Fig 1.
Chromatography fractions isolated from TSWV-infected plant contain RISC associated nuclease activity that specifically cleave S RNA in vitro.
(A) Schematical diagram of the tospoviral tripartite RNA genome, encoding five open reading frames. Genomic RNA (gRNA) molecules are tightly associated with N protein (in green). Viral transcripts of the five ORFs are aligned below the gRNA strands. Those for the RdRP, Gn/Gc and N (in grey) result from primary transcription of the gRNA, whereas the additional NSm and NSs transcripts from the ambisense M- and S RNA (in orange), respectively, are produced by secondary transcription, i.e. after gRNA has been copied into a complementary strand that serves as a template for secondary transcription (and replication of progeny gRNA). Small dots at the 5’ end of the viral mRNAs present heterogenous, host derived, capped-RNA leader sequence resulting from cap-snatching. Minus sign (+) represents the vc strand of genomic RNA. Minus sign (−) and 3′ to 5′ designation represent the v strand of genomic RNA. (B) Diagram of antiviral RISC isolation and in vitro cleavage assay for RISC associated nuclease activity. Leaves were harvested from TSWV-infected or mock-treated N. benthamiana plant (~40 g). The extract of leaves was loaded onto Hydroxyapatite column followed by Superdex S-200 HR gel column. Sixteen Superdex S-200 fractions were finally collected and every fraction (600 μL) was incubated with in vitro transcribed DIG-labelled RNA for the activity of RISC associated nuclease. (C) DIG-labelled full length S genomic RNA was incubated with isolated chromatography fraction 6 (S1A Fig) from TSWV-infected N. benthamiana plants (Lane 1–3) or with chromatography fraction 6 (S1B Fig) from mock-inoculated control N. benthamiana plants (Lane 4–6). As a negative control, DIG-labelled full length GFP RNA was incubated with isolated chromatography fraction 6 from TSWV-infected N. benthamiana plants (Lane 7–9).
Fig 2.
TSWV RNPs protect viral genomic RNA by the cleavage of host antiviral RISC in vitro.
(A) The carton crystal structure showing trimer of TSWV N:RNA complexes. Protein database bank No. 5ip2 of TSWV N was used to display the structure by PyMol. Chains A, B and C in the trimeric nucleocapsid protein (NP) structure are colored in green, cyan and purple, respectively. (B) The crystal structure of TSWV N-RNA complexes showing the embedding of viral RNA in the cleft of TSWV N protein. (C) In vitro cleavage assay of TSWV genomic S RNA complexed with N protein by isolated chromatography fractions from TSWV infected N. benthamiana plants. DIG-labelled full length genomic S RNA was incubated with TSWV N protein to form N-RNA complexes. All 16 fractions tested in S1A Fig were mixed with N-RNA complexes for the RNA protection assay. (D) In vitro cleavage assay of naked genomic M RNA by the fraction containing RISC-activity. DIG-labelled full length M genomic RNA was incubated with fraction 6 (S1A Fig) containing the RISC-activity. (E) In vitro cleavage assay of TSWV genomic M RNA complexed with N protein by fraction 6 containing antiviral RISC-activity. DIG-labelled full length M genomic RNA was incubated with TSWV N protein to form N-RNA complexes. The RISC fraction was then added into N-RNA complexes to test the RNA protection activity. The signals on the blot were detected by AP-labeled anti-digoxigenin antibodies and followed with BCIP/NBT staining.
Fig 3.
TRV-induced RISC mainly targets viral mRNA but not genomic RNA of TSWV S segment in planta.
(A) The TRV-NSs, TRV-N and TRV-GUS constructs were used to infect N. benthamiana plants by agro-infiltration, respectively. At 10 days post agroinfitration, the leaves of TRV-NSs, TRV-N and TRV-GUS pre-infected plants were rub inoculated with sap of TSWV from freshly-infected leaves. The phenotype of TSWV infection was monitored and photographed at 9 days post TSWV inoculation. The white arrow indicates the typical TSWV symptoms in systemically-infected leaves of N. benthamiana plants. The numbers showing in the image are the total number of the inoculated plants versus the infected plants observed from the treatments. (B) and (C) Detection of TSWV accumulation in systemically-infected leaves of TRV-NSs, TRV-N and TRV-GUS pre-infected N. benthamiana plant by western blot and qRT-PCR analysis, respectively. TSWV N-specific antibody and TSWV-N specific primers were used to detect accumulation of TSWV N protein and viral RNAs, respectively. The Ponceau S stained gel was used to show the sample loadings. Error bars represent SD (n = 3). (D) Northern blot analysis of genomic vRNA, vcRNA and NSs mRNA of TSWV S segment targeted by pre-assembled RISC from TRV-GUS and TRV-NSs in N. benthamiana plants using strand specific DIG-labelled NSs probe. Samples were collected at 3 dpi from TSWV inoculated leaves of TRV-GUS and TRV-NSs pre-infected plants. Genomic vRNA and viral NSs mRNA bands are indicated by the arrows in green and red, respectively. Ethidium bromide staining was used to show RNA loading. (E) and (F) Quantification of the accumulation level of genomic vRNA and NSs mRNA, respectively, shown in Fig 3D. (G) Northern blot analysis of genomic vcRNA and N mRNA of TSWV S segment targeted by pre-assembled RISC from TRV-GUS and TRV-N in N. benthamiana plants using a strand-specific DIG-labeled N probe. Samples were collected at 3 dpi from TSWV inoculated leaves of TRV-GUS and TRV-N pre-infected plants. Genomic vcRNA and N mRNA bands are indicated by the arrows in green and red, respectively. Ethidium bromide staining was used to show RNA loading. (H) and (I) Quantification of the accumulation level of genomic vcRNA and N mRNA, respectively, shown in Fig 3G. All experiments were repeated more than three times.
Fig 4.
TRV-induced RISC mainly targets viral mRNA but not genomic RNA of TSWV M segment in planta.
(A) The TRV-GUS, TRV-NSm and TRV-Gn constructs were used to infect N. benthamiana plants through agro-infiltration, respectively. At 10 days post agroinfitration, the leaves of TRV-GUS, TRV-NSm and TRV-Gn pre-infected plants were challenged with TSWV inoculum. The phenotype of N. benthamiana plants was monitored and photographed at 9 days post TSWV inoculation. The white arrow indicates the typical TSWV symptoms in systemically-infected leaves of TRV-GUS and TRV-Gn pre-infected N. benthamiana plants. The numbers showing in the image are the total number of the inoculated plants verses the infected plants observed from the different treatments. (B) and (C) Detection of TSWV accumulation in systemically-infected leaves of TRV-NSm, TRV-Gn and TRV-GUS pre-infected N. benthamiana plant by western blot and qRT-PCR analysis, respectively. TSWV N-specific antibody and TSWV-N specific primers were used to detect accumulation of TSWV N protein and viral RNAs, respectively. The Ponceau S stained gel was used to show the protein loading for samples. Error bars represent SD (n = 3). (D) Northern blot analysis of genomic vRNA and NSm mRNA of TSWV M segment targeted by pre-assembled RISC from TRV-GUS and TRV-NSm in N. benthamiana plants using a strand-specific DIG-labelled NSm probe. Samples were collected at 3 dpi from TSWV inoculated leaves of TRV-GUS and TRV-NSm treated plants. Genomic vRNA and viral NSm mRNA bands are indicated by the arrows in green and red, respectively. Ethidium bromide staining was used to show RNA loading. (E) and (F) Quantification of the accumulation level of genomic vRNA and NSm mRNA, respectively, shown in Fig 4D. (G) Northern blot analysis of genomic vcRNA and GP mRNA of TSWV M segment targeted by pre-assembled RISC from TRV-GUS and TRV-Gn in N. benthamiana plants using a strand-specific DIG-labelled Gn antisense probe. Samples were collected at 3 dpi from TSWV inoculated leaves of TRV-GUS and TRV-Gn pre-infected plants. Genomic vcRNA and GP mRNA bands are indicated by the arrows in green and red, respectively. Ethidium bromide staining was used to show RNA loading. (H) and (I) Quantification of the expression level of genomic vcRNA and GP mRNA, respectively, shown in Fig 4G. All experiments were repeated more than three times.
Fig 5.
RISC fraction from TZSV-infected plant targets naked genomic RNA but not the genomic RNA complexed with N proteins in vitro.
(A) and (C) In intro cleavage assay of naked genomic RNA of TZSV S and M segment by isolated RISC chromatography fractions. Chromatography fraction 5 (S6 Fig) from TZSV-infected N. benthamiana were incubated with DIG-labeled in vitro full-length RNA transcripts of TZSV S (100 ng; A) and M (100 ng; B) segment, respectively. (B) and (D) In vitro cleavage assay of TZSV genomic S or M RNA complexed with N protein by the RISC fraction. DIG-labelled full-length S (B) or M (D) genomic RNA was incubated with TZSV N protein to form N-RNA complexes. RISC fraction 5 isolated from TZSV-infected plants was added to N-S RNA or N-M RNA complexes. The blots were developed by AP-labeled anti-digoxigenin antibodies and followed with BCIP/NBT staining.
Fig 6.
TRV-induced RISC mainly targets viral mRNA but not genomic RNA of TZSV in planta.
(A) TRV-N, TRV-NSs, TRV-NSm, TRV-Gn, TRV-RdRp and TRV-GUS were used to pre-infect N. benthamiana plants via agro-infiltration, respectively. At 10 days post agroinfitration, the infiltrated leaves of TRV pre-infected plants were challenged with TZSV inoculum. The phenotype of N. benthamiana plants challenged by TZSV was monitored and photographed at 9 days post TZSV inoculation. The white arrow indicates the typical TZSV symptoms in systemically-infected leaves of TRV-NSs, TRV-Gn and TRV-GUS pre-infected N. benthamiana plants. The numbers showing in the image are the total number of the inoculated plants versus the infected plants observed from the various treatments. (B) and (C) Detection of TZSV accumulation in systemically-infected leaves of TRV-N, TRV-NSs, TRV-NSm, TRV-GP, TRV-RdRp and TRV-GUS pre-infected N. benthamiana plant by western blot and qRT-PCR analysis, respectively. TZSV N-specific antibody was used to detect accumulation of TSWV N protein on western blot. The Ponceau S stained gel shows the equal protein loading of samples. TZSV-N specific primers were used to detect viral RNAs by qRT-PCR. Error bars represent SD (n = 3). (D) Detection of genomic vcRNA and N mRNA of TZSV S segment targeted by antiviral RISC from TRV-N, TRV-NSs and TRV-GUS pre-infected plant by northern blot. Samples were collected at 3 dpi from the TZSV inoculated leaves of TRV pre-infected plants. Genomic vcRNA and viral mRNA bands are indicated by the arrows in green and red, respectively. Ethidium bromide staining was used to show equal RNA loading. (E) and (F) Quantification of the expression level of genomic vcRNA and N mRNA, respectively, shown in Fig 6D. (G) Northern blot analysis of viral genomic vRNA and NSs mRNA of TZSV S segment targeted by pre-assembled RISC from TRV-GUS and TRV-NSs in N. benthamiana plants using strand specific DIG-labelled NSs probe. Samples were collected at 3 dpi from TZSV inoculated leaves of TRV-GUS and TRV-NSs pre-infected plants. Genomic vRNA and viral NSs mRNA bands are indicated by the arrows in green and red, respectively. Ethidium bromide staining was used to show equal RNA loading. (H) and (I) Quantification of the accumulation level of viral genomic vRNA and NSs mRNA, respectively, shown in Fig 6G.
Fig 7.
TRV-induced RISC targets genomic RNA and subgenomic mRNA of CMV in planta.
(A) TRV-2a, TRV-2b and TRV-GUS constructs were used to pre-infect N. benthamiana plants by agro-infiltration, respectively. At 10 days post agroinfitration, the leaves of TRV-2a, TRV-2b and TRV-GUS pre-infected plants were rub inoculated with sap of CMV-Fny from freshly-infected leaf tissues. The phenotype of N. benthamiana plants was photographed at 12 days post CMV inoculation. The white arrow indicates the typical CMV symptoms in systemically-infected leaves of TRV-GUS pre-infected N. benthamiana plants. The numbers showing in the image are the total number of the inoculated plants versus the infected plants observed from the various treatments. (B) and (C) Detection of CMV accumulation in systemically-infected leaves of TRV-2a, TRV-2b and TRV-GUS pre-infected N. benthamiana plants by western blot and qRT-PCR analysis, respectively. CMV-CP specific antibody was used to detect the accumulation of CMV on western blot. The Ponceau S stained gel was used to show the equal protein loading for samples. CMV CP specific primers were used to detect viral RNAs in qRT-PCR. Error bars represent SD (n = 3). (D) Northern blot analysis of genomic(g) RNA2 targeted by pre-assembled RISC from TRV-2a in N. benthamiana plants using a strand specific DIG-labeled CMV 2a probe. Samples were collected at 3 dpi from the CMV inoculated leaves of TRV-GUS and TRV-2a pre-infected plants. The band of genomic viral (v) strand RNA is indicated by the arrow in green. Ethidium bromide staining was used to show RNA loading. (E) Quantification of the expression level of genomic vRNA in Fig 7D. (F) Northern blot analysis of genomic vRNA2 and subgenomic (sg) RNA of CMV targeted by pre-assembled antiviral RISC from TRV-2b in N. benthamiana plants using strand-specific DIG-labeled CMV 2b probe. Samples were collected at 5 dpi from the CMV inoculated leaves of TRV-GUS and TRV-2b pre-infected plant. Ethidium bromide staining was used to check and show equal loading of RNA. A 190 bp fragment (143–333 nt) from the C-terminal region of 2b was used for the construction of TRV-2b and a 170 bp fragment (164–333 nt) from the C-terminal region of 2b was used as template to prepare the DIG-labeled probe. The red asterisk indicates the band of TRV RNA2 from TRV-2b infected plants detected by the strand-specific DIG-labeled CMV 2b probe. (G) and (H) Quantification of the expression level of genomic vRNA2 and sgRNA for 2b, respectively, shown in Fig 7F.
Fig 8.
Model on antiviral RISC targeting of tospoviruses, a representative segmented plant NSVs: antiviral RISC mainly targets viral mRNA but not genomic RNA.
Upon tospovirus infection of plants, host antiviral RISC effector complexes become activated. The viral (anti)genomic RNA of tospoviruses associates with N protein and assembles into RNP complexes that are resistant to host antiviral RISC. Viral mRNAs do not assemble into RNPs and are fully accessible for vsiRNA-mediated degradation by the Argonaute slicer activity of RISC.