Virome release of an invasive exotic plant species in southern France.

The increase in human-mediated introduction of plant species to new regions has resulted in a rise of invasive exotic plant species (IEPS) that has had significant effects on biodiversity and ecosystem processes. One commonly accepted mechanism of invasions is that proposed by the enemy release hypothesis (ERH), which states that IEPS free from their native herbivores and natural enemies in new environments can outcompete indigenous species and become invasive. We here propose the virome release hypothesis (VRH) as a virus-centered variant of the conventional ERH that is only focused on enemies. The VRH predicts that vertically transmitted plant-associated viruses (PAV, encompassing phytoviruses and mycoviruses) should be co-introduced during the dissemination of the IEPS, while horizontally transmitted PAV of IEPS should be left behind or should not be locally transmitted in the introduced area due to a maladaptation of local vectors. To document the VRH, virome richness and composition as well as PAV prevalence, co-infection, host range, and transmission modes were compared between indigenous plant species and an invasive grass, cane bluestem (Bothriochloa barbinodis), in both its introduced range (southern France) and one area of its native range (Sonoran Desert, Arizona, USA). Contrary to the VRH, we show that invasive populations of B. barbinodis in France were not associated with a lower PAV prevalence or richness than native populations of B. barbinodis from the USA. However, comparison of virome compositions and network analyses further revealed more diverse and complex plant-virus interactions in the French ecosystem, with a significant richness of mycoviruses. Setting mycoviruses apart, only one putatively vertically transmitted phytovirus (belonging to the Amalgaviridae family) and one putatively horizontally transmitted phytovirus (belonging to the Geminiviridae family) were identified from B. barbinodis plants in the introduced area. Collectively, these characteristics of the B. barbinodis-associated PAV community in southern France suggest that a virome release phase may have immediately followed the introduction of B. barbinodis to France in the 1960s or 1970s, and that, since then, the invasive populations of this IEPS have already transitioned out of this virome release phase, and have started interacting with several local mycoviruses and a few local plant viruses.

A newly emerging alphasatellite affects banana bunchy top virus replication, transcription, siRNA production and transmission by aphids.

Banana bunchy top virus (BBTV) is a six-component ssDNA virus (genus Babuvirus, family Nanoviridae) transmitted by aphids, infecting monocots (mainly species in the family Musaceae) and likely originating from South-East Asia where it is frequently associated with self-replicating alphasatellites. Illumina sequencing analysis of banana aphids and leaf samples from Africa revealed an alphasatellite that should be classified in a new genus, phylogenetically related to alphasatellites of nanoviruses infecting dicots. Alphasatellite DNA was encapsidated by BBTV coat protein and accumulated at high levels in plants and aphids, thereby reducing helper virus loads, altering relative abundance (formula) of viral genome components and interfering with virus transmission by aphids. BBTV and alphasatellite clones infected dicot Nicotiana benthamiana, followed by recovery and symptomless persistence of alphasatellite, and BBTV replication protein (Rep), but not alphasatellite Rep, induced leaf chlorosis. Transcriptome sequencing revealed 21, 22 and 24 nucleotide small interfering (si)RNAs covering both strands of the entire viral genome, monodirectional Pol II transcription units of viral mRNAs and pervasive transcription of each component and alphasatellite in both directions, likely generating double-stranded precursors of viral siRNAs. Consistent with the latter hypothesis, viral DNA formulas with and without alphasatellite resembled viral siRNA formulas but not mRNA formulas. Alphasatellite decreased transcription efficiency of DNA-N encoding a putative aphid transmission factor and increased relative siRNA production rates from Rep- and movement protein-encoding components. Alphasatellite itself spawned the most abundant siRNAs and had the lowest mRNA transcription rate. Collectively, following African invasion, BBTV got associated with an alphasatellite likely originating from a dicot plant and interfering with BBTV replication and transmission. Molecular analysis of virus-infected banana plants revealed new features of viral DNA transcription and siRNA biogenesis, both affected by alphasatellite. Costs and benefits of alphasatellite association with helper viruses are discussed.

Genome characterization and diversity of trifolium virus 1: identification of a novel legume-infecting capulavirus.

A novel geminivirus was identified in France and Spain in asymptomatic plants of white clover (Trifolium repens) and shrub medick (Medicago arborea). Its genome has the hallmarks of a capulavirus, and its relationship to other capulaviruses was confirmed by phylogenetic analysis. White clover isolates formed a tight cluster in the phylogenetic tree, while shrub medick isolates formed two distinct, more divergent groups with sequence identity values close to the species cutoff. These three groups have likely participated in recombination events involving alfalfa leaf curl virus and French bean severe leaf curl virus. The name "trifolium virus 1" (TrV1) is proposed for this new Capulavirus. Three TrV1 genotypes (TrV1-A, TrV1-B, and TrV1-C) were clearly distinguished.

Badnaviruses and banana genomes: a long association sheds light on Musa phylogeny and origin.

Badnaviruses are double-stranded DNA pararetroviruses of the family Caulimoviridae. Badnaviral sequences found in banana are distributed over three main clades of the genus Badnavirus and exhibit wide genetic diversity. Interestingly, the nuclear genome of many plants, including banana, is invaded by numerous badnaviral sequences although badnaviruses do not require an integration step to replicate, unlike animal retroviruses. Here, we confirm that banana streak viruses (BSVs) are restricted to clades 1 and 3. We also show that only BSVs from clade 3 encompassing East African viral species are not integrated into Musa genomes, unlike BSVs from clade 1. Finally, we demonstrate that sequences from clade 2 are definitively integrated into Musa genomes with no evidence of episomal counterparts; all are phylogenetically distant from BSVs known to date. Using different molecular approaches, we dissected the coevolution between badnaviral sequences of clade 2 and banana by comparing badnavirus integration patterns across a banana sampling representing major Musa speciation events. Our data suggest that primary viral integrations occurred millions of years ago in banana genomes under different possible scenarios. Endogenous badnaviral sequences can be used as powerful markers to better characterize the Musa phylogeny, narrowing down the likely geographical origin of the Musa ancestor.

Nanopore-based detection and characterization of yam viruses.

We here assessed the capability of the MinION sequencing approach to detect and characterize viruses infecting a water yam plant. This sequencing platform consistently revealed the presence of several plant virus species, including Dioscorea bacilliform virus, Yam mild mosaic virus and Yam chlorotic necrosis virus. A potentially novel ampelovirus was also detected by a complimentary Illumina sequencing approach. The full-length genome sequence of yam chlorotic necrosis virus was determined using Sanger sequencing, which enabled determination of the coverage and sequencing accuracy of the MinION technology. Whereas the total mean sequencing error rate of yam chlorotic necrosis virus-related MinION reads was 11.25%, we show that the consensus sequence obtained either by de novo assembly or after mapping the MinION reads on the virus genomic sequence was >99.8% identical with the Sanger-derived reference sequence. From the perspective of potential plant disease diagnostic applications of MinION sequencing, these degrees of sequencing accuracy demonstrate that the MinION approach can be used to both reliably detect and accurately sequence nearly full-length positive-sense single-strand polyadenylated RNA plant virus genomes.

The Westward Journey of Alfalfa Leaf Curl Virus.

Alfalfa leaf curl virus (ALCV), which causes severe disease symptoms in alfalfa (Medicago sativa L.) and is transmitted by the widespread aphid species, Aphis craccivora Koch, has been found throughout the Mediterranean basin as well as in Iran and Argentina. Here we reconstruct the evolutionary history of ALCV and attempt to determine whether the recent discovery and widespread detection of ALCV is attributable either to past diagnostic biases or to the emergence and global spread of the virus over the past few years. One hundred and twenty ALCV complete genome sequences recovered from ten countries were analyzed and four ALCV genotypes (ALCV-A, ALCV-B, ALCV-C, and ALCV-D) were clearly distinguished. We further confirm that ALCV isolates are highly recombinogenic and that recombination has been a major determinant in the origins of the various genotypes. Collectively, the sequence data support the hypothesis that, of all the analyzed locations, ALCV likely emerged and diversified in the Middle East before spreading to the western Mediterranean basin and Argentina.

From Spatial Metagenomics to Molecular Characterization of Plant Viruses: A Geminivirus Case Study.

The number of plant viruses that are known likely remains only a vanishingly small fraction of all extant plant virus species. Consequently, the distribution and population dynamics of plant viruses within even the best-studied ecosystems have only ever been studied for small groups of virus species. Even for the best studied of these groups very little is known about virus diversity at spatial scales ranging from an individual host, through individual local host populations to global host populations. To date, metagenomics studies that have assessed the collective or metagenomes of viruses at the ecosystem scale have revealed many previously unrecognized viral species. More recently, novel georeferenced metagenomics approaches have been devised that can precisely link individual sequence reads to both the plant hosts from which they were obtained, and the spatial arrangements of these hosts. Besides illuminating the diversity and the distribution of plant viruses at the ecosystem scale, application of these "geometagenomics" approaches has enabled the direct testing of hypotheses relating to the impacts of host diversity, host spatial variations, and environmental conditions on plant virus diversity and prevalence. To exemplify how such top-down approaches can provide a far deeper understanding of host-virus associations, we provide a case-study focusing on geminiviruses within two complex ecosystems containing both cultivated and uncultivated areas. Geminiviruses are a highly relevant model for studying the evolutionary and ecological aspects of viral emergence because the family Geminiviridae includes many of the most important crop pathogens that have emerged over the past century. In addition to revealing unprecedented degrees of geminivirus diversity within the analyzed ecosystems, the geometagenomics-based approach enabled the focused in-depth analysis of the complex evolutionary dynamics of some of the highly divergent geminivirus species that were discovered.

Nanovirus-alphasatellite complex identified in Vicia cracca in the Rhône delta region of France.

Nanoviruses are multi-component plant-infecting single-stranded DNA viruses. Using a viral metagenomics-informed approach, a new nanovirus and two associated alphasatellite molecules have been identified in an uncultivated asymptomatic Vicia cracca plant in the Rhône region of France. This novel nanovirus genome includes eight genomic components (named DNA-R, DNA-S, DNA-M, DNA-C, DNA-N, DNA-U1, DNA-U2 and DNA-U4) and, across all components, shares < 66% pairwise sequence identity with other nanovirus genomes. The two associated alphasatellites share 62% identity with each other and < 81% identity will all other nanovirus-associated alphasatellites.

Complete genome sequences of cowpea polerovirus 1 and cowpea polerovirus 2 infecting cowpea plants in Burkina Faso.

The full-length genome sequences of two novel poleroviruses found infecting cowpea plants, cowpea polerovirus 1 (CPPV1) and cowpea polerovirus 2 (CPPV2), were determined using overlapping RT-PCR and RACE-PCR. Whereas the 5845-nt CPPV1 genome was most similar to chickpea chlorotic stunt virus (73% identity), the 5945-nt CPPV2 genome was most similar to phasey bean mild yellow virus (86% identity). The CPPV1 and CPPV2 genomes both have a typical polerovirus genome organization. Phylogenetic analysis of the inferred P1-P2 and P3 amino acid sequences confirmed that CPPV1 and CPPV2 are indeed poleroviruses. Four apparently unique recombination events were detected within a dataset of 12 full polerovirus genome sequences, including two events in the CPPV2 genome. Based on the current species demarcation criteria for the family Luteoviridae, we tentatively propose that CPPV1 and CPPV2 should be considered members of novel polerovirus species.

Molecular characterization and prevalence of two capulaviruses: Alfalfa leaf curl virus from France and Euphorbia caput-medusae latent virus from South Africa.

Little is known about the prevalence, diversity, evolutionary processes, genomic structures and population dynamics of viruses in the divergent geminivirus lineage known as the capulaviruses. We determined and analyzed full genome sequences of 13 Euphorbia caput-medusae latent virus (EcmLV) and 26 Alfalfa leaf curl virus (ALCV) isolates, and partial genome sequences of 23 EcmLV and 37 ALCV isolates. While EcmLV was asymptomatic in uncultivated southern African Euphorbia caput-medusae, severe alfalfa disease symptoms were associated with ALCV in southern France. The prevalence of both viruses exceeded 10% in their respective hosts. Besides using patterns of detectable negative selection to identify ORFs that are probably functionally expressed, we show that ALCV and EcmLV both display evidence of inter-species recombination and biologically functional genomic secondary structures. Finally, we show that whereas the EcmLV populations likely experience restricted geographical dispersion, ALCV is probably freely moving across the French Mediterranean region.

Alfalfa Leaf Curl Virus: an Aphid-Transmitted Geminivirus.

The family Geminiviridae comprises seven genera differentiated by genome organization, sequence similarity, and insect vector. Capulavirus, an eighth genus, has been proposed to accommodate two newly discovered highly divergent geminiviruses that presently have no known vector. Alfalfa leaf curl virus, identified here as a third capulavirus, is shown to be transmitted by Aphis craccivora. This is the first report of an aphid-transmitted geminivirus.

Metagenomics Approaches Based on Virion-Associated Nucleic Acids (VANA): An Innovative Tool for Assessing Without A Priori Viral Diversity of Plants.

This chapter describes an efficient approach that combines quality and yield extraction of viral nucleic acids from plants containing high levels of secondary metabolites and a sequence-independent amplification procedure for both the inventory of known plant viruses and the discovery of unknown ones. This approach turns out to be a useful tool for assessing the virome (the genome of all the viruses that inhabit a particular organism) of plants of interest. We here show that this approach enables the identification of a novel Potyvirus member within a single plant already known to be infected by two other Potyvirus species.

The genomes of many yam species contain transcriptionally active endogenous geminiviral sequences that may be functionally expressed.

Endogenous viral sequences are essentially 'fossil records' that can sometimes reveal the genomic features of long extinct virus species. Although numerous known instances exist of single-stranded DNA (ssDNA) genomes becoming stably integrated within the genomes of bacteria and animals, there remain very few examples of such integration events in plants. The best studied of these events are those which yielded the geminivirus-related DNA elements found within the nuclear genomes of various Nicotiana species. Although other ssDNA virus-like sequences are included within the draft genomes of various plant species, it is not entirely certain that these are not contaminants. The Nicotiana geminivirus-related DNA elements therefore remain the only definitively proven instances of endogenous plant ssDNA virus sequences. Here, we characterize two new classes of endogenous plant virus sequence that are also apparently derived from ancient geminiviruses in the genus Begomovirus. These two endogenous geminivirus-like elements (EGV1 and EGV2) are present in the Dioscorea spp. of the Enantiophyllum clade. We used fluorescence in situ hybridization to confirm that the EGV1 sequences are integrated in the D. alata genome and showed that one or two ancestral EGV sequences likely became integrated more than 1.4 million years ago during or before the diversification of the Asian and African Enantiophyllum Dioscorea spp. Unexpectedly, we found evidence of natural selection actively favouring the maintenance of EGV-expressed replication-associated protein (Rep) amino acid sequences, which clearly indicates that functional EGV Rep proteins were probably expressed for prolonged periods following endogenization. Further, the detection in D. alata of EGV gene transcripts, small 21-24 nt RNAs that are apparently derived from these transcripts, and expressed Rep proteins, provides evidence that some EGV genes are possibly still functionally expressed in at least some of the Enantiophyllum clade species.

Appearances can be deceptive: revealing a hidden viral infection with deep sequencing in a plant quarantine context.

Comprehensive inventories of plant viral diversity are essential for effective quarantine and sanitation efforts. The safety of regulated plant material exchanges presently relies heavily on techniques such as PCR or nucleic acid hybridisation, which are only suited to the detection and characterisation of specific, well characterised pathogens. Here, we demonstrate the utility of sequence-independent next generation sequencing (NGS) of both virus-derived small interfering RNAs (siRNAs) and virion-associated nucleic acids (VANA) for the detailed identification and characterisation of viruses infecting two quarantined sugarcane plants. Both plants originated from Egypt and were known to be infected with Sugarcane streak Egypt Virus (SSEV; Genus Mastrevirus, Family Geminiviridae), but were revealed by the NGS approaches to also be infected by a second highly divergent mastrevirus, here named Sugarcane white streak Virus (SWSV). This novel virus had escaped detection by all routine quarantine detection assays and was found to also be present in sugarcane plants originating from Sudan. Complete SWSV genomes were cloned and sequenced from six plants and all were found to share >91% genome-wide identity. With the exception of two SWSV variants, which potentially express unusually large RepA proteins, the SWSV isolates display genome characteristics very typical to those of all other previously described mastreviruses. An analysis of virus-derived siRNAs for SWSV and SSEV showed them to be strongly influenced by secondary structures within both genomic single stranded DNA and mRNA transcripts. In addition, the distribution of siRNA size frequencies indicates that these mastreviruses are likely subject to both transcriptional and post-transcriptional gene silencing. Our study stresses the potential advantages of NGS-based virus metagenomic screening in a plant quarantine setting and indicates that such techniques could dramatically reduce the numbers of non-intercepted virus pathogens passing through plant quarantine stations.

The genome of African yam (Dioscorea cayenensis-rotundata complex) hosts endogenous sequences from four distinct Badnavirus species.

Several endogenous viral elements (EVEs) have been identified in plant genomes, including endogenous pararetroviruses (EPRVs). Here, we report the first characterization of EPRV sequences in the genome of African yam of the Dioscorea cayenensis-rotundata complex. We propose that these sequences should be termed 'endogenous Dioscorea bacilliform viruses' (eDBVs). Molecular characterization of eDBVs shows that they constitute sequences originating from various parts of badnavirus genomes, resulting in a mosaic structure that is typical of most EPRVs characterized to date. Using complementary molecular approaches, we show that eDBVs belong to at least four distinct Badnavirus species, indicating multiple, independent, endogenization events. Phylogenetic analyses of eDBVs support and enrich the current taxonomy of yam badnaviruses and lead to the characterization of a new Badnavirus species in yam. The impact of eDBVs on diagnosis, yam germplasm conservation and movement, and breeding is discussed.

The prevalence of badnaviruses in West African yams (Dioscorea cayenensis-rotundata) and evidence of endogenous pararetrovirus sequences in their genomes.

Yam (Dioscorea spp.) is an important vegetatively-propagated staple crop in West Africa. Viruses are pervasive in yam worldwide, decreasing growth and yield, as well as hindering the international movement of germplasm. Badnaviruses have been reported to be the most prevalent in yam, and genomes of some other badnaviruses are known to be integrated in their host plant species. However, it was not clear if a similar scenario occurs in Dioscorea yam. This study was conducted to verify the prevalence of badnaviruses, and determine if badnavirus genomes are integrated in the yam genome. Leaf samples (n=58) representing eight species of yam from global yam collections kept at CIRAD, France, and 127 samples of D. rotundata breeding lines (n=112) and landraces (n=15) at IITA, Nigeria, were screened using generic badnavirus PCR primers. Positive amplification of an expected ca. 579bp fragment, corresponding to a partial RT-RNaseH region, was detected in 47 (81%) of 58 samples analysed from CIRAD collections, and 100% of the 127 IITA D. rotundata samples. All the D. cayenensis and D. rotundata samples from the CIRAD and IITA collections tested PCR-positive, and sequencing of a selection of the PCR products confirmed they were typical of the genus Badnavirus. A comparison of serological and nucleic acid techniques was used to investigate whether the PCR-positives were sequences amplified from badnavirus particles or putative endogenous badnavirus sequences in the yam genome. Protein A sandwich-enzyme-linked immunosorbent assay (PAS-ELISA) with badnavirus polyclonal antisera detected cross-reacting viral particles in only 60% (92 of 153) of the CIRAD collection samples analysed, in contrast to the aforementioned 81% by PCR. Immunosorbent electron microscopy (ISEM) of virus preparations of a select set of 16 samples, representing different combinations of positive and negative PCR and PAS-ELISA results, identified bacilliform particles in 11 of these samples. Three PCR-positive yam samples from Burkina Faso (cv. Pilimpikou) were identified in which no viral particles were detected by either PAS-ELISA or ISEM. Southern hybridisation results using a yam badnavirus RT-RNaseH sequence (Gn155Dr) as probe, supported a lack of badnavirus particles in the cv. Pilimpikou and identified their equivalent sequences to be of plant genome origin. Probe Gn155Dr, however, hybridised to viral particles and plant genomic DNA in three D. rotundata samples from Guinea. These results represent the first data demonstrating the presence of integrated sequences of badnaviruses in yam. The implications of this for virus-indexing, breeding and multiplication of seed yams are discussed.

Micropropagation by tissue culture triggers differential expression of infectious endogenous Banana streak virus sequences (eBSV) present in the B genome of natural and synthetic interspecific banana plantains.

The genome of Musa balbisiana spp. contains several infectious endogenous sequences of Banana streak virus (eBSV). We have shown previously that in vitro micropropagation triggers the activation of infectious eBSOLV (endogenous sequences of Banana streak Obino l'Ewai virus) in the synthetic tetraploid interspecific hybrid FHIA21 (AAAB). In this work, we show that another synthetic tetraploid (AAAB) hybrid and two natural triploid (AAB) plantains are equally prone to the activation of infectious eBSOLV during tissue culture. These results are a strong indication that such activation is a general phenomenon in interspecific Musa cultivars, whether synthetic or natural. We also report the first in-depth study of the correlation between the duration of tissue culture and the level of activation of infectious eBSOLV, and show that specific and common activation patterns exist in these banana plants. We hypothesize that these patterns result from the concomitant activation of infectious eBSOLV and a decrease in the virus titre in neoformed plantlets, resulting from cell multiplication outcompeting virus replication. We provide experimental data supporting this hypothesis. No activation of infectious eBSGFV (endogenous sequences of Banana streak Goldfinger virus) by tissue culture was observed in the two natural AAB plantain cultivars studied here, whereas such activation occurred in the AAAB synthetic hybrid studied. We demonstrate that this differential activation does not result from differences in the structure of eBSGFV, as all banana genomes harbour eaBSGFV-7.

A reliable IC One-step RT-PCR method for the detection of BBrMV to ensure safe exchange of Musa germplasm.

An immunocapture (IC) One-step RT-PCR assay was developed to improve the detection of Banana bract mosaic virus (BBrMV) in single and bulked samples of banana plants. In this paper, an atypical strain of BBrMV was described, the BBrMV "Ref" strain, and we showed that detection with available BBrMV tools using ELISA and RT-PCR approaches was not reliable. Primer sets Bract N1/NR and N2/NR specific to BBrMV were designed and used in RT-PCR and IC-RT-PCR assays with two commercial kits that allow the RT and the PCR reactions to take place simultaneously in the same tube. The new assay enabled detection of BBrMV in leaf extract diluted up to 1 x 10(-10) and in bulked samples of 10 plants, and was proposed as a new international standard to index BBrMV.