1 Instituto de Biología Molecular y Celular de Plantas (UPV-CSIC), Universidad Politécnica de Valencia, Avenida de los Naranjos s/n, 46022 Valencia, Spain
2 Dipartimento di Arboricoltura, Botanica e Patologia Vegetale, Università di Napoli, 80055 Portici, Italy
3 Department of Biological Sciences, Imperial College London, Imperial College Road, London SW7 2AZ, UK
4 Dipartimento di Protezione delle Piante e Microbiologia Applicata, Università degli Studi and Istituto di Virologia Vegetale del CNR, Sezione di Bari, 70126 Bari, Italy
5 Tekirdag Ziraat Fakültesi, Trakya Universitesi, 59030 Tekirdag, Turkey
6 Adnan Menderes University, Agricultural Faculty, Plant Pathology Department, 09100 Aydin, Turkey
Correspondence
Ricardo Flores
rflores{at}ibmcp.upv.es
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers of the sequences reported in this paper are AJ781397, AJ781398, AJ781399 and AJ781400 for CCRS-associated dsRNAs 1, 2, 3 and 4 and AJ781166, AJ781165, AJ781164 and AJ781163 for ACD-associated dsRNAs 1, 2, 3 and 4.
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INTRODUCTION |
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From a symptomatological perspective, CCRS disease resembles only the Amasya cherry disease (ACD) described previously in the region of Turkey with this name (Blodgett et al., 1970; Citir, 1987
). Interestingly, ACD is also associated with a complex set of presumably viral dsRNAs (Aç
kgöz et al., 1994
). Although the symptoms as well as the number and sizes of the dsRNAs associated with both cherry disorders present clear parallels, a direct comparison of the two diseases has been lacking.
Here, we present the results of research that addressed two questions. Firstly, whether CCRS and ACD are indeed closely related diseases and, secondly, whether the molecular characterization of some of the CCRS- and ACD-associated dsRNAs could provide some insight into the nature of these RNAs. Our data showed that both diseases were very similar on the basis of symptoms and, particularly, of the sequence of six of their associated dsRNAs, which are the genomic components of two new tentative species, one in the genus Chrysovirus and one in the genus Partitivirus (see accompanying paper by Coutts et al., 2004, in this issue). Since members of the genera Chrysovirus and Partitivirus typically infect fungi, these results, combined with previous observations, suggest a fungal aetiology for CCRS and ACD.
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METHODS |
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Extraction and fractionation of nucleic acids.
Total nucleic acid preparations were obtained by extracting leaves with buffer-saturated phenol as described previously (Pallás et al., 1987). These preparations were then partitioned by chromatography on non-ionic cellulose with STE (50 mM Tris/HCl, pH 7·2, 100 mM NaCl, 1 mM EDTA) containing 16 % ethanol and the dsRNA-rich fraction was recovered by ethanol precipitation (Morris & Dodds, 1979
).
PAGE and molecular hybridization analyses.
The CCRS- and ACD-associated dsRNAs were separated by PAGE in non-denaturing (5 %) gels that were stained with ethidium bromide (and, when indicated, with silver). Some of the CCRS dsRNAs generating the individual bands were eluted, recovered by ethanol precipitation and used for dot-blot hybridization or for cDNA synthesis (see below). Pre-hybridization, hybridization with digoxigenin-labelled riboprobes (at 68 °C in 50 % formamide) and washing were as recommended by the supplier (Roche) and membranes were analysed by autoradiography or using a Fuji Bas 1500 apparatus.
Reverse transcription, PCR amplification, cloning and sequencing.
The first cDNA clones of CCRS chrys-dsRNAs 1 and 4 were obtained by a random-primer RT-PCR protocol for cloning RNAs of unknown sequence (Navarro et al., 1998), with their identity being confirmed by dot-blot hybridization with purified preparations of these dsRNAs. For the synthesis of additional cDNAs covering their complete sequence, purified CCRS chrys-dsRNAs 1 and 4 (200300 ng) were denatured with 10 mM methyl mercuric hydroxide and reverse transcribed with Superscript II reverse transciptase as recommended by the supplier (Invitrogen) and specific primers derived from the initial clone. The resulting cDNAs were 3'-tailed with terminal transferase (Roche) and dATP or dCTP, and PCR amplified with Taq DNA polymerase (Roche) and the specific primers used in the reverse transcriptase reaction combined with others containing a dT25 or dG25 tail at their 3' termini (with the last base degenerated). For obtaining cDNA clones corresponding to the 5' and 3' ends, denatured CCRS chrys-dsRNAs 1 and 4 were polyadenylated with yeast poly(A) polymerase (USB Corporation) and ATP, reverse transcribed with a primer containing a 3'-dT25 tail with the last base degenerated and PCR amplified with this primer combined with others derived from internal regions.
Complete cDNA clones of the CCRS chrys-dsRNAs 2 and 3 were obtained using, with minor modifications, the single-primer method for cloning dsRNAs of unknown sequence (Isogai et al., 1998; Vreede et al., 1998
). In brief, the purified dsRNA was denatured, polyadenylated and reverse transcribed with a primer containing a 3'-dT25 tail with the last base degenerated. Following treatment with RNase H (Roche), the cDNA strands were annealed for 10 min at 80 °C, for 16 h at 65 °C and for 3 h at 30 °C and the resulting hybrid was filled in with T4 DNA polymerase (Roche) and PCR amplified using the Expand High Fidelity system (Roche) and the same primer with the 3'-dT25 tail and the last base degenerated. The PCR profile consisted of an initial denaturation at 94 °C for 2 min and 35 cycles of 50 s at 94 °C, 50 s at 58 °C and 3 min at 68 °C, with a final extension of 15 min at 72 °C. RT-PCR amplification products were cloned in bacterial plasmids following standard techniques (Sambrook et al., 1989
) and the insert sequences were determined automatically using an ABI PRISM DNA 377 apparatus (Perkin-Elmer). Proper assignation of the sequences to specific dsRNAs was established by dot-blot hybridization using purified preparations of the three dsRNAs spotted on to membranes and probes derived from internal cDNA clones.
Synthesis of cDNA clones for the ACD-associated dsRNAs was performed essentially as described for the CCRS-associated dsRNAs but used an RNA ligase-mediated (RLM)-RACE PCR (Coutts & Livieratos, 2003) procedure to determine the 5'- and 3'-terminal sequences. Sequences of the dsRNAs were completed and confirmed by overlapping of cDNA clones prepared with internal primers, and assignment to specific dsRNAs was made by comparison with the CCRS-associated dsRNAs.
Sequence analysis.
The coding capacity of the nucleotide sequences was determined using the program TRANSLATE (www.expasy.ch). Comparisons with sequences deposited in databases (GenBank, EMBL, SWPROT and PIR) were executed using the programs BLASTN, BLASTP and BLASTX (www.genome.ad.jp, www.ncbi.nlm.nih.gov and www.ebi.ac.uk). Pairwise alignments were done using the programs BESTFIT and GAP of the GCG package v. 10 and multiple alignments with CLUSTAL X v. 1.81 (Thompson et al., 1997) and visualized with GENEDOC v. 2.6 (www.psc.edu). The subsequent phylogenetic analysis was obtained with the MEGA program v. 2.1 (Kumar et al., 2001
) and the phylogenetic relationships of the unrooted tree were based on the neighbour-joining method with a 10 000 replicate bootstrap search. The BOOTSTRAP option in the MEGA program was used for the bootstrap analysis.
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RESULTS |
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To obtain additional evidence, CCRS- and ACD-affected leaves of Italian and Turkish origin, respectively, were examined side by side. The symptoms appeared very similar, if not identical (data not shown), and specific, suggesting that both disorders might be the same and have a common causal agent.
CCRS and ACD are associated with a common pattern of 1012 dsRNAs
PAGE analysis of dsRNA-rich preparations from cherry trees displaying the typical ACD symptoms showed a complex profile of dsRNAs that were absent from healthy-looking trees (Açkgöz et al., 1994
). Similar analysis showed ten dsRNAs, which ranged in size from approximately 1700 to 5500 bp, and two smaller dsRNAs of approximately 500 and 650 bp that were diagnostic for CCRS disease (Di Serio et al., 1996
) (Fig. 1
a). Moreover, when CCRS-affected leaves were divided prior to extraction, with one fraction consisting of chlorotic-rusty lesions and the other the asymptomatic surrounding areas, the dsRNAs were found predominantly in the first fraction (Di Serio et al., 1996
). To determine whether the dsRNAs associated with CCRS and ACD had a common distribution of sizes, a direct comparison by electrophoresis on the same polyacrylamide gel was performed. This analysis revealed a remarkable similarity in the mobility of the ten individual components of higher size (Fig. 1b
), thus suggesting that if they were components of the causal agent, this agent might be the same for both diseases. The CCRS- and ACD-associated dsRNAs could in principle represent the components of one or more viruses with a multipartite dsRNA genome(s) or the genomic and subgenomic dsRNAs resulting from infection by one or more single-stranded RNA (ssRNA) viruses.
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CCRS chrys-dsRNA 2 comprised 3125 bp and in one strand showed 5'- and 3'-UTRs of 95 and 48 nt, respectively, flanking an ORF potentially encoding a protein of 993 aa with a molecular mass of approximately 112 kDa and similarities to the major coat protein of chrysoviruses (Fig. 2b). CCRS chrys-dsRNA 3 comprised 2833 bp and in one strand showed a 5'-UTR of 94 nt, which increased to 304 nt if it was assumed that the second-most 5'-AUG codon was in a better initiation context (Kozak, 1986
; Lütcke et al., 1987
), and a 3'-UTR of 84 nt, flanking an ORF potentially encoding a protein of 884 aa with a molecular mass of approximately 98 kDa (Fig. 2b
). CCRS chrys-dsRNA 4 comprised 2499 bp and in one strand showed a 5'-UTR of 106 nt, which increased to 151 nt if it was assumed that the second-most 5'-AUG codon was in a better initiation context, and a 3'-UTR of 359 nt, flanking an ORF potentially encoding a protein of 677 aa with a molecular mass of approximately 77 kDa (Fig. 2b
). The functions of the putative products of CCRS chrys-dsRNA 3 and 4, like those of the dsRNAs 3 and 4 of other chrysoviruses (Jiang & Ghabrial, 2004
; Ghabrial & Castón, 2004
), which are similar in size and sequence, are currently unknown, although CCRS chrys-dsRNA 3 contains a motif conserved in other ssRNA and dsRNA viruses (see below).
Assignment of numbers 1 to 4 to the dsRNAs of the CCRS-associated chrysovirus was made according to their decreasing size, following the same criterion used for PcV and Hv145SV chrysoviruses. Yet, sequence comparisons indicated that dsRNAs 3 of the CCRS-associated chrysovirus and that of Hv145SV were more closely related to PcV dsRNA 4 than to dsRNA 3. In particular, the proteins potentially encoded by these three dsRNAs contained the motif PGDGXCXXHX. This motif (I), along with motifs II (with a conserved K), III and IV (with a conserved H), form the conserved core of the ovarian tumour gene-like superfamily of predicted proteases (Makarova et al., 2000). Multiple alignments showed that motifs I to IV were also present in the ORF of other viruses including Agaricus bisporus virus 1 associated with La France disease, a tentative chrysovirus; Potato virus M, Hop latent virus and Blueberry scorch virus, three carlaviruses; and Rupestris stem pitting-associated virus 1, a foveavirus (data not shown). Whether the RNAs of these viruses indeed code for the predicted proteases remains to be investigated. On the other hand, a phytoreo S7 domain shared with a family of several phytoreovirus S7 proteins has recently been found in PcV dsRNA 3 (Jiang & Ghabrial, 2004
). This domain is also present in dsRNAs 4 of CCRS-associated chrysovirus and Hv145SV [the consensus for the three chysoviruses is X(V/I)V(M/L)P(A/M)G(C/H)GK(T/S)T(L/I)], indicating that they are the likely counterparts of PcV dsRNA 3.
The 5'- and 3'-UTRs of the four CCRS chrys-dsRNAs presented strictly conserved GAAAAUUAUGG and AUAUGC termini, respectively. Whereas the 5' termini were similar to those of the sense strand of all four dsRNAs from PcV and Hv145SV (GAUAAAAA), the situation differed with the 3' termini of the four dsRNAs from PcV and Hv145SV (UAAGUGU). We do not believe that these discrepancies could have resulted from the procedures used in our case not allowing proper cloning of the exact termini (see below). Additionally, the 5'- and 3'-UTRs of the four CCRS chrys-dsRNAs showed strong sequence similarities in their internal parts. This was specifically the case with the 5'-UTRs, which contained a highly conserved stretch of 3637 nt, the so-called Box 1 of chrysoviruses (Ghabrial & Castón, 2004), followed by a less-conserved stretch of 6364 nt rich in CAA, CAAA and CAAAA repeats (Fig. 4a
). These repeats are also characteristic of the 5'-UTRs of chrysovirus dsRNAs (Jiang & Ghabrial, 2004
; Ghabrial & Castón, 2004
) and have been previously identified in the 5'-leader sequence of Tobacco mosaic virus RNA as a binding site for the heat-shock protein HSP101, which is required for translational enhancement (Gallie & Walbot, 1992
; Gallie, 2002
). Therefore, the sequence characteristics of these four CCRS-associated dsRNAs strongly suggested that they were the genomic components of a new chrysovirus species.
Sequence analysis of the CCRS- and ACD-associated chrysovirus dsRNAs supports the close similarity of both diseases
From dsRNA-rich preparations of ACD-affected tissue, cDNA clones were synthesized using a combination of random priming, RT-PCR with internal primers and RLM-RACE PCR. Assignment of specific dsRNAs was made by comparison with the CCRS-associated dsRNAs. Comparative analysis of the four chrysovirus dsRNAs associated with CCRS and ACD showed that they had almost identical sequences (nucleotide identity greater than 93 % and predicted amino acid sequences more than 95 % identical). This was particularly the case for the dsRNAs 1 and 3, which had 3399 and 2833 bp, respectively, and contained ORFs of 1087 aa and 884 aa, respectively. The CCRS chrys-dsRNA 2 was 3 bp shorter with respect to its ACD counterpart (3125 vs 3128 bp) because of a deletion in a region within the ORF, which was thus 1 aa shorter in the former. The CCRS chrys-dsRNA 4 was 1 bp longer with respect to its ACD counterpart (2499 vs 2498 bp) as a consequence of the insertion of 1 bp in the 5'-UTR of the former. Therefore, not only were the electrophoretic patterns of and the apparent sizes inferred for the CCRS- and ACD-associated dsRNAs very similar, but the sequences of four of these dsRNAs, which corresponded to the genomic components of a new species in the genus Chrysovirus, were also very similar. This similarity could be extended to other two dsRNAs (Fig. 2), which were the genomic components of a new species in the genus Partitivirus (see accompanying paper by Coutts et al., 2004
, in this issue).
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DISCUSSION |
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The molecular characteristics of four of the CCRS- and ACD-associated dsRNAs (Fig. 2) closely paralleled those of the genus Chrysovirus and, on the basis of their size, sequence and structural features of their 5'-UTRs, which are demarcation criteria in the genus (Ghabrial & Castón, 2004
), we propose that they are the genomic components of a new species of this genus. Although the species of genus Chrysovirus described so far have been found infecting only fungi (Ghabrial & Castón, 2004
), the possibility exists that other members could infect plants, as is the case with cryptoviruses (the members of the family Partitiviridae that infect plants) that are included in this family under the genera Alphacryptovirus and Betacryptovirus (Ghabrial et al., 2004
). However, there is circumstantial evidence suggesting a fungal origin for the CCRS- and ACD-associated chrysoviruses (and, indirectly, a fungal aetiology for the diseases). Firstly, electron microscopic examinations of thin sections have revealed a fungus-like organism in CCRS and ACD symptomatic leaves, and fluorescent microscopy observations have confirmed the presence in affected leaf areas of a mycelium growing between the lower epidermis, palisade and spongy mesophyll cells that was not found in vascular bundles or in asymptomatic samples (Alioto et al., 2003
). Secondly, although an initial report provided some data suggesting that ACD disease was induced by a graft-transmissible agent, presumably a virus (Blodgett et al., 1970
), this has not been further confirmed and attempts to transmit the CCRS agent by grafting have been unsuccessful (Alioto et al., 2003
). Lastly, treatment with systemic fungicides diminished the adverse effects of ACD (A. Citir, unpublished data). Collectively, these results support the involvement of a fungus as the causal agent, although efforts to isolate and grow it on artificial media have failed, with the saprophytic Alternaria alternata being sporadically detected (Alioto et al., 2003
). No direct attempts to observe or isolate chrysovirus-like virions from the leaf material containing CCRS- or ACD-associated dsRNAs have been made (D. Alioto, personal communication).
Chrysovirus virions are isometric, 3540 nm, non-enveloped particles with a capsid formed by 60 protein subunits arranged on a genuine T=1 icosahedral lattice that separately encapsidate the four genomic dsRNAs (Castón et al., 2003; Ghabrial & Castón, 2004
). From the topology of a phylogenetic tree obtained with the fragments containing the eight motifs conserved in the RdRps of a series of mycoviruses (Fig. 3b
), the CCRS- and ACD-associated chrysoviruses form a separate group with PcV and Hv145SV, the two other sequenced species in the genus Chrysovirus (Jiang & Ghabrial, 2004
; Ghabrial & Castón, 2004
). Moreover, the CCRS- and ACD-associated chrysoviruses appear to be more closely related to Hv145SV than to PcV (Fig. 3b
). This relationship, based on the ORFs contained in the chrysovirus dsRNAs 1, is also supported by a phylogenetic reconstruction derived from the ORFs contained in the chrysovirus dsRNAs 2 (data not shown). The 5'-UTRs of the four dsRNAs of the CCRS- and ACD-associated chrysoviruses are considerably shorter than those of their Hv145SV and PcV counterparts, whereas the 3'-UTR of the dsRNA 4 of the CCRS- and ACD-associated chrysoviruses is longer. Despite these length differences, we believe we have determined the real termini of the genomic components of the CCRS- and ACD-associated chrysoviruses because: (i) the 5' and 3' termini were identical for the four dsRNAs from the two diseases; (ii) diverse RT-PCR approaches were used for their amplification and cloning; (iii) the 5' terminus of one of the dsRNAs was confirmed by primer-extension analysis (data not shown); and (iv) there are nucleotide residues strictly conserved in both terminal regions of all chrysovirus dsRNAs (Fig. 4b
). In any case, the high conservation of the 5' and 3' termini in the four dsRNAs of the CCRS- and ACD-associated chrysoviruses (Fig. 4
) suggested that they may be replicated by the same RdRp, as previously advanced for other chrysoviruses (Ghabrial et al., 2002
).
It must be finally noted that we do not know yet the nature of the four CCRS- and ACD-associated dsRNAs of higher size (Fig. 1). They could be the replicative intermediates of a ssRNA virus(es) or, more likely, the genomic components of additional dsRNA viruses infecting the same fungus. In support of the second view, the size of some of the largest CCRS- and ACD-associated dsRNAs is consistent with that of totivirus dsRNAs, and a mixed infection of a chrysovirus and a totivirus has been previously reported in H. victoriae (Sanderlin & Ghabrial, 1978
; Ghabrial et al., 2002
). Furthermore, the possibility of more than one fungus being involved in CCRS and ACD cannot be dismissed. On the other hand, it is unlikely that the two dsRNAs of lower size found in association at least with CCRS (Fig. 1
) might contain functional ORFs, because they would be at most 100 aa long. The alternative suggestion that they could represent defective or satellite RNAs appears more plausible. A proposal of this kind has been advanced for the viroid-like RNAs associated with CCRS (Di Serio et al., 1997
). In any case, whether the CCRS- and ACD-associated dsRNAs are avirulent for their fungal hosts, as with PcV (Ghabrial & Castón, 2004
), or whether they modulate fungal pathogenicity as previously reported for other mycoviruses (Ghabrial, 1994
; Nuss, 1996
; McCabe et al., 1999) remains an intriguing issue to be explored.
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ACKNOWLEDGEMENTS |
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Received 8 April 2004;
accepted 19 July 2004.