Virp1 is a host protein with a major role in Potato spindle tuber viroid infection in Nicotiana plants

Abstract

Viroids are small, circular, single-stranded RNA molecules that, while not coding for any protein, cause several plant diseases. Viroids rely for their infectious cycle on host proteins, most of which are likely to be involved in endogenous RNA-mediated phenomena. Therefore, characterization of host factors interacting with the viroid may contribute to the elucidation of RNA-related pathways of the hosts. Potato spindle tuber viroid (PSTVd) infects several members of the Solanaceae family. In an RNA ligand screening we have previously isolated the tomato protein Virp1 by its ability to specifically interact with PSTVd positive-strand RNA. Virp1 is a bromodomain-containing protein with an atypical RNA binding domain and a nuclear localization signal. Here we investigate the role of Virp1 in the viroid infection cycle by the use of transgenic lines of Nicotiana tabacum and Nicotiana benthamiana that either overexpress the tomato Virp1 RNA or suppress the orthologous Nicotiana genes through RNA silencing. Plants of the Virp1-suppressed lines were not infected by PSTVd or Citrus exocortis viroid through mechanical inoculation, indicating a major role of Virp1 in viroid infection. On the other hand, overexpression of tomato Virp1 in N. tabacum and N. benthamiana plants did not affect PSTVd KF 440-2 infectivity or symptomatology in these species. Transfection experiments with isolated protoplasts revealed that Virp1-suppressed cells were unable to sustain viroid replication, suggesting that resistance to viroid infection in Virp1-suppressed plants is likely the result of cell-autonomous events.

FIG. 1.

(A and B) Schematic representation of the tomato Virp1 protein (A, top); the location of the primers used for the RT-PCR (A, bottom); and the constructs used (B) for overexpression of Virp1, GFP-Virp1 fusion, FLAG-NbVirp1, FLAG-NbVirp1Δ, and suppression of Virp1 (from top to bottom, respectively) in N. benthamiana and N. tabacum transgenic plants. (A) (Top) Virp1 contains a nuclear localization signal (amino acids 43 to 48), a bromodomain (amino acids 204 to 276), and a potential RNA binding domain (amino acids 313 to 403). (B) nptII, selection gene; ocs3′, ocs terminator sequence; LB and RB, T-fragment left and right border sequences, respectively; 35S, cauliflower mosaic virus 35S promoter; B, BamHI sites delimiting the bromodomain. The truncated FLAG-NbVirp1Δ contains the first 902 bp of the NbVirp1 sequence. (C) Comparison of PSTVd strains KF 440-2, NT, and NB, showing the relative locations of the sequence differences between these three strains with respect to the loop E (dotted-line boxes) and RY motifs (solid-line boxes). In addition to a C259U substitution in the loop E motif of PSTVd-NT (shown by an oversized letter), strain PSTVd-NB also contains five additional mutations (shown by oversized letters).

FIG. 2.

PSTVd coprecipitates with FLAG-NbVirp1. PSTVd-infected leaf tissue was infiltrated with FLAG-NbVirp1, FLAG-LeVirp1, and FLAG-NbVirp1Δ. An anti-FLAG antibody was then used to immunoprecipitate the tagged proteins, and RT-PCR using PSTVd-specific primers was then performed to detect coprecipitated viroid RNA. Lane 1, noninfiltrated tissue; lane 2, FLAG-LeVirp1; lane 3, FLAG-NbVirp1; lane 4, FLAG-NbVirp1Δ; lane 5, 100-bp DNA ladder.

FIG. 3.

GFP-Virp1 is localized in the nucleus. Leaf area agroinfiltrated with GFP-Virp1 fusion construct. (A and B) GFP fluorescence reveals fusion protein localization in the nucleus (A), unlike unfused GFP, which can be found all over the cell (B). (C and D) Same areas as those in panels A and B, respectively, viewed for DAPI fluorescence. The red background is due to the natural fluorescence of chlorophyll. Bars, 25 μm.

FIG. 4.

Overexpression of Virp1 in N. benthamiana plants. Lane 1, wt; lanes 2 to 5, transgenic lines BX1.2, BX1.7, BX1.10, and BX1.13, respectively. (Top) Virp1-specific DNA probe. (Bottom) Hybridization of the same membrane with an 18S rRNA probe as a loading control.

FIG. 5.

Generation of Virp1-suppressed lines with the use of hairpin constructs. Virp1 mRNA levels are strongly reduced in most N. benthamiana (A) and N. tabacum (B) transgenic lines. (A) (Top) Northern blot analysis of Virp1 mRNA in N. benthamiana. Lane 1, line BX1.13 overexpressing Virp1; lane 2, wt N. benthamiana; lanes 3 to 5, transgenic N. benthamiana in which expression of Virp1 is suppressed (lines ph5.2nb, ph10nb, and ph11nb, respectively). A 5′ region of the Virp1 sequence was used as a probe to avoid detection of the hairpin transcript. (Bottom) Hybridization of the same membrane with an 18S rRNA probe as a loading control. (B) (Top) Northern blot analysis of Virp1 expression levels in transgenic N. tabacum lines. Lane 1, wt N. tabacum; lanes 2 and 3, transgenic N. tabacum in which expression of Virp1 is suppressed (lines ph10nt and ph4nt, respectively). A 5′ region of the Virp1 sequence was used as a probe to avoid detection of the hairpin transcript. (Bottom) Loading control (18S rRNA).

FIG. 6.

Transgenic N. benthamiana and N. tabacum lines suppressed for Virp1 cannot be infected by PSTVd. Detection of viroid infection by Northern blot analysis of RNA extracted from the upper leaves of transgenic lines 6 weeks p.i. Samples were separated by polyacrylamide gel electrophoresis on 6% gels. The probe was negative-strand RNA transcript of PSTVd KF 440-2 strain. (A) Lane 1, in vitro-transcribed PSTVd; lanes 2 and 3, wt N. benthamiana plants infected with 30 and 100 μl of inoculum, respectively; lanes 4 to 14, Virp1-suppressed transgenic lines, two or three plants each; lanes 4 to 6, line ph5.2nb; lanes 7 to 9, line ph10nb; lanes 10 to 12, line ph11nb; lanes 13 and 14, line ph20nb. (Bottom) The same membrane was hybridized with a U6 snRNA riboprobe as a loading control. (B) Lane 1, ph4nt; lane 2, ph10nt; lane 3, wt N. tabacum; lane 4, in vitro-transcribed PSTVd. (Bottom) Loading control (18S rRNA). C, size of circular form of viroid; L, size of linear form of viroid. The same source of inoculum was used for all experiments.

FIG. 7.

CEVd is unable to infect Virp1-suppressed N. benthamiana. (Top) Northern blot analysis of Virp1-suppressed (ph11nb, lanes 1 and 2) and wt (lanes 3 and 4) plants inoculated with CEVd. Total RNA extracted from noninoculated leaves 6 weeks p.i. was separated on a 1.5% agarose gel; a CEVd-specific DNA probe was used. (Bottom) The same membrane was hybridized with a U1 snRNA riboprobe as a loading control. nt, nucleotide.

FIG. 8.

Virp1-suppressed N. tabacum protoplasts are unable to sustain viroid infection. Protoplasts from wt (lanes 1 and 2) and Virp1-suppressed (line ph10nt, lanes 3 and 4) N. tabacum were electroporated with total RNA extracted from the leaves of a tomato plant that had been infected with PSTVd strain PSTVd^Nb. Protoplasts were cultured for 6 days postelectroporation, and total RNA was analyzed by Northern blotting. The probe was negative-strand RNA transcript of PSTVd strain PSTVd^Nb. (Bottom) Loading control (18S rRNA).