Functional Studies On Poplar MiR472a And MiR6445 In Cytspora Chrysosperma Infection And Drought Stress Response

Populus is an important silvicultural tree species in north China,which has high economic and ecological value.However,poplar growth and industrialization are limited by its sensitivity to water deficiency and pathogen infection.Exploring the response mechanism of poplar to disease and drought stress and identifying the key regulatory genes have important theoretical significance and application prospects for rapid breeding of new poplar varieties with improved resistance through genetic engineer-ing technology.In this study,based on the analysis of RNA-seq data from drought treatment of poplar,microRNA472 and poplar-specific microRNA6445,which were up-regulated in response to drought stress,were overexpressed or silencd in poplar(P.alba × P.glandulosa,84K),respectively,and then explore their function in response to drought stress and pathogen infection.This study provided genetic resources and theoretical basis for improving poplar resistance through genetic engineering.Specific research contents are as follows:1.In order to study the role of miR472a in plant disease and drought resistance,the overexpression and silencing vectors of miR472a were constructed in this study,and Agrobacterium-mediated trans-formation was used to overexpress and silence miR472a in P.alba × P.glandulosa,and the overexpres-sion lines miR472aOE and silencing lines STTM472a were obtained.The target gene of miR472a,NBL1,was identified and verified by the degradation data,5 'RACE and tobacco co-expression.At the same time,qRT-PCR showed that NBL1,NBL2,NBL3 and NBL4 were down-regulated in miR472aOE lines,and up-regulated in STTM472a lines,which also indicated that they may be targeted by miR472a.In addition,we found that miR472a triggered the production of phasiRNAs(SIR14,SIR16,SIR17,SIR21 and SIR25)via the result of qRT-PCR.The generated phasiRNAs(such as SIR16,SIR21 and SIR25)target other NBS-LRR genes(such as NBL2),thus making NBS-LRR at an extremely low ex-pression level under normal growth conditions.2.In this study,the wild type,over-expressed lines miR472aOE and silent lines STTM472a were all treated with flg22.Colletotrichum gloeosporioides and Cytospora chrysosperma,seperately.The results showed that,after treated with flg22,the miR472aOE lines had the lowest H2O2 burst and callose deposition compared with wild type and STTM472a lines;after treated with C.gloeosporioides,the expression of miR472a decreased while that of NBL1-NBL4 increased in wild type leaves,and the le-sion area of miR472aOE lines was significantly larger than that of wild type and STTM472a lines;when treated with C.chrysosperma,the miR472aOE lines had the smallest lesion area,and the JA/ET maker gene ERF1 was quickly up-regulated in the miR472aOE lines.These results indicated that miR472a negatively regulates the immune response triggered by flg22 and C.gloeosporioides,while enhances plant resistance to C.chrysosperma.3.MiR472a was up-regulated in leaves(especially young leaves)and down-regulated in roots of P.euphratica after drought treatment.In order to explore the role of miR472a responding to drought stress in plant,drought treatments were carried out in different transgenic poplar lines.The result showed that miR472aOE lines wilted on the fourth day of drought treatment,while STTM472a lines and wild type wilted on the sixth day,which indicated that miR472a negatively regulated the drought stress response in populus.However,under normal growth conditions,the miR472aOE lines showed larger leaf eara and biomass than the wild type and STTM472a lines,suggesting that miR472a positively regulated poplar growth and development.To explore whether NBS-LRR genes is involved in the drought stress response,we selected the mutants of NBL1 and NBL2 homologous genes in Arabidopsis,nbll and nbl2,for germination rate under mannitol treatment.The results showed that under normal and mannitol treatment,the germination rate has no significant difference between the mutants and the wild type.At the same time,we found that a prodicted miR472a target gene,FBI,was down-regulated in miR472aOE lines and up-regulated in STTM472a lines,and tobacco co-expression indicated that it is a target gene of miR472a.The seed germination rate and root growth of the mutant in mannitol treatment is significantly lower than that of the wild type,and fb1 exhibted wilting more quickly under natural drought conditions,these results indicated that FBI positively regulated plant drought stress response.Compared with wild type,the mutant has the larger root enlongation,leaf area and biomass under nor-mal conditions,indicating that FBI negatively regulated plant growth and development.In summary,miR472a may positively regulate plant growth and development while negatively regulate drought stress response by targeting FBI.4.In order to study the role of poplar specific miR6445 on plant disease and drought resistance,in this study,the promoter sequence of MIR6445b was analyzed,and MBS,CGTCA,TGACG-motif and other cis-acting elements which related to drought stress and JA signal were present in this promoter sequence.Later,the silencing vector of miR6445 was constructed,and the transgenic silencing lines STTM6445 were obtained.The data of qRT-PCR indicated that the expression of miR6445 prodicted target gene(NAC112?NAC097?NAC152?NAC088)were up-regulated in the silencing lines(STTM6445).Then the wild type and STTM6445 lines were treated with drought stress.The results showed that the STTM6445 lines wilted on the fourth day after drought,while the wild type wilted on the sixth day,and the relative electrical conductivity rate was significantly higher than wild type on the sixth day.At last,the wild type and STTM6445 lines were treated with C.chrysosperma.The results showed that STTM6445 lines exhibited the larger lesion area.In summary,miR6445 may positively regulate plant drought tolerance and C.chrysosperma resistance.Taken together,miR472a negatively regulated drought resistance and C.gloeosporioides resistance by targeting the NBS-LRR genes and triggering phasiRNAs,while enhanced plant resistance to C.chrysosperma;on the other hand,miR472a negatively regulated plant drouhgt stress response while posetivly regulated plant growth and development by targeting the FBI gene.Secondly,miR6445 posi-tively regulated poplar drought resistance and C.chrysosperma defense by targeting NAC transcription factors.This study provides gene resources and theoretical basis for drought-resistant and dis-ease-resistant breeding of plants.