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.

Endogenous viral elements are targeted by RNA silencing pathways in banana.

Endogenous banana streak virus (eBSV) integrants derived from three distinct species, present in Musa balbisiana (B) but not Musa acuminata (A) banana genomes are able to reconstitute functional episomal viruses causing banana streak disease in interspecific triploid AAB banana hybrids but not in the diploid (BB) parent line, which harbours identical eBSV loci. Here, we investigated the regulation of these eBSV. In-depth characterization of siRNAs, transcripts and methylation derived from eBSV using Illumina and bisulfite sequencing were carried out on eBSV-free Musa acuminata AAA plants and BB or AAB banana plants with eBSV. eBSV loci produce low-abundance transcripts covering most of the viral sequence and generate predominantly 24-nt siRNAs. siRNA accumulation is restricted to duplicated and inverted viral sequences present in eBSV. Both siRNA-accumulating and nonaccumulating sequences of eBSV in BB plants are heavily methylated in all three CG, CHG and CHH contexts. Our data suggest that eBSVs are controlled at the epigenetic level in BB diploids. This regulation not only prevents their awakening and systemic infection of the plant but is also probably involved in the inherent resistance of the BB plants to mealybug-transmitted viral infection. These findings are thus of relevance to other plant resources hosting integrated viruses.

Extrachromosomal viral DNA produced by transcriptionally active endogenous viral elements in non-infected banana hybrids impedes quantitative PCR diagnostics of banana streak virus infections in banana hybrids.

The main edible and cultivated banana varieties are intra- and interspecific hybrids of the two main Musa species, Musa acuminata and Musa balbisiana, having diploid genomes denoted A and B, respectively. The B genome naturally hosts sequences of banana streak virus (BSV) named endogenous BSV (eBSV). Upon stress, eBSVs are identified as the origin of BSV infection for at least three BSV species, causing banana streak disease. For each of the three species, BSV and eBSV share >99.9 % sequence identity, complicating PCR-based diagnosis of viral infection in the B genome-containing bananas. Here, we designed a quantitative PCR-based method to only quantify episomal BSV particles produced, overcoming the limitation of eBSV also being detected by qPCR by using it as a 'calibrator'. However, our results revealed unexpected variation of eBSV amplification in calibrator plants composed of a clonal population of 53 replicating virus-free banana hybrids with the same AAB genotype. Our in-depth molecular analyses suggest that this calibrator variation is due to the variable abundance of non-encapsidated extrachromosomal viral DNA, likely produced via the transcription of eBSVs, followed by occasional reverse transcription. We also present evidence that accumulation of viral transcripts in AAB plants is downregulated both at post-transcriptional and transcriptional levels by an RNA interference mechanism that keeps the plants free of virus infection. Finally, we recommend that such eBSV amplification variation be taken into account to establish a quantitative viral diagnostic for banana plants with the B genome.

How endogenous plant pararetroviruses shed light on Musa evolution.

BACKGROUND AND AIMS: Banana genomes harbour numerous copies of viral sequences derived from banana streak viruses (BSVs) - dsDNA viruses belonging to the family Caulimoviridae.These viral integrants (eBSVs) are mostly defective, probably as a result of 'pseudogenization' driven by host genome evolution. However, some can give rise to infection by releasing a functional viral genome following abiotic stresses. These distinct infective eBSVs correspond to the three main widespread BSV species (BSOLV, BSGFV and BSIMV), fully described within the Musa balbisiana B genomes of the seedy diploid 'Pisang Klutuk Wulung' (PKW). METHODS: We characterize eBSV distribution among a Musa sampling including seedy BB diploids and interspecific hybrids with Musa acuminate exhibiting different levels of ploidy for the B genome (ABB, AAB, AB). We used representative samples of the two areas of sympatry between M. acuminate and M. balbisiana species representing the native area of the most widely cultivated AAB cultivars (in India and in East Asia, ranging from the Philippines to New Guinea). Seventy-seven accessions were characterized using eBSV-related PCR markers and Southern hybridization approaches. We coded both sets of results to create a common dissimilarity matrix with which to interpret eBSV distribution. KEY RESULTS: We propose a Musa phylogeny driven by the M. balbisiana genome based on a dendrogram resulting from a joint neighbour-joining analysis of the three BSV species, showing for the first time lineages between BB and ABB/AAB hybrids. eBSVs appear to be relevant phylogenetic markers that can illustrate theM. balbisiana phylogeography story. CONCLUSION: The theoretical implications of this study for further elucidation of the historical and geographical process of Musa domestication are numerous. Discovery of banana plants with B genome non-infective for eBSV opens the way to the introduction of new genitors in programmes of genetic banana improvement.

Evasion of short interfering RNA-directed antiviral silencing in Musa acuminata persistently infected with six distinct banana streak pararetroviruses.

UNLABELLED: Vegetatively propagated crop plants often suffer from infections with persistent RNA and DNA viruses. Such viruses appear to evade the plant defenses that normally restrict viral replication and spread. The major antiviral defense mechanism is based on RNA silencing generating viral short interfering RNAs (siRNAs) that can potentially repress viral genes posttranscriptionally through RNA cleavage and transcriptionally through DNA cytosine methylation. Here we examined the RNA silencing machinery of banana plants persistently infected with six pararetroviruses after many years of vegetative propagation. Using deep sequencing, we reconstructed consensus master genomes of the viruses and characterized virus-derived and endogenous small RNAs. Consistent with the presence of endogenous siRNAs that can potentially establish and maintain DNA methylation, the banana genomic DNA was extensively methylated in both healthy and virus-infected plants. A novel class of abundant 20-nucleotide (nt) endogenous small RNAs with 5'-terminal guanosine was identified. In all virus-infected plants, 21- to 24-nt viral siRNAs accumulated at relatively high levels (up to 22% of the total small RNA population) and covered the entire circular viral DNA genomes in both orientations. The hotspots of 21-nt and 22-nt siRNAs occurred within open reading frame (ORF) I and II and the 5' portion of ORF III, while 24-nt siRNAs were more evenly distributed along the viral genome. Despite the presence of abundant viral siRNAs of different size classes, the viral DNA was largely free of cytosine methylation. Thus, the virus is able to evade siRNA-directed DNA methylation and thereby avoid transcriptional silencing. This evasion of silencing likely contributes to the persistence of pararetroviruses in banana plants. IMPORTANCE: We report that DNA pararetroviruses in Musa acuminata banana plants are able to evade DNA cytosine methylation and transcriptional gene silencing, despite being targeted by the host silencing machinery generating abundant 21- to 24-nucleotide short interfering RNAs. At the same time, the banana genomic DNA is extensively methylated in both healthy and virus-infected plants. Our findings shed light on the siRNA-generating gene silencing machinery of banana and provide a possible explanation why episomal pararetroviruses can persist in plants whereas true retroviruses with an obligatory genome-integration step in their replication cycle do not exist in plants.

Three infectious viral species lying in wait in the banana genome.

Plant pararetroviruses integrate serendipitously into their host genomes. The banana genome harbors integrated copies of banana streak virus (BSV) named endogenous BSV (eBSV) that are able to release infectious pararetrovirus. In this investigation, we characterized integrants of three BSV species-Goldfinger (eBSGFV), Imove (eBSImV), and Obino l'Ewai (eBSOLV)-in the seedy Musa balbisiana Pisang klutuk wulung (PKW) by studying their molecular structure, genomic organization, genomic landscape, and infectious capacity. All eBSVs exhibit extensive viral genome duplications and rearrangements. eBSV segregation analysis on an F1 population of PKW combined with fluorescent in situ hybridization analysis showed that eBSImV, eBSOLV, and eBSGFV are each present at a single locus. eBSOLV and eBSGFV contain two distinct alleles, whereas eBSImV has two structurally identical alleles. Genotyping of both eBSV and viral particles expressed in the progeny demonstrated that only one allele for each species is infectious. The infectious allele of eBSImV could not be identified since the two alleles are identical. Finally, we demonstrate that eBSGFV and eBSOLV are located on chromosome 1 and eBSImV is located on chromosome 2 of the reference Musa genome published recently. The structure and evolution of eBSVs suggest sequential integration into the plant genome, and haplotype divergence analysis confirms that the three loci display differential evolution. Based on our data, we propose a model for BSV integration and eBSV evolution in the Musa balbisiana genome. The mutual benefits of this unique host-pathogen association are also discussed.

A new genus of alphasatellites associated with banana bunchy top virus in Southeast Asia.

Autonomously replicating alphasatellites (family Alphasatellitidae) are frequently associated with plant single-stranded (ss)DNA viruses of the families Geminiviridae, Metaxyviridae, and Nanoviridae. Alphasatellites encode a single replication-initiator protein (Rep) similar to Rep proteins of helper viruses and depend on helper viruses for encapsidation, movement, and transmission. Costs versus benefits of alphasatellite-helper virus association are poorly understood. Our surveys in Southeast Asia (SEA) for wild and cultivated banana plants infected with banana bunchy top virus (BBTV, Nanoviridae) and Illumina sequencing reconstruction of their viromes revealed, in addition to a six-component BBTV genome, one to three distinct alphasatellites present in sixteen of twenty-four BBTV-infected plants. Comparative nucleotide and Rep protein sequence analyses classified these alphasatellites into four distinct species: two known species falling into the genus Muscarsatellite (subfamily Petromoalphasatellitinae) previously identified in SEA and two novel species falling into the tentative genus Banaphisatellite (subfamily Nanoalphasatellitinae) so far containing a single species recently identified in Africa. The banaphisatellites were found to be most related to members of the genus Fabenesatellite of subfamily Nanoalphasatellitinae and the genus Gosmusatellite of subfamily Geminialphasatellitinae, both infecting dicots. This suggests a dicot origin of banaphisatellites that got independently associated with distinct strains of monocot-infecting BBTV in Africa and SEA. Analysis of conserved sequence motifs in the common regions driving replication and gene expression of alphasatellites and BBTV strains revealed both differences and similarities, pointing at their ongoing co-evolution. An impact of alphasatellites on BBTV infection and evasion of RNA interference-based antiviral defences was evaluated by measuring relative abundance of BBTV genome components and alphasatellites and by profiling BBTV- and alphasatellite-derived small interfering RNAs. Taken together, our findings shed new light on the provenance of alphasatellites, their co-evolution with helper viruses, and potential mutual benefits of their association.

Evidence for Dicot Plants as Alternative Hosts of Banana Bunchy Top Virus and Its Alphasatellites in South-East Asia.

Banana bunchy top virus is a multicomponent circular ssDNA virus (family Nanoviridae) that causes one of the most devastating diseases of cultivated bananas and plantains (family Musaceae). It is transmitted by the aphids Pentalonia nigronervosa and P. caladii among host plants of Musaceae and some other families of monocots. Our Illumina sequencing reconstruction of virome components of BBTV-infected banana plants and their neighbor non-banana plants sampled in Vietnam and Laos revealed the monocot Commelina sp. (Commelinaceae) and the dicots Bidens pilosa and Chromolaena odorata (both Asteraceae) as hosts of BBTV and circular ssDNA alphasatellites (family Alphasatellitidae). Counting the proportions and relative abundances of Illumina reads representing BBTV genome components and alphasatellites suggested that Chromolaena and Commelina are poor hosts for BBTV and one to three alphasatellite species, whereas Bidens is a permissive host for BBTV and four alphasatellite species representing two genera of Alphasatellitidae. Our findings provide evidence for the dicot plants of family Asteraceae as alternative hosts of BBTV and its alphasatellites, which warrants further investigation of these and other dicots as a potential refuge and source of BBTV and multiple alphasatellites that become associated with this virus and likely affect its replication, transmission, and host range.

Evolution of endogenous sequences of banana streak virus: what can we learn from banana (Musa sp.) evolution?

Endogenous plant pararetroviruses (EPRVs) are viral sequences of the family Caulimoviridae integrated into the nuclear genome of numerous plant species. The ability of some endogenous sequences of Banana streak viruses (eBSVs) in the genome of banana (Musa sp.) to induce infections just like the virus itself was recently demonstrated (P. Gayral et al., J. Virol. 83:6697-6710, 2008). Although eBSVs probably arose from accidental events, infectious eBSVs constitute an extreme case of parasitism, as well as a newly described strategy for vertical virus transmission in plants. We investigated the early evolutionary stages of infectious eBSV for two distinct BSV species-GF (BSGFV) and Imové (BSImV)-through the study of their distribution, insertion polymorphism, and structure evolution among selected banana genotypes representative of the diversity of 60 wild Musa species and genotypes. To do so, the historical frame of host evolution was analyzed by inferring banana phylogeny from two chloroplast regions-matK and trnL-trnF-as well as from the nuclear genome, using 19 microsatellite loci. We demonstrated that both BSV species integrated recently in banana evolution, circa 640,000 years ago. The two infectious eBSVs were subjected to different selective pressures and showed distinct levels of rearrangement within their final structure. In addition, the molecular phylogenies of integrated and nonintegrated BSVs enabled us to establish the phylogenetic origins of eBSGFV and eBSImV.

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.

A four-partner plant­virus interaction: enemies can also come from within.

Plant viruses are disseminated by either vertical (vegetative multiplication or sexual reproduction) or horizontal (vector-mediated) propagation. Plant pararetroviruses—members of the Caulimoviridae family—have developed an alternative strategy for vertical propagation via integration within the host plant genome, although integration is not required for viral replication. Integrated endogenous pararetrovirus (EPRV) sequences have undergone extensive viral genome rearrangements and contain more than one copy of the viral genome. Furthermore, EPRV can become infectious upon spontaneous escape of active virus following stresses such as wounding, tissue culture, or interspecific crosses. Such infectious EPRV are of great importance, not only in terms of their ability to precipitate epidemic outbreaks but also because of their effect on breeding of numerous plant genomes in temperate and tropical crops. This is especially true for banana, a crop susceptible to banana streak viruses, the causative agents of banana streak disease. Thus, the classical three-component banana–Banana streak virus (BSV)–mealybug pathosystem can be expanded to include endogenous BSV as an alternative source of active virions. The BSV-banana pathosystem is one of only three pathosystems known to date to harbor this remarkable feature, and the present review focuses exclusively on it to illustrate this four-partner interaction.

Phylogeny of Banana Streak Virus reveals recent and repetitive endogenization in the genome of its banana host (Musa sp.).

Banana streak virus (BSV) is a plant dsDNA pararetrovirus (family Caulimoviridae, genus badnavirus). Although integration is not an essential step in the BSV replication cycle, the nuclear genome of banana (Musa sp.) contains BSV endogenous pararetrovirus sequences (BSV EPRVs). Some BSV EPRVs are infectious by reconstituting a functional viral genome. Recent studies revealed a large molecular diversity of episomal BSV viruses (i.e., nonintegrated) while others focused on BSV EPRV sequences only. In this study, the evolutionary history of badnavirus integration in banana was inferred from phylogenetic relationships between BSV and BSV EPRVs. The relative evolution rates and selective pressures (d(N)/d(S) ratio) were also compared between endogenous and episomal viral sequences. At least 27 recent independent integration events occurred after the divergence of three banana species, indicating that viral integration is a recent and frequent phenomenon. Relaxation of selective pressure on badnaviral sequences that experienced neutral evolution after integration in the plant genome was recorded. Additionally, a significant decrease (35%) in the EPRV evolution rate was observed compared to BSV, reflecting the difference in the evolution rate between episomal dsDNA viruses and plant genome. The comparison of our results with the evolution rate of the Musa genome and other reverse-transcribing viruses suggests that EPRVs play an active role in episomal BSV diversity and evolution.

A single Banana streak virus integration event in the banana genome as the origin of infectious endogenous pararetrovirus.

Sequencing of plant nuclear genomes reveals the widespread presence of integrated viral sequences known as endogenous pararetroviruses (EPRVs). Banana is one of the three plant species known to harbor infectious EPRVs. Musa balbisiana carries integrated copies of Banana streak virus (BSV), which are infectious by releasing virions in interspecific hybrids. Here, we analyze the organization of the EPRV of BSV Goldfinger (BSGfV) present in the wild diploid M. balbisiana cv. Pisang Klutuk Wulung (PKW) revealed by the study of Musa bacterial artificial chromosome resources and interspecific genetic cross. cv. PKW contains two similar EPRVs of BSGfV. Genotyping of these integrants and studies of their segregation pattern show an allelic insertion. Despite the fact that integrated BSGfV has undergone extensive rearrangement, both EPRVs contain the full-length viral genome. The high degree of sequence conservation between the integrated and episomal form of the virus indicates a recent integration event; however, only one allele is infectious. Analysis of BSGfV EPRV segregation among an F1 population from an interspecific genetic cross revealed that these EPRV sequences correspond to two alleles originating from a single integration event. We describe here for the first time the full genomic and genetic organization of the two EPRVs of BSGfV present in cv. PKW in response to the challenge facing both scientists and breeders to identify and generate genetic resources free from BSV. We discuss the consequences of this unique host-pathogen interaction in terms of genetic and genomic plant defenses versus strategies of infectious BSGfV EPRVs.

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.

Improved detection of episomal Banana streak viruses by multiplex immunocapture PCR.

Banana streak viruses (BSV) are currently the main viral constraint to Musa germplasm movement, genetic improvement and mass propagation. Therefore, it is necessary to develop and implement BSV detection strategies that are both reliable and sensitive, such as PCR-based techniques. Unfortunately, BSV endogenous pararetrovirus sequences (BSV EPRVs) are present in the genome of Musa balbisiana. They interfere with PCR-based detection of episomal BSV in infected banana and plantain, such as immunocapture PCR. Therefore, a multiplex, immunocapture PCR (M-IC-PCR) was developed for the detection of BSV. Musa sequence tagged microsatellite site (STMS) primers were selected and used in combination with BSV species-specific primers in order to monitor possible contamination by Musa genomic DNA, using multiplex PCR. Furthermore, immunocapture conditions were optimized in order to prevent Musa DNA from interfering with episomal BSV DNA during the PCR step. This improved detection method successfully allowed the accurate, specific and sensitive detection of episomal DNA only from distinct BSV species. Its implementation should benefit PCR-based detection of viruses for which homologous sequences are present in the genome of their hosts, including transgenic plants expressing viral sequences.

Biology, etiology, and control of virus diseases of banana and plantain.

Banana and plantain (Musa spp.), produced in 10.3 million ha in the tropics, are among the world's top 10 food crops. They are vegetatively propagated using suckers or tissue culture plants and grown almost as perennial plantations. These are prone to the accumulation of pests and pathogens, especially viruses which contribute to yield reduction and are also barriers to the international exchange of germplasm. The most economically important viruses of banana and plantain are Banana bunchy top virus (BBTV), a complex of banana streak viruses (BSVs) and Banana bract mosaic virus (BBrMV). BBTV is known to cause the most serious economic losses in the "Old World," contributing to a yield reduction of up to 100% and responsible for a dramatic reduction in cropping area. The BSVs exist as episomal and endogenous forms are known to be worldwide in distribution. In India and the Philippines, BBrMV is known to be economically important but recently the virus was discovered in Colombia and Costa Rica, thus signaling its spread into the "New World." Banana and plantain are also known to be susceptible to five other viruses of minor significance, such as Abaca mosaic virus, Abaca bunchy top virus, Banana mild mosaic virus, Banana virus X, and Cucumber mosaic virus. Studies over the past 100 years have contributed to important knowledge on disease biology, distribution, and spread. Research during the last 25 years have led to a better understanding of the virus-vector-host interactions, virus diversity, disease etiology, and epidemiology. In addition, new diagnostic tools were developed which were used for surveillance and the certification of planting material. Due to a lack of durable host resistance in the Musa spp., phytosanitary measures and the use of virus-free planting material are the major methods of virus control. The state of knowledge on BBTV, BBrMV, and BSVs, and other minor viruses, disease spread, and control are summarized in this review.

A possible scenario for the evolution of Banana streak virus in banana.

Outbreaks of Banana streak virus (BSV) have been recorded worldwide where Musa spp. is grown during the last 20 years with no convincing evidence of epidemics. Epidemics were previously reported in Uganda where BSV is currently endemic. BSV is a plant pararetrovirus of the family Caulimoviridae, genus Badnavirus it causes chlorosis leaf streak disease. The information currently available on banana streak disease makes it possible to identify a complex of distinct BSV species each causing the same disease. BSV exists in two states: one as an episomal form, infecting plant cells; the other as viral DNA integrated within the B genome of banana (endogenous BSV-eBSV) forming a viral genome for de novo viral particles. Both forms can be infectious in banana plants. The BSV phylogeny is polyphyletic with BSV distributed in two clades. Clade 1 clusters BSV species that occur worldwide and may have an eBSV counterpart, whereas Clade 3 only comprises BSV species from Uganda. Clearly, two distinct origins explain such BSV diversity. However, the epidemiology/outbreaks of BSV remains unclear and the role of eBSV needs to be clarified. In this review, the biodiversity of BSV is explained and discussed in the light of field and molecular epidemiology data. A scheme is proposed for the co-evolution of BSV and banana based on old or recent infection hypotheses related to African domestication sites and banana dissemination to explain the disease context.

The common evolutionary history of badnaviruses and banana.

Recent plant genome sequencing efforts have revealed myriad viral sequences suggesting a cryptic interaction between both partners. Interestingly, no integration step has ever been reported as an obligatory step in the life cycle of plant viruses. Circular dsDNA viruses belonging to the family Caulimoviridae are the most abundant among integrated plant viral sequences. In this review, we describe how this hitherto hidden interaction could inform the evolutionary history of both partners badnaviruses and banana plants.

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.