Cherry chlorotic rusty spot and Amasya cherry diseases are associated with a complex pattern of mycoviral-like double-stranded RNAs. II. Characterization of a new species in the genus Partitivirus

Robert H. A. Coutts1, Laura Covelli2,3, Francesco Di Serio4, Ahmet Citir5, Serap Açikgöz6, Carmen Hernández2, Antonio Ragozzino3 and Ricardo Flores2

1 Department of Biological Sciences, Imperial College London, Imperial College Road, London SW7 2AZ, UK
2 Instituto de Biología Molecular y Celular de Plantas (UPV-CSIC), Universidad Politécnica de Valencia, Avenida de los Naranjos s/n, 46022 Valencia, Spain
3 Dipartimento di Arboricoltura, Botanica e Patologia Vegetale, Università di Napoli, 80055 Portici, Italy
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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Two dsRNAs from cherry trees affected with cherry chlorotic rusty spot (CCRS) in Italy and Amasya cherry disease (ACD) in Turkey were sequenced and found to be essentially identical. The larger dsRNA 1 (2021 or 2006 bp, respectively) potentially encoded a protein of 621 aa containing the conserved motifs of the RNA-dependent RNA polymerases (RdRp) of dsRNA mycoviruses, having highest similarity with those in the genus Partitivirus. The smaller dsRNA 2 (1841 or 1839 bp, respectively) had properties consistent with the second genomic component of a partitivirus and potentially encoded the coat protein (CP) of 504 aa. Phylogenetic analysis based on the RdRp and CP was coincidental and indicated that species in the genus Partitivirus could be separated into two subgroups. Because species of this genus only infect fungi, these observations suggest a fungal aetiology for CCRS and ACD, further substantiating a previous proposal (see accompanying paper by Covelli et al., 2004, in this issue).

The GenBank/EMBL/DDBJ accession numbers of the sequences reported in this paper are AJ781168 and AJ781167 for ACD-associated dsRNAs 1 and 2 and AJ781401 and AJ781402 for CCRS-associated dsRNAs 1 and 2.


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In the accompanying paper (Covelli et al., 2004), we illustrated that two cherry diseases, cherry chlorotic rusty spot (CCRS) from Italy and Amasya cherry disease (ACD) from Turkey are very similar diseases probably caused by a fungus (Alioto et al., 2003), which, in turn, is associated with multiple species of double-stranded RNAs (dsRNAs), presumably of viral origin (Covelli et al., 2004; Di Serio et al., 1996, 1998). In confirmation of the mycoviral nature of these dsRNAs, we reported that four of them comprised the genome of a new species in the genus Chrysovirus (Covelli et al., 2004; Ghabrial & Castón, 2004) and here we have shown that two other dsRNAs comprise the genome of a new species in the genus Partitivirus.

Members of the family Partitiviridae (Ghabrial et al., 2004), which together with the Chrysoviridae, Hypoviridae and Totiviridae constitute the four fungal dsRNA virus families (Ghabrial, 2001; Ghabrial & Castón, 2004), are isometric, non-enveloped viruses, 30–40 nm in diameter, with a genome composed of two linear dsRNAs of approximately 1400–3000 bp (Ghabrial et al., 2004). The smaller dsRNA encodes the coat protein (CP) and the larger the virion-associated RNA-dependent RNA polymerase (RdRp) (Ghabrial et al., 2004). Additional satellite or defective RNAs may also be present. The family Partitiviridae has been divided into four genera, Partitivirus and Chrysovirus for viruses that infect fungi, and Alphacryptovirus and Betacryptovirus for viruses that infect plants (Ghabrial, 2001). Recently, the genus Chrysovirus was removed from the family Partitiviridae and placed into the new family Chrysoviridae (Ghabrial & Castón, 2004), which, in addition to Penicillium chrysogenum virus (PcV) (Jiang & Ghabrial, 2004), the type species, and Helminthosporium victoriae 145S virus (Hv145SV) (Ghabrial & Castón, 2004), now includes a tentative new species present in dsRNA extracts produced from ACD- and CCRS-affected cherry trees (Covelli et al., 2004). Most of these viruses cause latent infections and may have originated from totiviruses (Ghabrial, 1998). We previously suggested that two of the 10–12 dsRNAs (of approx. 1800 and 2000 bp) associated with both of the above diseases probably comprised the genome of a partitivirus (see accompanying paper by Covelli et al., 2004, in this issue) and here we report its molecular characterization.

The dsRNAs were isolated from infected sweet cherry leaves collected in Turkey and Italy exhibiting typical symptoms of ACD and CCRS, respectively, and after separation by electrophoresis on polyacrylamide or agarose gels they were either collectively or individually used for reverse transcription, PCR amplification, cloning and sequencing as described previously (Covelli et al., 2004). cDNA clones of the presumed ACD-associated partitivirus dsRNAs 1 and 2 were obtained by random priming of methyl mercuric hydroxide-denatured dsRNA, with further DNA manipulations being performed as previously described (Sambrook et al., 1989). For the synthesis of additional cDNAs covering their complete sequence, purified dsRNAs 1 and 2 of the presumed ACD-associated partitivirus were denatured with methyl mercuric hydroxide and subjected to a single-primer, genome-walking RT-PCR protocol developed previously to sequence plant virus RNAs (Livieratos et al., 1999). An RNA ligase-mediated (RLM)-RACE PCR procedure was used to determine the 5'- and 3'-terminal sequences of the dsRNAs (Coutts & Livieratos, 2003). The sequences of the equivalent CCRS-associated dsRNAs were obtained by the single-primer method as described previously (Covelli et al., 2004). Nucleotide and amino acid sequences were manipulated (Deveraux et al., 1984), aligned using CLUSTAL X (Thompson et al., 1997) and drawn using the MEGA 2 program (Kumar et al., 2001).

The complete dsRNA 1 sequences of the presumed ACD- and CCRS-associated partitiviruses were 2006 and 2021 bp, respectively, and each included one ORF potentially encoding a putative protein of 621 aa with a molecular mass of approximately 73 kDa. The two predicted proteins were 98 % identical to one another and contained the conserved motifs characteristic of the RdRps of partitiviruses and dsRNA viruses infecting simple eukaryotes (Bruenn, 1993) (Fig. 1a). A phylogenetic reconstruction using the fragments containing these conserved motifs from some representative toti-, chryso- and partitiviruses provided corroboration that the presumed ACD- and CCRS-associated partitiviruses clustered together with members of this group (Covelli et al., 2004). To delimit more precisely the relationships within the genus Partitivirus, a phylogenetic tree was constructed based on multiple alignment of the fragments containing the RdRp conserved motifs from all species of this genus deposited in databases using Beet cryptic virus (BCV-3) as an outgroup. The tree showed that ACD- and CCRS-associated partitiviruses formed a subgroup with the Helicobasidium mompa virus strain V70 (HmV-V70) (Osaki et al., 2002), the Heterobasidion annosum virus P-type (HaV-Pt) (Ihrmark, 2001), the Atkinsonella hypoxylon virus isolate 2H (AhV-2H) (Oh & Hillman, 1995), the Fusarium poae virus 1 (FpV-1) (Compel et al., 1999) and the Rhizoctonia solani virus 717 (RhsV-717) (Strauss et al., 2000), with the remaining five members of the genus forming a second subgroup (Fig. 1b).



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Fig. 1. Relationships between the CCRS- and ACD-associated partitiviruses and the other species in the genus Partitivirus. (a) Multiple alignment of a fragment of the putative RdRp encoded by CCRS-and ACD-associated partitivirus dsRNA 1 with the RdRps of the other partitiviruses. GenBank accession numbers were as follows. Upper subgroup: Amasya cherry disease associated partitivirus (ACD-PV; AJ781168), HmV-V70 (AB025903), FpV-1 (AF047013), RhsV-717 (AF133290), AhV-2H (L39125) and HaV-Pt (AF473549). Lower subgroup: Fusarium solani virus 1 (FsV-1; D55668), Gremmeniella abietina RNA virus MS1 (GaRV-MS1; AY089993), Penicillium stoloniferum virus S (PsV-S; AY156521), Discula destructiva virus 1 (DdV-1; AF316992) and DdV-2 (AY033436). BCV-3 (S63913), a member of the family Partitiviridae infecting plants, was used as an outgroup sequence. Numbers on top refer to the conserved motifs characteristic of the RdRps of partitiviruses and dsRNA viruses of simple eukaryotes (Bruenn, 1993; Ghabrial, 1998) (motifs 1 and 2 were not included because they are poorly defined in partitiviruses). The numbers of residues between the conserved motifs are indicated. In the consensus sequence, asterisks denote identical residues and letters denote residues conserved in at least five of the sequences. Within each subgroup, which are separated by a space, residues conserved in at least 50 % of the sequences are indicated in light grey (upper subgroup) or dark grey (lower subgroup). (b) Phylogenetic tree based on the neighbour-joining method with a 10 000 replicate bootstrap search. Bootstrap values are indicated at the branch points.

 
The complete dsRNA 2 sequences of the ACD- and CCRS-associated partitiviruses were 1839 and 1841 bp, respectively, and each included one ORF potentially encoding a putative CP of 504 aa with a molecular mass of approximately 55·5 kDa. The two predicted proteins were 94 % identical to one another, confirming the very close relationship between the two partitiviruses, which can be considered as different isolates of the same virus. A comparison with the dsRNA 2 (and the putative encoded proteins) of the other partitiviruses deposited in databases showed that ACD- and CCRS-associated partitiviruses clustered in a subgroup characterized by having larger dsRNAs and predicted proteins also larger and with lower isoelectric points (Table 1). Multiple alignments of the corresponding amino acid sequences also supported the existence of two subgroups, one with extensive sequence identity/similarity in several regions of the protein (Fig. 2a), and a second subgroup with considerably lower sequence conservation (Fig. 2b). Within this second subgroup, the putative CP of the ACD- and CCRS-associated partitiviruses, which shared an identity/similarity of 32/41 % with that of AhV-2H, the type species of the genus Partitivirus (Ghabrial et al., 2004), appeared as the most closely related to the proteins of the other subgroup.


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Table 1. Physical properties of dsRNA 2 and its potentially encoded proteins for members of the genus Partitivirus

 


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Fig. 2. Relationships between the CCRS- and ACD-associated partitiviruses and the other species in the genus Partitivirus inferred from the predicted sequences of their putative CPs. Regions derived from multiple alignments of the available sequences that allowed the distinction of the two subgroups are shown. For virus names and accession numbers, see legend to Fig. 1 and Table 1, respectively. Regions displaying different sequence conservation are presented within each subgroup. Asterisks denote identical residues and letters denote conserved residues in at least three (a) or two (b) of the sequences.

 
For both ACD- and CCRS-associated partitiviruses, the sizes of the 5'-untranslated regions (5'-UTRs) of dsRNAs 1 and 2 were 89 and 120 nt, respectively, and contained the CAA repeats reported previously in another partitivirus (Strauss et al., 2000). The lengths of the 3'-UTRs of the same molecules were 50 and 203 nt for the ACD isolate and 66 and 206 nt for the CCRS isolate. The 5'-terminal 12 nt were identical in the two dsRNAs of both partitiviruses but the 3'-UTRs were apparently less conserved. Nevertheless, the 3'-UTRs of both partitiviruses contained polyadenylated stretches in dsRNAs 1 and 2 and were A/U rich, a situation similar to that found in the 3'-UTRs of the dsRNAs of other partitiviruses (Oh & Hillman, 1995; Osaki et al., 2002; Strauss et al., 2000).

In summary, we have characterized the two dsRNA components of the ACD- and CCRS-associated partitiviruses. The phylogenetic analysis derived from the RdRp potentially encoded by dsRNA 1 of the known partitiviruses was essentially coincidental with the current taxonomy of the genus (Ghabrial et al., 2004) and located the ACD- and CCRS-associated partitiviruses in one specific subgroup. Interestingly, a parallel analysis based on the more limited data available for the putative CP potentially encoded by dsRNA 2 also led to the same subgroups, thus providing additional support for the present classification scheme. Within this scheme, ACD- and CCRS-associated partitiviruses clustered in the same subgroup with a predominance of dsRNA viruses that infect basidiomycetous fungi (HmV-V70, HaV-Pt and RhsV-717). What significance in evolutionary terms this has necessitates the isolation and molecular characterization of further partitiviruses from a variety of fungal species in the future.


   ACKNOWLEDGEMENTS
 
Work in the laboratories of R. F. and A. R. was partially supported by the Generalidad Valenciana (España) and the Ministero delle Politiche Agricole e Forestali (Italia), respectively. We thank Dr D. Alioto for detailed information about the fungus-like organism found in CCRS and ACD symptomatic leaves. During this work, L. C. was the recipient of pre-doctoral fellowships from the Marie Curie Program (European Union) and from the Università ‘Federico II’ di Napoli (Italia).


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Received 8 April 2004; accepted 19 July 2004.



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