| Apple Alternaria leaf spot is one of the most devastating diseases in apple,and it happens in lots of areas of China,especially in Bohai bay and the Yellow River area.Apple Alternaria Blotch is a kind of Alternaria alternaria f.sp mali(ALT1),which mainly happened in apple leaves,fruit and new branch.This will affect tree body and reduce fruit quality and yield.Nowadays,the most common method of controlling disease is chemical control.However,this will cause environmental pollution and endanger human health.The most effective ways to solve the problem is to breed resistant apple species by crossbreeding and transformation.According to the result by many years of investigation,we found that different apple species perform variety resistant to ALT1,species like Golden Delicious,Delicious,Straking Delicious,Ralls Janet and so on showed weak resistant,but species like Hanfu,Jiguan,Hongxia and so on showed strong resistant.However,the molecular mechanism is unclear.In recently,people have showed that miRNA is a significant regulator in plant defend pathway,hence,we use miRNA next-generation sequencing to detect miRNAs in a weak resistant cultivar,and find some miRNAs which are induced by ALT1,after a deep research,we obtained some molecular mechanism among them,and find a SNP which was closely related to apple resistance.These result provide a guidance in apple resistant breeding.The main result is as follow:1.We performed next-generation sequencing of small RNAs(sRNAs)and globally profiled miRNAs in the apple cultivar Golden Delicious(GD)infected or not with the apple leaf spot fungus Alternaria alternaria f.sp.mali(ALT 1),and identified 60 miRNAs that exhibited more than a 2-fold upregulation upon ALT1 infection.We identified a pair of miRNAs,Md-miR156ab(miRBase MIMAT0025980)and Md-miR395(miRBase MIMAT0025894),which target two WRKY transcription factors involved in the defense response against fungal pathogens.2.We identified a novel miRNA in next-generation sequencing,Md-miRLn12,which was most highly expressed in inoculated Alternaria leaf spot(ALT1)apple leaves than in the control.Md-miRLn12 targets MdRNL-1,MdRNL-2,MdRPW8-3 and MdRPW8-4 four Resistance(R)genes verified by 5'RACE.We found Md-miRLn12,Md-miRLn12 precursor and four target genes is expressed differentially in the ALT 1-susceptible(GD)versus ALT1-resistant(HF)apple cultivar,Md-miRLn12,Md-MIRLn12-277 was highly induced by ALT1 inoculation in GD but not in HF,and the target genes opposite.Thus,silencing of Md-miRLn12 and overexpressing MdRNL1 and MdRNL2 increases ALT1 resistance in the susceptible apple cultivar GD;overexpressing of Md-miRLn12 and silencing MdRNL1 and MdRNL2 decreases ALT1 resistance in the resistant apple cultivar HF.These results indicate that MdRNL-1 and MdRNL-2 are target genes of Md-miRLn12 and are responsive to ALT1 infection in apple.3.To explain why Md-miRLn12 is expressed differentially in the ALT 1-susceptible(GD)versus ALT 1-resistant(HF)apple cultivar,we cloned the promoter sequence of Md-MIRLn12-277(1711 bp),revealing the presence of three SNPs at-1506 bp,-1186 bp,and-175 bp in the promoters of Md-MIRLn12-277 isolated from GD(pMd-MIRLn12-277-GD)and HF(pMd-MIRLn12-277-HF).And we have verified motif b is critical for the activity of the Md-MIRLn12-277 promoter and that a G-to-T mutation destroys the activity of the Md-MIRLn12-277 promoter.Additional three cross populations from Han Fu × Yue Shuai?Golden Delicious × Nagafu 2 and Yue Guan × Nagafu 2 were performed and analyzed the SNP in pMd-MIRLn12-277 is bound up with Alternaria leaf spot resistance.We combination of EMSA and liquid chromatography-mass spectrometry(LC-MS)identification and we have got MdWHy is unable to bind to pMd-MIRLn12-277-HF and Md-MIRLn12-277 transcription is suppressed,resulting in low levels of Md-miRLn12,high levels of MdRNL-1 and MdRNL-2 expression,and strong resistance to Alternaria leaf spot.4.One of Md-miRLn12 target genes,MdRNL2 which is a coiled-coil(CCR)-NB-LRR protein induced in an leaf blotch-resistant cultivar of apple('Hanfu',HF).Protein-protein interaction and liquid chromatography-mass spectrometry analyses revealed that MdNL1(an NB-LRR protein),MdPR10-1(a PR10 protein)and MdPR10-2(a PR10 protein)potentially interacts with MdRNL2.Yeast two-hybrid(Y2H)and bimolecular fluorescence complementation assays(BiFC)indicated that MdRNL2 physically interacts with MdNL1.We suggested a mechanism for resistance to ALT1 in apple in which pairs of CCR-NB-LRR(MdRNL2)and NB-LRR(MdNL1)proteins interact,leading to upregulate the expression of MdPR10-1 and MdPR10-2,and then MdPR10-1,MdPR10-2 binding to MdRNL2,MdNL1 take part in the defense response to ALT1.In conclusion,we have obtained a novel miRNA,Md-miRLn12.The expression of Md-miRLn12 is different in resistant cultivar(HF)and susceptible cultivar(GD),the resistance difference is determined by the SNP of pMd-MIRLn12-277.MdWHy is unable to bind to pMd-MIRLn12-277-HF and Md-MIRLn12-277 transcription is suppressed,resulting in low levels of Md-miRLn12,high levels of MdRNL-1 and MdRNL-2 expression,and a target gene of Md-miRLn12,MdRNL2 is paired with MdNLl upregulated the the expression of MdPR10-1 and MdPR10-2,then MdPR10-1,MdPR10-2 binding to MdRNL2,MdNLl in the defense response to ALT1 in resistant cultivar(HF),and strong resistance to Alternaria leaf spot. |
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