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Identification and Characterization of a Variant of Citrus viroid V (CVd-V) in Seminole Tangelo

Previous studies on Atalantia citroides, a citrus relative that appeared to be immune to viroid infection, revealed the existence of a new viroid, which was designated tentatively as Citrus viroid-V (CVd-V) and has been proposed as a new member of the genus Apscaviroid within the family Pospiviroidae. Biological indexing of a Seminole tangelo tree on the Etrog citron indicator followed by sPAGE analysis revealed the presence of a viroid that has been characterized as a new variant of CVd-V. The variant has 97.6% sequence identity with the reference sequence of CVd-V and shows three characteristics: (i) two compensatory changes that modify an A-U base pair between the upper and lower “strands” into a G-C base pair, (ii) a U→A change located in a loop that does not seem to disturb the viroid secondary structure, and (iii) two sets of changes located in the upper and lower strands that result in a rearrangement of the base pairing between the upper and lower strands. Infectivity studies performed with an artificial mutant revealed the existence of structural constraints in the region in which major differences between CVd-V and the Seminole tangelo variant were identified. Index words: Citrus viroids, Apscaviroid, structural motifs. Studies performed to define the response of several species of citrus and citrus related genera to viroid infection showed that Atalantia citroides appears to be immune to infection with CEVd, HSVd, CBLVd, CVd-III and CVd-IV (1), and highlighted the presence of a viroid not described previously (2). This new viroid, tentatively named Citrus viroid V (CVd-V) has a GC-rich genome of 293294 nucleotide residues, and its predicted secondary structure contains the central conserved region (CCR) characteristic of members of the genus Apscaviroid, and the terminal conserved region (TCR) present in this and other genera of the family Pospiviroidae (10). Viroid indexing of a Seminole tangelo tree by graft-inoculation on citron Etrog followed by sequential polyacrylamide gel electrophoresis (sPAGE) (3) revealed the presence of a viroid-like RNA with a mobility similar to the circular forms of CVd-III. Molecular hybridization assays using viroid-specific probes failed to reveal a relationship between this viroid-like RNA and any of the viroids known at that time (unpublished results). Here, we describe the characterization of this viroid-like RNA as a sequence variant of CVd-V. MATERIALS AND METHODS Extraction procedure and viroid analysis. Tissue samples (5 g) of young leaves (Etrog citron) or bark (Seminole tangelo) were homogenized in 5 ml of extraction buffer (0.4M Tris-HCl pH=8.9; 1% (w/v) SDS; 5mM EDTA; 4% (v/v) 2mercaptoethanol) and water-saturated phenol, and the total nucleic acids were partitioned in 2M LiCl (9). The soluble fraction was concentrated by ethanol precipitation and resuspended in TKM buffer (10 mM Tris-HCl; 10 mM KCl; 0.1mM MgCl2 pH 7.4). Aliquots of these preparations were used for sPAGE, Northern blot hybridization and RT-PCR analysis. For sPAGE (5%, 39:1) analysis, aliquots (20 μl equivalent to 300 mg of fresh tissue) were subjected to electrophoresis under non-denaturing conditions (2 h, 60 mA) and 8M urea denaturing conditions (4 h, 18 mA) (7) and the circular forms of the viroid were