| There are various plant pathogens in nature including bacteria,fungi,oomycetes,and viruses.They have developed different pathogenisis strategies during evolution and interfere with the normal growth of plants and cause severe damage to plant.To defend against pathogenic infection,plants have evolved and formed innate immune system,that mainly rely on two layers of defense:pathogen-associated molecular patterns（PAMPs）triggered immunity（PTI）and effector triggered immunity（ETI）.In both layers of immunity,protein post-translational modifications including ADP-ribosyltion which was studied in this thesis play critical roles.Protein ADP-ribosylation is the covalent transfer of either single or multiple ADP-ribose moieties from nicotinamide adenine dinucleotide catalyzed（NAD+）to target protein catalyzed by PARP polymerase.Protein ADP-ribosylation plays an important role in Arabidopsis innate immunity as reported recently.This study aims to explore the potential function of PARylation in disease resistance in monocot rice.It mainly addressed three questions:（1）Does PARylation exist in monocots rice?（2）Does PARylation function in rice innate immunity?（3）Has PARylation in monocots evolved divergent function compared to its counterpart in dicots?By genetic,cellular and biochemical experiments,we have following findings:1、Rice genome encodes OsPARPs that catalyze PARylation and OsPARG1 that degrade PARylation.Similar to their homologous proteins from Arabidopsis,we found that OsPARP1、OsPARP2A and OsPARP3are exclusively localized in nuclear,and OsPARG1 could be detected in both nuclear and cytoplasm.OsPARP1 and OsPARP2A and OsPARG1could catalyze PARylation and degradation of PAR respectively.Through prokaryotic expression and purification of OsPARP1,OsPARP2A,OsPARP3 and OsPARG1 proteins and in vitro PARylation and hydrolase activity experiments,we found OsPARP1 and OsPARP2A have PARylation activity,and OsPARG1 showed the activity of degrading PAR.We mutated Glycine 269 to Leucine(the 3rdG of the catalytic core,GGG-X7-QEE)in OsPARG1,which partially compromise its enzyme activity.This indicates that OsPARG1 has a conserved enzyme activity center similar to that of At PARG1.OsPARP1 and OsPARP2A expressed in protoplasts also showed enzyme activity,which implicates that OsPARP1 and OsPARP2A are also active in vivo.Interestingly,the enzymatic activity of OsPARP2A seem to be higher than that of At PARP2,implicating divergence of their functions.We also carried out function complementation assay,in which OsPARP2A and OsPARG1driven by the native promoter of their homolog in Arabidopsis were expressed in atparp1/2 and parg1 respectively.The results revealed that OsPARG1 exhibit even stronger transcription inhibition activity than that of At PARG1 towards immune genes,implying difference between PAR metabolism in rice and that in Arabidopsis.2、In order to study the immune function of PARylation in monocotyledonous rice,we used CRISPR-Cas9 technology to knock out OsPARP1,OsPARP2A,OsPARP2B,OsPARP3 and OsPARG1 in ZH11background.After inoculation with Magnapothe oryzae Guy11 and Xanthomonas oryzae pv.oryzae P6,the causing agent of rice blast and bacterial blight respectively,the osparp1,osparp2b,osparp3 and osparp1/2a/3 mutants are more susceptible than ZH11.There was no significant difference between osparp2a and ZH11.Therefore,OsPARPs are shown to be involved in rice resistance. |
