SiRomics for universal diagnostics of plant viral disease and virus diversity studies

Abstract

Traditional methods of viral diagnostics using specific antibodies and PCR often fail to identify a viral pathogen. In our EU Marie-Curie IDP bridges project, we used an alternative novel approach called siRomics which allows not only to detect the virus but also to de novo reconstruct a complete consensus master genome in the viral quasispecies population. The main plant antiviral defense system is based on RNA silencing mediated by small RNAs. In plants infected with DNA and RNA viruses, host Dicer enzymes generate 21-24 nucleotide (nt) viral small interfering RNAs (siRNAs) that restrict virus replication and systemic spread. Growing evidence indicates that viral siRNAs are derived from the entire genome sequence of RNA and DNA viruses and accumulate at high levels. Hence it appears feasible to reconstruct a complete viral genome simply from viral siRNA species. Current bioinformatics algorithms enable de novo assembly of genomes and transcriptomes from short sequencing reads. In the past years, the siRomics pipeline, developed by Seguin et al. (2014b) in model plants, was further applied in crop plants (Seguin et al. 2014b, 2016, Rajeswaran et al. 2014a, 2014b, Fuentes et al. 2016). Thus, our siRomics approach has the potential for universal diagnostics of plant virus disease and de novo reconstruction of viral genomes in mixed infections. In this study we applied siRomics for virus detection and virome reconstruction in several case studies of economically-important viral diseases in Switzerland. In naturally-infected Solanum tuberosum (potato), one case study revealed a virome comprising Potato virus Y (genus Potyvirus) and Potato virus X (genus Potexvirus), which was reconstructed by de novo assembling separate genome-size sRNA contigs. Another case study revealed a virome comprising NTN and O strains of Potato virus Y, whose sRNAs assembled in chimeric contigs which could be disentangled on the basis of reference genome sequences. Both viromes were stable in vegetative potato progeny. In a cross-protection trial of Solanum lycopersicum (tomato), the supposedly protective mild strain CH2 of Pepino mosaic virus (Potexvirus) was tested for protection against the strain LP of the same virus. Reciprocal mechanical inoculations eventually resulted in co-infection of all individual plants with CH2 and LP strains, reconstructed as separate sRNA contigs. LP invasions into CH2-preinfected plants and vice versa were accompanied by alterations of consensus genome sequences in viral quasispecies, indicating a potential risk of cross-protection measures. Additionally, the study also revealed, by reconstruction from sRNAs, the presence of the mechanically non- transmissible Southern tomato virus (Amalgavirus) in some plants. Our in-depth analysis of sRNA sizes, 5'-nucleotide frequencies and hotspot maps revealed similarities in sRNA- generating mechanisms in potato and tomato, differential silencing responses to virome components and potential for sRNA-directed cross-targeting between viral strains which could not, however, prevent the formation of stable viromes. Furthermore, by siRomics we characterized the virome present in cultivated and non-cultivated perennial plants including grapevine, cherry, fig, privet and larch. As expected, grapevine samples showed a complex virome, including viroids, in particular Grapevine Fanleaf virus, Grapevine virus A, Grapevine leafroll associated virus, Yellow speckle viroid 1, Yellow speckle viroid 2, Hopstunt viroid and Australian grapevine viroid. In cherry trees affected by little cherry disease, we confirmed that the presence of two Little cherry virus (1 and 2, respectively) in one of the samples, induces more severe symptoms compared with the sample where only Little cherry virus 1 was present. In a fig tree exhibiting virus-like symptoms coming from a private garden, new isolates of Fig mosaic virus and Fig Badnavirus-1 were identified and reconstructed. In the forest bush plant privet (Ligustrum vulgare) showing yellow mosaic disease, a novel virus distantly related to Barley yellow strip virus and Lychnis ringspot virus was identified, fully reconstructed and named Ligustrum mosaic virus. Our work combined multi-disciplinary approaches ranging from advanced molecular methods of next generation sequencing to sophisticated bioinformatics algorithms for virus genome reconstruction. The results of our study are informative for further understanding the mechanisms of RNA silencing-based antiviral defense, which would contribute to basic research in the field of plant-pathogen interaction, and for developing novel strategies of virus control, which could potentially be implemented in the future in Swiss agriculture though our recommendations to the policy makers. In modern agriculture, horticulture and (bio-) farming, it becomes critical to assess the risk of emerging plant infections and to control the spread of plant viral diseases.