Brassica yellows virus P0 protein impairs the antiviral activity of NbRAF2 in Nicotiana benthamiana

Plant material and growth conditions

Wild-type Nicotiana benthamiana and transgenic lines with the ferredoxin NADP(H) oxidoreductase transit peptide fused to enhanced green fluorescent protein (FNR–EGFP) (Schattat et al., 2011) were grown at 24 °C with a 16-h light/8-h dark cycle.

Plasmid constructs

All of the primers used in this study are listed in Supplementary Table S1 at JXB online.

The vectors pGD and pGDG (Goodin et al., 2002) were used for transient expression. The P38 protein encoded by Turnip crinkle virus was cloned into pGD for transient expression. P0^BrA was cloned into to pGD–3Flag, a modified version of vector pGD that has a C-terminal-fused 3×Flag tag. For co-immunoprecipitation (Co-IP) and confocal microscopy, pGD–3G-mCherry was constructed. A DNA fragment of GGG-mCherry was amplified and cloned into the vector pGD to produce pGD–3G-mCherry. Full-length AtRAF2, NbRAF2, and their mutants were independently cloned into the vector pGDGm [a modified version of pGD that allows the production of a C-terminal green fluorescent protein (GFP)-fused protein] and pGD–3G-mCherry. NbSKP1 was cloned into the vector pGDGm. For its subcellular localization, P0^BrA was amplified and cloned into pSuper1300–GFP (Yang et al., 2010). Wild-type BrYV full-length cDNAs were cloned into the pCB301-2 × 35S-MCS-HDV_RZ-NOS vector (Yao et al., 2011; Zhang et al., 2015b).

Agrobacterium-mediated transient expression in N. benthamiana

Plasmids were transformed into the Agrobacterium tumefaciens strain EHA105 or C58CI using the freeze–thaw method (Holsters et al., 1978). The recombinant EHA105 or C58CI was grown overnight, resuspended in infiltration buffer (10 mM MgCl₂, 10 mM MES, and 100 μM acetosyringone), and incubated at room temperature for at least 3 h before infiltration. The A. tumefaciens cultures were infiltrated into N. benthamiana leaves and the infiltrated leaves were detached for the corresponding assays.

Yeast two-hybrid (Y2H) screen and interaction assays

For the Y2H assay, the Clontech Matchmaker GAL4 Two-Hybrid System 3 (Clontech, Mountain View, CA, USA) was used. Protein-interacting screens were performed with the Matchmaker GAL4 two-hybrid system according to the manufacturer’s protocol. The full-length of P0^BrA was cloned into pGDBKT7 containing a binding domain (BD) to generate BD–P0^BrA and then transformed into the yeast host strain Y187. The Arabidopsis cDNA library was used to screen P0^BrA-binding proteins. The full-length AtRAF2 was cloned into pGDADT7 containing an activating domain (AD) to generate AD–AtRAF2 and then transformed into the yeast host strain AH109. Co-transformants were plated onto synthetic dropout (SD) media lacking Trp and Leu (SD/−WL) and SD media lacking Ade, His, Trp, and Leu (SD/−AHLW). Full-length NbRAF2 (GenBank accession MG560271), AtRAF2 (GenBank accession AED96036.1), NbRbcL, P0^MA, and P0^Sc were independently cloned into pGDBKT7. Full-length NbRAF2, truncated NbRAF2 mutants, AtRAF2 and AtRAF2 mutants were independently cloned into pGDADT7. Protein interactions were tested using the yeast mating assay. The interaction of NbRAF2 with P0^BrA was identified using the split ubiquitin membrane-bound yeast two-hybrid (MbY2H) system. NbRAF2 was cloned into the MbY2H bait vector pBT3-STE, and P0^BrA was cloned into the prey vector pPR3-N. All combinations were transformed to the yeast strain NMY51 for mating and the transformed yeast cells were then transferred to SD/−WL and SD/−AHLW plates for 3–5 d.

Protein extraction and western blots

Total proteins were extracted from infiltrated lamina of N. benthamiana leaves using 2× sodium dodecyl sulfate (SDS) sample buffer [100 mM Tris (pH 6.8), 4% (w/v) SDS, 20% (v/v) glycerol, and 0.2% (w/v) bromophenol blue]. Proteins were separated with SDS polyacrylamide gel electrophoresis. Western blots were performed with the primary anti-Flag antibody (Sigma-Aldrich) diluted at 1:1000, anti-NbRAF2 antibody diluted at 1:500, anti-GFP antibody diluted at 1:3000, anti-H3 antibody (Sigma-Aldrich) diluted at 1:3000, anti-PEPC antibody diluted at 1:3000, or anti-BrYV-A coat protein (CP) antibody diluted at 1:500, and then incubated with anti-rabbit alkaline phosphatase (Sigma-Aldrich) or anti-rabbit goat HRP secondary antibody (1:3000; Bio-Rad, Hercules, CA, USA). Finally, the membrane was detected with an enhanced chemiluminescence detection kit (GE Healthcare, Buckinghamshire, UK) according to the manufacturer’s instructions.

Co-IP assay

The Co-IP assays were performed as previously reported (Win et al., 2011) with minor modifications. Total proteins were extracted from N. benthamiana leaf tissues that were ground in liquid nitrogen and homogenized in 2 ml g⁻¹ extraction buffer [10% (v/v) glycerol, 25 mM Tris-HCl (pH 7.5), 1 mM EDTA, 150 mM NaCl, 2% (w/v) PVPP, 10 mM DTT, 1× protease inhibitor cocktail (Sigma-Aldrich), and 0.1% (v/v) Triton X-100 (Sigma-Aldrich)]. After centrifugation at 3000 g for 10 min at 4 °C and filtration with a 0.45-mm filter, the clarified lysate was incubated with 4% BSA-pre-blocked anti-Flag M2 agarose beads (Sigma-Aldrich) for 3 h, and the complex was washed three times with IP buffer [10% (v/v) glycerol, 25 mM Tris-HCl (pH 7.5), 1 mM EDTA, 150 mM NaCl, and 0.1% (v/v) Triton X-100]. The immunoprecipitates were denatured and subjected to immunoblotting using corresponding antibodies.

RNA extraction and RT-PCR

Total RNA was extracted using TRIzol Reagent (Invitrogen) according to the manufacturer’s protocol. A 4-μg of total RNA was treated with RNase-free DNase I (TaKaRa). First-strand cDNA was synthesized using 2 μg of treated RNA, oligo d(T) primer, or gene-specific primer and M-MLV Reverse Transcriptase (Promega), as instructed by the protocol. The mRNAs of EF1A, NbRAF2, and BrYV-A RNA were determined using specific primers (see Supplementary Table S1).

Fluorescence microscopy

Agro-infiltrated leaf tissue from N. benthamiana was detached 2 d post infiltration (dpi), and confocal laser scanning microscopy was performed using a Leica SP8 laser-scanning microscope. GFP, mCherry, and chloroplast fluorescence were obtained using laser excitation at 488, 552, and 638 nm, respectively. For bimolecular fluorescence complementation (BiFC) assays, NbRAF2 and AtRAF2 were respectively cloned into pSPYNE–35S and pSPYCE–35S (provided by J. Kudla, Universität Münster, Germany). Yellow fluorescent protein (YFP) fluorescence was obtained using 488-nm laser excitation. To determine its distribution, the nuclear-localized NbRAF2 was quantified from Z stacks and expressed as the total number of nuclear-localized NbRAF2 particles per total number of nuclei.

VIGS assay

For the VIGS assay (Liu et al., 2002a), a 360-bp fragment of NbRAF2 and full-length GFP were cloned and inserted into pTRV2. Each of the TRV2-based constructs was transformed into A. tumefaciens strain GV3101. Agrobacterium harboring TRV1- or TRV2-derived vectors were mixed at a 1:1 ratio and infiltrated into N. benthamiana plants.

Nucleocytoplasmic fractionation assay

Nucleocytoplasmic fractionation was performed using a CelLytic^TM PN Isolation/Extraction kit following the manufacturer’s instructions (Sigma-Aldrich). Total, nuclear, and cytoplasmic proteins were detected by immunoblotting with corresponding antibodies. The PEPC and H3 proteins were used as cytosolic and nuclear markers, respectively.

P0^BrA interacts with NbRAF2

To study the function of the P0 protein, we performed a Y2H screen of an Arabidopsis cDNA library using P0^BrA as the bait to identify P0^BrA-interacting proteins. Among the screened positive clones, one contained an intact ORF that shared 100% identity with AtRAF2 (At5g51110; GenBank accession AED96036.1). To explore the role of interactions between P0^BrA and RAF2 in N. benthamiana, we searched for AtRAF2 homologs in the N. benthamiana genome (http://solgenomics.net). The homolog that we identified encoded a 211-amino acid protein, shared approximately 50% identity with AtRAF2 and ZmRAF2 (GenBank accession NP_001144391) (see Supplementary Fig. S1), and was named NbRAF2 (GenBank accession MG560271). However, the traditional directed Y2H assay did not reveal a P0^BrA–NbRAF2 interaction, although it did detect AtRAF2–P0^BrA (Supplementary Fig. S2). Hence, we used the split ubiquitin MbY2H system to test for an interaction (McGee, 2006; Brückner et al., 2009). The P0^BrA was cloned into either the MbY2H bait vector to express a TF::Cub::P0^BrA (P0^BrA–Cub)-fusion protein or into the MbY2H prey vector to express a NubG::P0^BrA (NubG–P0^BrA)-fusion protein. NbRAF2 was also cloned into either the MbY2H bait vector to express a TF::Cub::NbRAF2 (NbRAF2–Cub)-fusion protein or into the MbY2H prey vector to express a NubG::NbRAF2 (NubG–NbRAF2)-fusion protein. A series of controls were performed. NubG constructs served as test proteins and NubI constructs were used as positive controls. All combinations were transformed into yeast cells for mating, and the transformed yeast cells were transferred to SD/−WL and SD/−AHLW plates for 2–3 d. However, the NubG–NbRAF2/Cub combination also grew on the SD/−AHLW plates (data not shown). Thus, we chose NbRAF2–Cub to identify the interaction. Yeast transformed with NbRAF2–Cub and NubG–P0^BrA grew on selective plates, as did the positive control; however, the negative controls did not (Fig. 1A). Thus, P0^BrA can directly interact with NbRAF2 in yeast.

Fig. 1.

P0^BrA interacts with NbRAF2. (A) Analysis of interactions between NbRAF2 and P0^BrA using the split ubiquitin membrane-bound yeast two-hybrid (MbY2H) system. NbRAF2 was cloned into the MbY2H bait vector pBT3-STE, and P0^BrA was cloned into the prey vector pPR3-N. NubG constructs served as test proteins and NubI constructs were positive controls. All the combinations were transformed into yeast NMY51. Yeast strains were grown on SD/–Leu/–Trp and SD/–Ade/–His/–Leu/–Trp, and maintained at 30 °C for 2–3 d. (B) Co-immunoprecipitation analyses of NbRAF2 and P0^BrA proteins in N. benthamiana leaves. P0^BrA–Flag was co-expressed with GFP, NbRAF2–GFP, or NbSKP1–GFP through agro-infiltration. GFP and NbSKP1 were used as negative and positive controls, respectively. Protein complexes were immunoprecipitated using anti-Flag beads. Immunoprecipitates were assessed with western blotting using anti-GFP and anti-Flag antibodies.

We used Co-IP assays to further confirm whether P0^BrA interacted with NbRAF2 in plant cells. Flag-tagged P0^BrA (P0^BrA–Flag) was co-expressed with GFP, GFP-tagged NbRAF2 (NbRAF2–GFP), or GFP-tagged NbSKP1 (NbSKP1–GFP) in N. benthamiana leaves through agro-infiltration. The interaction between P0^BrA and NbSKP1 was used as a positive control. At 2 dpi, protein complexes were immunoprecipitated using anti-Flag beads. The P0^BrA–Flag was co-immunoprecipitated with NbRAF2–GFP, but not with GFP (Fig. 1B). These experiments demonstrated that P0^BrA interacts with NbRAF2 in plant cells.

Subcellular localization of NbRAF2

To investigate the subcellular localization of NbRAF2, we generated mCherry-tagged NbRAF2 (NbRAF2–mCherry) (see Supplementary Fig. S3), and then transiently co-expressed it with free GFP in N. benthamiana leaves through agro-infiltration. The infiltrated leaves were collected at 2 dpi and observed with confocal microscopy. The mCherry signal revealed that NbRAF2 was present in the chloroplast, cell periphery, and nucleus of the same cells, similar to the subcellular localization of AtSDIRIP1/AtRAF2. Surprisingly, NbRAF2 was localized to the chloroplast stromules (Fig. 2A). To confirm this, we transiently expressed NbRAF2–mCherry in FNR–EGFP transgenic N. benthamiana through agro-infiltration. The FNR–EGFP was used here as a stromule-localized marker (Schattat et al., 2011). Confocal microscopy showed that NbRAF2–mCherry could perfectly co-localize with FNR–EGFP in chloroplasts and stromules (Fig. 2B). Thus, NbRAF2–mCherry was localized not only to the nucleus and cell periphery but also to the chloroplasts and stromal fraction. Similarly, AtRAF2 was also localized to the stromules (Supplementary Fig. S4).

Fig. 2.

Subcellular localization of NbRAF2 and its mutants. (A) The subcellular localization of NbRAF2 and its mutants in N. benthamiana leaf cells as assessed by confocal microscopy. NbRAF2–mCherry and its mutants were transiently co-expressed with GFP in leaves through agro-infiltration and imaged at 2 d post-inoculation. The white arrowhead indicates that the protein is localized to the stromules of chloroplasts. A magnification of the selected area is shown in the first image on the top row. (B) The subcellular localization of NbRAF2 and its mutants in FNR–GFP transgenic N. benthamiana leaf cells as assessed with confocal microscopy. NbRAF2–mCherry and its mutants were independently transiently expressed in leaves of FNR–GFP transgenic N. benthamiana through agro-infiltration. White arrowheads indicate that the protein is localized to the stromules of chloroplasts. The scale bars represent 10 μm.

The C-terminal residues of NbRAF2 are required for its self-interaction

The Y2H and BiFC assays showed that NbRAF2 self-interacted. The Y2H results demonstrated that the combinations of pGBKT7–NbRAF2 and pGADT7–NbRAF2 could not grow on the SD/−AHLW selective plates (Fig. 3A), but NbRAF2 could interact with NbRAF2–ΔTP, a truncated mutant of NbRAF2 that contained a deleted transit peptide (TP) of 1–45 aa in the N-terminus (Figs 2, 3A, Supplementary Fig. S3). For the BiFC assays, NbRAF2 was fused to N- and C-terminal halves of YFP at the N-termini to generate NbRAF2–YN and NbRAF2–YC, respectively. The reconstituted YFP fluorescence was observed in N. benthamiana leaf epidermal cells co-infiltrated with NbRAF2–YN and NbRAF2–YC, which co-localized with chloroplast autofluorescence (Fig. 3B). In addition, YFP signals occurred in the stromules. Fluorescence could not be visualized in the negative controls NbRAF2-YN/YC and YN/NbRAF2-YC (Supplementary Fig. S5). Arabidopsis and maize RAF2 can form dimers (Valkai, 2004; Feiz et al., 2014), consistent with our observations for NbRAF2. In addition, the C-terminal truncated mutant NbRAF2–ΔCter, which contained a deleted C-terminal 183–211 aa and localized to the nucleus and chloroplast (Supplementary Fig. S3, Fig. 2), failed to interact with full-length NbRAF2 (Fig. 3A, B).

Fig. 3.

The C-terminal residues of NbRAF2 are required for its self-interaction. (A) Self-interaction analysis of NbRAF2 in yeast. Activating domain (AD) constructs containing NbRAF2 and its variants were independently transformed with BD–NbRAF2. Yeast cells transformed separately with binding domain (BD) and AD vectors showed no growth on selective plates. Yeast strains were grown on SD/–Leu/–Trp and SD/–Ade/–His/–Leu/–Trp, and maintained at 30 °C for 5–7 d. The yeast two-hybrid results showed that C-terminal residues are required for the self-interaction of NbRAF2. (B) BiFC assays for NbRAF2 self-interaction were performed on N. benthamiana leaves through agro-infiltration. YFP is an indicator of protein–protein interactions. NbRAF2–NbRAF2 interactions are apparent in the chloroplasts and chloroplast stromules, and the self-interaction requires C-terminal residues.

Thus, NbRAF2 self-interacted in chloroplasts and stromules, and the C-terminal residues were required for self-interaction but not for chloroplastic and nuclear localization. Similarly, the C-terminal 192–220 aa of AtRAF2 was required for its self-interaction but not for its chloroplastic and nuclear localization (Supplementary Figs S4, S6).

NbRAF2 co-localizes with P0^BrA in the nucleus and cell periphery

To investigate the subcellular localization of P0^BrA, we constructed a vector expressing the P0^BrA protein with a GFP-tag fused to its C-terminus (P0^BrA–GFP). P0^BrA–GFP was co-expressed with free mCherry (which was used as a marker to delineate the nucleus and cytoplasm) in N. benthamiana leaves through agro-infiltration. Confocal microscopy showed that P0^BrA–GFP co-localized with mCherry, suggesting that P0^BrA–GFP can localize to the nucleus and the cell periphery (Fig. 4A). Moreover, P0^BrA–GFP also formed punctate structures in the cytosol (Fig. 4A). To further investigate where P0^BrA and NbRAF2 co-localized in vivo, the P0^BrA–GFP was transiently co-expressed with NbRAF2–mCherry in N. benthamiana leaves through agro-infiltration. Confocal microscopy showed that they co-localized in the nucleus and cell periphery (Fig. 4B).

Fig. 4.

NbRAF2 co-localizes with P0^BrA in the nucleus and cell periphery. (A) P0^BrA localizes to the nucleus and the cytoplasm. GFP-tagged P0^BrA was transiently co-expressed with mCherry in N. benthamiana leaves. (B) Co-localization of NbRAF2 and P0^BrA. The co-localization of the proteins is shown in the merged image. Confocal images show that NbRAF2 co-localizes with P0^BrA in the nucleus and cell periphery. Images were taken at 2 d post-inoculation. The scale bars represent 10 μm.

The nuclear pool of NbRAF2 decreases in the presence of P0^BrA or BrYV-A

Although P0^BrA–GFP co-localized with NbRAF2–mCherry in the nucleus, the cells demonstrating nuclear co-localization were difficult to find. Interestingly, the distribution of NbRAF2–mCherry to the nucleus was significantly reduced in the presence of P0^BrA–GFP compared with the GFP control (Fig. 5A). The relative numbers of nuclear-localized NbRAF2 were calculated based on the confocal images, and there was a reduction of approximately 30% in nuclear-localized NbRAF2 in the P0^BrA–GFP co-expressed cells compared with GFP co-expressed cells. Nucleocytoplasmic fractionation assays were performed to further identify the nuclear accumulation of the NbRAF2 protein. The NbRAF2–mCherry with P0^BrA–GFP or GFP were transiently co-expressed in N. benthamiana leaves, and protein samples were prepared at 2 dpi. A western blot analysis revealed that the nuclear pool of NbRAF2 protein significantly decreased after leaves were inoculated with P0^BrA–GFP (Fig. 5B). However, no significant difference was observed in the total amounts of NbRAF2 protein.

Fig. 5.

The nuclear pool of NbRAF2 decreases in the presence of P0^BrA or BrYV-A. (A) The localization of NbRAF2 when co-expressed with GFP or P0^BrA–GFP. Images were taken at 2 d post-inoculation (dpi). The scale bars represent 20 μm. Z stacks of cells were imaged. Values for relative nuclear NbRAF2 are presented in the table, and are calculated as the ratio of the number of nuclear-localized NbRAF2 to the total number of nuclei, for each combination as determined by confocal microscopy. (B, C) Western blotting of NbRAF2 in nuclei-enriched and nuclei-depleted fractions, and total protein when co-expressed with GFP or P0^BrA–GFP (B), and when co-expressed with Mock or BrYV-A (C) in N. benthamiana leaves. Protein samples were prepared from plant leaves at 2 dpi. All fractions were subjected to immunoblot analyses and were assessed using the corresponding antibodies. PEPC and histone H3 were used as cytosolic and nuclear markers, respectively.

NbRAF2–ΔTP, the TP deletion mutant, localized to the nucleus but not to the chloroplast; however, NbRAF2–ΔCter, the C-terminal residue deletion mutant, localized to the nucleus and the chloroplast (Fig. 2). We tested the effect of P0^BrA on the nuclear accumulation of these two mutants. Confocal microscopy showed that P0^BrA inhibited the accumulation of a nuclear pool of NbRAF2–ΔCter–mCherry, but not that of NbRAF2–ΔTP–mCherry (see Supplementary Fig. S7). Thus, the P0^BrA-mediated reduction of nuclear NbRAF2 required the dual localization of NbRAF2 in the chloroplast and nucleus.

The effect of BrYV-A on the nuclear accumulation of NbRAF2 was assayed using nucleocytoplasmic fractionation assays and confocal microscopy. NbRAF2–mCherry was co-expressed with the mock or BrYV-A through agro-infiltration in N. benthamiana leaves. The samples were prepared from inoculated leaves at 2 dpi. The nuclear pool of NbRAF2 decreased after BrYV-A infection (Fig. 5C, Supplementary Fig. S8).

NbRAF2 expression is down-regulated during BrYV-A infection

We investigated whether the NbRAF2 expression level was affected during BrYV-A infection. Total RNA was obtained from BrYV-A-inoculated N. benthamiana leaves at 2 dpi. Semi-quantitative RT-PCR showed that the mRNA level of NbRAF2 decreased during the BrYV-A infection (Fig. 6A).

Fig. 6.

Silencing NbRAF2 increases local BrYV-A accumulation levels in inoculated leaves and enhances systemic infection of the virus. (A) Analysis of NbRAF2 gene expression in leaves of mock-inoculated (empty vector, EV) and BrYV-infected plants at 2 d post-inoculation (dpi). The relative accumulation of NbRAF2 mRNA was determined using semi-quantitative RT-PCR. Images are agarose gels loaded with 26-, 28-, 30-, and 32-cycle PCR products. (B) Silencing of NbRAF2 causes leaf chlorosis compared with TRV–GFP-infected control plants. Images were taken ~2–3 weeks after infiltration for VIGS. (C) NbRAF2 mRNA levels were confirmed by RT-PCR for TRV–NbRAF2 and TRV–GFP systemic leaves. (D) Silencing NbRAF2 increases local BrYV-A accumulation in inoculated leaves at 2 dpi. Proteins and RNA were extracted and subjected to western blotting and RT-PCR. The coat protein (CP) was detected with BrYV-A CP polyclonal antiserum. RbcL is the Rubisco large subunit. The actin protein was used as a loading control. ImageJ software was used to quantify the bands. RT-PCR confirmed that suppression of NbRAF2 enhanced BrYV-A RNA levels. Representative gels of RT-PCR products are shown. (E) Silencing NbRAF2 promotes systemic infection by BrYV-A. Photographs were taken at 14 d after BrYV-A inoculation. RT-PCR showed that NbRAF2-silenced plants became systemically infected with BrYV-A at 14 dpi, but non-silenced plants did not. For each RT-PCR, EF-1A mRNA levels were used as internal controls. The ratios below the images show the number of systemic infection plants out of the total number of infiltrated plants. The systemic infection efficiency was scored in three independent experiments.

Silencing NbRAF2 increases the local accumulation level of BrYV-A in inoculated leaves and enhances systemic infection of the virus

To investigate the possible role of NbRAF2 during BrYV-A infection, we used the TRV-VIGS vector (Liu et al., 2002b) to knock down the expression of NbRAF2. A partial cDNA fragment of NbRAF2 was cloned into the RNA2-derived vector of TRV to generate pTRV2–NbRAF2, and GFP was cloned into pTRV2 to generate pTRV2–GFP, which was used as a negative control. At 2–3 weeks post-inoculation, NbRAF2-silenced plants showed leaf chlorosis compared with TRV–GFP-infected control plants (Fig. 6B).The NbRAF2 expression level was then determined with RT-PCR. The NbRAF2 mRNA level was significantly reduced in NbRAF2-silenced plants compared with non-silenced controls (Fig. 6C). The RbcL protein level correspondingly decreased in NbRAF2-silenced plants (Fig. 6D) and RbcL interacted with NbRAF2 (see Supplementary Fig. S9), consistent with the role of ZmRAF2 and AtRAF2 in Rubisco accumulation (Feiz et al., 2014; Fristedt et al., 2018). Subsequently, the NbRAF2-silenced and non-silenced N. benthamiana plants were inoculated with BrYV-A using agro-infiltration. Western blot and RT-PCR analyses showed that BrYV-A CP protein and BrYV-A RNA levels increased in the NbRAF2-silenced plants compared with non-silenced control plants at 2 dpi (Fig. 6D). BrYV-A can cause necrosis in inoculated leaves, but symptoms are not visible in systemic leaves. However, we observed no obvious difference in necrosis of NbRAF2-silenced and non-silenced plants in the inoculated leaves at 3 and 4 dpi (Supplementary Fig. S10). At 14 dpi, BrYV-A was able to spread to the upper non-inoculated leaves of the NbRAF2-silenced plants but not easily into those of non-silenced plants. BrYV-A RNA was detected in the systemic leaves of NbRAF2-silenced and non-silenced plants (Fig. 6E). RT-PCR detection showed that 31.6% of non-silenced control plants had systemic BrYV-A infection at 14 dpi, but 63.2% of NbRAF2-silenced plants became systemically infected with BrYV-A (Fig. 6E, lower panel). These findings suggested that silencing of NbRAF2 increases the local accumulation of BrYV-A in inoculated leaves and promotes the systemic infection of the virus. Thus, our results showed that NbRAF2 negatively regulates the BrYV-A infection.

Overexpression of nuclear RAF2 enhances resistance to BrYV-A

Silencing NbRAF2 increased BrYV-A accumulation, and the nuclear accumulation of NbRAF2 decreased during BrYV-A infection (Figs 5C, 6D). Therefore, we investigated the function of nuclear NbRAF2 in BrYV. A nuclear localization sequence (NLS; Wen et al., 1995) was fused to the N-terminus of NbRAF2–ΔTP–mCherry to generate NLS–NbRAF2–ΔTP–mCherry (see Supplementary Fig. S3). Confocal microscopy showed that NLS–NbRAF2–ΔTP–mCherry was exclusively redirected to the nucleus (Fig. 7A). Hence, we transiently expressed NLS–NbRAF2–ΔTP–mCherry and mCherry through agro-infiltration in N. benthamiana leaves. Then, at 1 dpi, we inoculated BrYV-A through agro-infiltration in the same leaves for 2 d. The accumulation of BrYV-A CP was examined with western blotting, which demonstrated that overexpressing nuclear NbRAF2 decreased the level of BrYV-A CP compared with mCherry (Fig. 7B). To determine whether nuclear AtRAF2 had the same role in resistance to BrYV-A, we constructed AtRAF2–ΔTP–mCherry, which was exclusively redirected to the nucleus (Supplementary Fig. S11). The results of western blotting demonstrated that overexpressing nuclear AtRAF2 also inhibited virus accumulation (Supplementary Fig. S11). These results suggested that the increased nuclear accumulation of NbRAF2 or AtRAF2 enhances resistance to BrYV-A.

Fig. 7.

Overexpression of nuclear RAF2 enhances resistance to BrYV-A. (A) Confocal microscopy images showing the subcellular localization of mCherry (mC) and NLS–NbRAF2–ΔTP–mCherry (NLS–NbRAF2–ΔTP–mC). Images were taken at 2 d post-inoculation. The scale bars represent 10 μm. N, nucleus. (B) BrYV-A was agro-infiltrated at 1 dpi in leaves separately overexpressing NLS–NbRAF2–ΔTP–mC and mC. At 3 dpi, protein was extracted and subjected to western blotting. Coat protein (CP) was detected with BrYV-A CP polyclonal antiserum. RbcL is the Rubisco large subunit. ImageJ software was used to quantify the bands.