Characterization of Spanish Olive Virome by High Throughput Sequencing Opens New Insights and Uncertainties.

The use of high throughput sequencing (HTS) for the analysis of Spanish olive trees showing leaf yellowing discoloration, defoliation, and/or decline has provided new insights into the olive viruses present in Spain and has opened discussions about the pros and cons of these technologies for diagnostic purposes. In this study, we report for the first time in Spanish orchards the presence of olive leaf yellowing-associated virus (OLYaV), for which the second full coding sequence has been determined. This virus has also been detected in a putative vector, the psyllid Euphyllura olivina. In addition, the presence in Spain of Olea europaea geminivirus (OEGV), recently reported in Italy, has been confirmed, and the full-length sequence of two isolates was obtained by HTS and Sanger sequencing. These results, as well as the detection of other viral sequences related to olive latent virus 3 (OLV-3) and olive viral satellite RNA, raises questions on the biological significance of the findings, about the requirement of standardization on the interpretation of HTS results, and the necessity of additional tests to confirm the relevance of the HTS detection of viral sequences.

A Bipartite Geminivirus with a Highly Divergent Genomic Organization Identified in Olive Trees May Represent a Novel Evolutionary Direction in the Family Geminiviridae.

Olea europaea Geminivirus (OEGV) was recently identified in olive in Italy through HTS. In this work, we used HTS to show the presence of an OEGV isolate in Portuguese olive trees and suggest the evolution direction of OEGV. The bipartite genome (DNA-A and DNA-B) of the OEGV-PT is similar to Old World begomoviruses in length, but it lacks a pre-coat protein (AV2), which is a typical feature of New World begomoviruses (NW). DNA-A genome organization is closer to NW, containing four ORFs; three in complementary-sense AC1/Rep, AC2/TrAP, AC3/REn and one in virion-sense AV1/CP, but no AC4, typical of begomoviruses. DNA-B comprises two ORFs; MP in virion sense with higher similarity to the tyrosine phosphorylation site of NW, but in opposite sense to begomoviruses; BC1, with no known conserved domains in the complementary sense and no NSP typical of bipartite begomoviruses. Our results show that OEGV presents the longest common region among the begomoviruses, and the TATA box and four replication-associated iterons in a completely new arrangement. We propose two new putative conserved regions for the geminiviruses CP. Lastly, we highlight unique features that may represent a new evolutionary direction for geminiviruses and suggest that OEGV-PT evolution may have occurred from an ancient OW monopartite Begomovirus that lost V2 and C4, gaining functions on cell-to-cell movement by acquiring a DNA-B component.

Olea Europaea Geminivirus: A Novel Bipartite Geminivirid Infecting Olive Trees.

In 2014, high-throughput sequencing of libraries of total DNA from olive trees allowed the identification of two geminivirus-like contigs. After conventional resequencing of the two genomic DNAs, their analysis revealed they belonged to the same viral entity, for which the provisional name of Olea europaea geminivirus (OEGV) was proposed. Although DNA-A showed a genome organization similar to that of New World begomoviruses, DNA-B had a peculiar ORF arrangement, consisting of a movement protein (MP) in the virion sense and a protein with unknown function on the complementary sense. Phylogenetic analysis performed either on full-length genome or on coat protein, replication associated protein (Rep), and MP sequences did not endorse the inclusion of this virus in any of the established genera in the family Geminiviridae. A survey of 55 plants revealed that the virus is widespread in Apulia (Italy) with 91% of the samples testing positive, although no correlation of OEGV with a disease or specific symptoms was encountered. Southern blot assay suggested that the virus is not integrated in the olive genome. The study of OEGV-derived siRNA obtained from small RNA libraries of leaves and fruits of three different cultivars, showed that the accumulation of the two genomic components is influenced by the plant genotype while virus-derived-siRNA profile is in line with other geminivirids reported in literature. Single-nucleotide polymorphism (SNP) analysis unveiled a low intra-specific variability.

Epigenetic Regulation of Verticillium dahliae Virulence: Does DNA Methylation Level Play A Role?

Verticillium dahliae is the etiological agent of Verticillium wilt of olive. The virulence of Defoliating V. dahliae isolates usually displays differences and high plasticity. This work studied whether an epigenetic mechanism was involved in this plasticity. An inverse correlation between virulence and DNA methylation of protein-coding genes was found. A set of 831 genes was selected for their highly consistent inverse methylation profile and virulence in the five studied isolates. Of these genes, ATP-synthesis was highly represented, which indicates that the more virulent D isolates are, the more energy requirements they may have. Furthermore, there were numerous genes in the protein biosynthesis process: genes coding for the chromatin structure, which suggests that epigenetic changes may also affect chromatin condensation; many transmembrane transporter genes, which is consistent with denser compounds, traffic through membranes in more virulent isolates; a fucose-specific lectin that may play a role in the attachment to plant cell walls during the host infection process; and pathogenic cutinases that facilitate plant invasion and sporulation genes for rapid spreading alongside plants. Our findings support the notion that differences in the virulence of the Defoliating V. dahliae isolates may be controlled, at least to some extent, by an epigenetic mechanism.

Virus-Induced Gene Silencing in Olive Tree (Oleaceae).

Research on gene functions in non-model tree species is hampered by a number of difficulties such as time-consuming genetic transformation protocols and extended period for the production of healthy transformed offspring, among others. Virus-induced gene silencing (VIGS) is an alternative approach to transiently knock out an endogenous gene of interest (GOI) by the introduction of viral sequences encompassing a fragment of the GOI and to exploit the posttranscriptional gene silencing (PTGS) mechanism of the plant, thus triggering silencing of the GOI. Here we describe the successful application of Tobacco rattle virus (TRV)-mediated VIGS through agroinoculation of olive plantlets. This methodology is expected to serve as a fast tracking and powerful tool enabling researchers from diversified fields to perform functional genomic analyses in the olive tree.

Molecular characterisation of a novel gemycircularvirus associated with olive trees in Italy.

A new gemycircularvirus sequence was obtained from total DNAs extracted from olive leaves and twigs tissues. Its complete genome consists of a single-stranded circular DNA of 2,145 nt, whose sequence was obtained by rolling circle amplification (RCA). Genome analysis identified three open reading frames, sharing homologies with the coat and replication-associated proteins, these latter in the anti-parallel strand, of known gemycircularvirus species. Search for homologies showed that the circular ssDNA sequence is distantly related to other gemycircularviruses thus originating from a new virus species, for which the name olive-associated gemycircularvirus 1 (OaGV1) is proposed. A survey in two different olive-growing areas of the Apulian region (Southern Italy), showed a limited distribution of OaGV1.

Genetic diversity of the coat protein of Olive mild mosaic virus (OMMV) and Tobacco necrosis virus D (TNV-D) isolates and its structural implications.

The genetic variability among 13 isolates of Olive mild mosaic virus (OMMV) and of 11 isolates of Tobacco necrosis virus D (TNV-D) recovered from Olea europaea L. samples from various sites in Portugal, was assessed through the analysis of the coat protein (CP) gene sequences. This gene was amplified through reverse transcriptase polymerase chain reaction (RT-PCR), cloned, and 5 clone sequences of each virus isolate, were analysed and compared, including sequences from OMMV and TNV-D isolates originally recovered from different hosts and countries and available in the GenBank, totalling 131 sequences. The encoded CP sequences consisted of 269 amino acids (aa) in OMMV and 268 in TNV-D. Comparison of the CP genomic and amino acid sequences of the isolates showed a very low variability among OMMV isolates, 0.005 and 0.007, respectively, as well as among TNV-D isolates, 0.006 and 0.008. The maximum nucleotide distances of OMMV and TNV-D sequences within isolates were also low, 0.013 and 0.031, respectively, and close to that found between isolates, 0.018 and 0.034, respectively. In some cases, less variability was found in clone sequences between isolates than in clone sequences within isolates, as also shown through phylogenetic analysis. CP aa sequence identities among OMMV and TNV-D isolates ranged from 84.3% to 85.8%. Comparison between the CP genomic sequences of the two viruses, showed a relatively low variability, 0.199, and a maximum nucleotide distance between isolates of 0.411. Analysis of comparative models of OMMV and TNV-D CPs, showed that naturally occurring substitutions in their respective sequences do not seem to cause significant alterations in the virion structure. This is consistent with a high selective pressure to preserve the structure of viral capsid proteins.

A degenerate pair of primers for simultaneous detection of four alpha- and betanecroviruses.

The high infection levels due to Olive latent virus 1 (OLV-1), Olive mild mosaic virus (OMMV) (alphanecrovirus) and Tobacco necrosis virus D (TNV-D) (betanecrovirus) in Portuguese olive orchards prompted us to develop a rapid PCR-based assay for the simultaneous detection of these viruses aimed at the sanitary selection and marketing of plant material in compliance with European Union regulations. A pair of degenerate oligonucleotide primers, parRdRp5' and parCoat3' was designed based on conserved regions located in the RNA-dependent RNA polymerase (RdRp) and coat protein (CP) genes of these viruses and one other alphanecrovirus, Tobacco necrosis virus A. Its use in RT-PCR assays generated a product of ca. 2000 bp for the 4 viral species tested. These primers were compared with virus specific primers in multiplex RT-PCR, and identical results were obtained. Its application to dsRNA extracted from 54 olive field growing trees originated the expected ca. 2000 bp amplicon in 17 trees. The virus identity was determined by sequencing the cloned RT-PCR products. No TNV-A was found. The RT-PCR assay using the degenerate primers described in this study were shown to be reliable in detecting any of the above-mentioned alpha- and betanecroviruses, and it is as sensitive as that which uses virus specific primers in multiplex assays. Therefore, this assay is well suited for the rapid screen of virus-free plant material in selection and improvement crop programmes. Additionally, it has the potential to reveal virus diversity and the presence of new viruses, provided the RT-PCR generated amplicon is further sequenced.

Complete nucleotide sequence and genome organization of Olive latent virus 3, a new putative member of the family Tymoviridae.

The complete nucleotide sequence and the genome organization were determined of a putative new member of the family Tymoviridae, tentatively named Olive latent virus 3 (OLV-3), recovered in southern Italy from a symptomless olive tree. The sequenced ssRNA genome comprises 7148 nucleotides excluding the poly(A) tail and contains four open reading frames (ORFs). ORF1 encodes a polyprotein of 221.6kDa in size, containing the conserved signatures of the methyltransferase (MTR), papain-like protease (PRO), helicase (HEL) and RNA-dependent RNA polymerase (RdRp) domains of the replication-associated proteins of positive-strand RNA viruses. ORF2 overlaps completely ORF1 and encodes a putative protein of 43.33kDa showing limited sequence similarity with the putative movement protein of Maize rayado fino virus (MRFV). ORF3 codes for a protein with predicted molecular mass of 28.46kDa, identified as the coat protein (CP), whereas ORF4 overlaps ORF3 and encodes a putative protein of 16kDa with sequence similarity to the p16 and p31 proteins of Citrus sudden death-associated virus (CSDaV) and Grapevine fleck virus (GFkV), respectively. Within the family Tymoviridae, OLV-3 genome has the closest identity level (49-52%) with members of the genus Marafivirus, from which, however, it differs because of the diverse genome organization and the presence of a single type of CP subunits.

Subcellular localization and immunodetection of movement proteins of olive latent virus 1.

Polyclonal sera raised to Escherichia coli-expressed movement proteins encoded by ORF 3 (p8K) and ORF 4 (p6K) of olive latent virus 1, were used for their immunodetection in infected Nicotiana benthamiana plants. In subfractionated locally infected tissues 4 days post inoculation (d.p.i.) that were analysed by Western blot, p8K was found in the fast-sedimenting fractions P1 and P30 containing membranous material and/or cell organelles and, likely, the fibrous structures mentioned below, but not in the soluble protein-containing supernatant. No p6K could be detected in these extracts. In locally inoculated leaves p8K began to accumulate from 2 d.p.i onwards reaching its peak at 4 d.p.i. Intracellular immunogold labelling of cells from locally and systemically infected tissues localized p8K primarily in fibrous inclusions made up of thin filaments with a helical structure present in the cytoplasm of locally and systemically infected cells. In systemic infections a light and scattered labelling was observed in the cytoplasm and near the cell wall. The specific serum to p6K did not label the fibrous structures and failed to recognize its antigen in systemically and locally infected tissues except at 4 d.p.i., when scattered labelling was observed in the cytoplasm and near plasmodesmata.

Viral properties, primary structure and phylogenetic analysis of the coat protein of an Olive latent virus 1 isolate from Olea europaea L.

An Olive latent virus 1 isolate designated GM6, obtained from a Portuguese olive tree, was characterized and the coat protein gene sequenced and analysed. The purified virus particles showed to be isometric with ca. 30 nm in diameter and contained a single-stranded RNA species with ca. 3.7 kb. The dsRNA profile obtained from infected tissues showed three major species with ca. 3.7, 1.5 and 1.3 kbp. SDS-PAGE analysis revealed a major peptide with an apparent molecular mass of 32 kDa identified as the coat protein. A viral genome region containing the coat protein gene was amplified by RT-PCR and the cDNA was cloned and sequenced. The coat protein gene revealed to be 813 nucleotides long and encode a peptide with 270 amino acid residues and an estimated Mr of 29,851. Alignment of the deduced amino acid sequence with that of other necroviruses showed a higher identity with OLV-1 tulip isolate (97.7%) than with OLV-1 citrus isolate (87.7%). The consensus pattern of the coat protein 'S' domain is conserved in GM6 isolate coat protein sequence, except in amino acid 151, leucine. This is the first report on the coat protein sequence of an OLV-1 olive isolate.

A Tobacco necrosis virus D isolate from Olea europaea L.: viral characterization and coat protein sequence analysis.

A virus isolated from Olea europaea L. grown in Portugal, was identified as a member of the species Tobacco necrosis virus D (TNV-D, genus Necrovirus, family Tombusviridae), based on the molecular and serological properties of the purified virus particles. The genomic region encoding the coat protein (CP) of this isolate (named GP isolate) was amplified by RT-PCR and the cDNA was cloned and sequenced. The CP gene encodes a predicted protein of 269 amino acids showing high identity (86.2%) to TNV-D coat protein sequence. Phylogenetic analysis based on necroviruses CP sequences, confirmed GP as a TNV-D isolate. The alignment with homologous TNV-D CP sequences revealed four conserved amino acids involved in Ca(2+) binding as well as the plant virus icosahedral capsid protein "S' signature. Based on the determined nucleotide sequence, specific primers were designed and successfully used in RT-PCR for virus diagnosis in naturally infected olive trees.