Tobacco calmodulin-like protein provides secondary defense by binding to and directing degradation of virus RNA silencing suppressors

Abstract

RNA silencing (RNAi) induced by virus-derived double-stranded RNA (dsRNA), which is in a sense regarded as a pathogen-associated molecular pattern (PAMP) of viruses, is a general plant defense mechanism. To counteract this defense, plant viruses express RNA silencing suppressors (RSSs), many of which bind to dsRNA and attenuate RNAi. We showed that the tobacco calmodulin-like protein, rgs-CaM, counterattacked viral RSSs by binding to their dsRNA-binding domains and sequestering them from inhibiting RNAi. Autophagy-like protein degradation seemed to operate to degrade RSSs with the sacrifice of rgs-CaM. These RSSs could thus be regarded as secondary viral PAMPs. This study uncovered a unique defense system in which an rgs-CaM-mediated countermeasure against viral RSSs enhanced host antiviral RNAi in tobacco.

Fig. 1.

Interaction of rgs-CaM with viral RSSs. (A) Purified RSS proteins were tested for binding to immobilized rgs-CaM protein and siRNA by SPR analysis. Plus sign, interaction detected; minus sign, no interaction detected. (Raw data are in Fig. S1A.) (B) CMV-R2b, -A2b, or -HL2b (at 150, 75, and 37.5 μg/mL) fused with maltose-binding protein (MBP) or MBP alone was tested for binding to immobilized rgs-CaM by SPR. (C) Interactions between transiently expressed Flag–rgs-CaM and Y2b, R2b, or A2b were detected in the proximity ligation assay (PLA) as fluorescent signals in BY2 cells. Each of these proteins was individually detected by single recognition PLA with anti-Flag (Flag–rgs-CaM) and anti-2b (Y2b) antibodies (Left). In cells expressing Flag-rgs-CaM and Y2b, a PLA signal means interactions between the two (Right, PLA). Weaker PLA signals (white arrowheads) were also detected for rgs-CaM combined with other 2bs (R2b and A2b). Hoechst 33342-stained nuclei (Nuclei) and merged images (Merged) are also shown. (Scale bar, 10 μm.) (D) Flag-tagged rgs-CaM (Flag–rgs-CaM) and its associated proteins were precipitated by anti-Flag antibody in crude extracts from tobacco infected with CMV/Y2b and either the PVX vector expressing Flag–rgs-CaM or the empty vector at 16 d postinoculation (dpi). Total (T) and precipitated proteins (IP) were fractionated by SDS/PAGE, and rgs-CaM and 2b were detected using specific antibodies. The 2b band in the IP fraction with PVX/Flag–rgs-CaM (arrowhead) indicates rgs-CaM interaction with 2b.

Fig. 2.

Effects of rgs-CaM–RSS interactions on the activity and stability of RSS proteins. (A–C) RSS activity of TAV 2b (A), CMV Y2b (B), and TuMV P1/HC-Pro (C) were measured by a dual luciferase assay in protoplasts of N. benthamiana. When endogenous rgs-CaM was silenced by a dsRNA cognate to rgs-CaM mRNA, RSS activities of all RSS proteins increased significantly, and even in the absence of RSS, RNAi activity was reduced (A; *P < 0.01, paired Student t test). Firefly and Renilla luciferase mRNAs (mFLuc and mRLuc, Middle) were also detected in RNA extracts from the same samples by Northern blotting (A, Middle), and results were consistent with the luciferase assay (A, Upper graph). The accumulation of endogenous rgs-CaM mRNA was monitored by real-time PCR to confirm its silencing (A, Lower graph). The x axis is the same as in Upper graph. Bars indicate SD. (D and E) CMV Y2b (D) or ClYVV P1/HC-Pro (E) and green fluorescent protein (GFP) were transiently expressed by agroinfiltration in transgenic T0 tobacco plants that either overexpressed rgs-CaM [rgs-CaM(+)] or silenced endogenous rgs-CaM [rgs-CaM(−)], and in nontransgenic plants (WT). Accumulated CMV 2b, ClYVV HC-Pro, GFP, and rgs-CaM proteins were detected by Western blotting. Arrowhead marks the 2b protein. Below each panel set, a Coomassie brilliant blue (CBB)-stained gel is shown as a loading control. Accumulation of the RSS proteins relative to GFP is shown in bar graphs in Fig. S4. (F) The same experiment shown in E was carried out using line 16 of T1 transgenic plants expressing rgs-CaM [rgs-CaM(+)16]. HC-Pro, GFP, and rgs-CaM mRNAs (mHC-Pro, mGFP, and mrgs-CaM) were additionally detected in total RNA extracts by Northern blotting. Lane control (Cont): Extracts from nontransgenic plant. Ribosomal RNA (rRNA) stained with ethidium bromide is shown as a loading control. We here note that the GFP mRNA levels were increased by the coinfiltrated HC-Pro in WT.

Fig. 3.

Control of rgs-CaM and RSS expression by proteolytic activities. (A) Western blots of transgenic BY tobacco leaves expressing ClYVV HC-Pro or CMV R2b and of nontransgenic tobacco leaves after 16-h treatment with inhibitors of host proteolytic pathways [20 μM clastolactacystin–lactone (Sigma-Aldrich) and 40 μM MG132 (MG132; Sigma-Aldrich) to inhibit 26S proteasome, 5 mM 3-methyladenine (3-MA; Sigma-Aldrich) to block autophagy or DMSO as control]; RSSs, α-tubulin and endogenous rgs-CaM were detected using urea as described in SI Materials and Methods and Fig. S8. Their mRNAs (mHC-Pro, mR2b, and mrgs-CaM) were also detected by Northern blotting of RNAs extracted from the same leaves. CBB-stained and ethidium-bromide-stained gels are shown as loading controls. (B–D) After treatment of BY2 cultured cells with inhibitors of host proteolytic pathways [20 μM MG132 to inhibit 26S proteasomes, 5 mM 3-MA and 10 μM E64 (Sigma-Aldrich) to block autophagy or DMSO as control] overnight, wild type (B) and Y2b-expressing (B–D) BY2 cultured cells were stained with LysoTracker (Lys; Life Technologies) and DAPI. Endogenous rgs-CaM (B and C), CMV Y2b (B and D), and α- and β-tubulin (tubulin) (B) were detected using specific antibodies. Differential interference contrast (DIC) images are also shown. (Scale bar, 10 μm.) (E) Western blots of inoculated leaves of tobacco infected with the PVX vector expressing antisense of beclin1 (PVX/asbeclin1) or not (PVX/empty) detected rgs-CaM, PVX CP, and α-tubulin at 14 dpi. The CBB stained gel was shown as loading control. The rgs-CaM and beclin1 mRNA levels were also investigated by real-time PCR. The relative accumulation of these mRNA to 18S ribosomal RNA was shown with bars of SD.

Fig. 4.

Resistance and response to CMV-Y infection in tobacco plants and schematic model of antiviral defense in tobacco. (A and B) T1 transgenic tobacco plants overexpressing rgs-CaM [rgs-CaM(+)16] or with knocked down rgs-CaM [rgs-CaM(−)1] and nontransgenic (WT) tobacco plants were inoculated with CMV-Y. Capsid (CP) and Y2b proteins, viral genome RNAs (gCMV), and pathogenesis-related protein 1a and rgs-CaM mRNAs (mPR1a, mrgs-CaM) were detected by Western and Northern blotting as in Fig. 2F. Lane H: healthy WT plants. (C) Presently accepted model of the viral counterdefense against the host’s RNAi-based defense. The viral RNA genome is configured into intermolecular and/or intramolecular dsRNA, which induces antiviral RNAi in the plants. To counteract this host defense, the virus expresses RSSs, many of which can bind to siRNA and/or dsRNA. Viral RSSs, in turn, may sequester viral siRNAs and long dsRNAs from inducing RNAi and eventually enhance viral fitness in the host. (D) The proposed model of the tobacco countermeasure against viral RSSs is depicted, as uncovered in the present study. Tobacco expresses the calmodulin-like protein rgs-CaM, which has an affinity for positively charged siRNA/dsRNA-binding surfaces of viral RSSs. The rgs-CaM prevents RSS from binding to dsRNAs/siRNAs and affects RSS protein stability by autophagy, resulting in a more potent RNAi defense against viral infection.