Tertiary structure and function of an RNA motif required for plant vascular entry to initiate systemic trafficking

Abstract

Vascular entry is a decisive step for the initiation of long-distance movement of infectious and endogenous RNAs, silencing signals and developmental/defense signals in plants. However, the mechanisms remain poorly understood. We used Potato spindle tuber viroid (PSTVd) as a model to investigate the direct role of the RNA itself in vascular entry. We report here the identification of an RNA motif that is required for PSTVd to traffic from nonvascular into the vascular tissue phloem to initiate systemic infection. This motif consists of nucleotides U/C that form a water-inserted cis Watson-Crick/Watson-Crick base pair flanked by short helices that comprise canonical Watson-Crick/Watson-Crick base pairs. This tertiary structural model was inferred by comparison with X-ray crystal structures of similar motifs in rRNAs and is supported by combined mutagenesis and covariation analyses. Hydration pattern analysis suggests that water insertion induces a widened minor groove conducive to protein and/or RNA interactions. Our model and approaches have broad implications to investigate the RNA structural motifs in other RNAs for vascular entry and to study the basic principles of RNA structure-function relationships.

Figure 1

Mutational analysis of PSTVd systemic infection. (A) A schematic representation of secondary structure of PSTVd, showing genomic location of the U43/C318 ‘loop' required for systemic infection. (B) A schematic representation showing the major cellular boundaries in inoculated and systemic leaves that PSTVd traffics through to establish systemic infection. The open spaces in the lines represent plasmodesmata. The arrows indicate direction of trafficking. For simplicity, the specific cell types in the phloem are not illustrated. (C) Enlarged view of nucleotide sequences of the short helices flanking the U43/C318 ‘loop' in the WT, C318A and U43G mutants.

Figure 2

Time course for PSTVd systemic infection in N. benthamiana plant. (A) RNA blots showing accumulation of both the (+)-circular (c-PSTVd) and linear (l-PSTVd) viroid RNAs at successive dpi. rRNA serves as loading control. (B) Quantification of the accumulation levels of c-PSTVd from RNA blot results, calculated by using the known amounts of loading size markers in the same gel as references. Each data point represents the mean value of three biological replicates.

Figure 3

RNA blots showing defects of C318A and U43G mutants in systemic infection in N. benthamiana. (A) Accumulation of WT, but not the two mutants, in systemic leaves. (B) Accumulation of the two mutants as well as the WT in inoculated leaves. (C) Accumulation of the two mutants as well as the WT in infected protoplasts. rRNA shows loading control. c-PSTVd, (+)-circular PSTVd; M, mock control.

Figure 4

Mutants C318A and U43G fail to traffic into the vascular tissue phloem at 12 dpi. (A) In situ hybridization shows trafficking of the two mutants as well as the WT in mesophyll. The purple hybridization signals show the presence of viroid RNAs in the nuclei (arrows). There is no hybridization signal in mock-inoculated samples. (B) In situ hybridization shows trafficking of the two mutants as well as the WT into the bundle sheath (BS). While the WT also accumulates in the phloem (Ph), the two mutants are absent from the phloem. Scale bars, 10 μm. (C) RT–PCR detects the presence of WT PSTVd (lanes 1 and 2), but absence of mutants C318A (lanes 3 and 4) and U43G (lanes 5 and 6), in petioles of inoculated leaves. Amplification of 18S rRNA serves as an internal control. (D) Hybridization with a PSTVd-specific probe confirms identity of the RT–PCR products as PSTVd.

Figure 5

Local distortion of an RNA helix due to the presence of a water-inserted cWW U/C base pair. The X-ray crystal structure of an RNA helix containing a U/C water-inserted base pair (nucleotides 763:765 and 899:901 from PDB File: 1s72) is superimposed on a helix that contains matching cWW G/C flanking base pairs and cWW A/U in place of U/C (Nucleotides 444:446 and 36:38 from PDB file: 1s72). (A) A stereo figure showing the U/C-containing helix (orange), with the inserted water molecule shown as an orange sphere, superimposed on the helix 5′-CAG-3′/3′-GUC-5′ (blue; see panel C). (B) A planar view of the superimposition of the central water-inserted cWW U/C base pair (orange) on the cWW A/U base pair (blue). Insertion of the water opens the U/C base pair toward the minor groove, with little distortion of the neighboring base pairs. (C) Nucleotide sequences of the superimposed nucleotides.

Figure 6

Mutational analyses of the trafficking motif. (A) A summary of mutagenesis results, with green and red boxes showing base pairs competent of and defective in systemic trafficking, respectively. Gray stars indicate mutants that do not replicate. WT indicates wild-type U43/C318 motif. (B) RNA blots showing presence or absence of the various mutants in systemic leaves of inoculated N. benthamiana. (C) RNA blots show presence or absence of the various mutants in inoculated N. benthamiana protoplasts. c-PSTVd, circular PSTVd; l-PSTVd, linear monomeric PSTVd. rRNA serves as loading controls.

Figure 7

Hydration patterns for the cWW and water-inserted cWW base pairs. Dark blue and light blue spheres represent high-density and medium-density hydration sites identified on the Solvation Web Service site or by structure search using FR3D. Background colors correspond to the experimentally determined trafficking function.

Figure 8

Covariation analyses of U/C motif in viroids of genus Pospivitoid, based on conservation of the overall secondary structures predicted by mfold. The analyses reveal conservation of U/C pair at the equivalent positions for Tomato planta macho viroid (TPMVd), Tomato chlorotic dwarf viroid (TCDVd), variation C/C for Citrus Exocortic viroid (CEVd), Tomato apical stunt viroid (TASVd) and Chrysanthemum stunt viroid (CSVd), and variation A/A for Mexican papita viroid (MPVd).

Figure 9

Covariation analysis for three conserved U/C base pairs found in 23S bacterial sequences. Nucleotide numbering is based on the E. coli sequence. The background colors indicate trafficking function in PSTVd variants for U43/C318.