Identification and characterization of privet leaf blotch-associated virus, a novel idaeovirus.

A novel virus has been identified by next-generation sequencing (NGS) in privet (Ligustrum japonicum L.) affected by a graft-transmissible disease characterized by leaf blotch symptoms resembling infectious variegation, a virus-like privet disease with an unclear aetiology. This virus, which has been tentatively named 'privet leaf blotch-associated virus' (PrLBaV), was absent in non-symptomatic privet plants, as revealed by NGS and reverse transcription-polymerase chain reaction (RT-PCR). Molecular characterization of PrLBaV showed that it has a segmented genome composed of two positive single-stranded RNAs, one of which (RNA1) is monocistronic and codes for the viral replicase, whereas the other (RNA2) contains two open reading frames (ORFs), ORF2a and ORF2b, coding for the putative movement (p38) and coat (p30) proteins, respectively. ORF2b is very probably expressed through a subgenomic RNA starting with six nucleotides (AUAUCU) that closely resemble those found in the 5'-terminal end of genomic RNA1 and RNA2 (AUAUUU and AUAUAU, respectively). The molecular signatures identified in the PrLBaV RNAs and proteins resemble those of Raspberry bushy dwarf virus (RBDV), currently the only member of the genus Idaeovirus. These data, together with phylogenetic analyses, are consistent with the proposal of considering PrLBaV as a representative of the second species in the genus Idaeovirus. Transient expression of a recombinant PrLBaV p38 fused to green fluorescent protein in leaves of Nicotiana benthamiana, coupled with confocal laser scanning microscopy assays, showed that it localizes at cell plasmodesmata, strongly supporting its involvement in viral movement/trafficking and providing the first functional characterization of an idaeovirus encoded protein.

Cydonia japonica, Pyrus calleryana and P. amygdaliformis: three new ornamental or wild hosts of Apple stem pitting virus.

Japanese quince, ornamental and wild pear symptomless samples were infected with Apple stem pitting virus (ASPV). Identification of ASPV was achieved by different PCR assays that amplified either the RNA polymerase or coat protein gene regions. For further confirmation, 312 bp amplicons within the polymerase gene were sequenced and compared with previously published ASPV sequences and additional sequences of isolates from ancient Italian cultivars. Comparison of the partial sequences isolated from wild/ornamental hosts and from cultivated species revealed significant divergence levels. Among the wild/ornamental isolates, the PCT88 isolate from Pyrus calleryana was the most divergent, having an amino acid deletion and incorporating a unique stretch of amino acids not present in any other isolate. Further to this preliminary partial sequence data, statistical analysis demonstrated that the isolates from wild or ornamental hosts were not more closely related to each other than to isolates from cultivated hosts. These results represent the first report of natural ASPV infection in these novel ornamental and wild Rosaceae hosts.

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

Cherry chlorotic rusty spot (CCRS) and Amasya cherry disease (ACD) display similar symptoms and are associated with a series of dsRNAs. However, a direct comparison has been lacking. Here, a side-by-side analysis confirmed that both diseases were symptomatologically very similar, as were the number (10-12) and size of their associated dsRNAs. Sequence determination of four of these dsRNAs revealed that they were essentially identical for CCRS and ACD. The largest (3399 bp), which potentially encoded a protein of 1087 aa with the eight motifs conserved in RNA-dependent RNA polymerases of dsRNA mycoviruses, had the highest similarity to those coded by dsRNA 1 of viruses belonging to the genus Chrysovirus and was termed CCRS or ACD chrys-dsRNA 1. The three closely migrating dsRNAs had the properties of the other components of a chrysovirus and in CCRS and ACD versions, respectively, were chrys-dsRNA 2 (3125 and 3128 bp), chrys-dsRNA 3 (2833 bp) and chrys-dsRNA 4 (2499 and 2498 bp), potentially encoding the major capsid protein (993 and 994 aa) and two proteins (884 and 677 aa, respectively) of unknown function. The four 5'- and 3'-UTRs shared internal similarities and had conserved GAAAAUUAUGG and AUAUGC termini, respectively. The 5'-UTRs contained the 'Box 1' motif followed by a stretch rich in CAA, CAAA and CAAAA repeats, characteristic of chrysovirus dsRNAs. Because species of the genus Chrysovirus have only been described as infecting fungi, this suggests a fungal aetiology for CCRS and ACD, a proposal supported by the properties of two other CCRS- and ACD-associated dsRNAs (see accompanying paper by Coutts et al., 2004, in this issue).

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.

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).

Characterization of Cestrum yellow leaf curling virus: a new member of the family Caulimoviridae.

Cestrum yellow leaf curling virus (CmYLCV) has been characterized as the aetiological agent of the Cestrum parqui mosaic disease. The virus genome was cloned and the clone was proven to be infectious to C. parqui. The presence of typical viroplasms in virus-infected plant tissue and the information obtained from the complete genomic sequence confirmed CmYLCV as a member of the Caulimoviridae family. All characteristic domains conserved in plant pararetroviruses were found in CmYLCV. Its genome is 8253 bp long and contains seven open reading frames (ORFs). Phylogenetic analysis of the relationships with other members of the Caulimoviridae revealed that CmYLCV is closely related to the Soybean chlorotic mottle virus (SbCMV)-like genus and particularly to SbCMV. However, in contrast to the other members of this genus, the primer-binding site is located in the intercistronic region following ORF Ib rather than within this ORF, and an ORF corresponding to ORF VII is missing.

Peach latent mosaic viroid variants inducing peach calico (extreme chlorosis) contain a characteristic insertion that is responsible for this symptomatology.

The involvement of Peach latent mosaic viroid (PLMVd) in an extensive chlorosis of peach known as calico (PC) has been advanced but ultimate proof is lacking. Sequencing of 16 full-length PLMVd cDNA clones of a PC isolate revealed two groups of variants. Nine had a size (336-338 nt) similar to that of typical PLMVd variants of nonsymptomatic and mosaic-inducing isolates, whereas the other 7 were longer (348-351 nt) due to an insertion of 12-13 nt. This insertion was always found in the hairpin loop capping the hammerhead arm, had a limited sequence variability, and folded itself into a hairpin. When three PLMVd dimeric transcripts, two with and the other without the insertion, were slash-inoculated on GF-305 peach seedlings, PC symptoms were produced exclusively by the RNAs containing the insertion, which was conserved in the progeny. These data demonstrate that the agent of PC is PLMVd. Direct support that the 12- to 13-nt insertion contains the PC pathogenicity determinant was obtained by its removal through site-directed mutagenesis from one of the PC-inducing variants. Inoculations with dimeric transcripts of the resulting variant showed that it could replicate but without eliciting symptoms. Our results also suggest that the insertion emerges sporadically de novo.

Cestrum yellow leaf curling virus (CmYLCV) promoter: a new strong constitutive promoter for heterologous gene expression in a wide variety of crops.

Appropriately regulated gene expression requires a suitable promoter. A number of promoters have been isolated and shown to be functional in plants, but only a few of them activate transcription of transgenes at high levels constitutively. We report here the cloning and characterization of a novel, constitutively expressed promoter isolated from Cestrum yellow leaf curling virus (CmYLCV), a double-stranded DNA plant pararetrovirus belonging to the Caulimoviridae family. The CmYLCV promoter is highly active in callus, meristems and vegetative and reproductive tissues in Arabidopsis thaliana, Nicotiana tabacum, Lycopersicon esculentum, Zea mays and Oryza sativa. Furthermore, the level of expression is comparable to, or higher than, that from the CaMV 35S, the 'super-promoter' or the maize ubiquitin 1 promoters, three frequently used promoters in agricultural biotechnology. The heritable, strong and constitutive activity in both monocotyledonous and dicotyledonous plants, combined with the extremely narrow CmYLCV host range, makes the CmYLCV promoter an attractive tool for regulating transgene expression in a wide variety of plant species.