Pelargonium Line Pattern Virus Research Articles

A Viral Suppressor of RNA Silencing May Be Targeting a Plant Defence Pathway Involving Fibrillarin.

To establish productive infections, viruses must be able both to subdue the host metabolism for their own benefit and to counteract host defences. This frequently results in the establishment of viral–host protein–protein interactions that may have either proviral or antiviral functions. The study of such interactions is essential for understanding the virus–host interplay. Plant viruses with RNA genomes are typically translated, replicated, and encapsidated in the cytoplasm of infected cells. Despite this, a significant array of their encoded proteins has been reported to enter the nucleus, often showing high accumulation at subnuclear structures such as the nucleolus and/or Cajal bodies. However, the biological significance of such a distribution pattern is frequently unknown. Here, we explored whether the nucleolar/Cajal body localization of protein p37 of Pelargonium line pattern virus (PLPV, genus Pelarspovirus, family Tombusviridae), might be related to potential interactions with the nucleolar/Cajal body marker proteins, fibrillarin and coilin. The results revealed that p37, which has a dual role as coat protein and as suppressor of RNA silencing, a major antiviral system in plants, is able to associate with these cellular factors. Analysis of (wildtype and/or mutant) PLPV accumulation in plants with up- or downregulated levels of fibrillarin or coilin have suggested that the former might be involved in an as yet unknown antiviral pathway, which may be targeted by p37. The results suggest that the growing number of functions uncovered for fibrillarin can be wider and may prompt future investigations to unveil the plant antiviral responses in which this key nucleolar component may take part.

Genetic evidence for the involvement of Dicer-like 2 and 4 as well as Argonaute 2 in the Nicotiana benthamiana response against Pelargonium line pattern virus.

In plants, RNA silencing functions as a potent antiviral mechanism. Virus-derived double-stranded RNAs (dsRNAs) trigger this mechanism, being cleaved by Dicer-like (DCL) enzymes into virus small RNAs (vsRNAs). These vsRNAs guide sequence-specific RNA degradation upon their incorporation into an RNA-induced silencing complex (RISC) that contains a slicer of the Argonaute (AGO) family. Host RNA dependent-RNA polymerases, particularly RDR6, strengthen antiviral silencing by generating more dsRNA templates from RISC-cleavage products that, in turn, are converted into secondary vsRNAs by DCLs. Previous work showed that Pelargonium line pattern virus (PLPV) is a very efficient inducer and target of RNA silencing as PLPV-infected Nicotiana benthamiana plants accumulate extraordinarily high amounts of vsRNAs that, strikingly, are independent of RDR6 activity. Several scenarios may explain these observations including a major contribution of dicing versus slicing for defence against PLPV, as the dicing step would not be affected by the RNA silencing suppressor encoded by the virus, a protein that acts via vsRNA sequestration. Taking advantage of the availability of lines of N. benthamiana with DCL or AGO2 functions impaired, here we have tried to get further insights into the components of the silencing machinery that are involved in anti-PLPV-silencing. Results have shown that DCL4 and, to lesser extent, DCL2 contribute to restrict viral infection. Interestingly, AGO2 apparently makes even a higher contribution in the defence against PLPV, extending the number of viruses that are affected by this particular slicer. The data support that both dicing and slicing activities participate in the host race against PLPV.

Epigenetic Changes in Host Ribosomal DNA Promoter Induced by an Asymptomatic Plant Virus Infection

DNA cytosine methylation is one of the main epigenetic mechanisms in higher eukaryotes and is considered to play a key role in transcriptional gene silencing. In plants, cytosine methylation can occur in all sequence contexts (CG, CHG, and CHH), and its levels are controlled by multiple pathways, including de novo methylation, maintenance methylation, and demethylation. Modulation of DNA methylation represents a potentially robust mechanism to adjust gene expression following exposure to different stresses. However, the potential involvement of epigenetics in plant-virus interactions has been scarcely explored, especially with regard to RNA viruses. Here, we studied the impact of a symptomless viral infection on the epigenetic status of the host genome. We focused our attention on the interaction between Nicotiana benthamiana and Pelargonium line pattern virus (PLPV, family Tombusviridae), and analyzed cytosine methylation in the repetitive genomic element corresponding to ribosomal DNA (rDNA). Through a combination of bisulfite sequencing and RT-qPCR, we obtained data showing that PLPV infection gives rise to a reduction in methylation at CG sites of the rDNA promoter. Such a reduction correlated with an increase and decrease, respectively, in the expression levels of some key demethylases and of MET1, the DNA methyltransferase responsible for the maintenance of CG methylation. Hypomethylation of rDNA promoter was associated with a five-fold augmentation of rRNA precursor levels. The PLPV protein p37, reported as a suppressor of post-transcriptional gene silencing, did not lead to the same effects when expressed alone and, thus, it is unlikely to act as suppressor of transcriptional gene silencing. Collectively, the results suggest that PLPV infection as a whole is able to modulate host transcriptional activity through changes in the cytosine methylation pattern arising from misregulation of methyltransferases/demethylases balance.

New Insights into the Nucleolar Localization of a Plant RNA Virus-Encoded Protein That Acts in Both RNA Packaging and RNA Silencing Suppression: Involvement of Importins Alpha and Relevance for Viral Infection.

Despite the fact that replication of plus-strand RNA viruses takes place in the cytoplasm of host cells, different proteins encoded by these infectious agents have been shown to localize in the nucleus, with high accumulation at the nucleolus. In most cases, the molecular determinants or biological significance of such subcellular localization remains elusive. Recently, we reported that protein p37 encoded by Pelargonium line pattern virus (family Tombusviridae) acts in both RNA packaging and RNA silencing suppression. Consistently with these functions, p37 was detected in the cytoplasm of plant cells, although it was also present in the nucleus and, particularly, in the nucleolus. Here, we searched for further insights into factors influencing p37 nucleolar localization and into its potential relevance for viral infection. Besides mapping the protein region containing the nucleolar localization signal, we have found that p37 interacts with distinct members of the importin alpha family-main cellular transporters for nucleo-cytoplasmic traffic of proteins-and that these interactions are crucial for nucleolar targeting of p37. Impairment of p37 nucleolar localization through downregulation of importin alpha expression resulted in a reduction of viral accumulation, suggesting that sorting of the protein to the major subnuclear compartment is advantageous for the infection process.

Nicotiana benthamiana plants asymptomatically infected by Pelargonium line pattern virus show unusually high accumulation of viral small RNAs that is neither associated with DCL induction nor RDR6 activity

Pelargonium line pattern virus (PLPV, Tombusviridae) normally establishes systemic, low-titered and asymptomatic infections in its hosts. This type of interaction may be largely determined by events related to RNA silencing, a major antiviral mechanism in plants. This mechanism is triggered by double or quasi double-stranded (ds) viral RNAs which are cut by DCL ribonucleases into virus small RNAs (vsRNAs). Such vsRNAs are at the core of the silencing process as they guide sequence-specific RNA degradation Host RNA dependent-RNA polymerases (RDRs), and particularly RDR6, strengthen antiviral silencing by promoting biosynthesis of secondary vsRNAs. To approach PLPV-host relationship, here we have characterized the vsRNAs that accumulate in PLPV-infected Nicotiana benthamiana. Such accumulation was found unprecedented high despite DCLs were not induced in infected tissue and neither vsRNA generation nor PLPV infection was apparently affected by RDR6 impairment. From the obtained data, triggers and host factors likely involved in anti-PLPV silencing are proposed.

Efficient Translation of Pelargonium line pattern virus RNAs Relies on a TED-Like 3´-Translational Enhancer that Communicates with the Corresponding 5´-Region through a Long-Distance RNA-RNA Interaction.

Cap-independent translational enhancers (CITEs) have been identified at the 3´-terminal regions of distinct plant positive-strand RNA viruses belonging to families Tombusviridae and Luteoviridae. On the bases of their structural and/or functional requirements, at least six classes of CITEs have been defined whose distribution does not correlate with taxonomy. The so-called TED class has been relatively under-studied and its functionality only confirmed in the case of Satellite tobacco necrosis virus, a parasitic subviral agent. The 3´-untranslated region of the monopartite genome of Pelargonium line pattern virus (PLPV), the recommended type member of a tentative new genus (Pelarspovirus) in the family Tombusviridae, was predicted to contain a TED-like CITE. Similar CITEs can be anticipated in some other related viruses though none has been experimentally verified. Here, in the first place, we have performed a reassessment of the structure of the putative PLPV-TED through in silico predictions and in vitro SHAPE analysis with the full-length PLPV genome, which has indicated that the presumed TED element is larger than previously proposed. The extended conformation of the TED is strongly supported by the pattern of natural sequence variation, thus providing comparative structural evidence in support of the structural data obtained by in silico and in vitro approaches. Next, we have obtained experimental evidence demonstrating the in vivo activity of the PLPV-TED in the genomic (g) RNA, and also in the subgenomic (sg) RNA that the virus produces to express 3´-proximal genes. Besides other structural features, the results have highlighted the key role of long-distance kissing-loop interactions between the 3´-CITE and 5´-proximal hairpins for gRNA and sgRNA translation. Bioassays of CITE mutants have confirmed the importance of the identified 5´-3´ RNA communication for viral infectivity and, moreover, have underlined the strong evolutionary constraints that may operate on genome stretches with both regulatory and coding functions.

Evidence supporting a premature termination mechanism for subgenomic RNA transcription in Pelargonium line pattern virus: identification of a critical long-range RNA-RNA interaction and functional variants through mutagenesis.

Pelargonium line pattern virus (PLPV) is a plus-strand RNA virus that has been proposed as type species of a tentative new genus, Pelarspovirus, in the family Tombusviridae. One of the singular traits of members of this prospective genus is the production of a unique subgenomic (sg) mRNA that is structurally and functionally tricistronic. Here, we have aimed to get insights into the mechanism that governs PLPV sg mRNA transcription. A long-range RNA-RNA interaction that is critical for the process has been identified through RNA folding predictions and mutational analysis of the viral genome. Such interaction seems to occur in the plus-strand, likely acts in cis, and specifically mediates the synthesis of sg RNA-sized minus-strand. The accumulation of this RNA species is easily detectable in plants and its generation can be uncoupled from that of the plus-strand sg mRNA. All these data together with the observation that 5' ends of PLPV genomic and sg mRNAs have sequence resemblances (as expected if both act as promoters in the corresponding minus-strand), support that premature termination is the mechanism underlying PLPV sg mRNA formation.

Key Importance of Small RNA Binding for the Activity of a Glycine-Tryptophan (GW) Motif-containing Viral Suppressor of RNA Silencing

Viruses express viral suppressors of RNA silencing (VSRs) to counteract RNA silencing-based host defenses. Although virtually all stages of the antiviral silencing pathway can be inhibited by VSRs, small RNAs (sRNAs) and Argonaute (AGO) proteins seem to be the most frequent targets. Recently, GW/WG motifs of some VSRs have been proposed to dictate their suppressor function by mediating interaction with AGO(s). Here we have studied the VSR encoded by Pelargonium line pattern virus (family Tombusviridae). The results show that p37, the viral coat protein, blocks RNA silencing. Site-directed mutagenesis of some p37 sequence traits, including a conserved GW motif, allowed generation of suppressor-competent and -incompetent molecules and uncoupling of the VSR and particle assembly capacities. The engineered mutants were used to assess the importance of p37 functions for viral infection and the relative contribution of diverse molecular interactions to suppressor activity. Two main conclusions can be drawn: (i) the silencing suppression and encapsidation functions of p37 are both required for systemic Pelargonium line pattern virus infection, and (ii) the suppressor activity of p37 relies on the ability to bind sRNAs rather than on interaction with AGOs. The data also caution against potential misinterpretations of results due to overlap of sequence signals related to distinct protein properties. This is well illustrated by mutation of the GW motif in p37 that concurrently affects nucleolar localization, efficient interaction with AGO1, and sRNA binding capability. These concomitant effects could have been overlooked in other GW motif-containing suppressors, as we exemplify with the orthologous p38 of turnip crinkle virus.

Complete nucleotide sequence of rose yellow leaf virus, a new member of the family Tombusviridae.

The genome of the rose yellow leaf virus (RYLV) has been determined to be 3918 nucleotides long and to contain seven open reading frames (ORFs). ORF1 encodes a 27-kDa peptide (p27). ORF2 shares a common start codon with ORF1 and continues through the amber stop codon of p27 to encode an 87-kDa (p87) protein that has amino acid similarity to the RNA-dependent RNA polymerase (RdRp) of members of the family Tombusviridae. ORFs 3 and 4 have no significant amino acid similarity to known functional viral ORFs. ORF5 encodes a 6-kDa (p6) protein that has similarity to movement proteins of members of the Tombusviridae. ORF5A has no conventional start codon and overlaps with p6. A putative +1 frameshift mechanism allows p6 translation to continue through the stop codon and results in a 12-kDa protein that has high homology to the carmovirus p13 movement protein. The 37-kDa protein encoded by ORF6 has amino acid sequence similarity to coat proteins (CP) of members of the Tombusviridae. ORF7 has no significant amino acid similarity to known viral ORFs. Phylogenetic analysis of the RdRp amino acid sequences grouped RYLV together with the unclassified Rosa rugosa leaf distortion virus (RrLDV), pelargonium line pattern virus (PLPV), and pelargonium chlorotic ring pattern virus (PCRPV) in a distinct subgroup of the family Tombusviridae.

ATTEMPTS TO ERADICATE TWO PELARGONIUM VIRUSES (PFBV AND PLPV) BY MERISTEM CULTURE AND SHOOT-TIP CRYOTHERAPY

Attempts to eradicate the Pelargonium flower break virus (PFBV) and Pelargonium line pattern virus (PLPV) by meristem culture and apex droplet-vitrification cryopreservation was carried out using 5 different cultivars. A simple meristem culture did not permit to eliminate PFBV and only 15% of Pelargonium x hortorum 'Stellar Artic' plants regenerated from meristems was PLPV-ELISA-negative. Plants regenerated from cryopreserved apices were tested by DAS-ELISA after a 3-month growing period. Viruses were not detected in 25 and 50% of the tested plants for PFBV and PLPV respectively. Immunolocalisations were carried out for virus localisation in apices from greenhouse plants (control) and vitroplants regenerated after meristem culture or cryopreservation. Immunolocalisations realised on control explants excised from DAS-ELISA positive plants showed that PFBV and PLPV were present in the apices, even in the meristematic dome. However, viral particles were more numerous in the cells of the basal zone than in the more meristematic ones. Immunolocalisations realised on apices from the DAS-ELISA negative cryoregenerated plants showed the viruses were still present. Our results firstly demonstrated that PFBV and PLPV are even present inside meristematic cells and secondly that cryopreservation could decrease their amount in Pelargonium plants but without eliminating them totally. More knowledge on virus behaviour during cryopreservation processes could optimize the management of genetic resources using this conservation method.

Population differentiation and selective constraints in Pelargonium line pattern virus

The genomic structure of Pelargonium line pattern virus (PLPV), a tentative member of a proposed new genus within the family Tombusviridae, has been recently determined. However, little is known about the genetic variability and population structure of this pathogen. Here, we have investigated the heterogeneity of PLPV isolates from different origins by sequence analysis of a 1817 nt fragment encompassing the movement (p7 and p9.7) and coat protein genes as well as flanking segments including the complete 3′ untranslated region. We have evaluated the selective pressures operating on both viral proteins and RNA genome in order to assess the relative functional and/or structural relevance of different amino acid or nucleotide sites. The results of the study have revealed that distinct protein domains are under different selective constraints and that maintenance of certain primary and/or secondary structures in RNA regulatory sequences might be an important factor limiting viral heterogeneity. We have also performed covariation analyses to uncover potential dependencies among amino acid sites of the same protein or of different proteins. The detection of linked amino acid substitutions has permitted to draw a putative network of intra- and interprotein interactions that are likely required to accomplish the different steps of the infection cycle. Finally, we have obtained phylogenetic trees that support geographical segregation of PLPV sequences.

Insights into the translational regulation of biologically active open reading frames of Pelargonium line pattern virus

Pelargonium line pattern virus (PLPV), a proposed member of a prospective genus ( Pelarspovirus) within family Tombusviridae, has a positive-sense, single-stranded genomic RNA. According to previous predictions, it contains six open reading frames (ORFs) potentially encoding proteins of 27 (p27), 13 (p13), 87 (p87), 7 (p7), 6 (p6), and 37 kDa (p37). Using a variety of techniques we demonstrate that all predicted ORFs are functional, with the exception of (p13) and (p6). We also characterize a previously unidentified ORF which encodes a 9.7 kDa protein (p9.7) that is essential for viral movement. Furthermore, we present evidence that the single subgenomic RNA (sgRNA) produced by the virus directs synthesis of p7, p9.7 and p37. Remarkably, the translation of these totally unrelated proteins is coordinated via leaky-scanning. This mechanism seems to be favoured by the poor translation context of the start codon of ORF(p7), the non-AUG weak initiation codon of ORF(p9.7) and the lack of additional AUG codons in any reading frame preceding ORF(p37). The results also suggest that precise regulation of protein production from the sgRNA is critical for virus viability. Altogether, the data supports the notion that PLPV belongs to a new genus of plant viruses.

Pelargonium chlorotic ring pattern virus: first report in Spain

A recent survey of symptomless ornamental geraniums ( Pelargonium zonale , P. peltatum and P. grandiflorum ) in Spain (2000–2003) revealed viral infections in 52·4% of the tested plants (Alonso & Borja, 2005). Most infected plants contained tombus-like viruses such as Pelargonium line pattern virus (PLPV), either alone or in combination with other viral pathogens. Several little-studied PLPV-related viruses have been reported in Pelargonium spp. but their distribution and importance to geranium growers is unknown since they are not usually included in phytosanitary programmes. One of these viruses, Pelargonium chlorotic ring pattern virus (PCRPV), was recently sequenced (GenBank Acc. No. AY038069) and found to be related to PLPV in nucleotide sequence and genome organization (Kinard & Jordan, 2002; Castano & Hernandez, 2005). To assess the occurrence of PCRPV in Spain 5 samples from the previous survey and 3 gathered in 2006 were tested. Total RNA was extracted using Plant RNA Reagent (Invitrogen) and reverse transcribed with Superscript II-RT (Invitrogen) and primer CH149 (5 ′ -CCGATTCTCGAGCTGCCAATC-3 ′ ), complementary to positions 1543–1523 of PCRPV. PCR used primers CH149 and CH148 (5 ′ -CGGTTTGAGCACTCTGCGTATC-3 ′ ), homologous to positions 1277–1298 of PCRPV. An amplicon of the expected size (267 bp comprising a region of the viral polymerase gene) was generated in five samples from different geographical origins (Zaragoza, Zamora, Aranjuez and Cordoba) and collecting times (2000, 2003 and 2006). The amplicons derived from two separate samples were sequenced (GenBank Acc. Nos. EF635383 and EF635384) and showed 95% nucleotide and 99–100% amino acid sequence identity with the PCRPV reference sequence. An Italian isolate of PCRPV was recovered from P. zonale with vein banding and leaf distortion (Lisa et al ., 1996; Kinard & Jordan, 2002); however from our results, PCRPV infection can also be asymptomatic. This is the first report of PCRPV in Spain.

High incidence of Pelargonium line pattern virus infecting asymptomatic Pelargonium spp. in Spain

Geraniums (Pelargonium spp.) are traditional ornamental plants largely cultivated in Europe and northern America. Vegetative propagation makes them prone to viral infections, which have detrimental effects on crop production and quality. Asymptomatic samples collected in Spain were tested for a range of viruses using ELISA. The tobamovirus, Tobacco mosaic virus (TMV), the cucumovirus, Cucumber mosaic virus (CMV), and several viruses in the family Tombusviridae, namely, Pelargonium line pattern virus (PLPV), Pelargonium flower break virus (PFBV), and Pelargonium leaf curl virus (PLCV), were detected either singly or in combination in 59.2% of 800 samples. PLPV and PFBV infections were confirmed by dot-blot hybridisation. The most relevant viral infection found on Spanish asymptomatic geraniums was by Pelargonium line pattern virus (PLPV). Symptoms did not develop for 3 years on most of the PLPV infected geranium plants under greenhouse conditions.

Complete nucleotide sequence and genome organization of Pelargonium line pattern virus and its relationship with the family Tombusviridae

Summary.The complete nucleotide sequence of Pelargonium line pattern virus (PLPV) has been determined. The PLPV genomic RNA comprises 3884 nt and contains six open reading frames (ORFs) potentially encoding proteins of 27 (p27), 13 (p13), 87 (p87), 7 (p7), 6 (p6), and 37 kDa (p37), respectively. The arrangement of these ORFs on the PLPV genome closely resembles that of members of the genus Carmovirus in the family Tombusviridae and, moreover, most of the putative PLPV gene products showed high identity with proteins of this viral group. However, several striking differences were noticed. Carmoviruses generate two subgenomic RNAs whereas PLPV produces a single one. In addition, only p7 showed similarity with movement proteins of carmoviruses whereas p6 (as p13) has no viral (or other) homologs. This protein might be expressed from a non-canonical start codon or, alternatively, through a −1 frameshift (FS) mechanism. Both, the production of one subgenomic RNA and the likely involvement of a −1 FS for expression of an internal ORF parallel the translation strategies reported for the unique species of the genus Panicovirus, belonging also to the family Tombusviridae. Overall, the results support the placement of PLPV in this family although its peculiar characteristics preclude its direct assignment to any of the current genera.

Development of a Non-radioactive Dot-blot Hybridisation Assay for the Detection of Pelargonium Flower Break Virus and Pelargonium line Pattern Virus

The ornamental geranium, Pelargonium×hortorum Bailey, is a traditional ornamental plant widely cultivated in Europe and Northern America. Vegetative propagation facilitates rapid spread of viral infections which have detrimental effects on the production and the quality of the crop. A non-radioactive nucleic acid hybridisation method was developed for detection of Pelargonium flower break virus (PFBV) and Pelargonium line pattern virus (PLPV) in infected host plants. This method was significantly more sensitive than the conventional ELISA test when using either purified viral preparations or crude plant extracts. The distribution of the viruses was studied by means of the non-isotopic hybridisation technique. The results indicated that the petioles and the apical blade regions of fully expanded leaves were the best source of test material. The hybridisation procedure enables the detection of PFBV and PLPV in a single assay, and its simplicity allows its application to routine large-scale indexing.

Intracellular Changes Provoked by Pelargonium Line Pattern Virus

Intracellular Changes Provoked by Pelargonium Line Pattern Virus