Virus Discovered by Deep Sequencing
A New Grapevine Virus Discovered by Deep Sequencing of Virus- And Viroid-Derived Small RNAs in Cv Pinot Gris
Content
Virus Research
Volume 163, Issue 1, January 2012, Pages 262–268
A new grapevine virus discovered by deep sequencing of virus- and viroid-derived small RNAs in Cv Pinot gris
Annalisa Giampetruzzia, 1, Vahid Roumia, 1, Roberta Robertoa, Umberto Malossinib, Nobuyuki Yoshikawac, Pierfederico La Nottea, Federica Terlizzid,
Rino Credid, Pasquale Saldarellia,
a
,
Dipartimento di Biologia e Chimica Agroforestale ed Ambientale, Università degli Studi di Bari ed Istituto di Virologia Vegetale Fondazione Edmund Mach – IASMA, via E. Mach 1 – 38010 San Michele a/A, Italy Faculty of Agriculture, Iwate University, Morioka 020-8550, Japan Dipartimento di Scienze e Tecnologie Agroambientali, Università di Bologna, Viale Fanin, 40, 40127 Bologna, Italia
del CNR UOS-Bari, via Amendola 165/A, 70126 Bari, Italy
b
c
d
http://dx.doi.org/10.1016/j.virusres.2011.10.010, How to Cite or Link Using DOI Permissions & Reprints
Abstract
Field symptoms of chlorotic mottling and leaf deformations were observed on the cv Pinot gris (PG) in the Trentino region (Italy). Extensive assays excluded the presence of widely distributed nepo-, ampelo- and vitiviruses. An analysis of small RNA populations from two PG grapevines showing or not symptoms was carried out by Illumina high throughput sequencing. The study disclosed the virus and viroids contents of the two vines that was composed by Grapevine rupestris stem pitting-associated
virus (GRSPaV), two viroids Hop stunt viroid (HSVd) and Grapevine yellow speckle viroid 1 (GYSVd1),
the marafivirusesGrapevine rupestris vein feathering virus (GRVFV) and Grapevine Syrah virus 1 (GSyV-1), and a hitherto unrecorded virus. This virus had a genome organization identical to that of Grapevine berry inner necrosis virus (GINV), a trichovirus reported only from Japan, with which it grouped in phylogenetic trees constructed with sequences of the RdRp domain and the coat protein gene. However, molecular differences with GINV are wide enough to warrant classification of the virus in question as a new species, for which the provisional name of Grapevine Pinot gris virus (GPGV) is proposed. A limited field survey for the presence of GPGV in diseased and symptomless plants from three different cultivars did not allow to clearly associating the virus to the observed symptoms.
Highlights
► Unknown viral symptoms were observed in cv. Pinot gris. ► Small RNAs from grapevine tissue were sequenced by Illumina technology. ► Viruses and viroids infecting Pinot gris plants and properties of their viral small RNAs were described. ► De novo assembling of small RNAs allowed assembling the genome of a new virus. ► This new virus is taxonomically related to trichoviruses. ► Limited field survey for the presence of this virus did not clearly associate the virus to the observed symptoms.
Keywords
Grapevine; Virus; Deep sequencing;
Contig;
Trichovirus
1. Introduction
Symptoms resembling those of a viral disease, i.e. chlorotic mottling, puckering and deformation of the leaves, reduced yield and low quality of the berries have been observed in 2003, in plants of cv Pinot
gris in vineyards of Trentino (northern Italy) (Fig. 1A and B). Successively (2009) similar symptoms
were described on the cvs Traminer and Pinot noir. Particularly on Traminer, symptoms were more accentuated, consisting in stunting and significant losses in production (Fig. 1C and D and unpublished information). No virus particles were detected with the electron microscope in dips from symptomatic PG leaves and no visible reactions were obtained with ELISA using commercial kits (Agritest, Italy) toGrapevine fanleaf virus (GFLV), Arabis mosaic virus (ArMV), Strawberry latent
ringspot virus (SLRSV),Grapevine leafroll-associated virus-1 (GLRaV-1), -2 (GLRaV-2) and -3
(GLRaV-3), Grapevine virus A(GVA) and Grapevine virus B (GVB) (unpublished results). Equally negative were RT-PCR assays using specific primers for the above reported ampelo- and vitiviruses and degenerate primers for the detection of members of the three subgroups of the genus Nepovirus (Digiaro et al., 2007).
Fig. 1. Chlorotic mottling and leaf deformations in the cvs Pinot gris (A and B) and Traminer (C). Shoot stunting in cv Traminer is shown in panel (D). Figure options
Deep sequencing is a powerful technology that provides rapid and exhaustive information on the infectious agents (viruses and viroids) present in plant tissues (Kreuze et al., 2009 and Wu et al., 2010). Therefore, this technology is being increasingly used for the quick identification of viruses replicating in plant tissues, either starting from the analysis of small interfering RNA (siRNAs) populations (Kreuze et al., 2009), or from sequenced libraries of fragmented double-stranded RNAs (dsRNAs) of viral origin (Al Rwahnih et al., 2009 and Coetzee et al., 2010), extracted from infected tissues. With a study of a vine of cv. Pinot Noir clone ENTAV 115, we have recently experienced the high potentiality of deep sequencing, and have described the “virome” [sensuCoetzee et al. (2010)] of the plant (Pantaleo et al., 2010). Thus, we have now deep sequenced and screened the small RNA population of a symptomatic and a symptomless PG vine, identifying, among other disease agents, a putatively new virus phylogenetically related to the trichovirus Grapevine berry inner necrosis
virus (GINV; Yoshikawa et al., 1997 and Kunugi et al., 2000), for which the provisional name
of Grapevine Pinot gris virus (GPGV) is proposed.
2. Materials and methods
2.1. Small RNA purification and sequencing
Leaves and petioles were collected in July 2010 from a symptomatic (plant S1+) and a symptomless (plant S2−) PG vine and stored at 4 °C for 5 days before using for total RNAs extraction. This was carried out as described by Pantaleo et al. (2010) and the low molecular weight RNA fraction (LMWRNA) was isolated by polyethylenglycol precipitation (Hamilton and Baulcombe, 1999). Small RNAs were separated by polyacrylamide gel electrophoresis and recovered from the gel as described by Lu et al. (2007). Libraries of small RNA populations from both vines were prepared for sequencing with an Illumina Genome Analyzer II (Institute of Applied Genomics, University of Udine, Italy) following the protocol of Lu et al. (2007), which generated 36–44 bp sequences. Adaptors, ribosomal and transfer RNA sequences were removed by the UEA siRNA toolkit (Moxon et al., 2008) and the small RNA sequences between 18 and 26 nt in size were initially screened for homology to known viruses by local BLASTN search against the GenBank Virus Reference Sequence database (RefSeq; http://www.ncbi.nlm.nih.gov/). Subsequently, libraries were searched with the SOAP (Short Oligonucleotide Alignment Program; Li et al., 2010) packages of software against reference sequences retrieved from the RefSeq database. Total small RNAs were then assembled into larger contigs using the Velvet Software 0.7.31 (Zerbino and Birney, 2008) with a k-mer of 17. GRSPaV, HSVd and GYSVd1 genomic RNAs were reconstructed by aligning and assembling contigson database-retrieved sequences, using the DNA Strider software 1.46 (Marck, 1988). Consensus sequences were finally obtained by the SOAPaligner software, which aligns small RNAs to
a reference genome, allowing a tolerance of two mismatches. Nucleotide polymorphisms having a frequency ≥80% and a minimum coverage of five valid small RNAs, as calculated by MapView (Bao et al., 2009), were allowed in the consensus sequence. Alignments and genome coverage were respectively visualized and estimated by MapView and Tablet (Milne et al., 2010). GPGV genome sequence was initially assembled on the available GINV genomic RNA (NC_015220) by aligning contigs obtained by Velvet. Sequence gaps were filled by sequencing cDNA fragments amplified with primers designed on the obtained contigs (Supplementary Table 1) cloned with the PCR cloning kit (Invitrogen, USA). A minimum of four clones per fragment was sequenced. Sequences of the extreme 3′ terminal nts were obtained by rapid amplification of cDNA ends (RACE) using two independent specific primers (GPgV6643f and GPgV7009f; Supplementary Table 1) and the 5′/3′ RACE kit from Roche Applied Sciences (Indianapolis, USA). Phylogenetic analysis was done with Clustal V (Larkin et al., 2007) using 1000 bootstrap replicates and the neighbor-joining method of analysis.
2.2. Field survey for the detection of GPGV
Forty grapevine plants of the cvs Pinot gris, Traminer and Pinot noir were identified in different commercial vineyards originating from the same cultivated area, either showing or not the described symptomatology. Total RNAs were purified from leaf and petiole tissues by a CTAB-based extraction protocol according toGambino et al. (2008). GPGV RT-PCR detection was performed by a two-step cDNA reverse transcription with random hexanucleotide primers and amplification according to Pantaleo et al. (2010)using specific primers (GPgV5619f/GPgV6668r; Supplementary Table 1) designed on the obtained sequence.
3. Results
3.1. High throughput sequencing and analysis of small RNAs
Libraries representative of the siRNAs population extracted from S1+ and S2 − vines and sequenced by Illumina technology, contained respectively 6.9 × 106 and 1.1 × 107 reads, after trimming adapters and filtering for transfer and ribosomal RNAs (Table 1). Only the data relevant for the identification of virus and viroid-related sequences were analyzed in the present study, whereas a detailed description of the library and of the small interfering RNA (siRNA) species will be the object of another paper.
Table 1. Virus- and viroid-related 18–26 siRNAs and contigs originated from both libraries. Plant S1+ Virus/genus HSVd/Hostuviroid GYSVd1/Apscaviroid GRSPaV/Foveavirus GRVFV/Marafivirus GSyV-1/Marafivirus GPGV Total virus reads Readsa 72,496 63,050 9378 451 368 112,184 257,559 %c 1.04 0.90 0.13 0.01 0.01 1.61 % v and vdd 28.15 24.48 3.64 0.18 0.14 43.56 Contigs 16 6 26 – – 36 Plant S2− Readsa 123,019 82,808 135,996 13,498 11,800 152,924 520,045 %c 1.11 0.75 1.23 0.12 0.11 1.38 % v and vdd 23.66 15.92 26.15 2.60 2.27 29.41 Contigs 15 13 189 25 28 48
Plant S1+ Virus/genus Other Total a
Redundant reads obtained by SOAPaligner.
e
Plant S2− %c % v and vdd Contigs Readsa 10,545,472 11,065,517b %c % v and vdd Contigs
Readsa 6,721,320 6,978,879b
b
Total reads after trimming of the adapters and filtering for ribosomal and transfer RNAs.
c
Percentages over the total reads.
d
Percentages over the total virus and viroidal reads.
e
Reads not matching with viral and viroidal sequences.
Table options
Preliminary BLASTN analysis of sequenced reads against the NCBI viral sequence database, identified siRNAs originating from the two grapevine viroids GYSVd1 and HSVd, the flexivirus GRSPaV and the two marafiviruses GRVFV and GSyV-1 (not shown). Libraries were therefore searched with SOAP using reference genome sequences of the above viruses and viroids. Vd-siRNAs originating from Hop stunt viroid (HSVd) and Grapevine yellow speckle viroid 1 (GYSVd1) were similarly represented in both plants either relatively to the total siRNAs or to v- and vd-siRNAs (Table 1). Consistently with Navarro et al. (2009), who reported that viroid RNAs are targeted by different Dicer enzymes producing 21, 22 and 24 nt siRNAs, we found a comparable size distribution of vd-siRNAs. In particular, the 21 and 24 nt populations were the most represented molecular species. However, differently from Navarro et al. (2009), equal (+) and (−) HSVd small RNAs populations were found whereas, in the case of GYSVd1, the ( −) orientation small RNA species prevailed (Supplementary Fig. 1).
Grapevine rupestris stem pitting-associated virus (GRSPaV) generated a significant amount of vsiRNAs which, however, considerably differed in number between the two plants, as they represented 3.64% and 26.15% of the total v-siRNAs and vd-siRNAs in vine S1+ and S2−, respectively (Table 1). The size distribution and polarity of GRSPaV-derived siRNAs, that showed the presence of a 21 prevailing v-siRNAs population followed by the 22 mer population both having a major (+) sense orientation, confirmedPantaleo et al. (2010) claim that the anti-viral component of RNA silencing in grapevine progresses, for this virus, as observed in other host–virus combinations (Donaire et al., 2009) (Supplementary Fig. 2). Additional siRNAs identified in both PG vines showed homologies to the marafiviruses Grapevine
Syrah virus-1 (GSyV-1) (Al Rwahnih et al., 2009) and Grapevine rupestris vein feathering virus (GRVFV) (Abou-Ghanem et al., 2003). Consistent with previous results (Pantaleo et al., 2010)
siRNAs from both marafiviruses showed a prevalence of ( −) sense orientation molecules for both viruses (Supplementary Figs. 2 and 3).
3.2. De novo assembly of small RNAs
De novo assembly of small RNAs originating from a replicating viral genome during an antiviral response to infection rests on their overlapping features that allow compiling large continuous fragments. Kreuze et al. (2009) approach for virus identification was adopted, following assemblage of 18–26 short reads into larger contigs by the Velvet specific software. This software generated 4158 and 4837 contigs from vine S1+ and S2−, respectively, with sizes ranging between 33 and 470 nts (S1+) and 33 and 505 nts (S2−). Following a search in the NCBI virus database, by BLASTN a BLASTX, five groups of contigs were identified using a cutoff e-value of 10−6 for BLASTN and 10−4 for BLASTX (Table 1).
3.3. Re-assembling GRSPaV genomic RNA
The first contigs group showed homology with several isolates of GRSPaV, whose presence in PG had already been ascertained by short reads analysis and RT-PCR. Vines S1+ and S2− yielded 26 and 189 homologous contigs, respectively, the large majority of which were identified by BLASTN analysis (Table 1). The almost complete GRSPaV genomic RNA (7731 of the 8725) was manually assembled from plant S2− on the template sequence of genomic RNA of isolate GRSPaV (Zhang et al., 1998; GenBank ID:AF026278). The choice of this RNA template was determined by the high number of contigs (149 of 189) showing homologies with the GRSPaV sequence. Of the remaining 40 contigs, 23 had a prevailing homology with GRSPaV-Syrah (GenBank ID: AY368590) throughout the entire genome, whereas 17 contigs could not be assigned but to a single viral strain. Contigs were distributed throughout the genomic RNA with the exceptions of 12 nts at the 5′ terminus and 63 nts at the 3′ end, and of a few interspersed regions. All contigs were correctly placed in each open reading frame (ORF), thus supporting their origin from the de novo assemblage of small RNAs coming from viable replicating molecules. A full length GRSPaV consensus sequence was obtained by SOAPaligner, which resulted in a 66× average coverage depth of the genome. This sequence was registered in the European Bioinformatics Institute (EBI) database under the accession number HE591388. These findings were interpreted as evidence that the GRSPaV population infecting vine S2− is made up of a “master” sequence related to GRSPaV and a minor variant with prevailing homology to GRSPaV-Syrah.
3.4. Discovery of a novel virus
The second group of contigs, composed of 36 from vine S1+ and 48 from vine S2 − (Table 1), showed homology to the 7241 nt genomic RNA of the trichovirus GINV. This finding prompted further investigations to confirm the presence of GINV-related sequences by alternative methods. RT-PCR analysis with flexivirid degenerate primers designed in the RdRp replicase domains (Saldarelli et al., 1999), amplified a 376-nt fragment from both vines, in line with the expectations because of the
alleged presence in them of the flexivirus GRSPaV. However, one of the five clones from vine S1+ contained a GINV-related sequence (BLASTN e-value 7e−81), whereas the remaining four clones and the five clones from vine S2− matched GRSPaV genomic RNA. The 84 contigs from both vines were mapped on the GINV genomic RNA for a total of 5744 nts, with the exception of the extreme 5′ and 3′ termini and a few internal gaps. These sequence gaps were covered by Sanger sequencing of cloned amplicons, obtained by RT-PCR with primers designed on selected contigs (Supplementary Table 1). These primers amplified cDNAs ranging from 941 to 1904 nt in size, from almost the entire genomic RNA length (Fig. 2). The 3′ untranslated sequence was obtained by RACE RT-PCR using the terminal-designed primers GPgV6643f and GPgV7009f (Supplementary Table 1), whereas repeated attempts to reach the 5′ terminal region with the same technique failed. An RNA consensus sequence was finally assembled by SOAP which resulted in a molecule 7258 nt in size, excluding the polyA tail which, as specified below, belongs to the apparently undescribed GINV-related virus, provisionally denoted GPGV. This nucleotide sequence was registered in the EBI database under the accession number FR877530. SOAP alignments of both S1+ and S2− libraries against the new genome identified 112,184 and 152,924 GPGV v-siRNA, respectively from plant S1+ and S2− (Table 1), represented by a majority of 21 and 22 nucleotides size length and an equal distribution of (+) and ( −) sense orientation (Supplementary Fig. 3). Although GPGV v-siRNAs were fewer in the symptomatic S1+ plant, their percentage, relatively to the total vand vd-siRNAs is (43.56%) higher than in the symptomless S2− plant (29.41%).
Fig. 2. Coding information of Grapevine Pinot Gris virus genomic RNA. Open reading frames coding for RNA dependent RNA polymerase (RdRp), movement protein (MP) and coat proteins (CP) are showed. Thin and thick bars below the figure indicate respectively, positions of contigs and cDNA amplified regions. Polyadenylated cDNAs obtained by 3′RACE are indicated. Figure options
GPGV genomic RNA, like that of members of the genus Trichovirus (Adams et al., 2004), consists of three overlapping open reading frames (ORFs) (Fig. 2). ORF1 is 1865 amino acid (aa) in size (214 kDa) and encodes, in the order, the replicase-associated proteins, methyltransferase (aa 44– 333), helicase (aa 1040–1277) and RdRp (aa 1447–1797). It also contains the AlkB domain (aa 730– 830), a partial HxD motif, plus all the other motifs and residues essential for Fe 2+ coordination (Bratlie and Drabløs, 2005). This protein starts with an AUG codon after a 5′ untranslated region of 94 nucleotides. ORF2 codes for a 376 aa polypeptide 42 kDa in size showing homologies with the movement proteins of GINV and Apple chlorotic leaf spot virus (ACLSV) (Table 2). ORF3 encodes the 195 aa (22 kDa) putative coat protein (CP). A 3′ untranslated region of 82 nucleotides terminates the genomic RNA before the polyA tail, whose addition is likely signaled by a AAUAAA sequence at position 7195. Analysis of these proteins showed strong similarities with the comparable GINV products with aa identities of 66.1% (ORF1), 64.9% (ORF2) and 71.4% (ORF3) (Table 2). Only the CP gene had the same length (588 nts) in both viruses whereas replicase and movement proteins are respectively 5604 nts 1053 nts long in GINV and 5538 nts and 1110 nt long in GPGV. Lower identity values were found with orthologous ORFs of other species in the family Flexiviridae that ranged
between 4 and 37.5% at the aa level (Table 2). These findings were confirmed by the phylogenetic analysis of RdRp and CP, which clearly grouped GPGV in the genus Trichovirus, in the same clade as GINV (Fig. 3A and B).
Table 2. Percentage amino acids identities between GPGV encoded proteins and orthologs of species of the family Flexiviridae. Genus Trichovirus Potexvirus Mandarivirus Allexivirus Tymovirus Vitivirus Trichovirus Capillovirus Citrivirus Foveavirus Carlavirus Unassigned Unassigned Virus GINV PVX ICRSV GarV-A TYMV GVA ACLSV ASGV CLBV ASPV PVS BVX BCV-F RdRpol 66.1 16.6 17.9 6.8 9.1 25.2 37.5 24.8 27.7 24.4 25 16.3 6.9 MP 64.9 – – – – 13 25 19 12 – – – – 24 28 23 8 5 5 4 4 Table options CP 71.4
Fig. 3. Phylogenetic trees constructed with RdRp (A) and CP (B) protein sequences of GPGV and members of the familyFlexiviridae. In both trees GPGV clusters with GINV in a separate clade. Numbers at branches indicate occurrence of bootstrap replicates out of the 1000 replicates performed in the test. Bars denote evolutionary distances corresponding to number of amino acids substitution per site. GenBank ID of the amino acid sequences are: TYMV AAB92649; ASGV AAP80757; GarV-ANC_003375; GarV-C NC_003376; BVX NC_005132; PVX AAA47167; PapMV NC_001748; ASPV NC_003462; NCLV NC_008266; PVM AAP76207; PVS NC_007289; APCLSV NC_006946; CNRMV NC_002468; ChMLV NC_002500; BVF NC_002604;ACLSV CAA68080;
PcMV NC_011552; GVB NC_003602; GVA NC_003604; LSV NC_005138; GRSPaV AF026278; CGRMVNC_001946; ICRSV NC_003093; ShVX NC_003557; ClYMV NC_001753; CLBV NC_003877; GINV NC_015220. Figure options
3.5. Contigs related to marafiviruses and viroids
BLAST search identified two other groups of contigs showing homologies to GRVFV and GSyV-1 only from PG vine S2−, which contained the highest number of specific reads (Table 1). However, due to the limited numbers of v-siRNAs, it was not possible to obtain a significant coverage of the genomic RNAs of the two viruses. These contigs covered 2743 out of 6617 and 1925 out of 6506 nucleotides of the GRVFV and GSyV-1 genomic RNAs, respectively, and were scattered along the entire genomes. Contigs homologous to sequences of GYSVd1 and HSVd composed the fifth group. Their analysis suggests that while the major HSVd variant is the same in the viroid populations of both plants with a prevalence of contigs homologous to isolate 09-2009-2140 hs (GenBank ID: HQ447057 (9 out of 16 and 9 out of 15 contigs, in vine S1+ and S2−, respectively), this is not true for GYSVd1, which occurred as a prevalent variant 09-2009-2140g1 (GenBank ID: HQ447058) in vine S2 − (9 out of 13 contigs) and as a mixture of variants in vine S1+. HSVd and GYSVd1 consensus sequences
completely covered the above reported variants with an average depth corresponding to 561 × and 230×, respectively. Whereas the presence of HSVd, GYSVd1, GRSPaV and GRVFV was confirmed by RT-PCR analysis (not shown) in both plants, all attempts to detect GSyV-1 using the primers designed by Al Rwahnih et al. (2009) did only reveal the virus in plant S2−. Moreover, RT-PCR runs were conducted using the universal tymovirid primer set RD (Sabanadzovic et al., 2000) and the degenerate primers TymZ-F/R for broad tymovirid detection (Sabanadzovic et al., 2009). Sequence analysis of 10 cloned fragments showed that these primers amplified 387 and 344 bp fragments from both plants, respectively, whose sequence, however, proved their GRVFV origin (BLASTN e-values ranging from e−95 and e−60). A further set of specific primers was therefore designed on GSyV-1 contigs sequences (GSyV-1f3743 and GSyV-1r4075;Supplementary Table 1). This new tool successfully amplified the expected amplicon from both plants although amplification in plant S1+ was obtained only from phloematic tissue (not shown). The 333 nt sequence of this Pinot gris isolate of GSyV-1, cloned from both plants, was registered in the EBI database under the accession number FR877531. BLASTN and BLASTX analysis of this sequence unequivocally identified GSyV-1.
3.6. Field survey for the presence of GPGV
An RT-PCR assay was developed for GPGV detection designing primers GPgV5619f and GPgV6668r (Supplementary Table 1) which amplify a 1049 bp product. A preliminary survey for the presence of GPGV was accomplished during June 2011 on 17 symptomatic (S+) and 23 (S−) symptomless plants, originating from different vineyards of the Trentino and belonging to the cvs Pinot gris, Traminer and Pinot noir. Results of RT-PCR detection showed that the virus was present in nearly all (16 out of 17) symptomatic analyzed plants whereas only 6 out of 23 symptomless plants were infected by GPGV (Table 3).
Table 3. Detection of GPGV in symptomatic (S+) and symptomless field plants of three different cultivars by RT-PCR. Plants S+ S− P. gris 6 out of 6 1 out of 9 Traminer 7 out of 8 3 out of 7 P. noir 3 out of 3 2 out of 7 Total 16 out of 17 6 out of 23 Table options
4. Discussion
Sequencing of a library of small RNAs extracted from infected PG grapevines using the Illumina technology cast a light on the entire “virome” and “viroidome” replicating in the analyzed plant tissues at sampling time, and proved decisive for the identification of a novel virus. Initial alignment of short reads to known viral sequences from the RefSeq database did not unveil the presence of GPGV due to its unknown genome sequence and the inadequacy of BLAST for analyzing the limited length (18 – 26 nt) of siRNAs. However, following Kreuze et al. (2009) strategy BLAST analysis of assembled contigs disclosed the presence of both GINV-like and other trichovirus-related sequences, which led to the assemblage of the entire genome of GPGV directly from grapevine tissues.
Contrary to GINV, repeated attempts to transmit GPGV to Nicotiana occidentalis and Chenopodium
quinoa failed. Moreover an antiserum to GINV (Yoshikawa et al., 1997) did not trap any virus particle
in immunosorbent electron miscroscope assays from symptomatic leaf dips (A. De Stradis, personal communication), nor reacted with structural viral proteins from infected grapevine tissues in Western blot analysis (unpublished information). These findings and the distant relationship at the aa level between the GPGV-encoded proteins and the orthologous proteins of members of the genus Trichovirus (Table 2) were the basis for identifying GPGV as a new tentative species of this genus. The use of a computational pipeline based on the analysis of siRNAs and de novo assembled contigs is an unbiased approach that is turning very advantageous for exploring the sanitary status of a plant and that, differently from similar dsRNA-based strategies ( Al Rwahnih et al., 2009 and Coetzee et al., 2010), provides additional siRNAs information useful for investigating the host genome and its interactions with viral and viroidal pathogens. In this frame, the analysis of the present data confirmed that, as previously reported (Pantaleo et al., 2010), in grapevine the antiviral silencing activity towards members of the genusMarafivirus, GRVFV and GSyV-1, generates a prevalence of minus sense viral siRNAs. However, whether or not the technique gave a complete overview of the virus and viroids infecting a perennial host is to be proved by the analysis of different tissues and phenological conditions. Whether GPGV is involved and to what extent in the disease shown by Pinot gris vines in the field remains an open question. The observed symptoms (i.e. chlorotic mottling, low vigour and short internodes) strongly resembled to those reported for GINV, in Japan. Moreover, a limited field survey, including cvs other than PG, showed an association of the virus with the symptoms although its presence in both symptomatic and symptomless deep sequenced plants is against its involvement. In agreement with Al Rwahnih et al. (2009) considerations, revealing viruses interactions by deep sequencing would help to decipher the role of different grapevine viruses and their possible antagonistic and/or synergistic effect in disease expression. Studies on the biological properties and distribution of GPGV are under way to clarify this new observed disease, keeping in mind that GINV, a close relative, is transmitted by grapevine erineum mites (Kunugi et al., 2000) that also thrive in the area of GPGV occurrence.
Acknowledgements
We thank Mezzacorona S.c.a. Winery (Mezzocorona, Italy) for the financial support for deep sequencing, and Prof. Giovanni Martelli for the critical revision of the manuscript. We also thanks Dr. Y. Terai and Dr. M. Digiaro for the useful information provided and Dr. Mauro Varner for the photos.
Appendix A. Supplementary data
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o Corresponding author at: Istituto di Virologia Vegetale del C.N.R., UOS-Bari, via Amendola 165/A, 70126 Bari, Italy. Tel.: +39 0805443065; fax: +39 0805443608. 1 These authors equally contributed to the work.
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Virus Research
Volume 163, Issue 1, January 2012, Pages 262–268
A new grapevine virus discovered by deep sequencing of virus- and viroid-derived small RNAs in Cv Pinot gris
Annalisa Giampetruzzia, 1, Vahid Roumia, 1, Roberta Robertoa, Umberto Malossinib, Nobuyuki Yoshikawac, Pierfederico La Nottea, Federica Terlizzid,
Rino Credid, Pasquale Saldarellia,
a
,
Dipartimento di Biologia e Chimica Agroforestale ed Ambientale, Università degli Studi di Bari ed Istituto di Virologia Vegetale Fondazione Edmund Mach – IASMA, via E. Mach 1 – 38010 San Michele a/A, Italy Faculty of Agriculture, Iwate University, Morioka 020-8550, Japan Dipartimento di Scienze e Tecnologie Agroambientali, Università di Bologna, Viale Fanin, 40, 40127 Bologna, Italia
del CNR UOS-Bari, via Amendola 165/A, 70126 Bari, Italy
b
c
d
http://dx.doi.org/10.1016/j.virusres.2011.10.010, How to Cite or Link Using DOI Permissions & Reprints
Abstract
Field symptoms of chlorotic mottling and leaf deformations were observed on the cv Pinot gris (PG) in the Trentino region (Italy). Extensive assays excluded the presence of widely distributed nepo-, ampelo- and vitiviruses. An analysis of small RNA populations from two PG grapevines showing or not symptoms was carried out by Illumina high throughput sequencing. The study disclosed the virus and viroids contents of the two vines that was composed by Grapevine rupestris stem pitting-associated
virus (GRSPaV), two viroids Hop stunt viroid (HSVd) and Grapevine yellow speckle viroid 1 (GYSVd1),
the marafivirusesGrapevine rupestris vein feathering virus (GRVFV) and Grapevine Syrah virus 1 (GSyV-1), and a hitherto unrecorded virus. This virus had a genome organization identical to that of Grapevine berry inner necrosis virus (GINV), a trichovirus reported only from Japan, with which it grouped in phylogenetic trees constructed with sequences of the RdRp domain and the coat protein gene. However, molecular differences with GINV are wide enough to warrant classification of the virus in question as a new species, for which the provisional name of Grapevine Pinot gris virus (GPGV) is proposed. A limited field survey for the presence of GPGV in diseased and symptomless plants from three different cultivars did not allow to clearly associating the virus to the observed symptoms.
Highlights
► Unknown viral symptoms were observed in cv. Pinot gris. ► Small RNAs from grapevine tissue were sequenced by Illumina technology. ► Viruses and viroids infecting Pinot gris plants and properties of their viral small RNAs were described. ► De novo assembling of small RNAs allowed assembling the genome of a new virus. ► This new virus is taxonomically related to trichoviruses. ► Limited field survey for the presence of this virus did not clearly associate the virus to the observed symptoms.
Keywords
Grapevine; Virus; Deep sequencing;
Contig;
Trichovirus
1. Introduction
Symptoms resembling those of a viral disease, i.e. chlorotic mottling, puckering and deformation of the leaves, reduced yield and low quality of the berries have been observed in 2003, in plants of cv Pinot
gris in vineyards of Trentino (northern Italy) (Fig. 1A and B). Successively (2009) similar symptoms
were described on the cvs Traminer and Pinot noir. Particularly on Traminer, symptoms were more accentuated, consisting in stunting and significant losses in production (Fig. 1C and D and unpublished information). No virus particles were detected with the electron microscope in dips from symptomatic PG leaves and no visible reactions were obtained with ELISA using commercial kits (Agritest, Italy) toGrapevine fanleaf virus (GFLV), Arabis mosaic virus (ArMV), Strawberry latent
ringspot virus (SLRSV),Grapevine leafroll-associated virus-1 (GLRaV-1), -2 (GLRaV-2) and -3
(GLRaV-3), Grapevine virus A(GVA) and Grapevine virus B (GVB) (unpublished results). Equally negative were RT-PCR assays using specific primers for the above reported ampelo- and vitiviruses and degenerate primers for the detection of members of the three subgroups of the genus Nepovirus (Digiaro et al., 2007).
Fig. 1. Chlorotic mottling and leaf deformations in the cvs Pinot gris (A and B) and Traminer (C). Shoot stunting in cv Traminer is shown in panel (D). Figure options
Deep sequencing is a powerful technology that provides rapid and exhaustive information on the infectious agents (viruses and viroids) present in plant tissues (Kreuze et al., 2009 and Wu et al., 2010). Therefore, this technology is being increasingly used for the quick identification of viruses replicating in plant tissues, either starting from the analysis of small interfering RNA (siRNAs) populations (Kreuze et al., 2009), or from sequenced libraries of fragmented double-stranded RNAs (dsRNAs) of viral origin (Al Rwahnih et al., 2009 and Coetzee et al., 2010), extracted from infected tissues. With a study of a vine of cv. Pinot Noir clone ENTAV 115, we have recently experienced the high potentiality of deep sequencing, and have described the “virome” [sensuCoetzee et al. (2010)] of the plant (Pantaleo et al., 2010). Thus, we have now deep sequenced and screened the small RNA population of a symptomatic and a symptomless PG vine, identifying, among other disease agents, a putatively new virus phylogenetically related to the trichovirus Grapevine berry inner necrosis
virus (GINV; Yoshikawa et al., 1997 and Kunugi et al., 2000), for which the provisional name
of Grapevine Pinot gris virus (GPGV) is proposed.
2. Materials and methods
2.1. Small RNA purification and sequencing
Leaves and petioles were collected in July 2010 from a symptomatic (plant S1+) and a symptomless (plant S2−) PG vine and stored at 4 °C for 5 days before using for total RNAs extraction. This was carried out as described by Pantaleo et al. (2010) and the low molecular weight RNA fraction (LMWRNA) was isolated by polyethylenglycol precipitation (Hamilton and Baulcombe, 1999). Small RNAs were separated by polyacrylamide gel electrophoresis and recovered from the gel as described by Lu et al. (2007). Libraries of small RNA populations from both vines were prepared for sequencing with an Illumina Genome Analyzer II (Institute of Applied Genomics, University of Udine, Italy) following the protocol of Lu et al. (2007), which generated 36–44 bp sequences. Adaptors, ribosomal and transfer RNA sequences were removed by the UEA siRNA toolkit (Moxon et al., 2008) and the small RNA sequences between 18 and 26 nt in size were initially screened for homology to known viruses by local BLASTN search against the GenBank Virus Reference Sequence database (RefSeq; http://www.ncbi.nlm.nih.gov/). Subsequently, libraries were searched with the SOAP (Short Oligonucleotide Alignment Program; Li et al., 2010) packages of software against reference sequences retrieved from the RefSeq database. Total small RNAs were then assembled into larger contigs using the Velvet Software 0.7.31 (Zerbino and Birney, 2008) with a k-mer of 17. GRSPaV, HSVd and GYSVd1 genomic RNAs were reconstructed by aligning and assembling contigson database-retrieved sequences, using the DNA Strider software 1.46 (Marck, 1988). Consensus sequences were finally obtained by the SOAPaligner software, which aligns small RNAs to
a reference genome, allowing a tolerance of two mismatches. Nucleotide polymorphisms having a frequency ≥80% and a minimum coverage of five valid small RNAs, as calculated by MapView (Bao et al., 2009), were allowed in the consensus sequence. Alignments and genome coverage were respectively visualized and estimated by MapView and Tablet (Milne et al., 2010). GPGV genome sequence was initially assembled on the available GINV genomic RNA (NC_015220) by aligning contigs obtained by Velvet. Sequence gaps were filled by sequencing cDNA fragments amplified with primers designed on the obtained contigs (Supplementary Table 1) cloned with the PCR cloning kit (Invitrogen, USA). A minimum of four clones per fragment was sequenced. Sequences of the extreme 3′ terminal nts were obtained by rapid amplification of cDNA ends (RACE) using two independent specific primers (GPgV6643f and GPgV7009f; Supplementary Table 1) and the 5′/3′ RACE kit from Roche Applied Sciences (Indianapolis, USA). Phylogenetic analysis was done with Clustal V (Larkin et al., 2007) using 1000 bootstrap replicates and the neighbor-joining method of analysis.
2.2. Field survey for the detection of GPGV
Forty grapevine plants of the cvs Pinot gris, Traminer and Pinot noir were identified in different commercial vineyards originating from the same cultivated area, either showing or not the described symptomatology. Total RNAs were purified from leaf and petiole tissues by a CTAB-based extraction protocol according toGambino et al. (2008). GPGV RT-PCR detection was performed by a two-step cDNA reverse transcription with random hexanucleotide primers and amplification according to Pantaleo et al. (2010)using specific primers (GPgV5619f/GPgV6668r; Supplementary Table 1) designed on the obtained sequence.
3. Results
3.1. High throughput sequencing and analysis of small RNAs
Libraries representative of the siRNAs population extracted from S1+ and S2 − vines and sequenced by Illumina technology, contained respectively 6.9 × 106 and 1.1 × 107 reads, after trimming adapters and filtering for transfer and ribosomal RNAs (Table 1). Only the data relevant for the identification of virus and viroid-related sequences were analyzed in the present study, whereas a detailed description of the library and of the small interfering RNA (siRNA) species will be the object of another paper.
Table 1. Virus- and viroid-related 18–26 siRNAs and contigs originated from both libraries. Plant S1+ Virus/genus HSVd/Hostuviroid GYSVd1/Apscaviroid GRSPaV/Foveavirus GRVFV/Marafivirus GSyV-1/Marafivirus GPGV Total virus reads Readsa 72,496 63,050 9378 451 368 112,184 257,559 %c 1.04 0.90 0.13 0.01 0.01 1.61 % v and vdd 28.15 24.48 3.64 0.18 0.14 43.56 Contigs 16 6 26 – – 36 Plant S2− Readsa 123,019 82,808 135,996 13,498 11,800 152,924 520,045 %c 1.11 0.75 1.23 0.12 0.11 1.38 % v and vdd 23.66 15.92 26.15 2.60 2.27 29.41 Contigs 15 13 189 25 28 48
Plant S1+ Virus/genus Other Total a
Redundant reads obtained by SOAPaligner.
e
Plant S2− %c % v and vdd Contigs Readsa 10,545,472 11,065,517b %c % v and vdd Contigs
Readsa 6,721,320 6,978,879b
b
Total reads after trimming of the adapters and filtering for ribosomal and transfer RNAs.
c
Percentages over the total reads.
d
Percentages over the total virus and viroidal reads.
e
Reads not matching with viral and viroidal sequences.
Table options
Preliminary BLASTN analysis of sequenced reads against the NCBI viral sequence database, identified siRNAs originating from the two grapevine viroids GYSVd1 and HSVd, the flexivirus GRSPaV and the two marafiviruses GRVFV and GSyV-1 (not shown). Libraries were therefore searched with SOAP using reference genome sequences of the above viruses and viroids. Vd-siRNAs originating from Hop stunt viroid (HSVd) and Grapevine yellow speckle viroid 1 (GYSVd1) were similarly represented in both plants either relatively to the total siRNAs or to v- and vd-siRNAs (Table 1). Consistently with Navarro et al. (2009), who reported that viroid RNAs are targeted by different Dicer enzymes producing 21, 22 and 24 nt siRNAs, we found a comparable size distribution of vd-siRNAs. In particular, the 21 and 24 nt populations were the most represented molecular species. However, differently from Navarro et al. (2009), equal (+) and (−) HSVd small RNAs populations were found whereas, in the case of GYSVd1, the ( −) orientation small RNA species prevailed (Supplementary Fig. 1).
Grapevine rupestris stem pitting-associated virus (GRSPaV) generated a significant amount of vsiRNAs which, however, considerably differed in number between the two plants, as they represented 3.64% and 26.15% of the total v-siRNAs and vd-siRNAs in vine S1+ and S2−, respectively (Table 1). The size distribution and polarity of GRSPaV-derived siRNAs, that showed the presence of a 21 prevailing v-siRNAs population followed by the 22 mer population both having a major (+) sense orientation, confirmedPantaleo et al. (2010) claim that the anti-viral component of RNA silencing in grapevine progresses, for this virus, as observed in other host–virus combinations (Donaire et al., 2009) (Supplementary Fig. 2). Additional siRNAs identified in both PG vines showed homologies to the marafiviruses Grapevine
Syrah virus-1 (GSyV-1) (Al Rwahnih et al., 2009) and Grapevine rupestris vein feathering virus (GRVFV) (Abou-Ghanem et al., 2003). Consistent with previous results (Pantaleo et al., 2010)
siRNAs from both marafiviruses showed a prevalence of ( −) sense orientation molecules for both viruses (Supplementary Figs. 2 and 3).
3.2. De novo assembly of small RNAs
De novo assembly of small RNAs originating from a replicating viral genome during an antiviral response to infection rests on their overlapping features that allow compiling large continuous fragments. Kreuze et al. (2009) approach for virus identification was adopted, following assemblage of 18–26 short reads into larger contigs by the Velvet specific software. This software generated 4158 and 4837 contigs from vine S1+ and S2−, respectively, with sizes ranging between 33 and 470 nts (S1+) and 33 and 505 nts (S2−). Following a search in the NCBI virus database, by BLASTN a BLASTX, five groups of contigs were identified using a cutoff e-value of 10−6 for BLASTN and 10−4 for BLASTX (Table 1).
3.3. Re-assembling GRSPaV genomic RNA
The first contigs group showed homology with several isolates of GRSPaV, whose presence in PG had already been ascertained by short reads analysis and RT-PCR. Vines S1+ and S2− yielded 26 and 189 homologous contigs, respectively, the large majority of which were identified by BLASTN analysis (Table 1). The almost complete GRSPaV genomic RNA (7731 of the 8725) was manually assembled from plant S2− on the template sequence of genomic RNA of isolate GRSPaV (Zhang et al., 1998; GenBank ID:AF026278). The choice of this RNA template was determined by the high number of contigs (149 of 189) showing homologies with the GRSPaV sequence. Of the remaining 40 contigs, 23 had a prevailing homology with GRSPaV-Syrah (GenBank ID: AY368590) throughout the entire genome, whereas 17 contigs could not be assigned but to a single viral strain. Contigs were distributed throughout the genomic RNA with the exceptions of 12 nts at the 5′ terminus and 63 nts at the 3′ end, and of a few interspersed regions. All contigs were correctly placed in each open reading frame (ORF), thus supporting their origin from the de novo assemblage of small RNAs coming from viable replicating molecules. A full length GRSPaV consensus sequence was obtained by SOAPaligner, which resulted in a 66× average coverage depth of the genome. This sequence was registered in the European Bioinformatics Institute (EBI) database under the accession number HE591388. These findings were interpreted as evidence that the GRSPaV population infecting vine S2− is made up of a “master” sequence related to GRSPaV and a minor variant with prevailing homology to GRSPaV-Syrah.
3.4. Discovery of a novel virus
The second group of contigs, composed of 36 from vine S1+ and 48 from vine S2 − (Table 1), showed homology to the 7241 nt genomic RNA of the trichovirus GINV. This finding prompted further investigations to confirm the presence of GINV-related sequences by alternative methods. RT-PCR analysis with flexivirid degenerate primers designed in the RdRp replicase domains (Saldarelli et al., 1999), amplified a 376-nt fragment from both vines, in line with the expectations because of the
alleged presence in them of the flexivirus GRSPaV. However, one of the five clones from vine S1+ contained a GINV-related sequence (BLASTN e-value 7e−81), whereas the remaining four clones and the five clones from vine S2− matched GRSPaV genomic RNA. The 84 contigs from both vines were mapped on the GINV genomic RNA for a total of 5744 nts, with the exception of the extreme 5′ and 3′ termini and a few internal gaps. These sequence gaps were covered by Sanger sequencing of cloned amplicons, obtained by RT-PCR with primers designed on selected contigs (Supplementary Table 1). These primers amplified cDNAs ranging from 941 to 1904 nt in size, from almost the entire genomic RNA length (Fig. 2). The 3′ untranslated sequence was obtained by RACE RT-PCR using the terminal-designed primers GPgV6643f and GPgV7009f (Supplementary Table 1), whereas repeated attempts to reach the 5′ terminal region with the same technique failed. An RNA consensus sequence was finally assembled by SOAP which resulted in a molecule 7258 nt in size, excluding the polyA tail which, as specified below, belongs to the apparently undescribed GINV-related virus, provisionally denoted GPGV. This nucleotide sequence was registered in the EBI database under the accession number FR877530. SOAP alignments of both S1+ and S2− libraries against the new genome identified 112,184 and 152,924 GPGV v-siRNA, respectively from plant S1+ and S2− (Table 1), represented by a majority of 21 and 22 nucleotides size length and an equal distribution of (+) and ( −) sense orientation (Supplementary Fig. 3). Although GPGV v-siRNAs were fewer in the symptomatic S1+ plant, their percentage, relatively to the total vand vd-siRNAs is (43.56%) higher than in the symptomless S2− plant (29.41%).
Fig. 2. Coding information of Grapevine Pinot Gris virus genomic RNA. Open reading frames coding for RNA dependent RNA polymerase (RdRp), movement protein (MP) and coat proteins (CP) are showed. Thin and thick bars below the figure indicate respectively, positions of contigs and cDNA amplified regions. Polyadenylated cDNAs obtained by 3′RACE are indicated. Figure options
GPGV genomic RNA, like that of members of the genus Trichovirus (Adams et al., 2004), consists of three overlapping open reading frames (ORFs) (Fig. 2). ORF1 is 1865 amino acid (aa) in size (214 kDa) and encodes, in the order, the replicase-associated proteins, methyltransferase (aa 44– 333), helicase (aa 1040–1277) and RdRp (aa 1447–1797). It also contains the AlkB domain (aa 730– 830), a partial HxD motif, plus all the other motifs and residues essential for Fe 2+ coordination (Bratlie and Drabløs, 2005). This protein starts with an AUG codon after a 5′ untranslated region of 94 nucleotides. ORF2 codes for a 376 aa polypeptide 42 kDa in size showing homologies with the movement proteins of GINV and Apple chlorotic leaf spot virus (ACLSV) (Table 2). ORF3 encodes the 195 aa (22 kDa) putative coat protein (CP). A 3′ untranslated region of 82 nucleotides terminates the genomic RNA before the polyA tail, whose addition is likely signaled by a AAUAAA sequence at position 7195. Analysis of these proteins showed strong similarities with the comparable GINV products with aa identities of 66.1% (ORF1), 64.9% (ORF2) and 71.4% (ORF3) (Table 2). Only the CP gene had the same length (588 nts) in both viruses whereas replicase and movement proteins are respectively 5604 nts 1053 nts long in GINV and 5538 nts and 1110 nt long in GPGV. Lower identity values were found with orthologous ORFs of other species in the family Flexiviridae that ranged
between 4 and 37.5% at the aa level (Table 2). These findings were confirmed by the phylogenetic analysis of RdRp and CP, which clearly grouped GPGV in the genus Trichovirus, in the same clade as GINV (Fig. 3A and B).
Table 2. Percentage amino acids identities between GPGV encoded proteins and orthologs of species of the family Flexiviridae. Genus Trichovirus Potexvirus Mandarivirus Allexivirus Tymovirus Vitivirus Trichovirus Capillovirus Citrivirus Foveavirus Carlavirus Unassigned Unassigned Virus GINV PVX ICRSV GarV-A TYMV GVA ACLSV ASGV CLBV ASPV PVS BVX BCV-F RdRpol 66.1 16.6 17.9 6.8 9.1 25.2 37.5 24.8 27.7 24.4 25 16.3 6.9 MP 64.9 – – – – 13 25 19 12 – – – – 24 28 23 8 5 5 4 4 Table options CP 71.4
Fig. 3. Phylogenetic trees constructed with RdRp (A) and CP (B) protein sequences of GPGV and members of the familyFlexiviridae. In both trees GPGV clusters with GINV in a separate clade. Numbers at branches indicate occurrence of bootstrap replicates out of the 1000 replicates performed in the test. Bars denote evolutionary distances corresponding to number of amino acids substitution per site. GenBank ID of the amino acid sequences are: TYMV AAB92649; ASGV AAP80757; GarV-ANC_003375; GarV-C NC_003376; BVX NC_005132; PVX AAA47167; PapMV NC_001748; ASPV NC_003462; NCLV NC_008266; PVM AAP76207; PVS NC_007289; APCLSV NC_006946; CNRMV NC_002468; ChMLV NC_002500; BVF NC_002604;ACLSV CAA68080;
PcMV NC_011552; GVB NC_003602; GVA NC_003604; LSV NC_005138; GRSPaV AF026278; CGRMVNC_001946; ICRSV NC_003093; ShVX NC_003557; ClYMV NC_001753; CLBV NC_003877; GINV NC_015220. Figure options
3.5. Contigs related to marafiviruses and viroids
BLAST search identified two other groups of contigs showing homologies to GRVFV and GSyV-1 only from PG vine S2−, which contained the highest number of specific reads (Table 1). However, due to the limited numbers of v-siRNAs, it was not possible to obtain a significant coverage of the genomic RNAs of the two viruses. These contigs covered 2743 out of 6617 and 1925 out of 6506 nucleotides of the GRVFV and GSyV-1 genomic RNAs, respectively, and were scattered along the entire genomes. Contigs homologous to sequences of GYSVd1 and HSVd composed the fifth group. Their analysis suggests that while the major HSVd variant is the same in the viroid populations of both plants with a prevalence of contigs homologous to isolate 09-2009-2140 hs (GenBank ID: HQ447057 (9 out of 16 and 9 out of 15 contigs, in vine S1+ and S2−, respectively), this is not true for GYSVd1, which occurred as a prevalent variant 09-2009-2140g1 (GenBank ID: HQ447058) in vine S2 − (9 out of 13 contigs) and as a mixture of variants in vine S1+. HSVd and GYSVd1 consensus sequences
completely covered the above reported variants with an average depth corresponding to 561 × and 230×, respectively. Whereas the presence of HSVd, GYSVd1, GRSPaV and GRVFV was confirmed by RT-PCR analysis (not shown) in both plants, all attempts to detect GSyV-1 using the primers designed by Al Rwahnih et al. (2009) did only reveal the virus in plant S2−. Moreover, RT-PCR runs were conducted using the universal tymovirid primer set RD (Sabanadzovic et al., 2000) and the degenerate primers TymZ-F/R for broad tymovirid detection (Sabanadzovic et al., 2009). Sequence analysis of 10 cloned fragments showed that these primers amplified 387 and 344 bp fragments from both plants, respectively, whose sequence, however, proved their GRVFV origin (BLASTN e-values ranging from e−95 and e−60). A further set of specific primers was therefore designed on GSyV-1 contigs sequences (GSyV-1f3743 and GSyV-1r4075;Supplementary Table 1). This new tool successfully amplified the expected amplicon from both plants although amplification in plant S1+ was obtained only from phloematic tissue (not shown). The 333 nt sequence of this Pinot gris isolate of GSyV-1, cloned from both plants, was registered in the EBI database under the accession number FR877531. BLASTN and BLASTX analysis of this sequence unequivocally identified GSyV-1.
3.6. Field survey for the presence of GPGV
An RT-PCR assay was developed for GPGV detection designing primers GPgV5619f and GPgV6668r (Supplementary Table 1) which amplify a 1049 bp product. A preliminary survey for the presence of GPGV was accomplished during June 2011 on 17 symptomatic (S+) and 23 (S−) symptomless plants, originating from different vineyards of the Trentino and belonging to the cvs Pinot gris, Traminer and Pinot noir. Results of RT-PCR detection showed that the virus was present in nearly all (16 out of 17) symptomatic analyzed plants whereas only 6 out of 23 symptomless plants were infected by GPGV (Table 3).
Table 3. Detection of GPGV in symptomatic (S+) and symptomless field plants of three different cultivars by RT-PCR. Plants S+ S− P. gris 6 out of 6 1 out of 9 Traminer 7 out of 8 3 out of 7 P. noir 3 out of 3 2 out of 7 Total 16 out of 17 6 out of 23 Table options
4. Discussion
Sequencing of a library of small RNAs extracted from infected PG grapevines using the Illumina technology cast a light on the entire “virome” and “viroidome” replicating in the analyzed plant tissues at sampling time, and proved decisive for the identification of a novel virus. Initial alignment of short reads to known viral sequences from the RefSeq database did not unveil the presence of GPGV due to its unknown genome sequence and the inadequacy of BLAST for analyzing the limited length (18 – 26 nt) of siRNAs. However, following Kreuze et al. (2009) strategy BLAST analysis of assembled contigs disclosed the presence of both GINV-like and other trichovirus-related sequences, which led to the assemblage of the entire genome of GPGV directly from grapevine tissues.
Contrary to GINV, repeated attempts to transmit GPGV to Nicotiana occidentalis and Chenopodium
quinoa failed. Moreover an antiserum to GINV (Yoshikawa et al., 1997) did not trap any virus particle
in immunosorbent electron miscroscope assays from symptomatic leaf dips (A. De Stradis, personal communication), nor reacted with structural viral proteins from infected grapevine tissues in Western blot analysis (unpublished information). These findings and the distant relationship at the aa level between the GPGV-encoded proteins and the orthologous proteins of members of the genus Trichovirus (Table 2) were the basis for identifying GPGV as a new tentative species of this genus. The use of a computational pipeline based on the analysis of siRNAs and de novo assembled contigs is an unbiased approach that is turning very advantageous for exploring the sanitary status of a plant and that, differently from similar dsRNA-based strategies ( Al Rwahnih et al., 2009 and Coetzee et al., 2010), provides additional siRNAs information useful for investigating the host genome and its interactions with viral and viroidal pathogens. In this frame, the analysis of the present data confirmed that, as previously reported (Pantaleo et al., 2010), in grapevine the antiviral silencing activity towards members of the genusMarafivirus, GRVFV and GSyV-1, generates a prevalence of minus sense viral siRNAs. However, whether or not the technique gave a complete overview of the virus and viroids infecting a perennial host is to be proved by the analysis of different tissues and phenological conditions. Whether GPGV is involved and to what extent in the disease shown by Pinot gris vines in the field remains an open question. The observed symptoms (i.e. chlorotic mottling, low vigour and short internodes) strongly resembled to those reported for GINV, in Japan. Moreover, a limited field survey, including cvs other than PG, showed an association of the virus with the symptoms although its presence in both symptomatic and symptomless deep sequenced plants is against its involvement. In agreement with Al Rwahnih et al. (2009) considerations, revealing viruses interactions by deep sequencing would help to decipher the role of different grapevine viruses and their possible antagonistic and/or synergistic effect in disease expression. Studies on the biological properties and distribution of GPGV are under way to clarify this new observed disease, keeping in mind that GINV, a close relative, is transmitted by grapevine erineum mites (Kunugi et al., 2000) that also thrive in the area of GPGV occurrence.
Acknowledgements
We thank Mezzacorona S.c.a. Winery (Mezzocorona, Italy) for the financial support for deep sequencing, and Prof. Giovanni Martelli for the critical revision of the manuscript. We also thanks Dr. Y. Terai and Dr. M. Digiaro for the useful information provided and Dr. Mauro Varner for the photos.
Appendix A. Supplementary data
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o Corresponding author at: Istituto di Virologia Vegetale del C.N.R., UOS-Bari, via Amendola 165/A, 70126 Bari, Italy. Tel.: +39 0805443065; fax: +39 0805443608. 1 These authors equally contributed to the work.
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