Small but mighty: Functional landscape of the versatile geminivirus-encoded C4 protein.

The fast-paced evolution of viruses enables them to quickly adapt to the organisms they infect by constantly exploring the potential functional landscape of the proteins encoded in their genomes. Geminiviruses, DNA viruses infecting plants and causing devastating crop diseases worldwide, produce a limited number of multifunctional proteins that mediate the manipulation of the cellular environment to the virus' advantage. Among the proteins produced by the members of this family, C4, the smallest one described to date, is emerging as a powerful viral effector with unexpected versatility. C4 is the only geminiviral protein consistently subjected to positive selection and displays a number of dynamic subcellular localizations, interacting partners, and functions, which can vary between viral species. In this review, we aim to summarize our current knowledge on this remarkable viral protein, encompassing the different aspects of its multilayered diversity, and discuss what it can teach us about geminivirus evolution, invasion requirements, and virulence strategies.

High-throughput sequencing reveals a novel closterovirus in arracacha (Arracacia xanthorrhiza).

High-throughput sequencing analysis detected a clostero-like virus from arracacha plants (Arracacia xanthorrhiza) in Brazil. The complete genome sequence, confirmed by RACE and Sanger sequencing, consists of 15,763 nucleotides with nine predicted open reading frames (ORFs) in a typical closterovirus genome organisation. The putative RNA-dependent RNA polymerase (RdRp), heat shock protein 70 homologue (Hsp70h), and coat protein showed 55-65, 38-44, and 20-36% amino acid sequence identity, respectively, to the homologous proteins of known closteroviruses. Phylogenetic analysis of Hsp70h showed that this putative novel arracacha plant virus was related to members of the genus Closterovirus in the family Closteroviridae. These results suggest that this virus, tentatively named "arracacha virus 1" (AV-1), is a novel member of the genus Closterovirus. This is the first closterovirus identified in arracacha plants.

A novel vitivirus-like sequence found in Arracacia xanthorrhiza plants by high throughput sequencing.

High throughput sequencing (HTS) is a very powerful tool for detecting and discovering novel viral-like sequences without prior knowledge of the sequence. Here we describe the complete genome of a new vitivirus-like sequence that was found in arracacha (Arracacia xanthorrhiza) plants using HTS technology. The complete genome sequence was validated by Sanger sequencing. The genomic organization of the new putative vitivirus resembles that of grapevine virus B (GVB) and grapevine virus D (GVD). The putative coat protein showed 41 to 49% identity with similar proteins of known vitiviruses, while the RNA-dependent RNA polymerase shared 52 to 55% identity with those encoded by grapevine vitiviruses. Based on the demarcation criteria for the genus Vitivirus, the virus described in this work, provisionally named as "Arracacha virus V", represents a novel species in this taxon.

Biological and molecular characterization of a highly divergent johnsongrass mosaic virus isolate from Pennisetum purpureum.

The complete genome sequence (9,865 nucleotides) of a highly divergent johnsongrass mosaic virus isolate (JGMV-CNPGL) was determined using Illumina sequencing. This isolate infected 10 genotypes of gramineous plants including maize. A comparative analysis of the complete genome showed 80 % nucleotide (nt) sequence identity (86 % amino acid (aa) sequence identity) to a johnsongrass mosaic virus isolate from Australia. The coat protein (CP) identity values, however, were lower than those for the whole genome (78 % and 80 % for nt and aa, respectively) and were close to the species demarcation values (77 % nt and 80 % aa). Unexpectedly, the amino-terminal portion of CP of JGMV-CNPGL showed only 38 % sequence identity to other JGMV isolates. The biological implications of this sequence divergence remain to be elucidated.

Subcellular localization of p29, a putative movement protein of pepper ringspot virus.

Pepper ringspot virus (PepRSV) is a member of the genus Tobravirus. It possesses a bipartite single-strand RNA genome in a positive-sense polarity. The p29 protein is encoded by RNA 1 and is presumed to be the movement protein (MP) of this virus. In this study, the intracellular distribution of the p29 protein was analyzed by confocal microscopy. Transient expression of the PepRSV p29 protein fused to green fluorescent protein was observed as punctate spots localized next to the cell wall. This protein partially co-localized with the eCFP-tagged tobacco mosaic virus 30K MP, which is known to associate with plasmodesmata. This result suggests that the p29 protein is most probably the movement protein for PepRSV.

Complete genome sequence of arracacha mottle virus.

Arracacha mottle virus (AMoV) is the only potyvirus reported to infect arracacha (Arracacia xanthorrhiza) in Brazil. Here, the complete genome sequence of an isolate of AMoV was determined to be 9,630 nucleotides in length, excluding the 3' poly-A tail, and encoding a polyprotein of 3,135 amino acids and a putative P3N-PIPO protein. Its genomic organization is typical of a member of the genus Potyvirus, containing all conserved motifs. Its full genome sequence shared 56.2 % nucleotide identity with sunflower chlorotic mottle virus and verbena virus Y, the most closely related viruses.

Specific Nucleotides in the Common Region of the Begomovirus Tomato Rugose Mosaic Virus (ToRMV) Are Responsible for the Negative Interference over Tomato Severe Rugose Virus (ToSRV) in Mixed Infection.

Mixed infection between two or more begomoviruses is commonly found in tomato fields and can affect disease outcomes by increasing symptom severity and viral accumulation compared with single infection. Viruses that affect tomato include tomato severe rugose virus (ToSRV) and tomato rugose mosaic virus (ToRMV). Previous work showed that in mixed infection, ToRMV negatively affects the infectivity and accumulation of ToSRV. ToSRV and ToRMV share a high degree of sequence identity, including cis-elements in the common region (CR) and their specific recognition sites (iteron-related domain, IRD) within the Rep gene. Here, we investigated if divergent sites in the CR and IRD are involved in the interaction between these two begomoviruses. ToSRV clones were constructed containing the same nucleotides as ToRMV in the CR (ToSRV-A(ToR:CR)), IRD (ToSRV-A(ToR:IRD)) and in both regions (ToSRV-A(ToR:CR+IRD)). When plants were co-inoculated with ToRMV and ToSRV-A(ToR:IRD), the infectivity and accumulation of ToSRV were negatively affected. In mixed inoculation of ToRMV with ToSRV-A(ToR:CR), high infectivity of both viruses and high DNA accumulation of ToSRV-A(ToR:CR) were observed. A decrease in viral accumulation was observed in plants inoculated with ToSRV-A(ToR:CR+IRD). These results indicate that differences in the CR, but not the IRD, are responsible for the negative interference of ToRMV on ToSRV.

Complete nucleotide sequence of a Spanish isolate of alfalfa mosaic virus: evidence for additional genetic variability.

Alfalfa mosaic virus (AMV) is a plant virus that is distributed worldwide and can induce necrosis and/or yellow mosaic on a large variety of plant species, including commercially important crops. It is the only virus of the genus Alfamovirus in the family Bromoviridae. AMV isolates can be clustered into two genetic groups that correlate with their geographic origin. Here, we report for the first time the complete nucleotide sequence of a Spanish isolate of AMV found infecting Cape honeysuckle (Tecoma capensis) and named Tec-1. The tripartite genome of Tec-1 is composed of 3643 nucleotides (nt) for RNA1, 2594 nt for RNA2 and 2037 nt for RNA3. Comparative sequence analysis of the coat protein gene revealed that the isolate Tec-1 is distantly related to subgroup I of AMV and more closely related to subgroup II, although forming a distinct phylogenetic clade. Therefore, we propose to split subgroup II of AMV into two subgroups, namely IIA, comprising isolates previously included in subgroup II, and IIB, including the novel Spanish isolate Tec-1.

The Association between New World Alphasatellites and Bipartite Begomoviruses: Effects on Infection and Vector Transmission.

Begomoviruses can be found in association with alphasatellites, which are capable of autonomous replication but are dependent on the helper begomovirus for systemic infection, encapsidation and vector transmission. Previous studies suggest that the presence of NW alphasatellites (genus Clecrusatellite) is associated with more severe symptoms. To better understand this interaction, we investigated the effects of two alphasatellites on infectivity, symptom development, viral DNA accumulation and vector transmission of three begomoviruses in three hosts. In tomato and Nicotiana benthamiana, all combinations were infectious. In Leonurus sibiricus, only the ToYSV/ToYSA combination was infectious. The presence of EuYMA increased symptom severity of EuYMV and ToYSV in N. benthamiana, and the presence of ToYSA was associated with more severe symptoms of ToYSV in N. benthamiana and L. sibiricus. EuYMA increased the accumulation of ToYSV in N. benthamiana but reduced the accumulation of EuYMV in tomato and of ToSRV in N. benthamiana. The presence of ToYSA decreased the accumulation of ToYSV in N. benthamiana and L. sibiricus. ToYSA negatively affected transmission of ToSRV by Bemisia tabaci MEAM1. Together, our results indicate that NW alphasatellites can interact with different begomoviruses, increasing symptom severity and interfering in the transmission of the helper begomovirus. Understanding this interaction is important as it may affect the emergence of diseases caused by begomovirus-alphasatellite complexes in the field.

Molecular Characterization of Hovenia Dulcis-Associated Virus 1 (HDaV1) and 2 (HDaV2): New Tentative Species within the Order Picornavirales.

In a systematic field survey for plant-infecting viruses, leaf tissues were collected from trees showing virus-like symptoms in Brazil. After viral enrichment, total RNA was extracted and sequenced using the MiSeq platform (Illumina). Two nearly full-length picorna-like genomes of 9534 and 8158 nucleotides were found associated with Hovenia dulcis (Rhamnaceae family). Based upon their genomic information, specific primers were synthetized and used in RT-PCR assays to identify plants hosting the viral sequences. The larger contig was tentatively named as Hovenia dulcis-associated virus 1 (HDaV1), and it exhibited low nucleotide and amino acid identities with Picornavirales species. The smaller contig was related to insect-associated members of the Dicistroviridae family but exhibited a distinct genome organization with three non-overlapping open reading frames (ORFs), and it was tentatively named as Hovenia dulcis-associated virus 2 (HDaV2). Phylogenetic analysis using the amino acid sequence of RNA-dependent RNA polymerase (RdRp) revealed that HDaV1 and HDaV2 clustered in distinct groups, and both viruses were tentatively assigned as new members of the order Picornavirales. HDaV2 was assigned as a novel species in the Dicistroviridae family. The 5' ends of both viruses are incomplete. In addition, a nucleotide composition analysis (NCA) revealed that HDaV1 and HDaV2 have similarities with invertebrate-infecting viruses, suggesting that the primary host(s) of these novel virus species remains to be discovered.

The complete nucleotide sequence of the RNA2 of the crinivirus tomato infectious chlorosis virus: isolates from North America and Europe are essentially identical.

The complete nucleotide sequences of the RNA2 of two isolates of Tomato infectious chlorosis virus (TICV, genus Crinivirus, family Closteroviridae) from the United States and Spain, respectively, were determined. The sequences of both isolates were found to be nearly identical. TICV RNA2 consisted of 7,914 nucleotides in both isolates and contains eight open reading frames that encompass the Closteroviridae hallmark gene array represented by a heat shock protein 70 family homologue, a protein of 59 kDa, the major coat protein, and a divergent copy of the coat protein. Phylogenetic analysis suggested that TICV is most similar to Lettuce infectious yellows virus (LIYV), the type species of the genus Crinivirus.

Infectivity, effects on helper viruses and whitefly transmission of the deltasatellites associated with sweepoviruses (genus Begomovirus, family Geminiviridae).

Begomoviruses (family Geminiviridae) are whitefly-transmitted viruses with single-stranded DNA genomes that are frequently associated with DNA satellites. These satellites include non-coding satellites, for which the name deltasatellites has been proposed. Although the first deltasatellite was identified in the late 1990s, little is known about the effects they have on infections of their helper begomoviruses. Recently a group of deltasatellites were identified associated with sweepoviruses, a group of phylogenetically distinct begomoviruses that infect plants of the family Convolvulaceae including sweet potato. In this work, the deltasatellites associated with sweepoviruses are shown to be transreplicated and maintained in plants by the virus with which they were identified, sweet potato leaf curl virus (SPLCV). These deltasatellites were shown generally to reduce symptom severity of the virus infection by reducing virus DNA levels. Additionally they were shown to be maintained in plants, and reduce the symptoms induced by two Old World monopartite begomoviruses, tomato yellow leaf curl virus and tomato yellow leaf curl Sardinia virus. Finally one of the satellites was shown to be transmitted plant-to-plant in the presence of SPLCV by the whitefly vector of the virus, Bemisia tabaci, being the first time a deltasatellite has been shown to be insect transmitted.

Infectious cDNA clones of the crinivirus Tomato chlorosis virus are competent for systemic plant infection and whitefly-transmission.

Tomato chlorosis virus (ToCV) (genus Crinivirus, family Closteroviridae) causes important emergent diseases in tomato and other solanaceous crops. ToCV is not transmitted mechanically and is naturally transmitted by whiteflies. The ToCV genome consists of two molecules of linear, positive-sense RNA encapsidated into long flexuous virions. We present the construction of full-length cDNA clones of the ToCV genome (RNA1 and RNA2) fused to the SP6 RNA polymerase promoter and under the control of the CaMV 35S promoter. RNA1 replicated in the absence of RNA2 in Nicotiana benthamiana and tomato protoplasts after inoculation with cDNA-derived in vitro transcripts. Agroinfiltration of RNA1 and RNA2 under the 35S promoter resulted in systemic infection in N. benthamiana plants. In addition, tomato plants were infected by grafting with agroinfected N. benthamiana scions, showing the typical ToCV symptoms. The viral progeny generated in tomato was transmissible by the whitefly Bemisia tabaci.

Sweepoviruses cause disease in sweet potato and related Ipomoea spp.: fulfilling Koch's postulates for a divergent group in the genus begomovirus.

Sweet potato (Ipomoea batatas) and related Ipomoea species are frequently infected by monopartite begomoviruses (genus Begomovirus, family Geminiviridae), known as sweepoviruses. Unlike other geminiviruses, the genomes of sweepoviruses have been recalcitrant to rendering infectious clones to date. Thus, Koch's postulates have not been fullfilled for any of the viruses in this group. Three novel species of sweepoviruses have recently been described in Spain: Sweet potato leaf curl Lanzarote virus (SPLCLaV), Sweet potato leaf curl Spain virus (SPLCSV) and Sweet potato leaf curl Canary virus (SPLCCaV). Here we describe the generation of the first infectious clone of an isolate (ES:MAL:BG30:06) of SPLCLaV. The clone consisted of a complete tandem dimeric viral genome in a binary vector. Successful infection by agroinoculation of several species of Ipomoea (including sweet potato) and Nicotiana benthamiana was confirmed by PCR, dot blot and Southern blot hybridization. Symptoms observed in infected plants consisted of leaf curl, yellowing, growth reduction and vein yellowing. Two varieties of sweet potato, 'Beauregard' and 'Promesa', were infected by agroinoculation, and symptoms of leaf curl and interveinal loss of purple colouration were observed, respectively. The virus present in agroinfected plants was readily transmitted by the whitefly Bemisia tabaci to I. setosa plants. The progeny virus population present in agroinfected I. setosa and sweet potato plants was isolated and identity to the original isolate was confirmed by sequencing. Therefore, Koch's postulates were fulfilled for the first time for a sweepovirus.