The Mechanisms Of Tomato Mir1916 And Mir396 In Plant Response To Stresses

microRNAs(miRNAs)are small non-coding RNA molecules,with length between 18 to 25 nucleotides.They negatively regulate the expression of target genes,and are important regulatory switches in plant growth and stress responses.The miR1916 and miR396 family is one of non-conserved and conserved microRNA families in plants,respectively.However,the molecular mechanisms of miR1916 and miR396 involved in biotic stresses responses are largely unknown.Especially,there are few studies on miR1916 and miR396 in the interaction between tomato(Solanum lycopersicum)and Phytophthora infestans or Botrytis cinerea,and the function of them respond to environmental stresses also has not been widespread concerned in Solanaceae.Based on the above problems,we systematically revealed the pleiotropic regulatory mechanism of miR1916 and miR396 in tomato stress resistance through genetic engineering strategy,secondary metabolism analysis and gene function identification.The main findings are as follows:(1)The expression of miR1916 in S.lycopersicum was down-regulated after infection with P.infestans or B.cinerea by qRT-PCR.The potential target genes STR-2,UGT,MYB12,CC-NBS-LRR,CC-NBS and HTPG were associated with respond to two kinds of pathogens infection.The cleavage sites of miR1916 on STR-2,UGT,MYB12 and CC-NBS-LRR were verified using PPM-RACE and 5'-RACE.Tomato plants that overexpressed miR1916 displayed significantly enhanced susceptibility to P.infestans and B.cinerea infection,and the tendency to produce reactive oxygen species(ROS)when subjected to pathogen-related biotic stress.Silencing of miR1916 using STTM and miRNAs strategies made the tomato plants more tolerant to adverse conditions.Additionally,Analyses of transgenic S.lycopersicum indicated that lower miR1916 expression could up-regulate the expression of STR-2,UGTs and MYB12,as manifested by accumulation of enhanced a-tomatine and anthocyanins accumulations.Overexpression of STR-2 in S.lycopersicum increased a-tomatine content and improved the adaptation of tomato to biotic stress,while ectopic expression of miR1916/STR-2 significantly changes tobacco tolerance to B.cinerea.Taken together,we demonstrated that miR1916 might regulate the expression of STR-2,UGT and MYB12 in tomato plant,conferring sensitivity to biotic stress via modulating a-tomatine or anthocyanins content.(2)The expression of miR396 in S.lycopersicum was down-regulated after infection with P.infestans or B.cinerea by qRT-PCR.The miR396 was targeted GRF1 and SAMT;miR396 was targeted GH and NBS-LRR in tomato.Those potential target genes were predicted by bioinformatics,which may be associated with respond to two kinds of pathogens infection.The expression of miR396 was upregulation in tomato reduced the resistance of tomato to P.infestans and B.xinerea infection,and and increased the production of ROS when infected by pathogens.Analysis of the target genes revealed that miR396 inhibited the expression of GRF1,SAMT,GH and NBS-LRR.This eventually led to suppression of the expression of disease-related genes TGA1,TGA2 and PR1 and JA-dependent PI I and ?,but enhanced endogenous SA content and NPR1 expression.Negative regulation of target genes and their downstream gene expression by miR396 through SA or JA signaling pathways is the key to the tomato response to biotic stress.(3)Abiotic stress experiments showed that miR1916 was a negative regulator in tomato and tobacco resistance to drought.Heterologous overexpression and silencing miR1916 regulat the expression of target genes HDAC and STR in tobacco,as well as affect proline,soluble sugar and accumulation of ROS after drought treatment.(4)Salt,drought and cold stress tolerance analysis confirmed that overexpression of miR396 inhibited GRF1,GRF3y GRF7 and GRF8,triggering the enhanced ability of osmotic regulation and reducing ROS accumulation.Therefore,miR396 as positive regulator increases tobacco resistance,which has comprehensive regulation of a variety of stress.In summary,this study found that tomato infected with P.infestans and B.cinerea showed reduced expression of miR1916 and miR396 compared to WT.Overexpression and silencing of miR1916 could regulate the expression of its target genes STR-2,UGT,MYB12,CC-NBS-LRR,CC-NBS and HTPG,and thus affect tomato resistance to pathogenic infections.miR396 as negative regulator participated in tomato defense pathogens by inhibiting defense-related genes associated with SA and JA signaling pathways.On the other hand,heterologous expression of miR1916 and miR396 revealed that miR1916 as negative regulator is involved in drought stress in tobacco,whereas miR396 a positive regulator is involved in salt,drought and cold stresses in tobacco.