Characterization of virus-derived small interfering RNAs and complete sequence of a strain of Soybean mosaic virus infected Pinellia pedatisecta

【Objective】To illuminate the virus silencing mechanism in Pinellia pedatisecta，performed an analysis of the sequence characteristics of virus-derived small interfering RNA（vsiRNA）in a Soybean mosaic virus（SMV）infecting P. pedatisecta.【Method】In this study，a plant of P. pedatisecta，which displayed typical virus infection symptoms， was found in Guangxi. The total RNA of the chlorotic and mottled leaves was extracted using the TRIzol method. A small RNA deep sequencing was employed to identify the vsiRNA. The full-length genome sequence of the virus was obtained by rapid amplification of cDNA ends（RACE）and segmented cloning. The phylogenetic relationships of the assembled virus sequence was analyzed using MEGA 7.0 software. The characterization of vsiRNA was performed using the cloned genome as a reference sequence.【Result】In total，4851249 high-quality reads，in which the relatively rich reads were 21， 22，and 24 nt with the percentage of 33.4%，13.7% and 11.3%，respectively，were obtained by small RNA deep sequencing. According to the small RNA sequencing results，the full-length genome sequence cloning of the virus was carried out. The virus genome was found to be 9735 nt in length，encoding a polyprotein that contained 3105 amino acids. Sequence analysis showed that the nucleotide sequence of the virus isolate genome shared 86.93% identical identity with the SMV HZ1 isolate. This virus isolate was named SMV NN tentatively. The phylogenetic relationship anlysis revealed that the SMV NN shared the closest genetic relation with the SMV HZ1 isolated from P. ternate. The small RNAs of the virus were mapped to the genome of SMV NN for vsiRNA characterization. The results showed that the vsiRNA with lengths of 21 nt and 22 nt was in high abundance；both positive and negative virus vsiRNA could cover the entire virus genome and had the strongest hot spots in the HC-Pro and P3 protein-coding regions，respectively.【Conclusion】P. pedatisecta mainly uses dicer like ribonuclease 4（DCL4）and Argonaute1（AGO1）to cleave the HC-Pro and P3 domain of SMV，and mainly produces vsiRNA with the lengths of 21 nt and 22 nt，resulting in the inhibition of SMV replication in plants.