No abstract

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.

Chromosome sizes of 71 phytoplasmas belonging to 12 major phylogenetic groups including several of the aster yellows subgroups were estimated from electrophoretic mobilities of full-length chromosomes in pulsed-field gels. Considerable variation in genome size, from 660 to 1,130 kilobases (kb), was observed among aster yellows phytoplasmas. Chromosome size heterogeneity was also observed in the stolbur phytoplasma group (range 860 to 1,350 kb); in this group, isolate STOLF contains the largest chromosome found in a phytoplasma to date. A wide range of chromosome sizes, from 670 to 1,075 kb, was also identified in the X-disease group. The other phytoplasmas examined, which included members of the apple proliferation, Italian alfalfa witches' broom, faba bean phyllody, pigeon pea witches' broom, sugarcane white leaf, Bermuda grass white leaf, ash yellows, clover proliferation, and elm yellows groups, all have chromosomes smaller than 1 megabase, and the size ranges within each of these groups is narrower than in the aster yellows, stolbur, and X-disease groups. The smallest chromosome, approximately 530 kb, was found in two Bermuda grass white leaf phytoplasma isolates. This not only is the smallest mollicute chromosome found to date, but also is the smallest chromosome known for any cell. More than one large DNA band was observed in several phytoplasma preparations. Possible explanations for the occurrence of more than one band may be infection of the host plant by different phytoplasmas, the presence of more than one chromosome in the same organism, or the presence of large extrachromosomal DNA elements.

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 59-and 39-UTRs shared internal similarities and had conserved GAAAAUUAUGG and AUAUGC termini, respectively. The 59-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).

RNA 3 of alfalfa mosaic virus (AMV) encodes the 5'-proximal movement protein (MP) gene and the 3'-proximal coat protein (CP) gene which is expressed from a subgenomic RNA. Several strategies were explored to use this RNA as a vector for expression of the green fluorescent protein (GFP) in Nicotiana tabaccum plants expressing the viral polymerase proteins P1 and P2 (P12 plants). Insertion of a subgenomic promoter (sgp)-GFP cassette between the CP gene and the 3'-untranslated region (UTR) interfered with RNA accumulation in protoplasts, indicating that cis-acting sequences required for replication were disrupted. When GFP was fused to the N-terminus of MP or CP, the chimeric RNAs accumulated in protoplasts but cell-to-cell movement in plants was blocked. Insertion of a GFP-sgp cassette immediately upstream of the CP gene caused a hypersensitive host response. However, insertion of a GFP-sgp cassette upstream of the MP gene did not affect symptom formation and yielded a vector that expressed GFP in inoculated but not in the systemic leaves of both P12 tobacco and non-transgenic N. benthamina plants. When the size of the GFP gene was reduced from 700 to 300 nucleotides, virus infection was observed in the non-inoculated leaves. Analysis of the progeny of some chimera revealed novel data on replication, encapsidation and recombination of AMV RNA 3.