Regulation Mechanism Of OsSCYL2 Gene On Innate Immunity In Rice(Oryza Sativa L.)

Clathrin-mediated trafficking（CMVT）is a very conserved intracellular material transport pathway in eukaryotes,which is involved in regulating the plant immune system,however,the molecular links between CMVT and plant immunity is remains elusive.In this study,a mutant scyl2-1 was obtained from japonica rice cultivar Xiushui 134（WT）treated with EMS（Ethy1 methane sulfonate）.The molecular and genetic mechanism of scyl2-1 were further studied.The main results are as follows:1.A new disease lesion mimic mutant scyl2-1 was obtained from the seeds of conventional japonica rice cultivar Xiushui 134 induced by EMS.Compared with the wild type,scyl2-1 leaf surface is dotted with reddish-brown spots,which will grow and increase with leaf growth and development until maturity,which is related to the mutant’s light-dependent characteristics.The results of related chemical tissue analysis showed that scyl2-1 had an abnormal active oxygen scavenging system,which resulted in the accumulation of H₂O₂.In addition,the expression of genes related to the defense signal pathway also changed,these results suggest that OsSCYL2 is associated with innate immune regulation in rice.2.Genetic analysis showed that scyl2-1 mutant was a recessive single gene mutation.map-based cloning found a 12bp-deletion in exon 4 of the candidate gene OsSCYL2（LOC＿Os01g42950）.Genetic complementation test and the CRISPR/Cas9 gene editing verified that OsSCYL2 is the causal gene of scyl2-1 mutant.3.Both qRT-PCR and Gus staining showed that OsSCYL2 gene was expressed in roots,stems,leaves,leaf sheaths and panicles,and the highest expression level was found in panicles.Subcellular localization showed that OsSCYL2 was localized in Golgi apparatus,Trans Golgi apparatus network and vacuolar precursors involved in vesicular transport.The results of RNA SEQ sequence analysis further confirmed that OsSCYL2plays a role in vesicle transport pathway.4.scyl2-1 mutant has similar phenotype to the reported rice lesion mimic mutant spl28.the results of yeast two-hybrid,Bi FC and split luciferase complementation assay showed that OsSCYL2 and OsSPL28 interacted indirectly in the medium of clathrin heavy chain Os CHC1,and they formed a functional module in regulating the innate immune system.