Characterization of the siRNAs associated with peach latent mosaic viroid infection.

The peach latent mosaic viroid (PLMVd) is a small, circular RNA species that has been shown to induce RNA silencing in plants. With the goal of better understanding the biological mechanism underlying this process, the siRNAs found in PLMVd infected peach leaves were isolated and sequenced. Analysis of the resulting data prompted several conclusions, including: i. PLMVd strands of both polarities are substrates for the Dicer-like enzymes found in peach leaves; ii. the more highly structured regions of PLMVd trigger the activity of the Dicer-like enzymes; and, iii. the circular PLMVd conformers appear to be favored for transport into the cytoplasm.

Genomic structure of new Tunisian peach latent mosaic viroid variants.

Peach latent mosaic viroid (PLMVd) is a single-stranded circular RNA that do not code for proteins and ranges in size from 335 to 351 nucleotides. It mainly infects peach. In this study, the sequence of 20 complete cDNA clones derived from seven PLMVd isolates detected in five Tunisian peach cultivars was analysed in 3 steps: primary structure, phylogeny and secondary structure. The analysis of the primary structure revealed that all the 20 cDNA clones sequences corresponded to different variants. They ranged in size from 336 to 341 nt. Sequence alignment of our variants with reference sequences revealed 81 polymorphic positions. Among them, 15 were never described in the literature so far. The variable positions are scattered all around the RNA molecules, but the majority of them were concentrated in the region corresponding to nucleotides 1 to 70 and 170 to 346 in the alignment. Sequence homologies between variants of the same isolate or variants of different isolates ranged from 96% to 100%. This confirms that a PLMVd isolate is composed by a complex mixture of closely related molecules. Moreover, some variants isolated from different cultivars were found to be similar, indicating that a sequence is not exclusive to a cultivar. Phylogenetic analysis of our sequences allowed their clustering into two groups: group I (16 variants) and group II (4 variants) that differed by 18 polymorphic positions. Further phylogenetic analysis and sequence alignment of our sequences and the reference sequences were done. It revealed that our sequences were similar to the reference sequence Hd8 in the regions delimited by nucleotides 1 to 69 (region 1) and 268 to 343 (region 5) and to the reference sequence Hd6 in the region between nucleotides 150 and 200 (region 3). The other regions corresponding to nucleotides 70 to 149 (region 2) and 201 to 267 (region 4) were similar for all the sequences. These observations revealed that our Tunisian PLMVd variants correspond to a new population never reported in the literature. Analysis of the secondary structure confirmed that all PLMVd Tunisian variants presented a branched secondary structure and revealed a new potential pseudoknot-like interaction between two loops.

Peach latent mosaic viroid Detected for the First Time on Almond Trees in Tunisia.

Almond (Prunus dulcis Mill) is an important crop in countries of the Mediterranean area. Until now, among viroids, only Hop stunt viroid (HSVd) is known to infect cultivated almond trees (2). In 2004, a survey of almond trees was carried out in orchards in different regions of Tunisia, a major producing and exporting country of almond. Symptoms such as mosaic and necrotic lesions, potentially caused by the Peach latent mosaic viroid (PLMVd), were observed on leaves of cultivated almond trees. Since PLMVd was recently detected in peach and pear trees in Tunisia (4), the presence of this viroid in almond trees was studied. The detection method on the basis of one-tube reverse transcription-polymerase chain reaction (RT-PCR) assays was previously described and validated for the detection of this viroid in fruit trees (4). Amplification products were obtained by using previously reported primer pairs of PLMVd (1). Positive controls included RNA preparations of twigs of PLMVd-infected GF 305 peach seedlings. These materials, provided by B. Pradier (Station de Quarantaine des Ligneux, Lempdes, France), were positive as revealed by chip budding on peach seedling indicator plants grown under greenhouse conditions. RT-PCR analysis of nucleic acid preparations from leaves of almond showed specific amplification products with the expected size of 337 bp for two almond trees among 17 trees tested. Nucleotide sequence analyses of cloned amplification products obtained with the PLMVd primers confirmed a size of 337 bp and revealed a sequence similar to sequences from other PLMVd isolates previously characterized. The sequences shared 94 to 98% identity with the reference isolates of PLMVd from peach (EMBL Accession No. M83545, AF170511, AF170514, and AY685181). The two infected almond trees are proximal to each other and peach trees infected with PLMVd. This suggests that one tree may have served as a source of inoculum for the other through agronomic practices such as pruning or the aphid Myzus percicae (3). Alternatively, PLMVd may have originated in an unknown host and was then transmitted to almond trees. Our investigation shows that almond is a new host for PLMVd. References: (1) N. Astruc. Eur. J. Plant Pathol. 102:837, 1996. (2) M. C. Cañizares et al. Eur. J. Plant Pathol. 105:553, 1999. (3) J. C. Desvignes et al. Phytoma 444:70, 1992. (4) I. Fekih Hassen et al. Plant Dis. 88:1164, 2004.