Plant Virus Discovery Using Next-Generation Sequencing Techniques

Losses caused by emerging plant diseases are often enormous and usually of great concern because of its effect on food security and sustenance.Diseases of viral origin are especially devastating because of the diversity of plant viruses and ability to adapt to wide host range.In order to combat plant disease outbreaks,strategies employed are either to stop the disease from spreading or to prevent the emergence of the disease.This can only be achieved through early detection of the disease in the host or the disease causing agent by adopting rapid,sensitive and accurate diagnostic method for the identification and characterization of the disease causing agent as well as other novel agents.The identification and characterization methods usually employed are physical,biological,serological and molecular methods which depends on prior knowledge of the etiological agent.However,these methods are not effective in identifying and characterizing new viruses for which primers or probes are not available.Next-generation sequencing techniques had become the method of choice for viral diagnostics due to its rapidity,accuracy,sensitivity and most importantly ability to determine etiological agents without prior knowledge of the agents.This study therefore aimed at using the fast advancing next-generation sequencing?NGS?techniques for rapid and accurate discovery and characterization of plant viruses.This method uses derivate of RNA silencing-based antiviral defense to identify viruses from virus-infected plants.When virus infects plant,the host Dicer-like?DCL?enzymes cleaves double stranded RNAs?dsRNAs?of the virus into 21-24 nucleotide?nt?short interfering RNAs?siRNAs?which then attaches to Argonaute?AGO?proteins and guides the formed RNA-induce silencing complexes?RISCs?to target complementary viral RNA for degradation.The cleaved 21-24 nt siRNAs from the RNA silencing mechanism can be reconstructed to get the full genome sequence of the virus causing infection in the plant.In this study,a bioinformatics pipeline was developed for rapid and accurate analysis of viral siRNAs from diseased plants for the detection and characterization of all disease causing yiruses without prior knowledge of the viruses.Leave samples from Tobacco,Maize and Strawberry were screened for plant viruses using next-generation sequencing techniques and the developed bioinformatics pipeline.Plant viruses infecting Tobacco were surveyed from 104 samples of Tobacco collected across Anhui Province of China.Nine different sRNA libraries were prepared from the samples and analyzed by the custom-made bioinformatics pipeline.Twenty two isolates from eight different virus species including Cucumber mosaic virus,Potato virus Y,Tobacco mosaic virus,Tobacco vein banding mosaic virus,Pepper mottle virus,Brassica yellow virus,Chilli venial mottle virus,Broad bean wilt virus 2 were identified in the Tobacco samples across the survey area.Phylogenetic and recombination analysis of the 22 virus isolates shows that the viruses are distantly related to known viruses.Also,in maize plant,a new member of the genus Totivirus was identified in the maize samples.Based on its genomic arrangement,phylogenetic relationships with other totiviruses,low sequence similarity to other viruses below 50%species demarcation for either coat protein?CP?or RNA-dependent RNA Polymerase?RdRP?genes of totiviruses,the virus was confirmed as a new member of the genus.Though a few plant-associated viruses have been identified in the genus Totivirus mostly dominated by fungi host,the identification of Maize-associated totivirus Anhui?MATV-Ah?and other few plant viruses in the genus suggests that viruses originating from plants might be emerging within this genus.Likewise in strawberry,a novel virus of the genus Potexvirus affecting strawberry was identified.The virus has only 64%nucleotide sequence similarity with SMYEV MY-18?D12517?and highest protein sequence similarity of 73%with SMYEV?NP₆20642?,which is below the 80%specie demarcation threshold for potexviruses.Therefore,it is provisionally named strawberry mild yellow edge-associated virus-DSH15?SMYEaV-DSH15?.This study using NGS techniques has revealed the effectiveness of the method in identifying multiple virus infections within a short time which can be harnessed for fast and accurate viral diagnosis.Also,our knowledge of plant virus diversity is limited and plants may be harboring viruses more than ever imagined resulting from coinfection.The interplay between viruses and different hosts must be closely monitored to prevent the emergence of new epidemic viruses.