Exploring the Multifunctional Roles of Odontoglossum Ringspot Virus P126 in Facilitating Cymbidium Mosaic Virus Cell-to-Cell Movement during Mixed Infection

Abstract

Synergistic interactions among viruses, hosts and/or transmission vectors during mixed infection can alter viral titers, symptom severity or host range. Viral suppressors of RNA silencing (VSRs) are considered one of such factors contributing to synergistic responses. Odontoglossum ringspot virus (ORSV) and cymbidium mosaic virus (CymMV), which are two of the most significant orchid viruses, exhibit synergistic symptom intensification in Phalaenopsis orchids with unilaterally enhanced CymMV movement by ORSV. In order to reveal the underlying mechanisms, we generated infectious cDNA clones of ORSV and CymMV isolated from Phalaenopsis that exerted similar unilateral synergism in both Phalaenopsis orchid and Nicotiana benthamiana. Moreover, we show that the ORSV replicase P126 is a VSR. Mutagenesis analysis revealed that mutation of the methionine in the carboxyl terminus of ORSV P126 abolished ORSV replication even though some P126 mutants preserved VSR activity, indicating that the VSR function of P126 alone is not sufficient for viral replication. Thus, P126 functions in both ORSV replication and as a VSR. Furthermore, P126 expression enhanced cell-to-cell movement and viral titers of CymMV in infected Phalaenopsis flowers and N. benthamiana leaves. Taking together, both the VSR and protein function of P126 might be prerequisites for unilaterally enhancing CymMV cell-to-cell movement by ORSV.

Keywords: Phalaenopsis; RNA silencing; cymbidium mosaic virus (CymMV); odontoglossum ringspot virus (ORSV); viral suppressor of RNA silencing (VSR); viral synergism.

Figure 1

Cy1 and OS4 infectivity and synergism in inoculated N. benthamiana plants. (A) Symptoms and synergistic effects upon co-infection with Cy1 and OS4 virions. Eighteen-day-old N. benthamiana plants were individually inoculated with 0.5 μg/leaf of Cy1 or OS4 virions or co-inoculated with 1 μg/leaf of Cy1 and OS4 (0.5 μg/leaf each). Symptoms on inoculated leaves (IL), systemic leaves (SL) and whole plants were photographed at 14 DPI. (B) Immuno-detection of Cy1 and OS4 CPs from leaf extracts of inoculated plants at 14 DPI using antibodies against the CP of CymMV or ORSV. Actin was detected by using anti-Actin monoclonal antibody as a protein loading control. (C) RNA blots for assessing Cy1 and OS4 viral RNA accumulation. Total RNAs were extracted from leaves as in (B). RNA was detected by using DIG-labeled probes against the CP and 3′ UTRs of Cy1 or OS4, respectively.

Figure 2

VSR assays of ORSV-encoded proteins and protein domains. (A) Schematics of expression cassettes for GFP (reporter), TBSV P19 and PVX P25 (VSR positive controls), as well as tested ORSV ORFs. (B) Individual ORFs and domains used for VSR assay. (C) Quantitative analysis of VSR activities. Overnight cultures of Agrobacterium carrying a corresponding plasmid were adjusted to final OD = 1. The co-infiltration of N. benthamiana leaves was performed by mixing Agrobacterium carrying pBIN61-GFP and Agrobacterium carrying plasmid positive for VSR or tested viral ORFs in a ratio of 1:1. GFP signals were detected and quantified using the IVIS Lumina III LT in vivo Imaging System at 4 DPA. Relative GFP intensity represents the mean of three independent experiments and was normalized to vector control. The significance was calculated by one-way ANOVA followed by post-hoc test and marked when p < 0.001 (***); ns: no significant difference. (D) Representative images of GFP signals as observed under hand-held UV lamp. (E) Accumulation of HA-tagged P126, GFP and actin in (D) was assessed by Western blotting by using antibodies against HA, GFP and actin, respectively.

Figure 3

VSR activity of single amino acid substitution mutants of ORSV P126. (A) GFP signals upon co-expression with vector control (Vec), P19, P126 and derived P126 mutants. (B) Total protein was extracted from the infiltrated leaf discs and then subjected to Western blot in order to detect GFP, actin, P126 and mutant variants. (C) Relative GFP protein level was normalized with actin. The GFP level upon co-expression with P126 was set as 1. Vec: vector control. Data represent results from three different experiments. Significant differences are marked as follows: *: p < 0.05; **: p < 0.01; **** p < 0.0001 (one-way ANOVA followed by a post-hoc test).

Figure 4

Replication of OS4 and its P126 mutants. (A) Symptoms of C. quinoa leaves inoculated with transcripts of OS4 and P126 mutants at 5 DPI. Mock represents leaves inoculated with phosphate buffer, which were used as a control. (B) Accumulation of ORSV CP in (A) as assessed by Western blot. Actin acted as a loading control. (C,D) Accumulations ORSV CP and viral RNAs in N. benthamiana at 2 DPA (C) and 8 DPA (D), respectively. Agrobacterium carrying pkn, pkOS4, pkOS4-P126-R, P126-S and P126-A mutants were adjusted to OD₆₀₀ = 1 and infiltrated into 17-day-old N. benthamiana leaves. Western blot detection of ORSV CP (upper two panels) and RNA blot for ORSV RNAs (lower two panels) were performed as described in Figure 1. Total RNA was used as RNA loading control.

Figure 5

CymMV cell-to-cell movement and accumulation upon overexpressing VSRs in N. benthamiana leaves. (A) Schematic construct of pkCy1GFP::mCherry. (B) Flowchart for protein expression and CymMV inoculation. Agrobacterium carrying pBIN61 (Vec), pBIN61-P126 (P126), pBIN19-P19 (P19) or pBIN61-P25 (P25) were adjusted to OD₆₀₀ = 0.5 and then infiltrated into 17-day-old N. benthamiana leaves. After 2 hours post-agroinfiltration, the Agrobacterium ooze carrying pkCy1GFP::mCherry was collected on a toothpick and pinpricked onto the infiltrated leaves. (C) Representative images of Agrobacterium primary infection sites (mCherry in red) and Cy1GFP spreading infected cells (GFP in green) using the IVIS Lumina III LT in vivo Imaging System at 6 DPA. Images were processed in ImageJ. (D) Quantification of CymMV movement. According to (C), the ratio of coverage areas was measured by the expanded Cy1GFP signals (GFP) against primary infection site (mCherry). (E) Effects of VSRs on accumulation of Cy1GFP. The relative Cy1GFP signals were measured by the total GFP signals per infection foci and quantification was normalized to vector control (black star) or P126 (red star). Data represent three experimental replicates, each of which encompassed analysis of six infection foci. Significant differences are marked as follows: **: p < 0.01 and ****: p < 0.0001 (one-way ANOVA followed by post-hoc test).