Submitted to: PLoS One
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/13/2012
Publication Date: 5/18/2012
Citation: Li, R., Shan, G., Hernamdez, A.G., Wechter, W.P., Fei, Z., Ling, K. 2012. Deep sequencing of small RNAs in tomato for virus and viroid identification and strain differentiation. PLoS One. 7(5):e37127. Interpretive Summary: Effective disease management in plants depends on timely and accurate pathogen identification. Most virus detection methods depend on prior knowledge of an antibody or nucleotide sequence in a virus genome. The advancement of next generation sequencing technologies has provided an exciting alternative for total pathogen characterization without prior knowledge. We describe here a powerful procedure by incorporating the concept of small RNA subtraction and assembly to efficiently identify known or unknown viruses and viroids in tomato. This technology was able to effectively distinguish sequences between a virus (Pepino mosaic virus) and a viroid (Potato spindle tuber viroid) in mixed infection and to achieve a strain-specific differentiation of virus isolates. By applying this strategy, we were able to identify a novel potyvirus without prior knowledge and then obtain its complete genome sequence for the first time. Furthermore, the authenticity of the siRNA generated viral genome sequences were validated through sequencing of viral RNAs. As more and more host genome sequences becoming available, the small RNA subtraction and assembly strategy will likely have greater applications in virus identification in plants, animals and other organisms.
Technical Abstract: Small RNAs (sRNA), including microRNAs (miRNA) and small interfering RNAs (siRNA), are produced abundantly in plants and animals and function in regulating gene expression or in defense against virus or viroid infection. Analysis of siRNA profiles upon virus infection in plant may allow for virus identification, strain differentiation, and de novo assembly of virus genomes. In the present study, four suspected virus-infected tomato samples collected in the U.S. and Mexico were used for sRNA library construction and deep sequencing. Each library generated between 5-7 million sRNA reads, of which more than 90% were from the tomato genome. Upon in-silico subtraction of the tomato sRNAs, the remaining highly enriched, virus-like siRNA pools were assembled with or without reference virus or viroid genomes. A complete viroid genome was assembled for Potato spindle tuber viroid (PSTVd) using siRNA alone. In addition, a near complete virus genome (98%) also was assembled for Pepino mosaic virus (PepMV). A common mixed infection of two strains of PepMV (EU and US1), which shared 82% of genome nucleotide sequence identity, also could be differentially assembled in their respective genomes. Using de novo assembly, a novel potyvirus with less than 60% overall genome nucleotide sequence identity to other known viruses was discovered for the first time and its full genome sequence obtained. Taken together, these data suggest that the sRNA deep sequencing technology will likely become an efficient and powerful generic tool for virus identification in plants and animals.