First report of Verbena latent virus infecting giant goldenrod in Belgium

peer reviewedGiant goldenrod (Solidago gigantea) was initially introduced from North America as an ornamental plant and is now considered highly invasive in Europe, negatively impacting native plant species. In summer 2023, a virome survey in Belgium collected asymptomatic goldenrod samples from 10 sites and detected Verbena latent virus (VeLV, Carlavirus, Betaflexiviridae) through high-throughput sequencing (HTS) techniques. VeLV was already detected in other plant species but its genome was not sequenced until the present work. The study also identified two novel plant-associated virus-like sequences and sequences of unknown mycoviruses in the samples. Further confirmation of VeLV infection was achieved through RT-PCR and Sanger analysis, with subsequent comparison to known VeLV sequences and the identification of one site infected in Belgium. This discovery underscores the importance of understanding VeLV biology and symptomatology, as VeLV presents a wide host range from in at least three plant families, the broad transmission profile of carlaviruses and their potential pathogenicity in single infection or mixed infection. To our knowledge, this is the first report of VeLV infecting S. gigantea worldwide. As giant goldenrod is widespread in Europe, it may serve as a significant asymptomatic reservoir for VeLV

Identification and Molecular Characterization of a Novel Hordeivirus Associated With Yellow Mosaic Disease of Privet (Ligustrum vulgare) in Europe.

Wild plants serve as a large reservoir of known and yet-unknown viruses and as a source of viral pathogens of cultivated plants. Yellow mosaic disease of forest shrub Ligustrum vulgare (privet) was recurrently observed in Europe for more than 100 years. Using a universal virus identification approach based on deep sequencing and de novo assembly of viral small interfering (si)RNAs we identified a causative agent of this disease in Switzerland and reconstructed its complete 3-segmented RNA genome. Notably, a short 3'-terminal common region (CR) attached to each segment via a ∼53-71 nucleotide poly(A) tract, as determined by RT-PCR sequencing, was initially identified as an orphan siRNA contig with conserved tRNA-like secondary structure. Phylogenomic analysis classified this virus as a novel member in the genus Hordeivirus of family Virgaviridae, which we named ligustrum mosaic virus (LigMV). Similar to other hordeiviruses, LigMV formed rod-shape virions (visualized by electron microscopy), was transmitted through seeds and could also be mechanically transmitted to herbaceous hosts Chenopodium quinoa and Nicotiana benthamiana. Blot hybridization analysis identified genomic and subgenomic RNAs, sharing the 3'-CR and likely serving as monocistronic mRNAs for seven evolutionarily-conserved viral proteins including two subunits of viral RNA-dependent RNA polymerase, coat protein, triple gene block proteins mediating viral movement and cysteine-rich suppressor of RNA silencing. Analysis of size, polarity, and hotspot profiles of viral siRNAs suggested that they are produced by the plant antiviral Dicer-like (DCL) proteins DCL2 and DCL4 processing double-stranded intermediates of genomic RNA replication. Whole genome sequencing of French and Austrian isolates of LigMV revealed its genetic stability over a wide geographic range (>99% nucleotide identity to Swiss isolates and each other), suggesting its persistence and spread in Europe via seed dispersal

What can viral metagenomics bring to our understanding of the diversity and ecology of plant viruses in agro ecological landscapes ?

peer reviewedHigh throughput sequencing (HTS) gave access for the first time to the viral metagenome or virome, by allowing to characterize, without a priori, all or nearly all viruses in a given sample. This broad-spectrum capability of HTS is raising a growing interest to study the diversity and ecology of plant viruses, in particular the richness and composition of viral communities at the agro-ecosystem scale, as well as the virus circulation among host reservoirs and the discovery of new and emerging viruses. Recent viromics studies revealed diversified and largely unknown phytoviromes in natural ecosystems, with high rates of co-infection and the abundance of so-called persistent (or cryptic) viruses representing more than half of the viruses identified in wild plants. The influence of plant traits (e.g., lifespan, height, occurrence) on the virome richness, and the relationships between host-pathogen richness in cultivated and non-cultivated plant communities were also unravelled. In particular, the richness and diversity of plant communities appeared as influencing the richness and composition of phytoviromes, especially the distribution of persistent and acute viruses. Other results demonstrated the stability of virome richness over time but the large viral intraspecific variability within and among plant communities. Thus, HTS technologies have highlighted and will continue to serve the exploration of the complex network of viral communities in nature for the times to come..

Exploring virus ecology and diversity in cultivated and wild Poaceae communities in Belgium

Ecology of plant viruses examines complex interactions among plant-associated viruses, their hosts and their vectors, and the environment. Starting at the end of the 19th century, virus ecology followed the development of new technologies for virus detection and characterization, from host symptomatology to biochemistry, microscopy, serological and molecular techniques. Most recently, high throughput sequencing (HTS) technologies allowed for the first time, to characterize all or nearly all viruses in a sample without a priori information about which viruses might be present, that is the study of the viral metagenome or virome. This thesis reviewed the major advances in virus ecology, in relation to the development of virus detection technologies. It focused on the viral metagenomics and detailed the opportunities and challenges associated to each step of a virome-based study using HTS technologies (field sampling, laboratory work and bioinformatics analyses). The research conducted in the framework of this thesis explored the diversity and ecology of viruses infecting a plant family of economical and ecological importance: the Poaceae plants or grasses. The study was performed in the region of the Belgian National Park “Burdinale-Mehaigne”. Combining untargeted HTS-based analysis and targeted virus detection by RT-PCR in thousands of grass samples, virome characterization was unrolled down to the species taxonomical rank to support an analysis at three different levels: in global plant communities, in plant populations from same species, and in individual plants. A global view of the Poaceae virome and its ecology could be obtained through various ecological and epidemiological analyses: examination of virus richness, prevalence and co-infection, network and clustering analyses, Akaike information corrected criterion (AICc) calculation, or Spatial Analysis by Distance IndicEs (SADIE). First, we investigated the virome in three different Poaceae communities presenting a contrasted biodiversity (in terms of grass species richness) and a gradient of human management (i.e. cereal crops, grazed pastures and mowed grasslands). A diversified and largely unknown virome was identified in cultivated and non-cultivated Poaceae, with at least seventy virus species, among which fifty previously unknown, from to eighteen virus families and twenty-nine genera. Viruses with persistent lifestyles belonging to Alphachrysovirus, Partitivirus and Totivirus genera represented a large part of this virome, reaching 60% of the viruses detected and mostly novel virus species. A positive correlation was found between virus species and grass species richness in the plant communities, with very few or no virus species detected in cereal crops while a diversified virome was observed in wilder communities with up 26 virus species in grasslands. It illustrated the influence of plant diversity and land use on the distribution of viral communities. In addition, virome comparison over years revealed complex virus-plant relationships within the Poaceae community, separating plant virus and mycovirus models. Second, virome was characterized and compared among Poaceae species, investigating the influence of plant traits (i.e. lifespan, height, occurrence) on virus richness observed. Significant higher virus richness was determined in perennial grasses compared to annuals, and low occurrence species presented specific virus species that were not observed in dominant species. Virome network and clustering analysis revealed the presence of both ubiquitous (or generalist) virus species and specialist viruses limited to one or a few host species, with specialist viruses dominating the Poaceae virome. Among all plant species examined, the perennial ryegrass (Lolium perenne) was demonstrated to represent an important virus reservoir and will thus constitute an interesting model for future studies in virus ecology. Analysis in individual plants showed contrasted prevalences, co-infections and spatial distributions among plant communities, plant species and virus species. Interactions between viruses were also explored and revealed positive and negative viral associations depending on the grass species. Third, a more-in-depth analysis was performed for two novel virus species belonging to Secoviridae family that were identified in abundance in Poaceae communities and species: Poaceae Liege nepovirus A (PoLNVA) and Poaceae Liege virus 1 (PoLV1). The analysis focused on rough bluegrass (Poa trivialis L.) for which almost complete virus genomes from both viruses could be obtained. Sequence and phylogenetic analyses placed PoLNVA in the genus Nepovirus, while low levels of amino acid identity for both Pro-Pol and CP regions could define PoLV1 as an unclassified secovirid, between the genera Waikavirus and Sequivirus. PoLNVA and PoLV1 were detected in eleven wild Poaceae species, sometimes with high prevalence (e.g. 86% of L. perenne and 74% of P. trivialis samples infected by PoLNVA), highlighting their significant presence and large host range within Poaceae. Virus transmission was also investigated and PoLNVA was found to be seed-transmitted. In summary, this thesis revealed the complex structure of viral communities in nature (in terms of richness, prevalence, co-infection, host range, spatial distribution and genetic structure) and improved our understanding of virus diversity ecology in agro-ecological landscapes, illustrated here with the Poaceae family. A couple of virus species were further characterized but dozens of other novel virus species remains to be examined and will likely provide new insights about the ecology and phylogeny of Poaceae viruses. Other fields of investigation concern the diversity of virus transmission agents and other plant pathogens (i.e. bacteria, fungi), in order to obtain a holistic view of the Poaceae phytobiome. A particular attention should be paid to fungi due to the abundance of so-called mycoviruses in Poaceae. Much remains to be done to unravel the secrets of the virus ecology in wild and cultivated Poaceae, a domain still in its infancy

PhD Thesis 3 minutes presentation: impact of ecosystem diversity on the Poaceae virome.

Before the domestication of plants, plant pathogens, including plant viruses, were co-evolving with wild plants growing in mixed species communities, thereby resulting in complex interactions. Plant viruses can be detrimental, but also commensal or even mutualistic for their hosts. Moreover, mixed-infections are very frequent in wild plants, which still increases the complexity of these relations (through antagonistic, mutualistic or coexistence interactions between viruses). The development of agriculture deeply modified the ecosystems, drastically reducing their biodiversity, raising their instability and disrupting the host-pathogen co-evolution. This instability impacts plants, vectors and viruses alike and alters the dynamics of virus-plant pathosystems which, in turn, drives the accelerated rate of virus evolution and emergence in response to changing circumstances. Moreover, the whole process is being exacerbated now by the climate change and the continuous world trade growth. Virus emergence, that is the introduction of new viruses in specific area, the emergence of new strains or the emergence of endemic viruses in newly introduced crops or cultivars, represents 47% of plant pathogen emergence worldwide. The global objective of this project is to study the impact of ecosystems with contrasted biodiversity on the virome of Poaceae plant species. These ecosystems are already localized in the National Park “Burdinale-Mehaigne” and include: monoculture crop, intensive pasture and pasture with high biological value. These ecosystems encompass cultivated, lawn, pasture, weed and pasture grasses at the interface between indigenous vegetation and cultivated areas. Virome comparison and standard population genetic analyses will provide information on the role of habitat biodiversity in shaping virome diversity, infection patterns and virus adaptation to host. The results will also inform on the role of wild or bordering ecosystems as reservoirs for infection of crops and pasture, and vice versa. This research has the potential to provide critical information not only on viral threats to biodiversity and cultivated species in contrasting environments, but also on virus evolution in diverse ecosystems

Towards mastering CRISPR-induced gene knock-in in plants: Survey of key features and focus on the model Physcomitrella patens

Beyond its predominant role in human and animal therapy, the CRISPR-Cas9 system has also become an essential tool for plant research and plant breeding. Agronomic applications rely on the mastery of gene inactivation and gene modification. However, if the knock-out of genes by non-homologous end-joining (NHEJ)-mediated repair of the targeted double-strand breaks (DSBs) induced by the CRISPR-Cas9 system is rather well mastered, the knock-in of genes by homology-driven repair or end-joining remains difficult to perform efficiently in higher plants. In this review, we describe the different approaches that can be tested to improve the efficiency of CRISPR–induced gene modification in plants, which include the use of optimal transformation and regeneration protocols, the design of appropriate guide RNAs and donor templates and the choice of nucleases and means of delivery. We also present what can be done to orient DNA repair pathways in the target cells, and we show how the moss Physcomitrella patens can be used as a model plant to better understand what DNA repair mechanisms are involved, and how this knowledge could eventually be used to define more performant strategies of CRISPR-induced gene knock-in

Deciphering the DSB repair mechanisms involved in CRISPR-induced mutagenesis and gene targeting in the model plant, Physcomitrella patens.

Site-directed nucleases make it very easy now to mutate (knock-out) any genomic sequence and to direct the insertion of a template DNA at a specific target (knock-in). The DNA repair mechanisms that are presumed to be mainly involved in these two types of events are respectively canonical non-homologous end-joining (c-NHEJ) and, if the template shares homology to the target, homologous recombination (HR). By using, the model plant Physcomitrella patens, where efficient gene editing (knock-out or knock-in) via SDN can be obtained, we demonstrated that it may not be as simple. By applying the CRISPR/Cas9 system to a series of mutants impacted in different DNA repair pathways, we progressed in the understanding of the different mechanisms that could be involved in CRISPR-induced mutagenesis and gene targeting in plants. Without template DNA, we showed that the mutation efficiency was not decreased in absence of key factors of c-NHEJ, which could indicate that CRISPR-induced mutations may not only be due to c-NHEJ but also to alternative end-joining like micro-homology mediated end joining (MMEJ) and other types of non-canonical end joining. Targeted insertion of a circular template DNA presenting homology to the target appeared to be mainly dependent on the RAD51-dependent HR pathway but not entirely, and other pathways, including single-strand annealing (SSA), could potentially be involved