Expression Profiling And Regulating Function Of MicroRNA In Poplar Upon Infection With The Foliar Rust Melampsora Larici-populina

Foliar rust disease is a serious disease occurs in poplar,induced through infections with the rust fungus Melampsora spp.,which may give rise to worldwide damage to the production of poplar.Mi RNAs are small non-coding RNAs that regulate the gene expression of target m RNAs involved in plant growth,development,and abiotic stress and pathogen responses.A majority of miRNAs in poplar were identified as it is a useful forest species model for genetic and ecological research.Previous studies have reported miRNAs in Populus that respond to abiotic stresses,such as cold,heat,drought,flooding,high salt and mechanical stress.However,few studies have focused on miRNA expression profiling in the response of poplars to biotic stress,especially the stress of foliar rust fungal infection.Here,we identified the miRNAs profile of Populus after inoculation with Melampsora larici-populina and predicted the target gene using high-throughput sequencing and bioinformatics analysis.The targets of disease resistance-related miRNA were validated by RLM-5'RACE,and full length of one of the target gene was amplified.Finally,we constructed the mi R482 overexpression vector and transfered it to poplar through the Agrobacterium tumefaciens.The results of the present study help elucidate the regulatory mechanisms of miRNA in Populus upon infection with pathogen.The main results were as followed:1.Expression profiling of miRNAs under foliar rust stressUsing Solexa sequencing,we constructed three libraries from uninoculated P.szechuanica leaves?control?and leaves inoculated with avirulent?Sb052?and virulent?Th053?isolates of M.larici-populina.A total of 90 known miRNAs were obtained in the Sb052,Th053 and control libraries,belonging to 42 families from 313 genomic loci.The dominant miRNA families were mi R156,mi R166,mi R167,mi R168,and most of these miRNAs were largely conserved in various plant species.378 novel miRNAs from 508 genome loci were identified using Mireap software.The highly expressed novel miRNAs were novel_mir₇5,novel_mir₁44,novel_mir₉1.To confirm the temporal dynamic expression of miRNAs involved in pathogen stress,we further analysed 10 miRNAs in P.szechuanica infected with M.larici-populina at 0,12,24,48,96,and 144 hpi using q RT-PCR.The results showed that the miRNA expression levels dramatically changed during the pathogenesis of foliar rust infection,and there were differences in different rust isolates.Most miRNAs in P.szechuanica infected with the avirulent isolate Sb052 and virulent isolate Th053 were up-regulated and down-regulated at 48 hpi,respectively.According to these results,we speculate that miRNAs could possibly regulate the expression of certain genes,and through the changing expression of miRNAs,could play vital roles in regulating disease-resistance in Populus infected with rust.2.Target prediction and functional annotationsA large number of targets were predicted for most miRNAs using the program developed by BGI.The results of KEGG pathway analysis revealed that the pathway that accounted for the largest percentage of the total targets was metabolic pathways,followed by plant-pathogen interaction as for the known miRNAs.However,regarding novel miRNAs,the pathway with the highest proportion of target genes was plant-pathogen interaction.Metabolic pathways took second place.We can see that plant-pathogen interaction and metabolic pathways were significantly over-represented in poplar upon rust fungal inoculation.The analysis of the differential expression of miRNAs in the inoculated and control libraries showed that 38 known and 92 novel differentially expressed miRNAs from the three libraries that might play important roles in response to pathogen stress.The expressions levels of the same miRNAs were significantly different,and the expression levels of poplar miRNAs were more suppressed during rust infection.Among the differentially expressed miRNAs,7 known and 20 novel miRNAs were relevant to the rust fungus infection,and according to KEGG pathway analysis,these miRNAs primarily regulate genes encoding disease-resistance proteins,serine/threonine protein kinases,transcription factors,and related proteins.3.Validation of predicted targets and the regulatory mechanism of mi R482 in plant response to pathogen stressWe validated the target gene RPM1 and RPS2 and their cleavage site through RLM-5'RACE.The results showed that mi R482 was complementary with the targets in specific site.RPM1 was cleaved at one specific site.However,it was capable of cleaving the RPS2 gene at two different sites.RPM1 and RPS2 were homologous to NBS-LRR class gene in P.trichocarpa.We amplified the full length of the RPS2 and conducted bioinformatics analysis.The full-length c DNA sequences were 3012 bp and 2256 bp with open reading frames,encoded 751 amino acids,contained typical NB-ARC and LRR domain.The encoded protein of full-length gene was conducted a Protein BLAST in NCBI.The results of sequence alignment revealed that the similarity of the target protein was 87% compared with putative disease resistance gene NBS-LRR family protein in P.trichocarpa.Therefore,the target gene maybe typical NBS-LRR disease resistance gene.Quantitative real time-PCR was used to analyse the expression of mi R482 and its target in poplars inoculated with avirulent and virulent isolates of M.larici-populina for different times.The expression level of mi R482 and target genes RPS2 present dynamic change over time.When poplar was under the infection of avirulent isolate Sb052,the expression level of mi R482 was higher in the early stage,but the expression of RPS2 was lower.In the later stage of infection,the expression level of mi R482 was decrease,but RPS2 was increase slightly.Under the stress of virulent strains Th053,the expression of mi R482 maintained in a low level in the early stage,and a dramatic rise in the later stage.However,the expression of RPS2 was decrease in the later stage of infection.The results showed that mi R482 maybe regulated negatively the expression of target gene in the later stage of inoculation?96 hpi¹44 hpi?and exhibited different temporal dynamics in incompatible and compatible libraries.4.Overpression vector construction and P.szechuanica transformationThe precursor sequence of mi R482 a was obtained by PCR and introduced it to plant expression vector p BI121.The mi R482-p BI121 vector was transferred to A.tumefaciens strain GV3101 using the freeze-thaw method.We successfully constructed the mi R482 a overexpression vector,used for poplar genetic transformation.The optimal concentration hormone combination in somaclone callus induction and rooting medium were MS+1.0 mg/L 6-BA+0.2 mg/L ?-NAA and1/2MS+0.02 mg/L ?-NAA,respectively.The leave of P.szechuanica was used to culture aseptic seedling as explant in MS medium with above concentration of hormone and transplant successfully.In the process of genetic transformation,the concentration of kanamycin in callus induction and rooting medium were 10 mg/L and 20 mg/L,respectively.It can inhibit agrobacterium pollution,effectively,and the leaf disks can differentiate into seedlings when the concentration of carbenicillin was 600 mg/L in selection medium.These provided experience for the further function validation of mi R482 regulate the disease resistance gene in P.szechuanica.