OsCul3a Mediated Cell Death And Broad-spectrum Disease Resistance In Rice(Oryza Sativa L.)

Cultivating crop varieties with broad-spectrum disease resistance is an effective solution to combat severe economic losses caused by plant diseases,and systematic research to develop varieties that balance growth and disease resistance is an ongoing need.We mainly applied TMT?Tandem Mass Tag?and PRM?Parallel Reaction Monitoring?proteomics to identify relationships between cell metabolism and cell death related growth and disease resistance from different aspects,such as phenotype,physiology and biochemistry,subcellular structure,metabolic pathway and protein interaction.We also employed CRISPR-Cas9 techniques to characterize OsCUL3a and its association with important immunity response genes OsNPR1/2/3 in controlling cell death.The main research results were as follows:1.We used stereo microscope to observe the DAB stained leaves of wild-type ZH11 and the oscul3a mutant,and found scattered local auto-activated cell death sites surrounded by reddish-brown color on leaves of the oscul3a mutant.So,the lesion mimic formation might be caused by the accumulation of H₂O₂ in the local tissue area of the leaf,and then gradually spread to the whole leaf.We then stained oscul3a leaves at different growth stages with DAB.This assay indicated that H₂O₂accumulation was initiated at 50 DPS and was highly accumulated at 60 DPS compared with the leaves of ZH11.The mutated oscul3a caused a late accumulation of H₂O₂ that enhanced disease resistance to rice pathogenic fungi Magnaporthe oryzae and pathogenic bacteria Xanthomonas oryzae pv.oryzae.2.We found that the oscul3a mutant had a relatively slower growth rate from 35 DPS to 55 DPS,coincident with H₂O₂ accumulation and lesion mimic formation.The oscul3a mutant was significantly shorter than the wild-type ZH11,p1300-OsCUL3a-1 and p1300-OsCUL3a-2 plants at 80 DPS,which was mainly reflected in the significantly shorter panicle and first internode length of the oscul3a mutant.So mutating OsCUL3a suppresses rice growth as a side effect of the enhanced disease resistance.3.TEM observation found high density accumulation of lipidosome in the cytoplasm and osmiophilic granules in the chloroplast of the oscul3a mutant compared with ZH11 at 70 DPS.The oscul3a mutant had significantly decreased 1000-grain weight,but no visible differences in grain shape compared to ZH11.Further grain quality analysis found that total lipids were increased,but total starch and protein content were reduced in the oscul3a mutant.The starch granules of the wild-type ZH11were rhomboidal polyhedrons of uniform size,but the starch granules of the oscul3a mutant were variable in size and shape.These differences in the grain quality could be a reason for reduced grain weight.4.A total of 4623 and 4572 functional proteins were identified before and after lesion mimic formation based on proteomic analysis.Among them,263 and 947 differentially expressed proteins in BLM?before lesion mimic formation?and ALM?after lesion mimic formation?were identified based on the fold change of protein level in the oscul3a mutant compared with wild-type ZH11,which included up-regulated proteins?fold change>1.20?and down-regulated proteins?fold change<0.83?and were used for functional annotation.The differentially expressed proteins were mainly located in the chloroplast and cytoplasm with catalytic activities and binding functions,and responsible for the disrupted cell metabolism with enhanced lipid metabolism,but suppressed carbon/nitrogen metabolism.The secondary metabolite salicylic acid?SA?was highly accumulated in leaves of the oscul3a mutant.Another secondary metabolite coumarin induced reactive oxygen species?ROS?in rice calli and inhibited the growth of rice blast disease.STRING analysis showed the interactions of OsCUL3a with down-regulated molecular switch proteins and up-regulated molecular switch proteins,and three signal layers were found in the core network.PRM approach confirmed that the OsCUL3a associated molecular switches include the lipoxygenases?CM-LOX1 and CM-LOX2?,now shown to play an important role in newly identified iron-dependent lipid peroxidation cell death?ferroptosis?and rice lesion mimic formation.5.Based on CRISPR-Cas9 technology,the role for OsCUL3a in cell death was confirmed with a new oscul3a knockout made in the wild-type ZH11 background.In addition to the genetic connection between OsCUL3a and NPR1 described previously during lesion mimic formation,I was also able to detect interactions amongst OsNPR1,OsNPR2,and OsNPR3 using the yeast-two-hybrid assay,and then knocked out OsNPR2 and OsNPR3 in the oscul3a mutant background.The rice immunity associated OsNPR1/2 and OsNPR3 proteins have opposing functions as oscul3a/osnpr1 or oscul3a/osnpr2knockouts suppress cell death,while oscul3a/osnpr3 knockouts enhance cell death.