Genetic Differentiation and Migration Fluxes of Viruses from Melon Crops and Crop Edge Weeds.

Weeds surrounding crops may act as alternative hosts, playing important epidemiological roles as virus reservoirs and impacting virus evolution. We used high-throughput sequencing to identify viruses in Spanish melon crops and plants belonging to three pluriannual weed species, Ecballium elaterium, Malva sylvestris, and Solanum nigrum, sampled at the edges of the crops. Melon and E. elaterium, both belonging to the family Cucurbitaceae, shared three virus species, whereas there was no virus species overlap between melon and the other two weeds. The diversity of cucurbit aphid-borne yellows virus (CABYV) and tomato leaf curl New Delhi virus (ToLCNDV), both in melon and E. elaterium, was further studied by amplicon sequencing. Phylogenetic and population genetics analyses showed that the CABYV population was structured by the host, identifying three sites in the CABYV RNA-dependent RNA polymerase under positive selection, perhaps reflecting host adaptation. The ToLCNDV population was much less diverse than the CABYV one, likely as a consequence of the relatively recent introduction of ToLCNDV in Spain. In spite of its low diversity, we identified geographical but no host differentiation for ToLCNDV. Potential virus migration fluxes between E. elaterium and melon plants were also analyzed. For CABYV, no evidence of migration between the populations of the two hosts was found, whereas important fluxes were identified between geographically distant subpopulations for each host. For ToLCNDV, in contrast, evidence of migration from melon to E. elaterium was found, but not the other way around. IMPORTANCE It has been reported that about half of the emerging diseases affecting plants are caused by viruses. Alternative hosts often play critical roles in virus emergence as virus reservoirs, bridging host species that are otherwise unconnected and/or favoring virus diversification. In spite of this, the viromes of potential alternative hosts remain largely unexplored. In the case of crops, pluriannual weeds at the crop edges may play these roles. Here, we took advantage of the power of high-throughput sequencing to characterize the viromes of three weed species frequently found at the edges of melon crops. We identified three viruses shared by melon and the cucurbit weed, with two of them being epidemiologically relevant for melon crops. Further genetic analyses showed that these two viruses had contrasting patterns of diversification and migration, providing an interesting example on the role that weeds may play in the ecology and evolution of viruses affecting crops.

How To Be a Successful Monopartite Begomovirus in a Bipartite-Dominated World: Emergence and Spread of Tomato Mottle Leaf Curl Virus in Brazil.

Begomoviruses are members of the family Geminiviridae, a large and diverse group of plant viruses characterized by a small circular single-stranded DNA genome encapsidated in twinned quasi-icosahedral virions. Cultivated tomato (Solanum lycopersicum L.) is particularly susceptible and is infected by >100 bipartite and monopartite begomoviruses worldwide. In Brazil, 25 tomato-infecting begomoviruses have been described, most of which are bipartite. Tomato mottle leaf curl virus (ToMoLCV) is one of the most important of these and was first described in the late 1990s but has not been fully characterized. Here, we show that ToMoLCV is a monopartite begomovirus with a genomic DNA similar in size and genome organization to those of DNA-A components of New World (NW) begomoviruses. Tomato plants agroinoculated with the cloned ToMoLCV genomic DNA developed typical tomato mottle leaf curl disease symptoms, thereby fulfilling Koch's postulates and confirming the monopartite nature of the ToMoLCV genome. We further show that ToMoLCV is transmitted by whiteflies, but not mechanically. Phylogenetic analyses placed ToMoLCV in a distinct and strongly supported clade with other begomoviruses from northeastern Brazil, designated the ToMoLCV lineage. Genetic analyses of the complete sequences of 87 ToMoLCV isolates revealed substantial genetic diversity, including five strain groups and seven subpopulations, consistent with a long evolutionary history. Phylogeographic models generated with partial or complete sequences predicted that the ToMoLCV emerged in northeastern Brazil >700 years ago, diversifying locally and then spreading widely in the country. Thus, ToMoLCV emerged well before the introduction of MEAM1 whiteflies, suggesting that the evolution of NW monopartite begomoviruses was facilitated by local whitefly populations and the highly susceptible tomato host. IMPORTANCE Worldwide, diseases of tomato caused by whitefly-transmitted geminiviruses (begomoviruses) cause substantial economic losses and a reliance on insecticides for management. Here, we describe the molecular and biological properties of tomato mottle leaf curl virus (ToMoLCV) from Brazil and establish that it is a NW monopartite begomovirus indigenous to northeastern Brazil. This answered a long-standing question regarding the genome of this virus, and it is part of an emerging group of these viruses in Latin America. This appears to be driven by widespread planting of the highly susceptible tomato and by local and exotic whiteflies. Our extensive phylogenetic studies placed ToMoLCV in a distinct strongly supported clade with other begomoviruses from northeastern Brazil and revealed new insights into the origin of Brazilian begomoviruses. The novel phylogeographic analysis indicated that ToMoLCV has had a long evolutionary history, emerging in northeastern Brazil >700 years ago. Finally, the tools used here (agroinoculation system and ToMoLCV-specific PCR test) and information on the biology of the virus (host range and whitefly transmission) will be useful in developing and implementing integrated pest management (IPM) programs targeting ToMoLCV.

Cnidoscolus mild mosaic virus: a new bipartite begomovirus isolated from Cnidoscolus urens in Brazil.

A novel bipartite begomovirus infecting Cnidoscolus urens (Euphorbiaceae) from Pernambuco State, Brazil, has been characterized. The complete DNA-A (2657 nt) and DNA-B (2622 nt) components of the viral isolates show the typical genome organization of New World bipartite begomoviruses. DNA-A of the isolates had the highest percentage of nucleotide sequence identity (88.6-88.9%) to cnidoscolus mosaic leaf deformation virus. Based on the current classification criteria for the genus Begomovirus, the virus infecting C. urens should be considered a new member of the genus, and the name "cnidoscolus mild mosaic virus" is proposed for the virus, and the name "Begomovirus caboniensis" is proposed for its species.

Contribution of historical herbarium small RNAs to the reconstruction of a cassava mosaic geminivirus evolutionary history.

Emerging viral diseases of plants are recognised as a growing threat to global food security. However, little is known about the evolutionary processes and ecological factors underlying the emergence and success of viruses that have caused past epidemics. With technological advances in the field of ancient genomics, it is now possible to sequence historical genomes to provide a better understanding of viral plant disease emergence and pathogen evolutionary history. In this context, herbarium specimens represent a valuable source of dated and preserved material. We report here the first historical genome of a crop pathogen DNA virus, a 90-year-old African cassava mosaic virus (ACMV), reconstructed from small RNA sequences bearing hallmarks of small interfering RNAs. Relative to tip-calibrated dating inferences using only modern data, those performed with the historical genome yielded both molecular evolution rate estimates that were significantly lower, and lineage divergence times that were significantly older. Crucially, divergence times estimated without the historical genome appeared in discordance with both historical disease reports and the existence of the historical genome itself. In conclusion, our study reports an updated time-frame for the history and evolution of ACMV and illustrates how the study of crop viral diseases could benefit from natural history collections.

Genomic properties of allamanda leaf mottle distortion virus, a new begomovirus from golden trumpet (Allamanda cathartica) in India.

Golden trumpet (Allamanda cathartica) plants were observed to exhibit mottling and distortion symptoms on leaves. The genome of an associated begomovirus (Al-K1) was amplified by rolling-circle amplification, cloned, and sequenced. The viral genome consisted of two circular ssDNA molecules, and the organization of the ORFs was similar to those of DNA-A and DNA-B components of bipartite begomoviruses. The size of DNA-A (KC202818) and DNA-B (MG969497) of the begomovirus was 2772 and 2690 nucleotides, respectively. Sequence analysis revealed that the DNA-A and DNA-B components shared the highest sequence identity with duranta leaf curl virus (MN537564, 87.8%) and cotton leaf curl Alabad virus (MH760452, 81.0%), respectively. Interestingly, the Al-K1 isolate shared significantly less nucleotide sequence identity with allamanda leaf curl virus (EF602306, 71.6%), the only monopartite begomovirus reported previously in golden trumpet from China. Al-K1 shared less than 91% sequence identity with other begomoviruses, and hence, according to the latest ICTV guidelines for species demarcation of begomoviruses, Al-K1 is proposed to be a member of a new species, and we propose the name "allamanda leaf mottle distortion virus" (AllLMoDV-[IN-Al_K1-12]) for this virus. AllLMoDV was detected in various golden trumpet samples from different locations by PCR with specific primers based on the genome sequence determined in this study. Our study provides evidence of the occurrence of a new bipartite begomovirus in a perennial ornamental plant in India.

A New Type of Satellite Associated with Cassava Mosaic Begomoviruses.

Cassava mosaic disease (CMD), which is caused by single-stranded DNA begomoviruses, severely limits cassava production across Africa. A previous study showed that CMD symptom severity and viral DNA accumulation increase in cassava in the presence of a DNA sequence designated SEGS-2 (sequence enhancing geminivirus symptoms). We report here that when SEGS-2 is coinoculated with African cassava mosaic virus (ACMV) onto Arabidopsis thaliana, viral symptoms increase. Transgenic Arabidopsis with an integrated copy of SEGS-2 inoculated with ACMV also display increased symptom severity and viral DNA levels. Moreover, SEGS-2 enables Cabbage leaf curl virus (CaLCuV) to infect a geminivirus-resistant Arabidopsis thaliana accession. Although SEGS-2 is related to cassava genomic sequences, an earlier study showed that it occurs as episomes and is packaged into virions in CMD-infected cassava and viruliferous whiteflies. We identified SEGS-2 episomes in SEGS-2 transgenic Arabidopsis. The episomes occur as both double-stranded and single-stranded DNA, with the single-stranded form packaged into virions. In addition, SEGS-2 episomes replicate in tobacco protoplasts in the presence, but not the absence, of ACMV DNA-A. SEGS-2 episomes contain a SEGS-2 derived promoter and an open reading frame with the potential to encode a 75-amino acid protein. An ATG mutation at the beginning of the SEGS-2 coding region does not enhance ACMV infection in A. thaliana. Together, the results established that SEGS-2 is a new type of begomovirus satellite that enhances viral disease through the action of an SEGS-2-encoded protein that may also be encoded by the cassava genome. IMPORTANCE Cassava is an important root crop in the developing world and a food and income crop for more than 300 million African farmers. Cassava is rising in global importance and trade as the demands for biofuels and commercial starch increase. More than half of the world's cassava is produced in Africa, where it is primarily grown by smallholder farmers, many of whom are from the poorest villages. Although cassava can grow under high temperature, drought, and poor soil conditions, its production is severely limited by viral diseases. Cassava mosaic disease (CMD) is one of the most important viral diseases of cassava and can cause up to 100% yield losses. We provide evidence that SEGS-2, which was originally isolated from cassava crops displaying severe and atypical CMD symptoms in Tanzanian fields, is a novel begomovirus satellite that can compromise the development of durable CMD resistance.

Kinship networks of seed exchange shape spatial patterns of plant virus diversity.

By structuring farmers' informal networks of seed exchange, kinship systems play a key role in the dynamics of crop genetic diversity in smallholder farming systems. However, because many crop diseases are propagated through infected germplasm, local seed systems can also facilitate the dissemination of seedborne pathogens. Here, we investigate how the interplay of kinship systems and local networks of germplasm exchange influences the metapopulation dynamics of viruses responsible for the cassava mosaic disease (CMD), a major threat to food security in Africa. Combining anthropological, genetic and plant epidemiological data, we analyzed the genetic structure of local populations of the African cassava mosaic virus (ACMV), one of the main causal agents of CMD. Results reveal contrasted patterns of viral diversity in patrilineal and matrilineal communities, consistent with local modes of seed exchange. Our results demonstrate that plant virus ecosystems have also a cultural component and that social factors that shape regional seed exchange networks influence the genetic structure of plant virus populations.

Interspecies Recombination Has Driven the Macroevolution of Cassava Mosaic Begomoviruses.

Begomoviruses (family Geminiviridae, genus Begomovirus) significantly hamper crop production and threaten food security around the world. The frequent emergence of new begomovirus genotypes is facilitated by high mutation frequencies and the propensity to recombine and reassort. Homologous recombination has been especially implicated in the emergence of novel cassava mosaic begomovirus (CMB) genotypes, which cause cassava mosaic disease (CMD). Cassava (Manihot esculenta) is a staple food crop throughout Africa and an important industrial crop in Asia, two continents where production is severely constrained by CMD. The CMD species complex is comprised of 11 bipartite begomovirus species with ample distribution throughout Africa and the Indian subcontinent. While recombination is regarded as a frequent occurrence for CMBs, a revised, systematic assessment of recombination and its impact on CMB phylogeny is currently lacking. We assembled data sets of all publicly available, full-length DNA-A (n = 880) and DNA-B (n = 369) nucleotide sequences from the 11 recognized CMB species. Phylogenetic networks and complementary recombination detection methods revealed extensive recombination among the CMB sequences. Six out of the 11 species descended from unique interspecies recombination events. Estimates of recombination and mutation rates revealed that all species experience mutation more frequently than recombination, but measures of population divergence indicate that recombination is largely responsible for the genetic differences between species. Our results support that recombination has significantly impacted the CMB phylogeny and has driven speciation in the CMD species complex. IMPORTANCE Cassava mosaic disease (CMD) is a significant threat to cassava production throughout Africa and Asia. CMD is caused by a complex comprised of 11 recognized virus species exhibiting accelerated rates of evolution, driven by high frequencies of mutation and genetic exchange. Here, we present a systematic analysis of the contribution of genetic exchange to cassava mosaic virus species-level diversity. Most of these species emerged as a result of genetic exchange. This is the first study to report the significant impact of genetic exchange on speciation in a group of viruses.

Complete genome sequence of hollyhock vein yellowing virus, a novel monopartite begomovirus infecting hollyhock in Pakistan.

Hollyhock (Alcea rosea, family Malvaceae) is an ornamental plant grown widely in gardens across South Asia. In a bed of ornamental plants near the village of Chakri (Punjab Province, Pakistan) in 2014, hollyhock plants showing two distinct symptom types were identified: yellow vein mosaic and leaf crumple. PCR amplification with universal primers amplified a begomovirus from separate nucleic acid extracts of single plants of each type but amplified a betasatellite only from the plant with the yellow vein mosaic symptoms. No potential begomovirus DNA B component or alphasatellite could be identified in either sample. After cloning, the genome sequences of two viruses, one from a plant of each symptom type, were determined and shown to share 99.9% nucleotide sequence identity with each other but less than 91% nucleotide sequence identity with all previously characterized begomoviruses, with the highest identity (90%) to an isolate of pedilanthus leaf curl virus (PeLCV). This indicates that the two hollyhock plants were infected with a newly identified begomovirus for which the name "hollyhock vein yellowing virus" (HoVYV) is proposed. HoVYV likely has a recombinant origin. The betasatellite showed the highest nucleotide sequence identity to an isolate of cotton leaf curl Multan betasatellite (CLCuMuB), a betasatellite associated with cotton leaf curl disease across Pakistan and northwestern India. These findings add to the diversity of known begomoviruses in South Asia and again highlight the role of hollyhock as a reservoir of the cotton leaf curl begomovirus betasatellite complex. The results also suggest that the yellow vein mosaic symptoms in hollyhock are due to the betasatellite rather than the virus.

Identification of the Begomoviruses Squash Leaf Curl Virus and Watermelon Chlorotic Stunt Virus in Various Plant Samples in North America.

Geminiviruses are a group of plant-infecting viruses with single-stranded DNA genomes. Within this family, viruses in the genus Begomovirus are known to have a worldwide distribution causing a range of severe diseases in a multitude of dicotyledonous plant species. Begomoviruses are transmitted by the whitefly Bemisia tabaci, and their ssDNA genomes can be either monopartite or bipartite. As part of a viral survey, various plants including those in the families Alliaceae, Amaranthaceae, Apiaceae, Asteraceae, Brassicaceae, Cactaceae, Cucurbitaceae, Lamiaceae, Lauraceae, Malvaceae, Oleaceae and Solanaceae were sampled and screened for begomoviruses using both a high-throughput sequencing and a begomovirus-specific primer pair approach. Based on the sequences derived using these approaches, the full-length genome of various begomoviruses were amplified from plants using abutting primers. Squash leaf curl virus (SLCV) and watermelon chlorotic stunt virus (WCSV) were identified in Cactaceae (n = 25), Solanaceae (n = 7), Cucurbitaceae (n = 2) and Lamiaceae (n = 1) samples. WCSV is an Old World bipartite begomovirus that has only recently been discovered infecting watermelons in the Americas. Our discovery of WCSV in the USA is the first indication that it has reached this country and indicates that this virus might be widespread throughout North America. Phylogenetic analysis suggests WCSV was introduced to the New World twice. The detection of begomoviruses in cactus plants suggests possible spillover events from agricultural areas into native vegetation. Since WCSV and SLCV have previously been found in mixed infections, pseudo-recombination infection experiments were conducted. We demonstrate that WCSV DNA-B is successfully trans-replicated by SLCV DNA-A despite very low degree of similarity between the replication-associated iterative sequences present in their common region, an essential feature for binding of the replication associated protein. This study highlights the importance of viral surveys for the detection of spillover events into native vegetation, but also suggests the need for more surveillance of WCSV in the USA, as this virus is a serious threat to watermelon cultivation in the Middle East.

Two strains of a novel begomovirus encoding Rep proteins with identical ß1 strands but different ß5 strands are not compatible in replication.

Geminiviruses have genomes composed of single-stranded DNA molecules and encode a rolling-circle replication (RCR) initiation protein ("Rep"), which has multiple functions. Rep binds to specific repeated DNA motifs ("iterons"), which are major determinants of virus-specific replication. The particular amino acid (aa) residues that determine the preference of a geminivirus Rep for specific iterons (i.e., the trans-acting replication "specificity determinants", or SPDs) are largely unknown, but diverse lines of evidence indicate that most of them are closely associated with the so-called RCR motif I (FLTYP), located in the first 12-19 aa residues of the protein. In this work, we characterized two strains of a novel begomovirus, rhynchosia golden mosaic Sinaloa virus (RhGMSV), that were incompatible in replication in pseudorecombination experiments. Systematic comparisons of the Rep proteins of both RhGMSV strains in the DNA-binding domain allowed the aa residues at positions 71 and 74 to be identified as the residues most likely to be responsible for differences in replication specificity. Residue 71 is part of the ß-5 strand structural element, which was predicted in previous studies to contain Rep SPDs. Since the Rep proteins encoded by both RhGMSV strains are identical in their first 24 aa residues, where other studies have mapped potential SPDs, this is the first study lending direct support to the notion that geminivirus Rep proteins contain separate SPDs in their N-terminal domain.

Biological and molecular characterization of bean bushy stunt virus, a novel bipartite begomovirus infecting common bean in northwestern Argentina.

Common bean plants (Phaseolus vulgaris L.) showing different virus-like symptoms were collected in northwestern Argentina. Dot-blot hybridization tests showed that the begomoviruses bean golden mosaic virus and tomato yellow vein streak virus were the most prevalent, but they also revealed the presence of unknown begomoviruses. The complete genome sequence of one of these unknown begomoviruses was determined. Sequence analysis showed that the virus is a typical New World begomovirus, for which the name "bean bushy stunt virus" (BBSV) is proposed. Biological assays based on biolistic inoculations showed that BBSV induced leaf roll and stunting symptoms similar to those observed in the field-collected common bean sample.

Metagenomic analyses and genetic diversity of Tomato leaf curl Arusha virus affecting tomato plants in Kenya.

BACKGROUND: Tomato production is threatened worldwide by the occurrence of begomoviruses which are associated with tomato leaf curl diseases. There is little information on the molecular properties of tomato begomoviruses in Kenya, hence we investigated the population and genetic diversity of begomoviruses associated with tomato leaf curl in Kenya. METHODS: Tomato leaf samples with virus-like symptoms were obtained from farmers' field across the country in 2018 and Illumina sequencing undertaken to determine the genetic diversity of associated begomoviruses. Additionally, the occurrence of selection pressure and recombinant isolates within the population were also evaluated. RESULTS: Twelve complete begomovirus genomes were obtained from our samples with an average coverage of 99.9%. The sequences showed 95.7-99.7% identity among each other and 95.9-98.9% similarities with a Tomato leaf curl virus Arusha virus (ToLCArV) isolate from Tanzania. Analysis of amino acid sequences showed the highest identities in the regions coding for the coat protein gene (98.5-100%) within the isolates, and 97.1-100% identity with the C4 gene of ToLCArV. Phylogenetic algorithms clustered all Kenyan isolates in the same clades with ToLCArV, thus confirming the isolates to be a variant of the virus. There was no evidence of recombination within our isolates. Estimation of selection pressure within the virus population revealed the occurrence of negative or purifying selection in five out of the six coding regions of the sequences. CONCLUSIONS: The begomovirus associated with tomato leaf curl diseases of tomato in Kenya is a variant of ToLCArV, possibly originating from Tanzania. There is low genetic diversity within the virus population and this information is useful in the development of appropriate management strategies for the disease in the country.

Diversity of tomato-infecting begomoviruses and spatiotemporal dynamics of an endemic viral species of the Brazilian Atlantic rain forest biome.

Yield losses induced by a complex of begomoviruses are observed across all major tomato-producing areas in Brazil. Tomato severe rugose virus (ToSRV) is the most widespread begomovirus in the country. Conversely, tomato common mosaic virus (ToCmMV) displays a more restricted geographical distribution to areas associated with the Atlantic Rain Forest (ARF) biome, encompassing the States of Espírito Santo-ES, Minas Gerais-MG, and Rio de Janeiro-RJ. Here, we characterized 277 tomato-infecting isolates collected in fields located within the ARF biome from 2006 to 2018. ToSRV displayed the highest prevalence (n = 157), followed by ToCmMV (n = 95) and tomato interveinal chlorosis virus (n = 14). Four other begomoviruses were also detected, but with very low incidences. ToCmMV was the predominant begomovirus in the ARF biome up to 2014-2015 with very low ToSRV incidence. Subsequently, ToSRV became the most prevalent species in ES and RJ, but ToCmMV was still predominating in the "Zona da Mata" meso-region in MG. Due to the remarkable endemic distribution of ToCmMV, we carried out phylogeographical studies of this virus using information from all 28 available isolates with complete DNA-A sequences. The closest common ancestor of ToCmMV was more likely originated around Coimbra-MG area ≈ 25 years before the formal report of this viral species. So far, all surveys indicated tomatoes as the only natural hosts of ToCmMV with outbreaks occurring mainly (but not exclusively) in highland areas. ToSRV shows a more widespread incidence across both highland and lowland areas of the ARF biome.

Tomato yellow vein streak virus and Tomato golden vein virus: a reappraisal of the classification status of two South American Begomovirus species based upon genome-wide pairwise identity of multiple isolates.

Tomato yellow vein streak virus (ToYVSV) and tomato golden vein virus (TGVV) are begomoviruses reported infecting tomatoes and other hosts across South America. However, their close phylogenetic relationship has generated uncertainties about their taxonomic status and nomenclature. In fact, genomic DNA-A identity levels of isolates reported with an identical virus name may range from 89-100%. In view of the potential inaccuracy regarding the classification status of these viruses (strains vs. distinct species), we carried out a comprehensive set of analyses employing all 45 available isolates with complete DNA-A sequences with either ToYVSV or TGVV designation. Two clear-cut clusters were identified and they were consistent with the current criteria for Begomovirus species demarcation. Moreover, our reappraisal confirmed a large array of misnamed isolates and recognized a distinctive set of virus species-specific genomic, biological, and ecological features. Hence, the present work gives support to the notion that these viruses are closely-related, but they are distinct and valid Begomovirus species. From the breeding standpoint, this information will be useful in guiding germplasm screening strategies searching for sources of large-spectrum resistance to isolates of both viruses.

A cassava protoplast system for screening genes associated with the response to South African cassava mosaic virus.

BACKGROUND: The study of transient gene expression in cassava plants during virus infection using existing protocols is laborious and may take approximately fifteen weeks due to cassava's recalcitrance to transformation. The combination of a protoplast system with CRISPR-mediated gene editing promises to shorten the turnaround time from plant tissue culture to high-throughput gene expression screening for candidate genes. Here, we detail a protocol for screening genes associated with the response to South African cassava mosaic virus (SACMV) in cassava protoplasts, with reference to the ubiquitin E3 ligase gene, MeE3L. METHODS: Cassava protoplasts of model, and SACMV-susceptible and -tolerant genotypes, were transformed with SACMV infectious clones and/or a CRISPR-editing construct targeting the MeE3L using PEG4000-mediated transfection. DNA and RNA were extracted from transformed protoplasts at 24 h post-transfection. Relative SACMV DNA accumulation was determined via qPCR using DpnI-digested total DNA, MeE3L relative expression was determined via reverse transcriptase qPCR, and results were analysed using one-way ANOVA, Tukey's HSD test and the 2-ΔΔCTstatistical method. The MeE3L exonic region was sequenced on the ABI 3500XL Genetic Analyzer platform; and sequences were analysed for mutations using MAFTT and MEGA-X software. Construction of a phylogenetic tree was done using the Maximum Likelihood method and Jones-Taylor-Thornton (JTT) matrix-based model. RESULTS: The differential expression of unedited and mutant MeE3L during SACMV infection of model, susceptible and tolerant cassava protoplasts was determined within 7 weeks after commencement of tissue culture. The study also revealed that SACMV DNA accumulation in cassava protoplasts is genotype-dependent and induces multiple mutations in the tolerant landrace MeE3L homolog. Notably, the susceptible cassava landrace encodes a RINGless MeE3Lwhich is silenced by SACMV-induced mutations. SACMV also induces mutations which silence the MeE3L RING domain in protoplasts from and tolerant cassava landraces. CONCLUSIONS: This protocol presented here halves the turnaround time for high-throughput screening of genes associated with the host response to SACMV. It provides evidence that a cassava E3 ligase is associated with the response to SACMV and forms a basis for validation of these findings by in planta functional and interaction studies.

Metagenomics of Neotropical Single-Stranded DNA Viruses in Tomato Cultivars with and without the Ty-1 Gene.

A complex of begomoviruses (Geminiviridae) can cause severe tomato yield losses in the neotropics. Here, next-generation sequencing was employed for large-scale assessment of single-stranded (ss)DNA virus diversity in tomatoes either harboring or lacking the large-spectrum begomovirus tolerance Ty-1 gene. Individual leaf samples exhibiting begomovirus-like symptoms (n = 107) were field-collected, circular DNA-enriched, subdivided into pools (with and without Ty-1), and Illumina-sequenced. Virus-specific PCR and Sanger dideoxy sequencing validations confirmed 15 distinct ssDNA virus/subviral agents (occurring mainly in mixed infections), which highlight the potential drawbacks of employing virus-specific resistance in tomato breeding. More viruses (14 versus 6 species) were observed in tomatoes without the Ty-1 gene. A gemycircularvirus (Genomoviridae), a new alpha-satellite, and two novel Begomovirus species were identified exclusively in samples without the Ty-1 gene. A novel begomovirus was found only in the Ty-1 pool, being the only species associated with severe symptoms in Ty-1 plants in our survey. Our work is the first step towards the elucidation of the potential begomovirus adaptation to Ty-1 and its specific filtering effects on a subset of ssDNA viral/subviral agents.

Association of a begomovirus-satellite complex with yellow vein and leaf curl disease of hollyhock (Alcea rosea) in India.

Geminiviruses cause considerable yield loss in several crop plants worldwide. In 2016, several hollyhock plants displaying yellow mosaic and leaf curling symptoms were noticed in a nursery of Jawaharlal Nehru University, New Delhi, India. Analysis of the collected samples indicated an association of monopartite and bipartite begomoviruses with satellites. Three begomoviruses (including a member of a new begomovirus species), two alphasatellites, and a betasatellite were isolated from yellow-mosaic-disease-affected plants. Similarly, a begomovirus, two alphasatellites, and a betasatellite were found to be associated with leaf curl disease of hollyhock. These begomoviruses and satellites were found to be recombinants. By harboring diverse begomoviruses and satellite DNAs, hollyhock may serve as a potential source of virus inoculum.

A new begomovirus isolated from a potyvirus-infected bean plant causes asymptomatic infections in bean and N. benthamiana.

In this work, a begomovirus isolated from a bean plant coinfected with the potyviruses bean common mosaic virus and bean common mosaic necrosis virus was characterized. The three viruses were detected by high-throughput sequencing and assembly of total small RNAs, but the begomovirus-related contigs did not allow precise identification. Molecular analysis based on standard DNA amplification techniques revealed the presence of a single bipartite virus, which is a novel begomovirus according to the current taxonomic criteria. Infectious clones were generated and agroinoculated into Phaseolus vulgaris and Nicotiana benthamiana plants. In all cases, viral DNA-A and DNA-B were detected in new growths, but no symptoms were observed, thus indicating that this virus produces asymptomatic infections in both host species.

Reconstruction and Characterization of Full-Length Begomovirus and Alphasatellite Genomes Infecting Pepper through Metagenomics.

In northwestern Argentina (NWA), pepper crops are threatened by the emergence of begomoviruses due to the spread of its vector, Bemisia tabaci (Gennadius). The genus Begomovirus includes pathogens that can have a monopartite or bipartite genome and are occasionally associated with sub-viral particles called satellites. This study characterized the diversity of begomovirus and alphasatellite species infecting pepper in NWA using a metagenomic approach. Using RCA-NGS (rolling circle amplification-next generation sequencing), 19 full-length begomovirus genomes (DNA-A and DNA-B) and one alphasatellite were assembled. This ecogenomic approach revealed six begomoviruses in single infections: soybean blistering mosaic virus (SbBMV), tomato yellow spot virus (ToYSV), tomato yellow vein streak virus (ToYVSV), tomato dwarf leaf virus (ToDfLV), sida golden mosaic Brazil virus (SiGMBRV), and a new proposed species, named pepper blistering leaf virus (PepBLV). SbBMV was the most frequently detected species, followed by ToYSV. Moreover, a new alphasatellite associated with ToYSV, named tomato yellow spot alphasatellite 2 (ToYSA-2), was reported for the first time in Argentina. For the Americas, this was the first report of an alphasatellite found in a crop (pepper) and in a weed (Leonurus japonicus). We also detected intra-species and inter-species recombination.