On the early identification and characterization of pear blister canker viroid, apple dimple fruit viroid, peach latent mosaic viroid and chrysanthemum chlorotic mottle viroid

Abstract

In the 90's, pear blister canker viroid (PBCVd), apple dimple fruit viroid (ADFVd), peach latent mosaic viroid (PLMVd) and chrysanthemum chlorotic mottle viroid (CChMVd) were identified and characterized in the Ricardo Flores' laboratory. In these studies, the autonomous replication of these infectious RNAs and their involvement in the elicitation of diseases in their natural hosts were also shown. Their discovery was achieved by classical approaches based on the physical purification of the viroid RNAs from polyacrylamide gels followed by the sequencing of their genomic RNAs and by bioassays to assess their autonomous replication and the fulfillment of Koch's postulates. The molecular characterization of these four viroids, including the study of their sequence variability, contributed to the establishment of the concept of quasispecies for viroids and to the development of reliable molecular diagnostic methods that have facilitated the control of the diseases they caused. Most importantly, some of these viroids became valuable experimental model systems that are still used nowadays to study structural-functional relationships in RNAs and to dissect evolutionary and pathogenic pathways underlying plant-viroid interaction. The differences between early viroid discovery strategies, relying on biological and pathogenic issues, and the current high-throughput sequencing-based approaches, that frequently allow the discovery of new viroids and viroid-like RNAs in symptomless hosts, is also discussed, clarifying why the traditional molecular and biological studies mentioned above are still required to conclusively define the nature of any novel viroid-like RNA.

Keywords: Apscaviroid; Koch's postulates; Pelamoviroid; Plant disease; Quasispecies, PBCVd, ADFVd, PLMVd, CChMVd; Viroid discovery.

Fig. 1

(A) Symptoms induced by PBCVd on bark and stems of pear cv. A20 (on the left), symptoms induced by PBCVd isolates P2098T and P1914T, respectively (in the middle), healthy control (on the right) (Courtesy of J.C. Desvignes). (B) Symptoms induced by ADFVd on apple fruits cv. Starking Delicious (on the left) compared with healthy control (on the right). (C) Symptoms of severe albinism (peach calico) induced by PLMVd (isolate PC—C40) in peach cv. GF305 (on the left), healthy control (on the right). (D) Symptoms of leaf chlorosis induced by CChMVd (isolate S) in chrysanthemum cv. Bonnie Jean (on the left) compared with a healthy control (on the right).

Fig. 2

Primary and secondary structure of lowest free energy for PBCVd reference variant NC_001830 (Hernández et al., 1992) (A), PLMVd variant PC-40 causing peach calico (B), and CChMVd symptomatic variant CM20 (C). In A, the terminal conserved region (TCR) is denoted by flags and the upper and lower strands of the central conserved region (CCR) by black and white circles, respectively. Positions of nucleotide insertions and deletions are marked by green arrows directed to or gray arrows starting from the sequence, respectively. Unique point mutations and other polymorphic positions, identified in the multiple sequence alignment performed with all the PBCVd variants deposited in databases, are denoted in red and blue colors, respectively. In B and C, sequences forming plus and minus hammerhead structures are delimited by flags, self-cleavage sites by arrows, and nucleotides conserved in most natural hammerhead structures are denoted in bold. Red and blue colors refer to plus and minus polarities, respectively. Sequences in green denote the pathogenicity determinant. The interaction between nucleotides in two loops forming a kissing-loop tertiary structure are indicated with broken lines.