Activity Identification And Molecular Mechanism Of Tomato MiRNA To Inhibit The Infection Of Botrytis Cinerea By Cross-species RNAi

MiRNA is a type of single-stranded small-molecule RNA that does not encode proteins.Through complementary binding with target RNA,it induces gene silencing and participates in the regulation of eukaryotic growth and stress response.The study found that miRNA can spread between interacting organisms and induce gene silencing in each other.This mechanism is called cross-speices RNAi.As a common pathogen of plants,Botrytis cinerea has great damage to the growth of crops and plants,and the economic loss caused by it every year is even more inestimable.The cross-speices RNAi effect can make plant miRNAs become environmentally friendly fungicides and inhibit the infection of Botrytis cinerea.In order to identify the miRNA molecules that regulate the cross-species regulation of Botrytis cinerea in tomato,based on the miRNAs identified by the tomato in response to the infection of Botrytis cinerea,this paper first predicted target genes by ps RNAtarget and selected three tomato miRNAs that can target the gene encoding Botrytis cinerea : mi R827,mi R5300 and miRNAn14 were used to identify the antibacterial activity and study the mechanism.The mycelium of Botrytis cinerea treated with miRNA drugs was inoculated on tobacco leaves.After 3 days,compared with Botrytis cinerea without miRNA drug treatment,it was found that the Botrytis cinerea treated with miRNAn14 had the smallest disease spots on plants,indicating that miRNAn14 had the strongest bacteriostatic efficiency;mi R827 followed,mi R5300 had the largest plaque.No significant difference from the control,indicating that mi R5300 has basically no antibacterial activity.In addition,we also studied the relationship between the bacteriostatic efficiency of different drug concentrations,and the results showed that the miRNAn14 drug’s inhibitory effect on Botrytis cinerea was the most obvious when the working concentration was 20 μM.In addition,we also found that the inhibitory effect of double-stranded miRNAn14 is more obvious than that of single-stranded miRNAn14.Moreover,this inhibition does not have the host specificity of plants,that is,it shows the same results on tobacco leaves or tomato leaves.Further research found that miRNAn14 drug can inhibit the germination of Botrytis cinerea spores.Through degradation group sequencing,we studied two potential target genes in Botrytis cinerea genes,which are the genes Bcin03g02170.1 and Bcin10g01400.1,respectively.Real-time RT-PCR analysis showed that after miRNAn14 treatment,the expression level of Bcin03g02170.1 gene in Botrytis cinerea decreased significantly at all time points,but the expression level of another gene Bcin10g01400.1 did not decrease significantly.Therefore,we believe that miRNAn14 may inhibit the germination or infection of Botrytis cinerea spores mainly by targeting the target gene in Bcin03g02170.1.Finally,we analyze miRNA stability and environmental safety.We found that when the mi drug was placed at room temperature,after 4 days,the miRNA could not be detected by RT-PCR.In order to study whether miRNA drugs will affect the organism,we conducted a toxicity test.The results showed that miRNA drugs were not toxic to tadpoles and bees.Our findings provide new evidence for the co-evolution of pathogens and host plants,as well as the use of plant-derived miRNAs to control pathogens.