The Mechanism Of HYL1 Modulates MiRNA-mediated Translation Inhibition And MiR319a In Regulating Plant Disease Resistance

Plant microRNAs(miRNA)are a class of?21 nt non-coding RNA.The miRNAs are first translated into pri-miRNA by RNA polymerase II,and then cleaved into miRNA:miRNA*duplex mainly by Dicer-Like 1(DCL1)accompanied by HYPONASTIC LEAVES 1(HYL1)and SERRATE(SE).Then the mature miRNAs are translocated into cytoplasm to target mRNA for further cleavage or translational repression under control of RNA-induced silencing complex(RISC).Through silencing target genes,miRNA is able to regulate plant development,biotic and abiotic stress responsing.The nuclear localized HYL1,an important partner of DCL1,is engaged in miRNA biogenesis,which is used to be considered as an important function of HYL1.Our lab has been engaged in the study of miRNA biogenesis and function of Arabidopsis HYL1 and its homologous BcpLH in cabbage for a long time.In our previous study,we found the types and abundance of miRNAs in hyl1 mutant cannot explain its phenotype establishing.We speculated that HYL1 may not only mediated miRNA biogenesis,but also regulate miRNA-mediated translational repression.To study the possible function of cytoplasmic HYL1,we construct nucleus-only HYL1 transgenic plant with strong nuclear localized signal(pHYL1::NLS40HYL1/hyl1-2;NLS40HYL1)and cytoplasm-only HYL1 transgenic plant with strong nuclear export signal(pHYL1::NESHYL1/hyl1-2;NESHYL1).The results of small RNA-sequencing,real-time quantitative PCR(RT-qPCR),northern blotting and phenotypic analysis showed that although NLS40HYL1 can completely restore the defects of miRNA processing in hyl1-2,it can only partially rescue the phenotype defects of hyl1-2.Furthermore,the NESHYL1 is not sufficient for miRNA processing,but it can partially rescue the phenotype defect of hyl1-2.These results indicate that cytoplasmic HYL1 is indeed function in plant development.We further find that although the mRNA levels of miRNA target genes are equal(NLS40HYL1 vs WT;NEsHYL1 vs hyl1-2),the protein levels are distinct(higher in NLS40HYL1 vs WT;lower in NESHYL1 vs hyl1-2),which means cytoplasmic HYL1 can inhibit translation of miRNA target genes.The results of Cu2+-limiting/replete condition and the reporter system experiments demonstrate that cytoplasmic HYL1 induce translational repression in a miRNA-dependent manner instead of a broad way.Furthermore,the cytoplasmic HYL1 is localized on endoplasmic reticulum(ER)and interacted with AGO1 and AMP1.RNA immunoprecipitation-quantitative PCR results showed that HYL1 binds to miRNA-targeted mRNA in cytoplasm specifically.In summary,our results demonstrate that cytoplasmic HYL1 interacts with AGOl and AMP1 on ER,binds to miRNA target mRNAs,represses their translation.This result decorates miRNA-mediated post-transcriptional silencing pathway and provides a new insight for further study on this mechanism.Plant are continuously colonized by microorganisms,some of which are pathogens that threaten plant survival and adversely affect productivity.To grow up healthily,plants have evolved two disease resistance modes(PTI and ETI)to avoid pathogen infection.miR319 is an ancient miRNA family which target TEOSINTE BRANCHED 1/CYCLOIDEA/PROLIFERATING CELL FACTOR(TCP)in Arabidopsis thaliana to regulate leaf development.Recent studies have showed that miR319 and its target TCP genes also participate in abiotic and biotic resistance.But how miR319 involved in plant immunity remains largely unknown.Brassica rapa spp.pekinesis is an important economic crop.Our studies find miR319a overexpressed B.rapa(eMIR3)9a)is more sensitive to Sclerotinia stem rot(SSR)both in the wild and growth chamber.The RT-qPCR results shows that JA-and SA-biogenesis and response related genes are induced after Sclerotinia sclerotiorum infection.The expression levels of pathogensis-related(PR)and plant defensing 1.2(PDF1.2)genes in eMIR319a are lower than that in wild-type plants after S.sclerotiorum infection.The expression of miR319a is elevated after pathogen infection,while the expressions of its target TCP genes are repressed,especially TCP3,TCP4 and TCP10.Subsequently,we construct BraTCP4-1 over-expressed B.rapa(5mTCP4-1)and test its pathogen resistance ability.After S.sclerotiorum infection,5mTCP4-1 plants harbor lower lesion area and disease rate compared to wild-type plants both in the wild and growth chamber.The expression levels of JA-and SA-biogenesis and responsive related genes are higher in 5mTCP4-1 than in wild-type plants after pathogen infection.These results suggest miR319a/TCP4 pathway regulate plant resistance to SSR through JA and SA responsive way.TCP4 is a translational factor and may influence plant immunity through regulate down stream pathway.We then employ RNA-seq to analysis the gene expressions in healthy rosette leaves of eMIR319a and Bre.The result shows many plant immunity related gene families are down regulated in eMIR319a,especially WRKY.Through electrophoretic mobility shift assay,chromatin immunoprecipitation-quantitative PCR and transient assay,we find BraTCP4-1 specifically bind to CATATG in the promoters of BraWRKY33-2,BraWRKY70-1 and BraWRKY75-1 and promote their translation.Finally,we test the expression levels of BraWRKY33-2,BraWRKY70-1 and BraWRKY75-1 after S.sclerotiorum infection.The expressions of those genes are significantly reduced in eMIR319a compared to that in wild type plants,while increased in 5mTCP4-1 In summary,this new miR319a-TCP4-WRKY pathway involved plant SSR resistance we find broaden the cognition of miR319a function and may provide a new insight on plant immunity.