RNA three-dimensional structure drives the sequence organization of potato spindle tuber viroid quasispecies.

RNA viruses and viroids exist and evolve as quasispecies due to error-prone replication. Quasispecies consist of a few dominant master sequences alongside numerous variants that contribute to genetic diversity. Upon environmental changes, certain variants within quasispecies have the potential to become the dominant sequences, leading to the emergence of novel infectious strains. However, the emergence of new infectious variants remains unpredictable. Using mutant pools prepared by saturation mutagenesis of selected stem and loop regions, our study of potato spindle tuber viroid (PSTVd) demonstrates that mutants forming local three-dimensional (3D) structures similar to the wild type (WT) are more likely to accumulate in PSTVd quasispecies. The selection mechanisms underlying this biased accumulation are likely associated with cell-to-cell movement and long-distance trafficking. Moreover, certain trafficking-defective PSTVd mutants can be spread by functional sister genomes in the quasispecies. Our study reveals that the RNA 3D structure of stems and loops constrains the evolution of viroid quasispecies. Mutants with a structure similar to WT have a higher likelihood of being maintained within the quasispecies and can potentially give rise to novel infectious variants. These findings emphasize the potential of targeting RNA 3D structure as a more robust approach to defend against viroid infections.

Deciphering the virome of Chunkung (Cnidium officinale) showing dwarfism-like symptoms via a high-throughput sequencing analysis.

BACKGROUND: Viruses have notable effects on agroecosystems, wherein they can adversely affect plant health and cause problems (e.g., increased biosecurity risks and economic losses). However, our knowledge of their diversity and interactions with specific host plants in ecosystems remains limited. To enhance our understanding of the roles that viruses play in agroecosystems, comprehensive analyses of the viromes of a wide range of plants are essential. High-throughput sequencing (HTS) techniques are useful for conducting impartial and unbiased investigations of plant viromes, ultimately forming a basis for generating further biological and ecological insights. This study was conducted to thoroughly characterize the viral community dynamics in individual plants. RESULTS: An HTS-based virome analysis in conjunction with proximity sampling and a tripartite network analysis were performed to investigate the viral diversity in chunkung (Cnidium officinale) plants. We identified 61 distinct chunkung plant-associated viruses (27 DNA and 34 RNA viruses) from 21 known genera and 6 unclassified genera in 14 known viral families. Notably, 12 persistent viruses (7 DNA and 5 RNA viruses) were exclusive to dwarfed chunkung plants. The detection of viruses from the families Partitiviridae, Picobirnaviridae, and Spinareoviridae only in the dwarfed plants suggested that they may contribute to the observed dwarfism. The co-infection of chunkung by multiple viruses is indicative of a dynamic and interactive viral ecosystem with significant sequence variability and evidence of recombination. CONCLUSIONS: We revealed the viral community involved in chunkung. Our findings suggest that chunkung serves as a significant reservoir for a variety of plant viruses. Moreover, the co-infection rate of individual plants was unexpectedly high. Future research will need to elucidate the mechanisms enabling several dozen viruses to co-exist in chunkung. Nevertheless, the important insights into the chunkung virome generated in this study may be relevant to developing effective plant viral disease management and control strategies.

The VP53 protein encoded by RNA2 of a fabavirus, broad bean wilt virus 2, is essential for viral systemic infection.

Plant viruses evolves diverse strategies to overcome the limitations of their genomic capacity and express multiple proteins, despite the constraints imposed by the host translation system. Broad bean wilt virus 2 (BBWV2) is a widespread viral pathogen, causing severe damage to economically important crops. It is hypothesized that BBWV2 RNA2 possesses two alternative in-frame translation initiation codons, resulting in the production of two largely overlapping proteins, VP53 and VP37. In this study, we aim to investigate the expression and function of VP53, an N-terminally 128-amino-acid-extended form of the viral movement protein VP37, during BBWV2 infection. By engineering various recombinant and mutant constructs of BBWV2 RNA2, here we demonstrate that VP53 is indeed expressed during BBWV2 infection. We also provide evidence of the translation of the two overlapping proteins through ribosomal leaky scanning. Furthermore, our study highlights the indispensability of VP53 for successful systemic infection of BBWV2, as its removal results in the loss of virus infectivity. These insights into the translation mechanism and functional role of VP53 during BBWV2 infection significantly contribute to our understanding of the infection mechanisms employed by fabaviruses.

The plant virus transmissions database.

Plant viruses are transmitted mechanically or by vegetative propagation, and by vectors such as arthropods, fungi, nematodes, or parasitic plants. Sources to access available information regarding plant virus transmissions are scattered and require extensive literature searches. Here, a recently created plant virus transmission database is described. This was developed to provide access to the modes of transmission and vectors of over 1600 plant viruses. The database was compiled using over 3500 publication records spanning the last 100 years. The information is publicly accessible via https://library.wur.nl/WebQuery/virus and fully searchable by virus name, taxonomic position, mode of transmission or vector.

Regulation of Disease-Resistance Genes against CWMV Infection by NbHAG1-Mediated H3K36ac.

Post-translational modification of proteins plays a critical role in plant-pathogen interactions. Here, we demonstrate in Nicotiana benthamiana that knockout of NbHAG1 promotes Chinese wheat mosaic virus (CWMV) infection, whereas NbHAG1 overexpression inhibits infection. Transcriptome sequencing indicated that a series of disease resistance-related genes were up-regulated after overexpression of NbHAG1. In addition, cleavage under targets and tagmentation (Cut&Tag)-qPCR results demonstrated that NbHAG1 may activate the transcription of its downstream disease-resistance genes by facilitating the acetylation level of H3K36ac. Therefore, we suggest that NbHAG1 is an important positive regulator of resistance to CWMV infestation.

Establishment of virus-induced gene-silencing system in Juglans sigillata Dode and functional analysis of JsFLS2 and JsFLS4.

Juglans sigillata Dode is one of the important tree species in southwest China, and it has significant economic and ecological value. However, there is still a lack of effective methods to identify the functional genes of J. sigillata. By verifying the model plant tobacco, the pTRV2::JsPDS vector was able to cause photobleaching. This study showed that photobleaching occurred 24 and 30 d after the silencing vector was infected with aseptic seedlings and fruits of J. sigillata, respectively. When the OD600 was 0.6, and the injection dose was 500 µL, the gene silencing efficiency of aseptic seedlings was the highest at 16.7 %, significantly better than other treatments. Moreover, when the OD600 was 0.8, and the injection dose was 500 µL, the gene silencing efficiency in the walnut fruit was the highest (20 %). In addition, the VIGS system was successfully used to silence JsFLS2 and JsFLS4 genes in J. sigillata. This study also showed that the flavonol content and gene expression in the treatment group were decreased compared to the control group. In addition, the proteins transcribed and translated from the JsFLS4 gene may have higher catalytic activity for dihydroquercetin. The above results indicate that the TRV-mediated VIGS system can be an ideal tool for studying J. sigillata gene function.

Old and New Aphid-Borne Viruses in Coriander in Chile: An Epidemiological Approach.

In Chile, edible herbs are mainly grown by small farmers. This type of horticultural crop typically requires intensive management because it is highly susceptible to insects, some of which transmit viruses that severely affect crop yield and quality. In 2019, in coriander plants tested negative for all previously reported viruses, RNA-Seq analysis of one symptomatic plant revealed a plethora of viruses, including one virus known to infect coriander, five viruses never reported in coriander, and a new cytorhabdovirus with a 14,180 nucleotide RNA genome for which the species name Cytorhabdovirus coriandrum was proposed. Since all the detected viruses were aphid-borne, aphids and weeds commonly growing around the coriander field were screened for viruses. The results showed the occurrence of the same seven viruses and the alfalfa mosaic virus, another aphid-borne virus, in aphids and weeds. Together, our findings document the presence of multiple viruses in coriander and the potential role of weeds as virus reservoirs for aphid acquisition.

Virus-induced changes in root volatiles attract soil nematode vectors to infected plants.

Plant-derived volatiles mediate interactions among plants, pathogenic viruses, and viral vectors. These volatile-dependent mechanisms have not been previously demonstrated belowground, despite their likely significant role in soil ecology and agricultural pest impacts. We investigated how the plant virus, tobacco rattle virus (TRV), attracts soil nematode vectors to infected plants. We infected Nicotiana benthamiana with TRV and compared root growth relative to that of uninfected plants. We tested whether TRV-infected N. benthamiana was more attractive to nematodes 7 d post infection and identified a compound critical to attraction. We also infected N. benthamiana with mutated TRV strains to identify virus genes involved in vector nematode attraction. Virus titre and associated impacts on root morphology were greatest 7 d post infection. Tobacco rattle virus infection enhanced 2-ethyl-1-hexanol production. Nematode chemotaxis and 2-ethyl-1-hexanol production correlated strongly with viral load. Uninfected plants were more attractive to nematodes after the addition of 2-ethyl-1-hexanol than were untreated plants. Mutation of TRV RNA2-encoded genes reduced the production of 2-ethyl-1-hexanol and nematode attraction. For the first time, this demonstrates that virus-driven alterations in root volatile emissions lead to increased chemotaxis of the virus's nematode vector, a finding with implications for sustainable management of both nematodes and viral pathogens in agricultural systems.

Meta-transcriptomic identification of groundnut RNA viruses in western Kenya and the novel detection of groundnut as a host for Cauliflower mosaic virus.

BACKGROUND: Groundnut (Arachis hypogaea L.) is the 13th most important global crop grown throughout the tropical and subtropical regions of the world. One of the major constraints to groundnut production is viruses, which are also the most economically important and most abundant pathogens among cultivated legumes. Only a few studies have reported the characterization of RNA viruses in cultivated groundnuts in western Kenya, most of which deployed classical methods of detecting known viruses. METHODS: We sampled twenty-one symptomatic and three asymptomatic groundnut leaf samples from farmers' fields in western Kenya. Total RNA was extracted from the samples followed by First-strand cDNA synthesis and sequencing on the Illumina HiSeq 2500 platform. After removing host and rRNA sequences, high-quality viral RNA sequences were de novo assembled and viral genomes annotated using the publicly available NCBI virus database. Multiple sequence alignment and phylogenetic analysis were done using MEGA X. RESULTS: Bioinformatics analyses using as low as ∼3.5 million reads yielded complete and partial genomes for Cauliflower mosaic virus (CaMV), Cowpea polerovirus 2 (CPPV2), Groundnut rosette assistor virus (GRAV), Groundnut rosette virus (GRV), Groundnut rosette virus satellite RNA (satRNA) and Peanut mottle virus (PeMoV) falling within the species demarcation criteria. This is the first report of CaMV and the second report of CPPV2 on groundnut hosts in the world. Confirmation of the detected viruses was further verified through phylogenetic analyses alongside reported publicly available highly similar viruses. PeMoV was the only seed-borne virus reported. CONCLUSION: Our findings demonstrate the power of Next Generation Sequencing in the discovery and identification of novel viruses in groundnuts. The detection of the new viruses indicates the complexity of virus diseases in groundnuts and would require more focus in future studies to establish the effect of the viruses as sole or mixed infections on the crop. The detection of PeMoV with potential origin from Malawi indicates the importance of seed certification and cross-boundary seed health testing.

Application of nanoparticles for management of plant viral pathogen: Current status and future prospects.

Plant viruses are responsible for nearly 47 % of all crop losses brought by plant diseases, which have a considerable negative impact on agricultural output. Nanoparticles have the potential to greatly raise agricultural output due to their wonderful applications in the fields of highly sensitive biomolecular detection, disease diagnostics, antimicrobials, and therapeutic compounds. The application of nanotechnology in plant virology is known as nanophytovirology, and it involves biostimulation, drug transport, genetic manipulation, therapeutic agents, and induction of plant defenses. The inactivation and denaturation of capsid protein, nucleic acids (RNA or DNA), and other protein constituents are involved in the underlying mechanism. To determine the precise mechanism by which nanoparticles affect viral mobility, reproduction, encapsidation, and transmission, more research is however required. Nanoparticles can be used to precisely detect plant viruses using nanobiosensors or as biostimulants. The varieties of nanoparticles employed in plant virus control and their methods of virus suppression are highlighted in this review.

In planta expression of specific single chain fragment antibody (scFv) against nucleocapsid protein of fig mosaic virus (FMV).

Fig mosaic virus (FMV) is recognized as the main viral agent associated with the mosaic disease (MD) of fig trees (Ficus carica). Due to its worldwide occurrence, FMV represents the most significant global threat to the production of fig fruit. A disease management strategy against the MD in fig orchards has never been effective; and therefore, expression of recombinant antibody in plant cells could provide an alternative approach to suppress FMV infections. In this study we focused on expressing a specific recombinant antibody, a single-chain variable fragment (scFv), targeting the nucleocapsid protein (NP) of FMV in planta. To accomplish this objective, we inserted the scFv gene into a plant expression vector and conducted transient expression in leaves of Nicotiana tabacum cv. Samson plants. The construct was transiently expressed in tobacco plants by agroinfiltration, and antibody of the anticipated size was detected by immunoblotting. The produced plantibody was then assessed for specificity using ELISA and confirmed by Western blot analysis. In this study, the plantibody developed against FMV could be considered as a potential countermeasure to the infection by conferring resistance to MD.

Predictive Modeling of Proteins Encoded by a Plant Virus Sheds a New Light on Their Structure and Inherent Multifunctionality.

Plant virus genomes encode proteins that are involved in replication, encapsidation, cell-to-cell, and long-distance movement, avoidance of host detection, counter-defense, and transmission from host to host, among other functions. Even though the multifunctionality of plant viral proteins is well documented, contemporary functional repertoires of individual proteins are incomplete. However, these can be enhanced by modeling tools. Here, predictive modeling of proteins encoded by the two genomic RNAs, i.e., RNA1 and RNA2, of grapevine fanleaf virus (GFLV) and their satellite RNAs by a suite of protein prediction software confirmed not only previously validated functions (suppressor of RNA silencing [VSR], viral genome-linked protein [VPg], protease [Pro], symptom determinant [Sd], homing protein [HP], movement protein [MP], coat protein [CP], and transmission determinant [Td]) and previously identified putative functions (helicase [Hel] and RNA-dependent RNA polymerase [Pol]), but also predicted novel functions with varying levels of confidence. These include a T3/T7-like RNA polymerase domain for protein 1AVSR, a short-chain reductase for protein 1BHel/VSR, a parathyroid hormone family domain for protein 1EPol/Sd, overlapping domains of unknown function and an ABC transporter domain for protein 2BMP, and DNA topoisomerase domains, transcription factor FBXO25 domain, or DNA Pol subunit cdc27 domain for the satellite RNA protein. Structural predictions for proteins 2AHP/Sd, 2BMP, and 3A? had low confidence, while predictions for proteins 1AVSR, 1BHel*/VSR, 1CVPg, 1DPro, 1EPol*/Sd, and 2CCP/Td retained higher confidence in at least one prediction. This research provided new insights into the structure and functions of GFLV proteins and their satellite protein. Future work is needed to validate these findings.

A plant virus manipulates the long-winged morph of insect vectors.

Wing dimorphism of insect vectors is a determining factor for viral long-distance dispersal and large-area epidemics. Although plant viruses affect the wing plasticity of insect vectors, the potential underlying molecular mechanisms have seldom been investigated. Here, we found that a planthopper-vectored rice virus, rice stripe virus (RSV), specifically induces a long-winged morph in male insects. The analysis of field populations demonstrated that the long-winged ratios of male insects are closely associated with RSV infection regardless of viral titers. A planthopper-specific and testis-highly expressed gene, Encounter, was fortuitously found to play a key role in the RSV-induced long-winged morph. Encounter resembles malate dehydrogenase in the sequence, but it does not have corresponding enzymatic activity. Encounter is upregulated to affect male wing dimorphism at early larval stages. Encounter is closely connected with the insulin/insulin-like growth factor signaling pathway as a downstream factor of Akt, of which the transcriptional level is activated in response to RSV infection, resulting in the elevated expression of Encounter. In addition, an RSV-derived small interfering RNA directly targets Encounter to enhance its expression. Our study reveals an unreported mechanism underlying the direct regulation by a plant virus of wing dimorphism in its insect vectors, providing the potential way for interrupting viral dispersal.

Production of Double-Stranded RNA in Planta by a Potato Mop-Top Virus (PMTV)-Based Vector for Inducing Gene Silencing.

RNA silencing (also known as gene silencing) is an evolutionary conserved mechanism that is involved in regulating gene expression, suppressing mobile elements, and defensing virus infection. RNA silencing is triggered by double-stranded RNA via Dicer or Dicer-like riboendonucleases. DsRNAs are also the replication intermediates of all RNA viruses; as a result, plant RNA viruses are ideal candidates to induce RNA silencing. A large body of plant viruses have been modified into vectors for RNA silencing in varied plant species. Here, we described a simple, time-saving, and operable system for gene function and genetic breeding study of potato and Nicotiana benthamiana using a potato mop-top (MPTV)-based vector.

MicroRNA750-3p Targets Processing of Precursor 7 to Suppress Rice Black-Streaked Dwarf Virus Propagation in Vector Laodelphax striatellus.

MicroRNAs (miRNAs) are non-coding RNAs, which, as members of the RNA interference pathway, play a pivotal role in antiviral infection. Almost 80% of plant viruses are transmitted by insect vectors; however, little is known about the interaction of the miRNAs of insect vectors with plant viruses. Here, we took rice black-streaked dwarf virus (RBSDV), a devastating virus to rice production in eastern Asia, and the small brown planthopper, (SBPH, Laodelphax striatellus) as a model to investigate the role of microRNA750-3p (miR750-3p) in regulating viral transmission. Our results showed that Ls-miR750-3p was downregulated in RBSDV-infected SBPH and predominately expressed in the midgut of SBPH. Injection with miR750-3p agomir significantly reduced viral accumulation, and the injection with the miR750-3p inhibitor, antagomir-750-3p, dramatically promoted the viral accumulation in SBPH, as detected using RT-qPCR and Western blotting. The processing of precursor 7 (POP7), a subunit of RNase P and RNase MRP, was screened, identified, and verified using a dual luciferase reporter assay as one target of miR750-3p. Knockdown of POP7 notably increased RBSDV viral propagation in SBPH and then increased the viral transmission rate by SBPH. Taken together, our data indicate that miR750-3p targets POP7 to suppress RBSDV infection in its insect vector. These results enriched the role of POP7 in modulating virus infection in host insects and shared new insight into the function of miRNAs in plant virus and insect vector interaction.

Naked-eye detection of plant viral disease using polymerase chain reaction amplification and DNAzyme.

Plant viral diseases can seriously affect the yield and quality of crops. In this work, a convenient and highly sensitive biosensor for the visual detection of plant viral disease is proposed by the PCR-induced generation of DNAzyme. In the absence of nucleic acid for a target plant virus, the primers prohibited the production of G-quadruplex by forming a hairpin structure. However, PCR amplification occurred and generated a number of specific PCR products with free G-quadruplex sequences at both ends in the presence of the target cDNA. A catalytically active G-quadruplex DNAzyme was formed with the help of K+ and hemin, resulting in the formation of colored products visible to the naked eye and a strong absorbance by the addition of ABTS2- and H2O2. The absorbance and the logarithm of target cDNA concentrations showed a good linear relationship in the range of 10 fM-1.0 nM, with a linear regression equation of A = 0.1402 lg c + 0.3761 (c: fM) and a detection limit of 0.19 fM. This method was successfully applied to the analysis of emerging tobacco mosaic virus (TMV) infections in tobacco leaf samples collected in the field due to its flexibility and convenience, indicating a potential application for the early detection of plant viral disease.

Revisiting the origins of the Sobemovirus genus: A case for ancient origins of plant viruses.

The discrepancy between short- and long-term rate estimates, known as the time-dependent rate phenomenon (TDRP), poses a challenge to extrapolating evolutionary rates over time and reconstructing evolutionary history of viruses. The TDRP reveals a decline in evolutionary rate estimates with the measurement timescale, explained empirically by a power-law rate decay, notably observed in animal and human viruses. A mechanistic evolutionary model, the Prisoner of War (PoW) model, has been proposed to address TDRP in viruses. Although TDRP has been studied in animal viruses, its impact on plant virus evolutionary history remains largely unexplored. Here, we investigated the consequences of TDRP in plant viruses by applying the PoW model to reconstruct the evolutionary history of sobemoviruses, plant pathogens with significant importance due to their impact on agriculture and plant health. Our analysis showed that the Sobemovirus genus dates back over four million years, indicating an ancient origin. We found evidence that supports deep host jumps to Poaceae, Fabaceae, and Solanaceae occurring between tens to hundreds of thousand years ago, followed by specialization. Remarkably, the TDRP-corrected evolutionary history of sobemoviruses was extended far beyond previous estimates that had suggested their emergence nearly 9,000 years ago, a time coinciding with the Neolithic period in the Near East. By incorporating sequences collected through metagenomic analyses, the resulting phylogenetic tree showcases increased genetic diversity, reflecting a deep history of sobemovirus species. We identified major radiation events beginning between 4,600 to 2,000 years ago, which aligns with the Neolithic period in various regions, suggesting a period of rapid diversification from then to the present. Our findings make a case for the possibility of deep evolutionary origins of plant viruses.

Structure of saguaro cactus virus 3' translational enhancer mimics 5' cap for eIF4E binding.

The genomes of several plant viruses contain RNA structures at their 3' ends called cap-independent translation enhancers (CITEs) that bind the host protein factors such as mRNA 5' cap-binding protein eIF4E for promoting cap-independent genome translation. However, the structural basis of such 5' cap-binding protein recognition by the uncapped RNA remains largely unknown. Here, we have determined the crystal structure of a 3' CITE, panicum mosaic virus-like translation enhancer (PTE) from the saguaro cactus virus (SCV), using a Fab crystallization chaperone. The PTE RNA folds into a three-way junction architecture with a pseudoknot between the purine-rich R domain and pyrimidine-rich Y domain, which organizes the overall structure to protrude out a specific guanine nucleotide, G18, from the R domain that comprises a major interaction site for the eIF4E binding. The superimposable crystal structures of the wild-type, G18A, G18C, and G18U mutants suggest that the PTE scaffold is preorganized with the flipped-out G18 ready to dock into the eIF4E 5' cap-binding pocket. The binding studies with wheat and human eIF4Es using gel electrophoresis and isothermal titration calorimetry, and molecular docking computation for the PTE-eIF4E complex demonstrated that the PTE structure essentially mimics the mRNA 5' cap for eIF4E binding. Such 5' cap mimicry by the uncapped and structured viral RNA highlights how viruses can exploit RNA structures to mimic the host protein-binding partners and bypass the canonical mechanisms for their genome translation, providing opportunities for a better understanding of virus-host interactions and non-canonical translation mechanisms found in many pathogenic RNA viruses.

Analysis of the relative frequencies of the multipartite BNYVV genomic RNAs in different plants and tissues.

Multipartite virus genomes are composed of two or more segments, each packaged into an independent viral particle. A potential advantage of multipartitism is the regulation of gene expression through changes in the segment copy number. Soil-borne beet necrotic yellow vein virus (BNYVV) is a typical example of multipartism, given its high number of genomic positive-sense RNAs (up to five). Here we analyse the relative frequencies of the four genomic RNAs of BNYVV type B during infection of different host plants (Chenopodium quinoa, Beta macrocarpa and Spinacia oleracea) and organs (leaves and roots). By successfully validating a two-step reverse-transcriptase digital droplet PCR protocol, we show that RNA1 and -2 genomic segments always replicate at low and comparable relative frequencies. In contrast, RNA3 and -4 accumulate with variable relative frequencies, resulting in distinct RNA1 : RNA2 : RNA3 : RNA4 ratios, depending on the infected host species and organ.

Mixed infection of ITPase-encoding potyvirid and secovirid in Mercurialis perennis: evidences for a convergent euphorbia-specific viral counterstrike.

BACKGROUND: In cellular organisms, inosine triphosphate pyrophosphatases (ITPases) prevent the incorporation of mutagenic deaminated purines into nucleic acids. These enzymes have also been detected in the genomes of several plant RNA viruses infecting two euphorbia species. In particular, two ipomoviruses produce replicase-associated ITPases to cope with high concentration of non-canonical nucleotides found in cassava tissues. METHOD: Using high-throughput RNA sequencing on the wild euphorbia species Mercurialis perennis, two new members of the families Potyviridae and Secoviridae were identified. Both viruses encode for a putative ITPase, and were found in mixed infection with a new partitivirid. Following biological and genomic characterization of these viruses, the origin and function of the phytoviral ITPases were investigated. RESULTS: While the potyvirid was shown to be pathogenic, the secovirid and partitivirid could not be transmitted. The secovirid was found belonging to a proposed new Comovirinae genus tentatively named "Mercomovirus", which also accommodates other viruses identified through transcriptome mining, and for which an asymptomatic pollen-associated lifestyle is suspected. Homology and phylogenetic analyses inferred that the ITPases encoded by the potyvirid and secovirid were likely acquired through independent horizontal gene transfer events, forming lineages distinct from the enzymes found in cassava ipomoviruses. Possible origins from cellular organisms are discussed for these proteins. In parallel, the endogenous ITPase of M. perennis was predicted to encode for a C-terminal nuclear localization signal, which appears to be conserved among the ITPases of euphorbias but absent in other plant families. This subcellular localization is in line with the idea that nucleic acids remain protected in the nucleus, while deaminated nucleotides accumulate in the cytoplasm where they act as antiviral molecules. CONCLUSION: Three new RNA viruses infecting M. perennis are described, two of which encoding for ITPases. These enzymes have distinct origins, and are likely required by viruses to circumvent high level of cytoplasmic non-canonical nucleotides. This putative plant defense mechanism has emerged early in the evolution of euphorbias, and seems to specifically target certain groups of RNA viruses infecting perennial hosts.