein Os PICI1 by yeast two hybrid assay combined with quantitative proteomics.Through pathogen inoculation assay on knock-out and over-expressed transgenic lines and further biochemical experiments,we found that this protein can be used as a novel deubiquitinating protease to regulate primary amino catabolism of rice and enhanced resistance to Xanthomonas oryzae pv.Oryzae(Xoo).To explore its conserved function of this new defense pathway in dicots and monocots,we cloned its homologous gene At ESP1/2in Arabidopsis which also encoded a protease and obtained two different mutant lines of atesp1/2 from the Arabidopsis mutant library.Interestingly,we found atesp1 show dramatic improved resistance to Pseudomonas Syringae Pst DC3000 compared with the wild type line after inoculation.Overexpression of At ESP1/2 show significantly enhanced susceptibility.These data demonstrated that At ESP1/2 served as a negative regulator in bacterial resistance in Arabidopsis.By the alignment of protein sequences and experiment verification,we concluded that the At ESP1/2 has ubiquitin activity.By IP-MS analysis,we found a highly conserved amino acid synthase in plants which are called At MTs.Then we test direct interaction between those proteins by Split-Luc experiment and Co-IP.At ESP1/2 affects the stability of the protein At MT1/2 through deubiquitinating enzyme activity.On the other hand,it also can regulate the biosynthesis of amino acids to suppress the resistance of Pseudomonas Syringae.This is verified to rescue atesp1 mutant resistance phenotypes by adding exogenous amino acid.Previously there have been reports that the hormone is involved in plant disease resistance signal path.The amino acid as a precursor for the synthesis of the hormone,we used gas chromatography and mass spectrometry to further confirm that the amino acids catalyzed by At MT1/2 may be involved in the immunity of Arabidopsis thaliana by regulating the biosynthesis of plant hormones.In addition,the At ESP1/2 is also interaction with Pseudomonas Syringae effector Hop AI1 and Hop B1,it may hint another promising research area.In conclusion,this study reveals a new At ESP1/2-At MT1/2-Hormone cascade signaling pathway,which clarifies the mechanism of regulation plant immunity by influencing primary production biosynthesis.Also,this may be a new approach in plant defense response,which will provide a reference for future plant comparative immunity research between monocots and dicots.