Tombusviruses orchestrate the host endomembrane system to create elaborate membranous replication organelles.

Positive-strand RNA viruses depend on intensive manipulation of subcellular organelles and membranes to create unique viral replication organelles (VROs), which represent the sites of robust virus replication. The host endomembrane-based protein-trafficking and vesicle-trafficking pathways are specifically targeted by many (+)RNA viruses to take advantage of their rich resources. We summarize the critical roles of co-opted endoplasmic reticulum subdomains and associated host proteins and COPII vesicles play in tombusvirus replication. We also present the surprising contribution of the early endosome and the retromer tubular transport carriers to VRO biogenesis. The central player is tomato bushy stunt virus (TBSV), which provides an outstanding system based on the identification of a complex network of interactions with the host cells. We present the emerging theme on how TBSV uses tethering and membrane-shaping proteins and lipid modifying enzymes to build the sophisticated VRO membranes with unique lipid composition.

Complete genome sequence of aucuba ringspot virus.

A new badnavirus, aucuba ringspot virus (AuRV), was identified in plants of Aucuba japonica showing mild mosaic, vein banding, and yellow ringspot symptoms on the leaves. The complete nucleotide sequence of the AuRV genome was determined and found to be 9,092 nt in length, and the virus was found to have a genome organization typical of members of the genus Badnavirus. ORF3 was predicted to encode a polyprotein containing conserved movement protein, coat protein, aspartic protease, reverse transcriptase (RT), and RNase H domains. Phylogenetic analysis suggested that this virus is most closely related to codonopsis vein clearing virus but belongs to a distinct species, based on only 69.6% nucleotide sequence identity within the part of ORF 3 encoding the RT and RNase H domains. The vector of AuRV is unknown, but based on phylogenetic relationships, it is predicted to be a type of aphid.

Genomic sequence and host range studies reveal considerable variation within the species Arracacha virus B.

Arracacha virus B type (AVB-T) and oca (AVB-O) strains from arracacha (Arracacia xanthorrhiza) and oca (Oxalis tuberosa) samples collected in 1975 and two additional isolates obtained from arracacha (AVB-PX) and potato (AVB-6A) in Peru in 1976 and 1978, respectively, were studied. In its host responses and serological properties, AVB-PX most resembled AVB-T, whereas AVB-6A most resembled AVB-O. Complete genomic sequences of the RNA-1 and RNA-2 of each isolate were obtained following high-throughput sequencing of RNA extracts from isolates preserved for 38 (AVB-PX) or 32 (the other 3 isolates) years, and compared with a genomic sequence of AVB-O obtained previously (PV-0082). RNA-2 was unexpectedly divergent compared to RNA-1, with the nucleotide (nt) sequence identity of different AVB isolates varying by up to 76% (RNA-2) and 89% (RNA-1). The coat protein amino acid sequences were the most divergent, with AVB-O and AVB-6A having only 68% identity to AVB-T and AVB-PX. Since the RNA2 sequence differences between the two isolate groupings also coincided with host range, symptom, and serological differences, AVB demonstrates considerable intraspecific divergence.

Characterization of the first tenuivirus naturally infecting dicotyledonous plants.

A mechanically transmissible virus tentatively named "melon chlorotic spot virus" (MeCSV) was isolated in southeastern France from a melon plant showing chlorotic spots and yellowing of the older leaves. Its complete sequence was obtained by Illumina and Sanger sequencing. The genome comprises eight RNAs for a total size of 20,079 nt and is distantly related to Ramu stunt virus and maize yellow stunt virus, two tentative tenuiviruses. MeCSV differs from other tenuiviruses by its number of genomic fragments, by being readily mechanically transmissible, and by infecting only dicotyledonous hosts. MeCSV should thus be considered a member of a tentative new species related to tenuiviruses.

Euphyllophyte Paleoviruses Illuminate Hidden Diversity and Macroevolutionary Mode of Caulimoviridae.

Endogenous viral elements (paleoviruses) provide "molecular fossils" for studying the deep history and macroevolution of viruses. Endogenous plant pararetroviruses (EPRVs) are widespread in angiosperms, but little is known about EPRVs in earlier-branching plants. Here we use a large-scale phylogenomic approach to investigate the diversity and macroevolution of plant pararetroviruses (formally known as Caulimoviridae). We uncover an unprecedented and unappreciated diversity of EPRVs within the genomes of gymnosperms and ferns. The known angiosperm viruses constitute only a minor part of the Caulimoviridae diversity. By characterizing the distribution of EPRVs, we show that no major euphyllophyte lineages escape the activity of Caulimoviridae, raising the possibility that many exogenous Caulimoviridae remain to be discovered in euphyllophytes. We find that the copy numbers of EPRVs are generally high, suggesting that EPRVs might define a unique group of repetitive elements and represent important components of euphyllophyte genomes. Evolutionary analyses suggest an ancient origin of Caulimoviridae and at least three independent origins of Caulimoviridae in angiosperms. Our findings reveal the remarkable diversity of Caulimoviridae and have important implications for understanding the origin and macroevolution of plant pararetroviruses.IMPORTANCE Few viruses have been documented in plants outside angiosperms. Viruses can occasionally integrate into host genomes, forming endogenous viral elements (EVEs). Endogenous plant pararetroviruses (EPRVs) are widespread in angiosperms. In this study, we performed comprehensive comparative and phylogenetic analyses of EPRVs and found that EPRVs are present in the genomes of gymnosperms and ferns. We identified numerous EPRVs in gymnosperm and fern genomes, revealing an unprecedented depth in the diversity of plant pararetroviruses. Plant pararetroviruses mainly underwent cross-species transmission, and angiosperm pararetroviruses arose at least three times. Our study provides novel insights into the diversity and macroevolution of plant pararetroviruses.

Characterization of a not so new potexvirus from babaco (Vasconcellea x heilbornii).

A new member of the genus Potexvirus was fully sequenced and characterized. The virus was isolated from babaco (Vasconcellea x heilbornii), a natural hybrid native to Ecuador. The virus contains a 6,692 nt long genome organized in five open reading frames in an arrangement typical of other potexviruses. Sequence comparisons revealed close relatedness with Papaya mosaic virus (PapMV), Alternathera mosaic virus (AltMV) and Senna mosaic virus (SenMV), exhibiting nucleotide identities up to 67% for the polymerase (Pol) and 68% for the coat protein (CP), with deduced amino acid identities of 70% and 72% for the Pol and CP, respectively. The presence of an AlkB domain, in the polymerase region, was observed. Terminal nucleotide sequences were conserved across potexviruses with characteristic motifs and predicted secondary structures at the 3' UTR. Although serologically undistinguishable from PapMV and AltMV, the new virus showed differences in host range and symptom induction. The name babaco mosaic virus is proposed for this newly characterized Potexvirus. The complete genome sequence of the new virus has been deposited in NCBI GenBank under accession number MF978248.

Changes in the composition of the RNA virome mark evolutionary transitions in green plants.

BACKGROUND: The known plant viruses mostly infect angiosperm hosts and have RNA or small DNA genomes. The only other lineage of green plants with a relatively well-studied virome, unicellular chlorophyte algae, is mostly infected by viruses with large DNA genomes. Thus RNA viruses and small DNA viruses seem to completely displace large DNA virus genomes in late branching angiosperms. To understand better the expansion of RNA viruses in the taxonomic span between algae and angiosperms, we analyzed the transcriptomes of 66 non-angiosperm plants characterized by the 1000 Plants Genomes Project. RESULTS: We found homologs of virus RNA-dependent RNA polymerases in 28 non-angiosperm plant species, including algae, mosses, liverworts (Marchantiophyta), hornworts (Anthocerotophyta), lycophytes, a horsetail Equisetum, and gymnosperms. Polymerase genes in algae were most closely related to homologs from double-stranded RNA viruses leading latent or persistent lifestyles. Land plants, in addition, contained polymerases close to the homologs from single-stranded RNA viruses of angiosperms, capable of productive infection and systemic spread. For several polymerases, a cognate capsid protein was found in the same library. Another virus hallmark gene family, encoding the 30 K movement proteins, was found in lycophytes and monilophytes but not in mosses or algae. CONCLUSIONS: The broadened repertoire of RNA viruses suggests that colonization of land and growth in anatomical complexity in land plants coincided with the acquisition of novel sets of viruses with different strategies of infection and reproduction.

Beet western yellows virus infects the carnivorous plant Nepenthes mirabilis.

Although poleroviruses are known to infect a broad range of higher plants, carnivorous plants have not yet been reported as hosts. Here, we describe the first polerovirus naturally infecting the pitcher plant Nepenthes mirabilis. The virus was identified through bioinformatic analysis of NGS transcriptome data. The complete viral genome sequence was assembled from overlapping PCR fragments and shown to share 91.1 % nucleotide sequence identity with the US isolate of beet western yellows virus (BWYV). Further analysis of other N. mirabilis plants revealed the presence of additional BWYV isolates differing by several insertion/deletion mutations in ORF5.

Genetic variation of eggplant mottled dwarf virus from annual and perennial plant hosts.

The genetic diversity of eggplant mottled dwarf virus (EMDV), a member of the family Rhabdoviridae, was studied using isolates collected from different herbaceous and woody plant species and remote geographic areas. Sequences corresponding to the N, X, P, Y, M and G ORFs as well as the untranslated regions (UTRs) between ORFs were determined from all isolates. Low genetic diversity was found in almost all genomic regions studied except for the X ORF and the UTRs, which were more variable, while interestingly, an EMDV isolate from caper possessed a truncated G gene sequence. Furthermore, low d N /d S ratios, indicative of purifying selection, were calculated for all genes. Phylogenetic analysis showed that the EMDV isolates clustered in three distinct subgroups based on their geographical origin, with the exception of one subgroup that consisted of isolates from northern Greece and Cyprus. Overall, the level of genetic diversity of EMDV differed between seed- and asexually propagated plants in our collection, and this could be related to the mode of transmission.

Evolution, functions, and mysteries of plant ARGONAUTE proteins.

ARGONAUTE (AGO) proteins bind small RNAs (sRNAs) to form RNA-induced silencing complexes for transcriptional and post-transcriptional gene silencing. Genomes of primitive plants encode only a few AGO proteins. The Arabidopsis thaliana genome encodes ten AGO proteins, designated AGO1 to AGO10. Most early studies focused on these ten proteins and their interacting sRNAs. AGOs in other flowering plant species have duplicated and diverged from this set, presumably corresponding to new, diverged or specific functions. Among these, the grass-specific AGO18 family has been discovered and implicated as playing important roles during plant reproduction and viral defense. This review covers our current knowledge about functions and features of AGO proteins in both eudicots and monocots and compares their similarities and differences. On the basis of these features, we propose a new nomenclature for some plant AGOs.

Pollen tubes introduce Raspberry bushy dwarf virus into embryo sacs during fertilization processes.

We developed a fertilization method in which pollen tubes entered into embryo sacs without any need to contact surrounding female sporophytic cells by using Torenia fournieri (Torenia) plants under the condition of hindering movement of the virus from a stigma, which is the first infection site leading to systemic infection. When RBDV-infected Torenia pollen grains were used for the developed fertilization method, the virus was transmitted to the seeds by pollen tubes germinating from them. On the other hand, no seeds were infected with the virus when Torenia plants were pollinated with healthy Torenia pollen grains in combination with RBDV-infected raspberry pollen grains, which caused the virus infection in the stigma by penetration of their pollen tubes arrested in its style. Our results indicate that vertical transmission of RBDV by pollen occurs in the transport of the virus into embryo sacs by pollen tubes reaching the embryo sacs.

Molecular characterization of the Tobacco rattle virus RNA2 genome isolated from Gladiolus.

Tobacco rattle virus (TRV-K) was first identified in a symptomatic Gladiolus plant cultivated in Korea. We analyzed the TRV-K genome and compared its phylogeny with other TRV isolates. After constructing of a full-length genomic RNA2 strand clone, a complete sequence was generated from several overlapping clones. The cloned genome was 3261 bases in length, identical to TRV-K, and had three open reading frames. TRV-K had the highest sequence identity with the American isolate TRV-ORY. Sequence analysis of the RNA2 genome showed that TRV-K contains an intact 2a, 2b, and 2c coding sequence and an RNA1-related 3' terminus, which is typical of TRV RNA2. Phylogenetic analysis revealed that TRV-K is in the same cluster as the American isolates and another Korean isolate, TRV-SK; however, it was in a different cluster than the European isolates.

Penetration of pollen tubes with accumulated Raspberry bushy dwarf virus into stigmas is involved in initial infection of maternal tissue and horizontal transmission.

Torenia fournieri (Torenia) plants were infected with Raspberry bushy dwarf virus (RBDV) by pollination with RBDV-infected raspberry pollen grains. The infected raspberry pollen grains germinated on Torenia stigmas, and then the pollen tubes penetrated into the stigma, even though the pollen tubes were arrested in the styles. In whole-mount in situ hybridization of germinating infected raspberry pollen grains, RBDV accumulated in the tips of the pollen tubes. Tissue blot hybridization of Torenia plants pollinated with infected raspberry pollen grains revealed that the first virus infection site leading to systemic infection is the stigma. When infected raspberry pollen grains that had lost germination capacity were pollinated on Torenia stigmas, RBDV could not infect the stigmas, and no horizontal transmission occurred. These results indicate that penetration of pollen tubes with accumulated RBDV into stigmas is essential in causing the first viral infection in the stigma to lead to systemic infection.

Plant-mediated whitefly-begomovirus interactions: research progress and future prospects.

Plant-mediated interactions between begomoviruses and whiteflies exert important influences on the population dynamics of vectors and the epidemiology of plant diseases. In this article, we synthesize the relevant literature to identify patterns to the interactions. We then review studies on the ecological, biochemical and molecular mechanisms underlying the interactions and finally elaborate on the most interesting issues for future research. The interactions between begomoviruses and the insect vector, the whitefly Bemisia tabaci, via their shared host plants can be mutualistic, neutral or negative. However, in contrast to a pattern of improved performance of vectors on virus-infected plants that has been observed with persistently transmitted RNA viruses, the number of cases exhibiting mutualistic, neutral or negative effects in the indirect interactions between begomoviruses and whiteflies appear evenly distributed. With regard to the mechanisms of plant-mediated positive effects on whiteflies, two case studies indicate that suppression of plant defence and/or alteration in plant nutrition as a result of virus infection can be important. Our review shows that we are only just beginning to understand the tripartite interactions between begomoviruses, whiteflies and plants. Future efforts in this area should try to expand the number and diversity of pathosystems for investigation to reveal the patterns of interactions, to investigate the molecular and biochemical mechanisms of the interactions using a multidisciplinary approach, and to examine the virus-plant-vector interactions in the field and in natural plant communities.

TRV-GFP: a modified Tobacco rattle virus vector for efficient and visualizable analysis of gene function.

Virus-induced gene silencing (VIGS) is a useful tool for functional characterization of genes in plants. Unfortunately, the efficiency of infection by Tobacco rattle virus (TRV) is relatively low for some non-Solanaceae plants, which are economically important, such as rose (Rosa sp.). Here, to generate an easy traceable TRV vector, a green fluorescent protein (GFP) gene was tagged to the 3' terminus of the coat protein gene in the original TRV2 vector, and the silencing efficiency of the modified TRV-GFP vector was tested in several plants, including Nicotiana benthamiana, Arabidopsis thaliana, rose, strawberry (Fragaria ananassa), and chrysanthemum (Dendranthema grandiflorum). The results showed that the efficiency of infection by TRV-GFP was equal to that of the original TRV vector in each tested plant. Spread of the modified TRV virus was easy to monitor by using fluorescent microscopy and a hand-held UV lamp. When TRV-GFP was used to silence the endogenous phytoene desaturase (PDS) gene in rose cuttings and seedlings, the typical photobleached phenotype was observed in 75-80% plants which were identified as GFP positive by UV lamp. In addition, the abundance of GFP protein, which represented the concentration of TRV virus, was proved to correlate negatively with the level of the PDS gene, suggesting that GFP could be used as an indicator of the degree of silencing of a target gene. Taken together, this work provides a visualizable and efficient tool to predict positive gene silencing plants, which is valuable for research into gene function in plants, especially for non-Solanaceae plants.

Endogenous pararetroviruses--a reservoir of virus infection in plants.

Endogenous pararetrovirus sequences (EPRV) belonging to the plant virus family Caulimoviridae have been discovered in the genomes of a wide range of Angiosperms. Although knowledge of EPRVs in plants is still in its infancy, it has been shown clearly in three different plant-virus pathosystems that these integrations are capable of generating functional circular viral genomes, and can thus trigger systemic infection. Here, we recapitulate information gathered over the last 15 years on how EPRVs contribute to virus replication in plants. We first present recent advances in our understanding of the molecular mechanisms involved in the transition from integrated to circular viral forms before addressing how EPRVs are controlled in planta.

Complete genome sequences of two begomoviruses infecting weeds in Venezuela.

The complete sequences of isolates of two new bipartite begomoviruses (genus Begomovirus, family Geminiviridae) found infecting weeds in Venezuela are provided. The names proposed for these new begomoviruses are "Datura leaf distortion virus" (DLDV), isolated from a Datura stramonium L. (family Solanaceae) plant, and "Dalechampia chlorotic mosaic virus" (DCMV), isolated from infected Dalechampia sp. (family Euphorbiaceae) and Boerhavia diffusa L. (family Nyctaginaceae) plants. Phylogenetic analysis showed that these new begomoviruses segregated in two distinct clades of New World begomoviruses. To our knowledge, this is the first record of a begomovirus infecting Dalechampia sp. plants. Also, this is the first report of a begomovirus infecting Boerhavia spp. and Datura spp. in Venezuela.

Diodia vein chlorosis virus is a group-1 crinivirus.

Members of the family Closteroviridae have emerged as a major problem in agricultural crops in the past two decades. Diodia vein chlorosis virus (DVCV) is an understudied whitefly-transmitted closterovirus. Given the presence of the primary host for the virus in major agricultural production areas in the United States, we characterized the virus at the molecular level, demonstrating that it belongs in the genus Crinivirus, developed detection protocols, evaluated its host range among hosts known to harbor viruses closely related to DVCV, and confirmed transmission by a second whitefly species, Trialeurodes vaporariorum.

Characterization of a new curtovirus, pepper yellow dwarf virus, from chile pepper and distribution in weed hosts in New Mexico.

Over 4,950 asymptomatic weed samples from more than 20 weed species that are host plants for curtoviruses were collected from ten chile pepper fields in southern New Mexico (NM) during 2003, 2004 and 2005 to identify whether they were infected with curtoviruses and to determine which curtoviruses were distributed in the weed population. Polymerase chain reaction using primers designed to detect a portion of the coat protein (cp) gene were used to detect curtoviruses, and infected plants were further tested for specific curtoviruses using primers designed to detect to a portion of the replication-associated protein (rep) gene. Amplification of the cp gene was successful from 3.7, 1.17, and 1.9% of the weed samples in 2003, 2004, and 2005, respectively. Seventy-three amplicons from those samples were sequenced and compared to well-characterized curtoviruses. Analysis of the rep nucleotide sequences showed that approximately 32.9% of the weed isolates tested were closely related to beet mild curly top virus (BMCTV). Approximately 12.4% were closely related to beet severe curly top virus (BSCTV). The rest of the weed isolates (54.7%), which shared a very high level of nucleotide sequence identity to each other, represent a new curtovirus species. Using eight primers designed for PCR, complete genomes of three curtoviruses isolated from chile pepper samples representing the three groups of curtoviruses in southern New Mexico were sequenced. Comparisons of whole sequences of the genomes revealed that the DG2SW171601 isolate (2,929 nucleotides) was nearly identical to BMCTV-W4 (approximately 98% nucleotide sequence identity). The LRME27601 isolate (2,927 nucleotides) was most closely related to BSCTV (approximately 92% nucleotide sequence identity). The LJN17601 isolate (2,959 nucleotides) shared only from 49.9 to 88.8% nucleotide sequence identity with other well-characterized curtoviruses. Based on the accepted cut-off of 89%, we propose that the LJN17601 isolate is a member of a new curtovirus species. Chile peppers infected with this virus in the field express chlorotic stunting symptoms, so we propose the name pepper yellow dwarf virus (PeYDV). This new curtovirus species may be the result of mutations in the genome and recombination between BMCTV-W4 and BSCTV.