Plant virus movement proteins originated from jelly-roll capsid proteins.

Numerous, diverse plant viruses encode movement proteins (MPs) that aid the virus movement through plasmodesmata, the plant intercellular channels. MPs are essential for virus spread and propagation in distal tissues, and several unrelated MPs have been identified. The 30K superfamily of MPs (named after the molecular mass of tobacco mosaic virus MP, the classical model of plant virology) is the largest and most diverse MP variety, represented in 16 virus families, but its evolutionary origin remained obscure. Here, we show that the core structural domain of the 30K MPs is homologous to the jelly-roll domain of the capsid proteins (CPs) of small RNA and DNA viruses, in particular, those infecting plants. The closest similarity was observed between the 30K MPs and the CPs of the viruses in the families Bromoviridae and Geminiviridae. We hypothesize that the MPs evolved via duplication or horizontal acquisition of the CP gene in a virus that infected an ancestor of vascular plants, followed by neofunctionalization of one of the paralogous CPs, potentially through the acquisition of unique N- and C-terminal regions. During the subsequent coevolution of viruses with diversifying vascular plants, the 30K MP genes underwent explosive horizontal spread among emergent RNA and DNA viruses, likely permitting viruses of insects and fungi that coinfected plants to expand their host ranges, molding the contemporary plant virome.

Plant virus evolution under strong drought conditions results in a transition from parasitism to mutualism.

Environmental conditions are an important factor driving pathogens' evolution. Here, we explore the effects of drought stress in plant virus evolution. We evolved turnip mosaic potyvirus in well-watered and drought conditions in Arabidopsis thaliana accessions that differ in their response to virus infection. Virus adaptation occurred in all accessions independently of watering status. Drought-evolved viruses conferred a significantly higher drought tolerance to infected plants. By contrast, nonsignificant increases in tolerance were observed in plants infected with viruses evolved under standard watering. The magnitude of this effect was dependent on the plant accessions. Differences in tolerance were correlated to alterations in the expression of host genes, some involved in regulation of the circadian clock, as well as in deep changes in the balance of phytohormones regulating defense and growth signaling pathways. Our results show that viruses can promote host survival in situations of abiotic stress, with the magnitude of such benefit being a selectable trait.

Revisiting Orthotospovirus phylogeny using full-genome data and testing the contribution of selection, recombination and segment reassortment in the origin of members of new species.

The family Tospoviridae of the order Bunyavirales is constituted of tri-segmented negative-sense single-stranded RNA viruses that infect plants and are also able to replicate in their insect vectors in a persistent manner. The family is composed of a single genus, Orthotospovirus, whose type species is Tomato spotted wilt orthotospovirus. Previous studies assessing the phylogenetic relationships within this genus were based on partial genomic sequences, resulting in unresolved clades and a poor assessment of the roles of recombination and segment reassortment during mixed infections. Full genome sequences of members of recognized Orthotospovirus species are now available at NCBI. In this study, we examined 67 complete genome sequences from members of 22 species. Our study confirms the existence of four phylogroups (A to D), grouped in two major clades (A-B and C-D) within the genus. We found strong evidence that within-segment recombination events and reassortment of segments during mixed infections have been involved in the origin of new orthotospoviruses. Also, selection pressures were analyzed for each gene, and evidence of positive selection was found in all genes.

Unveiling the hidden viromes across the animal tree of life: insights from a taxonomic classification pipeline applied to invertebrates of 31 metazoan phyla.

Invertebrates constitute the majority of animal species on Earth, including most disease-causing agents or vectors, with more diverse viromes when compared to vertebrates. Recent advancements in high-throughput sequencing have significantly expanded our understanding of invertebrate viruses, yet this knowledge remains biased toward a few well-studied animal lineages. In this study, we analyze invertebrate DNA and RNA viromes for 31 phyla using 417 publicly available RNA-Seq data sets from diverse environments in the marine-terrestrial and marine-freshwater gradients. This study aims to (i) estimate virome compositions at the family level for the first time across the animal tree of life, including the first exploration of the virome in several phyla, (ii) quantify the diversity of invertebrate viromes and characterize the structure of invertebrate-virus infection networks, and (iii) investigate host phylum and habitat influence on virome differences. Results showed that a set of few viral families of eukaryotes, comprising Retroviridae, Flaviviridae, and several families of giant DNA viruses, were ubiquitous and highly abundant. Nevertheless, some differences emerged between phyla, revealing for instance a less diverse virome in Ctenophora compared to the other animal phyla. Compositional analysis of the viromes showed that the host phylum explained over five times more variance in composition than its habitat. Moreover, significant similarities were observed between the viromes of some phylogenetically related phyla, which could highlight the influence of co-evolution in shaping invertebrate viromes.IMPORTANCEThis study significantly enhances our understanding of the global animal virome by characterizing the viromes of previously unexamined invertebrate lineages from a large number of animal phyla. It showcases the great diversity of viromes within each phylum and investigates the role of habitat shaping animal viral communities. Furthermore, our research identifies dominant virus families in invertebrates and distinguishes phyla with analogous viromes. This study sets the road toward a deeper understanding of the virome across the animal tree of life.

Genetic basis of Arabidopsis thaliana responses to infection by naïve and adapted isolates of turnip mosaic virus.

Plant viruses account for enormous agricultural losses worldwide, and the most effective way to combat them is to identify genetic material conferring plant resistance to these pathogens. Aiming to identify genetic associations with responses to infection, we screened a large panel of Arabidopsis thaliana natural inbred lines for four disease-related traits caused by infection by A. thaliana-naïve and -adapted isolates of the natural pathogen turnip mosaic virus (TuMV). We detected a strong, replicable association in a 1.5 Mb region on chromosome 2 with a 10-fold increase in relative risk of systemic necrosis. The region contains several plausible causal genes as well as abundant structural variation, including an insertion of a Copia transposon into a Toll/interleukin receptor (TIR-NBS-LRR) coding for a gene involved in defense, that could be either a driver or a consequence of the disease-resistance locus. When inoculated with TuMV, loss-of-function mutant plants of this gene exhibited different symptoms than wild-type plants. The direction and severity of symptom differences depended on the adaptation history of the virus. This increase in symptom severity was specific for infections with the adapted isolate. Necrosis-associated alleles are found worldwide, and their distribution is consistent with a trade-off between resistance during viral outbreaks and a cost of resistance otherwise, leading to negative frequency-dependent selection.

Evolution of anelloviruses from a circovirus-like ancestor through gradual augmentation of the jelly-roll capsid protein.

Anelloviruses are highly prevalent in diverse mammals, including humans, but so far have not been linked to any disease and are considered to be part of the 'healthy virome'. These viruses have small circular single-stranded DNA (ssDNA) genomes and encode several proteins with no detectable sequence similarity to proteins of other known viruses. Thus, anelloviruses are the only family of eukaryotic ssDNA viruses currently not included in the realm Monodnaviria. To gain insights into the provenance of these enigmatic viruses, we sequenced more than 250 complete genomes of anelloviruses from nasal and vaginal swab samples of Weddell seal (Leptonychotes weddellii) from Antarctica and a fecal sample of grizzly bear (Ursus arctos horribilis) from the USA and performed a comprehensive family-wide analysis of the signature anellovirus protein ORF1. Using state-of-the-art remote sequence similarity detection approaches and structural modeling with AlphaFold2, we show that ORF1 orthologs from all Anelloviridae genera adopt a jelly-roll fold typical of viral capsid proteins (CPs), establishing an evolutionary link to other eukaryotic ssDNA viruses, specifically, circoviruses. However, unlike CPs of other ssDNA viruses, ORF1 encoded by anelloviruses from different genera display remarkable variation in size, due to insertions into the jelly-roll domain. In particular, the insertion between ß-strands H and I forms a projection domain predicted to face away from the capsid surface and function at the interface of virus-host interactions. Consistent with this prediction and supported by recent experimental evidence, the outermost region of the projection domain is a mutational hotspot, where rapid evolution was likely precipitated by the host immune system. Collectively, our findings further expand the known diversity of anelloviruses and explain how anellovirus ORF1 proteins likely diverged from canonical jelly-roll CPs through gradual augmentation of the projection domain. We suggest assigning Anelloviridae to a new phylum, 'Commensaviricota', and including it into the kingdom Shotokuvirae (realm Monodnaviria), alongside Cressdnaviricota and Cossaviricota.

Production of Potyvirus-Derived Nanoparticles Decorated with a Nanobody in Biofactory Plants.

Viral nanoparticles (VNPs) have recently attracted attention for their use as building blocks for novel materials to support a range of functions of potential interest in nanotechnology and medicine. Viral capsids are ideal for presenting small epitopes by inserting them at an appropriate site on the selected coat protein (CP). VNPs presenting antibodies on their surfaces are considered highly promising tools for therapeutic and diagnostic purposes. Due to their size, nanobodies are an interesting alternative to classic antibodies for surface presentation. Nanobodies are the variable domains of heavy-chain (VHH) antibodies from animals belonging to the family Camelidae, which have several properties that make them attractive therapeutic molecules, such as their small size, simple structure, and high affinity and specificity. In this work, we have produced genetically encoded VNPs derived from two different potyviruses-the largest group of RNA viruses that infect plants-decorated with nanobodies. We have created a VNP derived from zucchini yellow mosaic virus (ZYMV) decorated with a nanobody against the green fluorescent protein (GFP) in zucchini (Cucurbita pepo) plants. As reported for other viruses, the expression of ZYMV-derived VNPs decorated with this nanobody was only made possible by including a picornavirus 2A splicing peptide between the fused proteins, which resulted in a mixed population of unmodified and decorated CPs. We have also produced tobacco etch virus (TEV)-derived VNPs in Nicotiana benthamiana plants decorated with the same nanobody against GFP. Strikingly, in this case, VNPs could be assembled by direct fusion of the nanobody to the viral CP with no 2A splicing involved, likely resulting in fully decorated VNPs. For both expression systems, correct assembly and purification of the recombinant VNPs was confirmed by transmission electron microscope; the functionality of the CP-fused nanobody was assessed by western blot and binding assays. In sum, here we report the production of genetically encoded plant-derived VNPs decorated with a nanobody. This system may be an attractive alternative for the sustainable production in plants of nanobody-containing nanomaterials for diagnostic and therapeutic purposes.

Defects in plant immunity modulate the rates and patterns of RNA virus evolution.

It is assumed that host genetic variability for susceptibility to infection conditions virus evolution. Differences in host susceptibility can drive a virus to diversify into strains that track different defense alleles (e.g. antigenic diversity) or to infect only the most susceptible genotypes. Here, we have studied how variability in host defenses determines the evolutionary fate of a plant RNA virus. We performed evolution experiments with Turnip mosaic potyvirus in Arabidopsis thaliana mutants that had disruptions in infection-response signaling pathways or in genes whose products are essential for potyvirus infection. Plant genotypes were classified into five phenogroups according to their response to infection. We found that evolution proceeded faster in more restrictive hosts than in more permissive ones. Most of the phenotypic differences shown by the ancestral virus across host genotypes were removed after evolution, suggesting the combined action of selection and chance. When all evolved viral lineages were tested in all plant genotypes used in the experiments, we found compelling evidences that the most restrictive plant genotypes selected for more generalist viruses, while more permissive genotypes selected for more specialist viruses. Sequencing the genomes of the evolved viral lineages, we found that selection targeted the multifunctional genome-linked protein VPg in most host genotypes. Overall, this work illustrates how different host defenses modulate the rates and extent of virus evolution.

A genome-wide association study identifies Arabidopsis thaliana genes that contribute to differences in the outcome of infection with two Turnip mosaic potyvirus strains that differ in their evolutionary history and degree of host specialization.

Viruses lie in a continuum between generalism and specialism depending on their ability to infect more or less hosts. While generalists are able to successfully infect a wide variety of hosts, specialists are limited to one or a few. Even though generalists seem to gain an advantage due to their wide host range, they usually pay a pleiotropic fitness cost within each host. On the contrary, a specialist has maximal fitness within its own host. A relevant yet poorly explored question is whether viruses differ in the way they interact with their hosts' gene expression depending on their degree of specialization. Using a genome-wide association study approach, we have identified host genes whose expression depends on whether hosts were infected with more or less specialized viral strains. Four hundred fifty natural accessions of Arabidopsis thaliana were inoculated with Turnip mosaic potyvirus strains with different past evolutionary histories and that shown different degrees of specialization. Three disease-related traits were measured and associated with different sets of host genes for each strain. The genetic architectures of these traits differed among viral strains and, in the case of the more specialized virus, also varied along the duration of infection. While most of the mapped loci were strain specific, one shared locus was mapped for both strains, a disease-resistance TIR-NBS-LRR class protein. Likewise, only putative cysteine-rich receptor-like protein kinases were involved in all three traits. The impact on disease progress of 10 selected genes was validated by studying the infection phenotypes of loss-of-function mutant plants. Nine of these mutants have altered the disease progress and/or symptoms intensity between both strains. Compared to wild-type plants six had an effect on both viral strains, three had an effect only on the more specialized, and two were significant during infection with the less specialized.

From foes to friends: Viral infections expand the limits of host phenotypic plasticity.

Phenotypic plasticity enables organisms to survive in the face of unpredictable environmental stress. Intimately related to the notion of phenotypic plasticity is the concept of the reaction norm that places phenotypic plasticity in the context of a genotype-specific response to environmental gradients. Whether reaction norms themselves evolve and which factors might affect their shape has been the object of intense debates among evolutionary biologists along the years. Since their discovery, viruses have been considered as pathogens. However, new viromic techniques and a shift in conceptual paradigms are showing that viruses are mostly non-pathogenic ubiquitous entities. Recent studies have shown how viral infections can even be beneficial for their hosts. This may happen especially in the context of stressed hosts, where the virus infection can induce beneficial changes in the host's physiological homeostasis, hence changing the shape of the reaction norm. Despite the fact that underlying physiological mechanisms and evolutionary dynamics are still not well understood, such beneficial interactions are being discovered in a growing number of plant-virus systems. Here, we aim to review these disperse studies and place them into the context of phenotypic plasticity and the evolution of reaction norms. This is an emerging field that is posing many questions that still need to be properly answered. The answers would clearly interest virologists, plant pathologists and evolutionary biologists and likely they will suggest possible future biotechnological applications, including the development of crops with higher survival rates and yield under adverse environmental situations.

Adaptation of turnip mosaic potyvirus to a specific niche reduces its genetic and environmental robustness.

Robustness is the preservation of the phenotype in the face of genetic and environmental perturbations. It has been argued that robustness must be an essential fitness component of RNA viruses owed to their small and compacted genomes, high mutation rates and living in ever-changing environmental conditions. Given that genetic robustness might hamper possible beneficial mutations, it has been suggested that genetic robustness can only evolve as a side-effect of the evolution of robustness mechanisms specific to cope with environmental perturbations, a theory known as plastogenetic congruence. However, empirical evidences from different viral systems are contradictory. To test how adaptation to a particular environment affects both environmental and genetic robustness, we have used two strains of turnip mosaic potyvirus (TuMV) that differ in their degree of adaptation to Arabidopsis thaliana at a permissive temperature. We show that the highly adapted strain is strongly sensitive to the effect of random mutations and to changes in temperature conditions. In contrast, the non-adapted strain shows more robustness against both the accumulation of random mutations and drastic changes in temperature conditions. Together, these results are consistent with the predictions of the plastogenetic congruence theory, suggesting that genetic and environmental robustnesses may be two sides of the same coin for TuMV.

Natural variation in Arabidopsis thaliana rosette area unveils new genes involved in plant development.

Growth is a complex trait influenced by multiple genes that act at different moments during the development of an organism. This makes it difficult to spot its underlying genetic mechanisms. Since plant growth is intimately related to the effective leaf surface area (ELSA), identifying genes controlling this trait will shed light on our understanding of plant growth. To find new genes with a significant contribution to plant growth, here we used the natural variation in Arabidopsis thaliana to perform a genome-wide association study of ELSA. To do this, the projected rosette area of 710 worldwide distributed natural accessions was measured and analyzed using the genome-wide efficient mixed model association algorithm. From this analysis, ten genes were identified having SNPs with a significant association with ELSA. To validate the implication of these genes into A. thaliana growth, six of them were further studied by phenotyping knock-out mutant plants. It was observed that rem1.2, orc1a, ppd1, and mcm4 mutants showed different degrees of reduction in rosette size, thus confirming the role of these genes in plant growth. Our study identified genes already known to be involved in plant growth but also assigned this role, for the first time, to other genes.

Role of host genetic diversity for susceptibility-to-infection in the evolution of virulence of a plant virus.

Predicting viral emergence is difficult due to the stochastic nature of the underlying processes and the many factors that govern pathogen evolution. Environmental factors affecting the host, the pathogen and the interaction between both are key in emergence. In particular, infectious disease dynamics are affected by spatiotemporal heterogeneity in their environments. A broad knowledge of these factors will allow better estimating where and when viral emergence is more likely to occur. Here, we investigate how the population structure for susceptibility-to-infection genes of the plant Arabidopsis thaliana shapes the evolution of Turnip mosaic virus (TuMV). For doing so we have evolved TuMV lineages in two radically different host population structures: (1) a metapopulation subdivided into six demes (subpopulations); each one being composed of individuals from only one of six possible A. thaliana ecotypes and (2) a well-mixed population constituted by equal number of plants from the same six A. thaliana ecotypes. These two populations were evolved for twelve serial passages. At the end of the experimental evolution, we found faster adaptation of TuMV to each ecotype in the metapopulation than in the well-mixed heterogeneous host populations. However, viruses evolved in well-mixed populations were more pathogenic and infectious than viruses evolved in the metapopulation. Furthermore, the viruses evolved in the demes showed stronger signatures of local specialization than viruses evolved in the well-mixed populations. These results illustrate how the genetic diversity of hosts in an experimental ecosystem favors the evolution of virulence of a pathogen.

Engineered Functional Redundancy Relaxes Selective Constraints upon Endogenous Genes in Viral RNA Genomes.

Functional redundancy, understood as the functional overlap of different genes, is a double-edge sword. At the one side, it is thought to serve as a robustness mechanism that buffers the deleterious effect of mutations hitting one of the redundant copies, thus resulting in pseudogenization. At the other side, it is considered as a source of genetic and functional innovation. In any case, genetically redundant genes are expected to show an acceleration in the rate of molecular evolution. Here, we tackle the role of functional redundancy in viral RNA genomes. To this end, we have evaluated the rates of compensatory evolution for deleterious mutations affecting an essential function, the suppression of RNA silencing plant defense, of tobacco etch potyvirus (TEV). TEV genotypes containing deleterious mutations in presence/absence of engineered functional redundancy were evolved and the pattern of fitness and pathogenicity recovery evaluated. Genetically redundant genotypes suffered less from the effect of deleterious mutations and showed relatively minor changes in fitness and pathogenicity. By contrast, nongenetically redundant genotypes had very low fitness and pathogenicity at the beginning of the evolution experiment that were fully recovered by the end. At the molecular level, the outcome depended on the combination of the actual mutations being compensated and the presence/absence of functional redundancy. Reversions to wild-type alleles were the norm in the nonredundant genotypes while redundant ones either did not fix any mutation at all or showed a higher nonsynonymous mutational load.