Mapping the molecular determinant of pathogenicity in a hammerhead viroid: a tetraloop within the in vivo branched RNA conformation

Abstract

Chrysanthemum chlorotic mottle viroid (CChMVd) is an RNA of 398-399 nt that can adopt hammerhead structures in both polarity strands. We have identified by Northern-blot hybridization a nonsymptomatic strain (CChMVd-NS) that protects against challenge inoculation with the symptomatic strain (CChMVd-S). Analysis of CChMVd-NS cDNA clones has revealed a size and sequence very similar to those of the CChMVd-S strain. Some of the mutations observed in CChMVd-NS molecular variants were previously identified in CChMVd-S RNA, but others were never found in this RNA. When bioassayed in chrysanthemum, cDNA clones containing the CChMVd-NS specific mutations were infectious but nonsymptomatic. Site-directed mutagenesis showed that one of the CChMVd-NS-specific mutations, a UUUC --> GAAA substitution, was sufficient to change the symptomatic phenotype into the nonsymptomatic one without altering the final accumulation level of the viroid RNA. The pathogenicity determinant-to our knowledge, a determinant of this class has not been described previously in hammerhead viroids-is located in a tetraloop of the computer-predicted branched conformation for CChMVd RNA. Analysis of the sequence heterogeneity found in CChMVd-S and -NS variants strongly supports the existence of such a conformation in vivo, showing that the rod-like or quasi-rod-like secondary structure is not a universal paradigm for viroids.

Figure 1

Identification of a nonsymptomatic CChMVd RNA. (A) Differential response of two chrysanthemum cultivars after inoculation with a nucleic acid preparation of the severe CChMVd-S strain. (B) Analysis of chrysanthemum RNAs by denaturing PAGE and ethidium bromide staining (Left) or Northern-blot hybridization with a probe complementary to CChMVd-S RNA (Right). Lane 1, DNA marker of 392 nt; lanes 2 and 3, RNAs from healthy and symptomatic “Bonnie Jean” plants, respectively; lanes 4 and 5, RNAs from symptomless “Yellow Delaware” plants uninoculated and inoculated with a CChMVd-S RNA preparation, respectively. The migration of the circular (c) and linear (l) viroid forms is indicated at the right.

Figure 2

Sequence alignment of 12 molecular variants from “Bonnie Jean” plants inoculated with the CChMVd-NS strain. For comparative purposes, the sequence of the symptomatic CM 20 variant is shown on the top. Dots indicate residues identical to the CM 20 sequence and dashes denote gaps. Variants CMNS 1, 6, 25, 26, 30, 35, 38, and 41 were synthesized with primers PI and PII, and the rest with PIII and PIV.

Figure 3

Secondary structure of lowest free energy of the symptomatic CM 20 variant. Numbering does not coincide exactly with that of the alignment because of the introduced gaps. The structure of the region between positions 70 and 93 is slightly different from that reported previously (5), because an updated version of the mfold program has been used. The most frequent mutations detected in CChMVd-NS variants are shown; those on a black background have never been found in CChMVd-S variants, whereas those on a white background were previously found in CChMVd-S variants. Sequences forming plus and minus hammerhead structures are indicated by flags, nucleotides conserved in most natural hammerhead structures by bars, and self-cleavage sites by arrows. Solid and open symbols refer to plus and minus polarities, respectively. (Upper Left Inset) Most stable secondary structure of the hairpin delimited by positions 70 to 94 in variant CMNS 2. (Right Inset) Most stable secondary structure of the hairpin delimited by positions 180–210 in variants CMNS 2, 4, 7, and 30. (Lower Left Inset) Hammerhead structures of plus and minus strands of the CM 20 variant. The most frequent mutations found in CChMVd-NS variants are on a black background and do not alter the stability of the hammerhead structures. Less frequent mutations of other CChMVd-NS variants, which affect the stability of their hammerhead structures, are on a white background with deletions indicated by −. Nucleotides conserved in most natural hammerhead structures are boxed, and self-cleavage sites are shown by arrows. The same numbering is used in both polarities and does not coincide exactly with numbering of the alignment because of the introduced gaps.

Figure 4

Infectivity of some representative CChMVd-NS variants and self-cleavage of their monomeric plus (+) and minus (−) RNAs. Dot-blot hybridization signals correspond to undiluted extracts and 1:10 dilutions (left and right spot columns, respectively). The extent of self-cleavage is expressed as the fraction (%) of the primary transcript that self-cleaved during in vitro transcription. The nonsymptomatic CM 20–1 variant was obtained from the symptomatic CM 20 variant by introducing the change UUUC82–85 → GAAA.

Figure 5

Effects of the sequence heterogeneity found in variants characterized so far from CChMVd-S and -NS strains on the secondary structure predicted for CChMVd RNA. Mutations of CMNS 6, 26, 38, and 41, which are presumably artifactual for the reasons stated in the text, are not included. Insertions and deletions are denoted by symbols + and −, respectively. For other details of the CChMVd reference variant, see Fig. 3.