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

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.