Molecular Mechanism Of MicroRNA168 And McroRNA137 Mediated Regulation In Malus Resistance Against Botryosphaeria Dothidea

MicroRNA(miRNA)-mediated post-transcriptional gene regulation plays important roles in various plant life process,such as growth and development,hormone signaling and stress response.Binding to ARGONAUTE1(AGOl)protein and forming an RNA-induced silencing complex(RISC),plant miRNA mediates direct cleavage or translation inhibition of target mRNAs in a base-pairing complementation manner to regulate their expression.In response to diverse pathogens infection,endogenous miRNAs participate in complex regulatory networks of plant defense response directly or indirectly,and play a key regulatory role in plant resistance.Apple is one of the most important fruit species worldwide.Botryosphaeria dothidea is a major fungal disease that endangers the safety of apple production in China.The resistance of apple germplasms against B.dothidea infection is significant different interspecies.Therefore,in-depth understanding the molecular mechanism of plant defense response is of great significance to prevent and control the damage caused by B.dothidea infection,and to improve the resistance of apple varieties to this disease.In this study,the function and regulation of miR168,which is the major regulator in the plant miRNA pathway,and its target gene AGO1,in the defense response of resistant germplasm Malus hupehensis against B.dothidea were revealed.The complex interactions and regulatory mechanisms of miR168 and MhAGO1 at transcriptional and posttranscriptional levels were identified through expression pattern analysis,promoter function studies,5'RLM RACE and tobacco co-expression assay.The role of miR168 and MhAGO1 in different layers of plant defense response and,and their effects on the resistance of M.hupehensis to the infection were investigated by means of transient expression,STTM and RNAi.By which,the evidence for the involvement of miRNAs in the regulation of the resistance of apple germplasm against B.dothidea was provided.Next,a novel miRNA(named miR137),which was in response to B.dothidea,was recognized in both resistant M.hupehensis and susceptible M.domestica 'Fuji'.The regulation of target gene ERF 14 by miR137 was clarified.The molecular function of ERF 14 as a transcription factor was identified using yeast one-hybridization,gel shift assay(EMSA)and transient expression.Furthermore,the molecular mechanism of miR137-ERF14 module in the regulation of plant pathogen resistance was analyzed by means of virus-assisted gene expression(VAGE).These studies laid the foundation for further elucidating the functional mechanism of key miRNAs in the Malus resistance against B.dothidea.The main findings are as follows:1.The precursor of M.hupehensis miR168a was isolated,and mature miR168 was located upstream of its folded hairpin structure.The induction of mature miR168 and pre-miR168a were revealed by RT-qPCR.Therefore,the accumulation of mature miR168 was at least partially caused by the induced of MIR168a.The target gene of miR168 was cloned from M.hupehensis,which encoded an AGO protein containing Piwi and Paz domains.Homologous analysis showed that this AGO protein has high homology to Arabidopsis AGO1 protein.5' RLM RACE confirmed that the cleavage site of MhAGO1 was between the 10th and 11th bases of the miR168 target site;tobacco co-transformation experiments showed that miR168 can inhibit the expression of the wild type MhAGO1 gene,but not the mMhAGO1 with mutated target site.Hence,MhAGO1 is the direct target of miR168,and miR168 regulates the expression of MhAGO1 by mediating the cleavage of target mRNA.Shown by Northern blot and RT-qPCR assay,the accumulation of uncleavaged AGO1 mRNA as well as 3' and 5'cleavage products were increased during B.dothidea infection,sharing a similar expression pattern with mature miR168.MhAGO1 gene was activated at the transcriptional level by the infection,and was inhibited by induced miR168 at the posttranscriptional level.DNA fragments of 2000 bp upstream of the transcription start sites were cloned as promoter regions of MIR168a and MhAGO1 respectively.There are large numbers of pathogen and salicylic acid(SA)-related cis-acting elements on the promoter of MIR168a,and some cis-acting elements that respond to pathogen infection exist in MhAGO1 promoter as well.Promoter function analyses showed that B.dothidea infection and SA treatment can active the transiently expression of GUS reporter gene driven by MIR 168a promoter,while the MhAGO1 promoter driven GUS can only be activated by B.dothidea infection but not SA treatment.Thus,although both miR168 and MhAGO1 were induced at transcriptional level by the infection of B.dothidea,they may be regulated via different mechanisms.2.Using Agrobacterium-mediated transformation approach,MIR168a was transient expressed in M.hupehensis leaves to overexpress miR168,and STTM168 and MhAGO1 RNAi vectors to silence endogenous miR168 and MhAGO1,respectively.Confirmed by Northern blot assay,the expression of miR168 and MhAGO1 could effectively change in the transformed leaves.Overexpression of miR168 and silencing of endogenous MhAGO1 significantly delayed the development of necrotic lesions caused by B.dothidea infection,whereas the inhibition of endogenous miR168 led to even more severe symptom.The biomass of the pathogen and the host was detected using RT-qPCR,to calculate the pathogen colonization coefficient.Compared with the control plants,the colonization coefficient of miR168 overexpressed and MhAGO1 RNAi leaves was lower,while the colonization of pathogen in STTM168 leaves was fastest.Overexpression of miR168 and silencing of endogenous MhAGO1 can inhibit H2O2 accumulation induced by B.dothidea,relieve cell death and disintegration caused by active oxygen burst,constitutively up-regulate the expression of several PR genes and PAL,and enhanced the sensitivity of PR1 expression to B.dothidea and SA treatment induction.The inhibition of endogenous miR168 led to the opposite result.Therefore,miR168 positively regulates the resistance of M.hupehensis to B.dothidea.By inhibiting the active oxygen burst and activating the SA-mediated defense response,miR168 not only delays the host disease progression,but also inhibits the growth and colonization of the pathogen.MhAGO1,as a negative regulator,plays the exact opposite role.3.In response to B.dothidea infection,miR137 was induced to upregulate in both resistant M.hupehensis and M.domestica 'Fuji' with varying degrees.Precursors of miR137 were isolated from M.hupehensis and M.domestica 'Fuji' respectively,which could be processed into 22nt mature miR137 upon being ectopically expressed in N.benthamiana.ERF14,the target gene of miR137,was cloned from these two apple germplasms.Although several different bases were found in ORFs of MhERF14 and MdERF14,they encoded an identical amino acid sequence.The 5' RLM RACE assay indicated that the major cleavage of ERF14 in both M.hupehensis and M.domestica 'Fuji' occurred between the 11th and 12th bases of the miR137 target site.Co-expressed miR137 inhibited the expression of wild type MhERF14 and MdERF14 in N.benthamiana,but not that of ERF14 with mutated target sites.The regulation of ERF14 by miR137 relied on base pairing within the binding site,and the base difference outside the complementary site did not affect the cleavage efficiency of miR137.The expression of ERF1 4 was induced by B.dothidea at the early stage of the infection,and this induction was partly eliminated by increased miR137 accumulation later.4.ERF14 gene encoded a protein that contains a conserved AP2/ERF DNA binding domain,which is a nuclear located ERF transcription factor.Demonstrated by yeast self-activation assay,ERF 14 protein is a transcriptional activator,and the 18 acidic amino acid residues at the C-terminus may be responsible for its activity.The dual binding activity of ERF 14 to GCC-box and DRE/CRT sequence was confirmed in vitro and in vivo by EMSA and yeast one-hybridization analysis respectively.Overexpression of ERF14 induced the expression of several PR genes constitutively in both M.hupehensis and M.domestica 'Fuji',whereas silencing of endogenous ERF14 had the opposite effect.Taking advantage of TRV-VAGE,wild type or mutated ERF14 was ectopically expressed in N.benthamiana,which induced the expression of several N.benthamiana disease-related genes and enhanced the plant resistance to B.cinerea.Co-expressed miR137 inhibited the accumulation of exogenous wild type ERF14 in N.benthamiana,resulting in partial elimination of the increased B.cinerea resistance;while the expression of mutated ERF14 were not affected.Therefore,miR137 involved in the resistance of N.benthamiana to B.cinerea through inhibiting the ectopic expression of exogenous ERF14.