Skip to main content
NCBI home page
Plant Physiology logoLink to Plant Physiology
. 2022 Apr 29;189(3):1848–1865. doi: 10.1093/plphys/kiac181

GLRaV-2 protein p24 suppresses host defenses by interaction with a RAV transcription factor from grapevine

Chenwei Zhang 1,†, Xianyou Wang 2, Hanwei Li 3, Jinying Wang 4, Qi Zeng 5, Wenting Huang 6, Haoqiang Huang 7, Yinshuai Xie 8, Shangzhen Yu 9, Qing Kan 10, Qi Wang 11, Yuqin Cheng 12,13,✉,‡
PMCID: PMC9237672  PMID: 35485966

Abstract

Grapevine leafroll-associated virus 2 (GLRaV-2) is a prevalent virus associated with grapevine leafroll disease, but the molecular mechanism underlying GLRaV-2 infection is largely unclear. Here, we report that 24-kDa protein (p24), an RNA-silencing suppressor (RSS) encoded by GLRaV-2, promotes GLRaV-2 accumulation via interaction with the B3 DNA-binding domain of grapevine (Vitis vinifera) RELATED TO ABSCISIC ACID INSENSITIVE3/VIVIPAROUS1 (VvRAV1), a transcription factor belonging to the APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) superfamily. Salicylic acid-inducible VvRAV1 positively regulates the grapevine pathogenesis-related protein 1 (VvPR1) gene by directly binding its promoter, indicating that VvRAV1 may function in the regulation of host basal defense responses. p24 hijacks VvRAV1 to the cytoplasm and employs the protein to sequester 21-nt double-stranded siRNA together, thereby enhancing its own RSS activity. Moreover, p24 enters the nucleus via interaction with VvRAV1 and weakens the latter’s binding affinity to the VvPR1 promoter, leading to decreased expression of VvPR1. Our results provide a mechanism by which a viral RSS interferes with both the antiviral RNA silencing and the AP2/ERF-mediated defense responses via the targeting of one specific host factor.

A viral RNA-silencing suppressor interferes with both the antiviral RNA silencing and host basal defense via the targeting of one specific host factor.

Introduction

Plants defend themselves from pathogen attacks by using various defense mechanisms. Plants possess a two-tiered innate immune system, called pathogen-associated molecular pattern (PAMP)-induced immunity (PTI) and effector-induced immunity (ETI). Both PTI and ETI induce a diverse array of immune responses such as reactive oxygen species generation, activation of mitogen-activated protein kinase, production of signaling hormones, and transcriptional reprogramming of defense genes (e.g. pathogenesis-related [(PR] genes) within the host (Tsuda and Somssich, 2015). Although there are certain differences between viruses and other pathogens in their molecular interactions with plant hosts, the concept of the zigzag model may be applied also to the molecular arms race between a virus and its host (Nakahara and Masuta, 2014; Niehl et al., 2016). Viral double-stranded RNAs (ds RNAs) produced during virus replication can be recognized as PAMPs that induce antiviral RNA silencing (PTI; Niehl et al., 2016). Then, viruses have consequently evolved RNA-silencing suppressors (RSSs) as effectors to overcome this antiviral defense mechanism. However, in the subsequent ETI-like phase, the plants activate counter-counterdefenses such as hypersensitive response and salicylic acid (SA)-mediated resistance against viral RSSs (Nakahara and Masuta, 2014).

Previous studies have shown that in addition to their function in silencing suppression, viral RSSs can also suppress additional host defense responses. For example, tobacco mosaic virus 126-kDa replicase suppresses basal host defenses via degradation of a NAC [no apical meristem (NAM), Arabidopsis transcription activation factor (ATAF), cup-shaped cotyledon (CUC)] domain transcription factor (TF; Wang et al., 2009). Viral RSS C2 proteins of a few geminiviruses have been shown to suppress jasmonic acid signaling through interaction with COP9 (constitutively photomorphogenic 9) signalosome subunit 5 (Lozano-Duran et al., 2011). HC-Pro (helper component proteinase) of turnip mosaic virus interacts with SA-binding protein 3 and compromises the latter’s function in the induction of SA accumulation (Poque et al., 2018). However, the molecular mechanisms by which viral RSSs suppress host defenses are still largely unclear.

APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) TFs have been divided into three separate subfamilies based on the number of AP2 DNA-binding domains and sequence similarity, namely AP2, RAV (related to ABI3/VP1) and ERF (Licausi et al., 2013). Increasingly, evidence shows that members of the AP2/ERF TF superfamily play complex regulatory roles in plants under biotic stress. They participate in different pathways of hormonal signaling and induce a set of PR genes in response to pathogen attack. For example, the members of the ERF subfamily activate PR genes by binding to the GCC box of their promoters in response to biotic stresses (Park et al., 2001; Pré et al., 2008). Pepper RAV1 induces the expression of some PR genes, conferring resistance to Pseudomonas syringae pv. tomato DC3000 (Hong et al., 2005; Sohn et al., 2006). Li et al. (2011) also reported that tomato (Solanum lycopersicum) RAV2 may comprise a key modulator in the plant defense signal pathway.

Grapevine leafroll disease is an economically important viral disease affecting grapevine (Vitis vinifera) worldwide (Song et al., 2021). Grapevine leafroll-associated virus 2 (GLRaV-2) is one of the prevalent GLRaVs in leafroll-affected grapevine. It is also associated with graft-incompatibility disorders (Angelini et al., 2017). GLRaV-2 is a member of the genus Closterovirus. Its positive-sense RNA genome is ∼16.5 kb in size, encoding nine open reading frames (Dolja et al., 2006; Liu et al., 2009); the 24-kDa protein (p24) encoded by the GLRaV-2 can block local RNA silencing (Chiba et al., 2006; Li et al., 2018). However, host factors that interact with GLRaV-2 proteins have not been identified, and the mechanism underlying GLRaV-2 infection remains unclear. Here we demonstrate that GLRaV-2 p24 interacts with grapevine RAV1 (VvRAV1), a TF that positively regulates VvPR1 transcription by the SA-dependent signaling pathway. p24 recruits VvRAV1 in the cytoplasm to bind 21-nt ds siRNA, thereby enhancing its silencing suppression capacity; at the same time, p24 enters the nucleus via interaction with VvRAV1, and reduces VvRAV1-mediated activation of VvPR1 transcription. Our results reveal a mechanism developed by a viral RSS to suppress host antiviral defenses.

Results

p24 suppresses systemic RNA silencing and binds to ds siRNA

Leaves of green fluorescent protein (GFP)-transgenic Nicotiana benthamiana line 16c (Voinnet and Baulcombe 1997) plants were agroinfiltrated with pGD–GFP/pGD–p24, and GFP silencing in the upper noninfiltrated leaves was monitored. Results from three independent experiments showed that ∼92% of the negative control plants displayed vein-proximal silencing of GFP in newly emerging leaves at 18-d postinfiltration (dpi) (Figure 1, A and B). In contrast, ∼93% and 95% of the upper noninfiltrated leaves in plants co-infiltrated with pGD–GFP/pGD–p24 or pGD–GFP/pGD–p19, respectively, retained GFP fluorescence for 18 dpi. Two p24 mutants, R2A and R86A which fail to efficiently inhibit local RNA silencing (Li et al., 2018), also had defects in suppressing systemic RNA silencing. The visual observations were confirmed by quantitative analyses of GFP protein and mRNA levels (Figure 1C).

Figure 1.

Figure 1

Open in a new tab

GLRaV-2 p24 can inhibit systemic RNA silencing and bind ds siRNA. A, Effect of p24 on systemic silencing. Leaves of N. benthamiana line 16c were agroinfiltrated with pGD–GFP plus the indicated construct. Co-infiltration of pGD–GFP and pGD–GUS (β-glucuronidase) or pGD–p19 was used as a negative and positive control, respectively. Photographs were taken under UV light 18 dpi. B, Table showing results from three independent experiments. C, Quantitative assessment of GFP and mRNA accumulation. Fifth leaf from the top of each plant as indicated by an arrow in (A) were sampled 18 dpi, and leaves of the same treatment within each experiment were mixed for total protein and RNA extraction. Coomassie brilliant blue (CBB) staining and ethidium-bromide-stained ribosomal RNA (rRNA) are shown as loading controls, respectively. D, EMSA analysis of the binding of p24 and its mutants to 21- and 24-nt siRNA duplexes. A constant amount (1 ng) of biotin-labeled 21- or 24-nt ds siRNA was incubated with 1 μg of the indicated His-tagged protein. GFP–His served as the negative control. Each assay was performed in three independent experiments.

Viral RSSs commonly adopt ds siRNA-binding strategy (Lakatos et al., 2006). Electrophoretic mobility shift assay (EMSA) demonstrated that p24–His was able to bind both 21-nt and 24-nt ds siRNA (Figure 1D). R2A–His and R86A–His could bind 21-nt ds siRNA, but much less efficiently than the wild-type (WT) p24; however, they failed to bind 24-nt ds siRNA (Figure 1D).

Taken together, p24 suppresses systemic RNA silencing and binds to both 21-nt and 24-nt ds siRNA; p24 mutants R2A and R86A bind very weakly to 21-nt ds siRNA but fail to bind 24-nt ds siRNA, and thus may fail to efficiently suppress local and systemic RNA silencing.

GLRaV-2 p24 interacts with the TF RAV1 from grapevine

Yeast two-hybrid (Y2H) screening was previously performed in our laboratory using GLRaV-2 p24 as bait and a V. vinifera L. (cv. Cabernet Sauvignon) cDNA library as prey. One positive clone encoded a peptide (amino acids [aa] 211–378) (Supplemental Figure S1A) that corresponded to the C terminus of grapevine TF VvRAV1, a member of the RAV subfamily. The sequence of VvRAV1 was cloned and sequence analysis revealed that it encodes a protein of 378 aa containing AP2 and B3 domains (Supplemental Figure S1).

Since VvRAV1 has transactivation activity (Supplemental Figure S2A), sequences encoding the complete VvRAV1, B3 domain of VvRAV1 (VvRAV1B3), the C-terminus of VvRAV1 (VvRAV1C) identified by yeast screening and the remaining N terminus of VvRAV1 (VvRAV1N) were fused with the GAL4 activation domain (AD) in the pGADT7 vector, and each was co-transformed with pGBK–p24 in yeast strain AH109 cells. p24 can interact with itself (Li et al., 2018) and the combination of pGAD–p24/pGBK–p24 (expressing AD–p24 and BD–p24, respectively) was therefore used as a positive control. As shown in Figure 2A, yeast cells co-transformed with pGAD–RAV1C/pGBK–p24 (expressing AD–VvRAV1C and BD–p24, respectively), pGAD–RAV1B3/pGBK–p24, or the positive control could grow on synthetic drop-out medium lacking Trp, Leu, His, and Ade (SD/–Leu/–Trp/–His/–Ade) medium, whereas cells harboring pGAD–RAV1N/pGBK–p24 or the negative control (pGADT7/pGBK–p24) could not. These results indicate that p24 interacts with both VvRAV1C and VvRAV1B3, but not with VvRAV1N. Interestingly, no interaction was observed between p24 and the complete VvRAV1 protein, because yeast cells co-transformed with pGAD–RAV1/pGBK–p24 were not able to grow on SD/–Leu/–Trp/–His/–Ade medium. This may be due to the limitation of Y2H.

Figure 2.

Figure 2

Open in a new tab

GLRaV-2 p24 interacts with VvRAV1 and its homologs. A, Analyses of interactions between p24 and VvRAV1 or VvRAV1 mutants in yeast. Yeast strain AH109 co-transformed with plasmids expressing the indicated fusion proteins were plated on SD/–Leu/–Trp/–His/–Ade. Transformation of pGADT7 and a plasmid expressing BD–p24 or plasmids expressing BD–p24/AD–p24 served as negative and positive controls, respectively. B, In-vitro pull-down analysis of the interaction between p24 and VvRAV1. p24–His (1 µg) was used to pull down 1 μg of GST–VvRAV1. The combination GFP–His/GST–VvRAV1 served as a negative control. Both the input and pull-down samples were detected by western blotting with anti-His and anti-GST antibodies. C, In-vivo BiFC analysis of protein–protein interactions. The indicated proteins were expressed in N. benthamiana leaves. Co-expression of p24–yellow fluorescent protein (YFP)N and p24–YFPC served as the positive control. CP and p24G1, RSSs of GINV and GLRaV-1, respectively. Confocal micrographs were taken 3 dpi. Bars = 50 μm. Each assay was repeated three times.

The interaction between p24 and the complete VvRAV1 protein was observed in pull-down assays: glutathione S-transferase (GST)–VvRAV1 was pulled down by p24–His, whereas no signal was observed when p24–His was replaced by GFP-His in the assay (Figure 2B).

Bimolecular fluorescence complementation (BiFC) demonstrated that p24 interacted with VvRAV1, VvRAV1C, and VvRAV1B3 in the cytoplasm of N. benthamiana cells. R2A and R86A could also interact with VvRAV1 (Figure 2C). No interaction between p24 and VvRAV1N was observed, possibly because VvRAV1N was not expressed (Supplemental Figure S2B).

NtRAV and AtRAV2 have been reported to interact with two unrelated viral RSSs, potyvirus HC-Pro, and p38 of turnip crinkle virus (Endres et al., 2010). The amino acid sequence of VvRAV1 shares ∼60% and 70% identity with that of AtRAV2 and NtRAV, respectively, and VvRAV1 has a closer genetic relationship with NtRAV (Supplemental Figure S1). BiFC demonstrated that p24 interacted with both NtRAV and AtRAV2 in the cytoplasm of N. benthamiana cells (Figure 2C).

p24G1 encoded by GLRaV-1 is an RSS (Zhang et al., 2020), and our unpublished data indicate that coat protein (CP) of grapevine berry inner necrosis virus (GINV) can inhibit RNA silencing and bind to 21-nt ds siRNA (Supplemental Figure S3). BiFC results demonstrated that VvRAV1 does not interact with GLRaV-1 p24G1 or GINV CP (Figure 2C).

Taken together, GLRaV-2 p24 and its silencing suppression-defective mutants can all interact with VvRAV1 in the cytoplasm of plant cells, and the B3 domain of VvRAV1 is responsible for the interaction. In addition, p24 can interact with VvRAV1 homologs from tobacco (Nicotiana tabacum) and Arabidopsis (Arabidopsis thaliana), whereas other RSSs of grapevine viruses, p24G1 of GLRaV-1 and GINV CP, cannot.

VvRAV1 promotes GLRaV-2 accumulation

Due to technical difficulties in grapevine transformation and agroinfiltration of grapevine leaves, grapevine callus and protoplast are widely used as model experimental systems (Jackson et al., 2001; Malnoy et al., 2016) for rapid analysis of gene function and regulation through transient gene expression and silencing. To investigate the role of VvRAV1 in GLRaV-2 infection, pMyc–RAV1 (expressing Myc–VvRAV1) and an infectious cDNA clone vector of GLRaV-2 (Liu et al., 2009) were co-introduced into grapevine callus by agroinfiltration. The transcript level of VvRAV1 at 3 dpi was 1,100% of that in controls, which led to a 2,200% increase in GLRaV-2 RNA accumulation in comparison to controls (Figure 3A). These results were further supported by western blot analysis (Figure 3A, right).

Figure 3.

Figure 3

Open in a new tab

VvRAV1 promotes the accumulation of GLRaV-2 but not GINV in grapevine. A and B, Transient overexpression of VvRAV1 promotes GLRaV-2 accumulation in grapevine. GLRaV-2 and pMyc–RAV1 or pGD–RAV1 were co-introduced into grapevine callus or in-vitro grown plantlets (B) by agroinfiltration. For agroinfiltration of plantlets, vacuum infiltration method was used. For visualization of GLRaV-2 replication under confocal microscope, detached leaves of plantlets 3 dpi were used. #1, #2, and #3 are representative images of more than 25 individual leaf samples from three individual experiments. Bar = 100 μm. C, Silencing of VvRAV1 decreased GLRaV-2 accumulation in grapevine callus. Grapevine callus was agroinfiltrated with pRAV1–hp to silence VvRAV1. VvRAV1-silenced callus 2 dpi was agrotransfected with GLRaV-2. D, Effects of overexpression (left) or silencing (right panel) of VvRAV1 on GINV RNA accumulation in grapevine callus. pGD–GUS (A, B, and D) or the empty vector pCsuper1300–hp (C and D) served as the negative control. Calli (A, C, and D) or leaves and stems of plantlets (B) from the same treatment in each experiment were sampled at 3 d after viral infection and pooled for extraction of total RNA and protein. Each assay was performed in three individual experiments. GLRaV-2 accumulation was detected using anti-p24 antiserum, and CBB staining confirmed equal loading (A, C). RT–qPCR results are shown as mean ± sd of three independent experiments. Statistically significant differences between means were determined by employing Tukey’s multiple range test. **P < 0.01.

The in vitro-grown whole grapevine plantlets were vacuum-agroinfiltrated with pGD–RAV1 and GLRaV-2. Plantlets inoculated with GLRaV-2 infectious clone did not develop obvious symptoms until sampling time. Reverse transcription–quantitative polymerase chain reaction (RT–qPCR) results showed that overexpression of VvRAV1 leads to a 50% increase in GLRaV-2 RNA accumulation 3 dpi compared to controls (Figure 3B). The infectious GFP-tagged clone of GLRaV-2 allowed us to visualize viral replication, as GFP intensity provides a reliable surrogate marker for measuring accumulation of viral RNAs in infected plant cells. Consistent with the RT–qPCR results, we observed that overexpression of VvRAV1 leads to stronger GFP signals than that in controls 3 dpi (Figure 3B, right). These results demonstrated that overexpression of VvRAV1 promotes GLRaV-2 accumulation in grapevine.

Next, grapevine callus was agroinfiltrated with an RNA interference (RNAi) hairpin construct targeting VvRAV1 (pRAV1–hp) to silencing VvRAV1. At 2 dpi, the transcript level of VvRAV1 in pRAV1–hp-infiltrated callus decreased by 90% relative to controls. VvRAV1-silenced callus was then agroinfiltrated with GLRaV-2 infectious clone. Silencing of VvRAV1 resulted in a 70% decrease in GLRaV-2 RNA accumulation 2 d after infection in comparison to controls (Figure 3C). These findings were further supported by western blot analysis (Figure 3C, right).

To assess whether there is a non-p24-mediated effect of VvRAV1 on virus accumulation, grapevine callus was agroinfiltrated with pMyc–RAV1 and an infectious cDNA clone vector of GINV (Fan et al., 2020). RT–qPCR results showed that overexpression of VvRAV1 leads to a slight decrease in GINV RNA accumulation 3 dpi (Figure 3D). Grapevine callus was agroinfiltrated with pRAV1–hp to silencing VvRAV1, and then agrotransfected with GINV at 2 dpi. GINV RNA accumulation in VvRAV1-silenced (VvRAV1 transcript level was 20% of that in controls) callus 3 d after GINV infection was slightly higher than that in controls (Figure 3D, right).

Our results revealed that VvRAV1 promotes GLRaV-2 accumulation but has no such effect on the accumulation of GINV, whose RSS CP is not able to interact with VvRAV1.

The p24–VvRAV1 interaction induces relocalization of the two proteins

Fusion proteins VvRAV1–GFP and GFP–VvRAV1 were expressed in N. benthamiana leaves. Leaf examination by confocal laser-scanning microscopy 3 dpi showed that both VvRAV1–GFP and GFP–VvRAV1 predominantly accumulate in the nucleus, as confirmed by 4',6-diamidino-2-phenylindole (DAPI) staining (Figure 4A). Red fluorescent protein (RFP)–p24 was distributed in the cytoplasm (Figure 4B), in agreement with our previous observation (Li et al., 2018). In contrast, free GFP and RFP accumulated in both the cytoplasm and nucleus (Supplemental Figure S4A).

Figure 4.

Figure 4

Open in a new tab

The p24–VvRAV1 interaction induces relocalization of both proteins. A, Confocal micrographs showing nuclear localization of fusion proteins VvRAV1–GFP and GFP–VvRAV1 in epidermal cells of N. benthamiana leaves. Nuclei were stained with DAPI. B, Cytoplasmic localization of fusion protein RFP–p24. C, Nuclear and cytoplasmic distribution of VvRAV1–GFP in epidermal cells of p24-expressing transgenic lines (p24-6/Nb and p24-8/Nb). D, Nuclear and cytoplasmic localization of both RFP–p24 and VvRAV1–GFP co-expressed in leaves of N. benthamiana. E, Nuclear–cytoplasmic fractionation analysis of the accumulation of p24 and VvRAV1–GFP. Detection of p24 and VvRAV1–GFP in the nuclear (upper) and cytoplasmic (lower) fractions of leaves of WT or p24-transgenic (line p24-6/Nb) N. benthamiana plants expressing the indicated protein(s). Agroinfiltrated tissues of each treatment within each experiment were sampled 3 dpi and pooled for protein extraction. Nuclear protein histone H3 and cytosolic protein cFBPase were detected as a quality control for the fractionation procedure. Western blot analysis was conducted with the indicated antibodies or antiserum (α-p24). CBB staining confirms equal loading. CF, cytoplasmic fraction; NF, nuclear fraction; No, p24-transgenic N. benthamiana plants were not agroinfiltrated. Leaves of WT or p24-transgenic N. benthamiana plants were expressed with the indicated protein(s), and confocal micrographs were taken 3 dpi (A–D). Bars = 50 μm. Each assay was repeated three times.

Observation of the p24/VvRAV1 interaction in the cytoplasm (Figure 1C) led us to hypothesize that VvRAV1 is relocalized by p24. This hypothesis was first tested using transgenic p24-expressing N. benthamiana lines (p24-6/Nb and p24-8/Nb). Plants of these transgenic lines displayed mild leaf chlorosis (Supplemental Figure S4B). GFP signal accumulated in both the cytoplasm and the nucleus in leaves of p24-transgenic lines that transiently expressed VvRAV1–GFP 3 dpi (Figure 4C). The altered distribution pattern of VvRAV1 by p24 was also observed in the leaves of WT N. benthamiana co-expressing RFP–p24 and VvRAV1–GFP (Figure 4D). Interestingly, RFP–p24 was also partially transported into the cell nucleus in the presence of VvRAV1–GFP (Figure 4D).

The visual observations were further supported by nuclear–cytoplasmic fractionation assays. As shown in Figure 4E, VvRAV1 was detected only in the nuclear fraction, not in cytoplasmic fraction of WT N. benthamiana leaves expressing VvRAV1–GFP. However, VvRAV1 was detected in both the nuclear and cytoplasmic fractions of p24-transgenic N. benthamiana leaves expressing VvRAV1–GFP or WT N. benthamiana leaves co-expressing p24 and VvRAV1–GFP. Similarly, p24 was detected only in the cytoplasmic fraction of p24-transgenic plant leaves or WT N. benthamiana leaves infiltrated with pGD–p24, but was detected in both the cytoplasmic and nuclear fractions of WT N. benthamiana leaves co-expressing p24 and VvRAV1–GFP or p24-transgenic plant leaves expressing VvRAV1–GFP. The nuclear protein histone H3 and cFBPase served as markers to assess the efficiency of subcellular fractionation and, as expected, they were detected only in the nuclear and cytoplasmic fractions, respectively.

Taken together, our results demonstrate that the p24–VvRAV1 interaction simultaneously changes the distribution pattern of the two proteins.

GLRaV-2 p24 recruits VvRAV1 for binding 21-nt ds siRNA to enhance its RSS activity

Redistribution of the nuclear protein VvRAV1 into the cytoplasm by p24 prompted us to investigate the effect of VvRAV1 on p24’s RSS activity. As shown in Figure 5, A and B, VvRAV1C–His could efficiently bind 21 nt but not 24-nt ds siRNA. VvRAV1B3–His had no siRNA-binding activity. Attempts to obtain His-tagged VvRAV1 failed (Supplemental Figure S5), and therefore GST-tagged VvRAV1 was used. GST–VvRAV1 had no siRNA binding activity; however, when p24–His together with either GST–VvRAV1 or VvRAV1C–His were incubated with 21-nt ds siRNA, a strong EMSA band appeared, whereas addition of GFP–His did not produce a similar effect. R2A–His and R86A–His had weak binding affinity to the 21-nt ds siRNA (Figure 1D), whereas with the protein mixtures R2A–His/GST–VvRAV1, R2A–His/VvRAV1C–His, R86A–His/GST–VvRAV1, and R86A–His/VvRAV1C–His, binding to the 21-nt ds siRNA obviously increased. No obvious beneficial effect of VvRAV1 or VvRAV1C on the binding of p24 to 24-nt siRNA duplexes was observed. VvRAV1N–His was not expressed in Escherichia coli (Supplemental Figure S5).

Figure 5.

Figure 5

Open in a new tab

GLRaV-2 p24 recruits VvRAV1 for its suppression of RNA silencing. A, EMSA showing the binding of the indicated protein or protein mixture to 21-nt ds siRNA. B, EMSA showing the binding of the indicated protein or protein mixture to 24-nt ds siRNA. Positive control was provided by the EMSA Kit. C, Analysis of suppression of RNA silencing in N. benthamiana leaves expressing the indicated protein(s). D, RNA silencing suppression by p24 mutants in the presence of VvRAV1. For EMSA (A and B), biotin-labeled 21- or 24-nt ds siRNA was incubated with the indicated His- or GST-tagged (only for VvRAV1) fusion protein(s), and GFP–His served as a control. For Agrobacterium infiltration-mediated RNA-silencing assays (C and D), leaves of N. benthamiana plants were expressed with the indicated protein(s), and expression of GUS served as a control. Myc–VvRAV1 and Myc–VvRAV1C are indicated with VvRAV1 and VvRAV1C, respectively (C and D). Each indicated GFP image is representative of more than 24 individual leaf samples from three individual experiments. Agroinfiltrated leaf patches from the same treatment in each experiment were sampled 3 dpi and pooled for RNA and/or protein extraction. GFP and mRNA levels were detected using α-GFP antibody and DIG-labeled cDNA probes. CBB staining and rRNA served as the loading control, respectively. The intensity of GFP protein and mRNA or EMSA bands was quantified by ImageJ software, and the relative intensity is shown. Each assay was repeated in three independent experiments.

Agrobacterium infiltration-mediated RNA-silencing assays were then performed. GFP fluorescence in tissues expressing Myc–VvRAV1 or Myc–VvRAV1C was negligible, accompanied by low levels of GFP mRNA, similar to that in patches expressing GUS (Figure 5C). However, in most cases (leaves showing enhanced GFP signal/total leaves, 11/14, 12/15, and 10/13), co-expression of p24 and Myc–VvRAV1 resulted in enhanced GFP fluorescence and increased levels of GFP and mRNA, as compared to co-expression of p24/GUS (Figure 5, C and D). These results indicated that VvRAV1 itself is not able to suppress RNA silencing, while its presence enhances p24’s RSS activity. Similar results were also obtained in grapevine callus: overexpression and silencing of VvRAV1, respectively, enhanced and decreased p24’s RSS activity (Supplemental Figure S6). Interestingly, however, VvRAV1C, which efficiently binds 21-nt ds siRNA in vitro, exerted a negative effect on the silencing suppression of p24 (Figure 5C).

Our findings also showed that GFP fluorescence in N. benthamiana leaf patches co-expressing Myc–VvRAV1/R2A or Myc–VvRAV1/R86A was obviously increased, almost equal to that in leaf tissues co-expressing p24 and GUS (Figure 5D). These visual observations were supported by quantitative analysis of GFP mRNA levels. The expression of VvRAV1, p24, and p24’s mutants was verified (Supplemental Figure S7).

The above results indicated that p24 recruits VvRAV1 to bind 21-nt ds siRNA for its suppression of RNA silencing.

EMSA also demonstrated that GST–NtRAV could not bind the 21-nt ds siRNA; however, protein mixtures of p24–His/GST–NtRAV and R2A–His/GST–NtRAV showed a more intense EMSA band than the respective controls (Figure 6A). AtRAV2 and NtRAV were unable to inhibit RNA silencing, but their presence enhanced p24’s RSS activity (Figure 6B, left and middle).

Figure 6.

Figure 6

Open in a new tab

Biological relevance of the interaction between p24 and RAVs. A, Binding of the indicated protein or protein mixture to 21-nt siRNA duplex. Biotin-labeled 21-nt siRNA duplex was incubated with the indicated His- or GST-tagged (only for NtRAV) fusion protein(s). GFP–His served as a control. B, Analysis of the suppression of RNA silencing in N. benthamiana leaves expressing the indicated protein(s). Leaves of line 16c N. benthamiana plants were expressed with GFP and the indicated protein(s) by agroinfiltration, and expression of GUS served as a control. Each indicated GFP image is representative of more than 20 individual leaf samples from three individual experiments. Agroinfiltrated leaf patches from the same treatment in each experiment were sampled 3 dpi and pooled for protein extraction. GFP was detected using α-GFP antibody, CBB staining served as the loading control. p24G1 and CP, RSSs encoded by GLRaV-1 and GINV, respectively. The intensity of GFP and EMSA bands was quantified by ImageJ software, and the relative intensity is shown. Each assay was repeated in three independent experiments.

In addition, the binding of CP–His/GST–VvRAV1 to 21-nt siRNA duplexes was similar to that of CP–His or CP–His/GFP–His (Figure 6A), and the presence of VvRAV1 did not influence the RSS activity of GLRaV-1 p24G1 or GINV CP (Figure 6B). These results highlight the biological relevance of the interaction between p24 and RAV homologs.

VvRAV1 directly upregulates VvPR1 in grapevine

The expression of RAVs is induced by SA (Sohn et al., 2006; Chen et al., 2021). The expression level of VvRAV1 in in vitro-grown grapevine plantlets was significantly induced 2 h after treatment with SA (Figure 7A, upper), but not with methyl jasmonate (MeJA) (Supplemental Figure S8A). VvPR1, which plays a positive role in plant defense against GLRaV-2 (Supplemental Figure S8C), was also significantly induced, but the induction occurred 4 h after SA treatment (Figure 7A, lower). These results demonstrated that both VvRAV1 and VvPR1 are induced by SA, with the induction of VvPR1 coming after that of VvRAV1.

Figure 7.

Figure 7

Open in a new tab

VvRAV1 directly upregulates the defense-related gene VvPR1. A, Expression of VvRAV1 and VvPR1 in response to SA. Grapevine cv. Black Summer in vitro-grown plantlets were sprayed with SA (0.1 mM) and sampled 2, 4, and 6 h after SA treatment. Treatment with ddH2O served as a control. B, Expression of VvPR1 in VvRAV1-overexpressing grapevine callus. Agroinfiltration of pGD–GFP served as a control. Calli were sampled 3 dpi. C, VvPR1 expression in VvRAV1-silenced callus. Agroinfiltration of the empty vector pCsuper1300-hp served as a negative control. Calli were sampled 2 dpi. D, VvRAV1 activates the transcription of GUS reporter gene under the control of the native VvPR1p. Grapevine callus was agroinfiltrated with the indicated construct(s) and GUS activity was determined 3 dpi. GUS activity induced by VvPR1p::GUS reporter alone was considered full promoter activity (set at 100%). E, EMSA showing that VvRAV1C–His binds to “TACCTG” element from VvPR1p. Binding specificity was analyzed using competitor unlabeled “TACCTG” probe. Leaves and stems of plantlets (A) or calli (B–D) from each treatment within each experiment were pooled for RNA or protein extraction. RT-qPCR results are shown as mean ± sd of three independent experiments. sd is denoted by error bars. Statistically significant differences between means were determined by employing Tukey’s multiple range test. **P < 0.01. Different letters indicate significant differences among different treatments (P < 0.05). Each assay was repeated in three individual experiments.

These findings, combined with the previous report that RAV1 induces PR genes (Hong et al., 2005; Sohn et al., 2006), led us to speculate that VvRAV1 might transcriptionally regulate VvPR1. To test this possibility, we first analyzed the effects of overexpression or silencing of VvRAV1 on VvPR1 expression. As shown in Figure 7B, expression of Myc–VvRAV1 resulted in a 144% increase in VvPR1 expression in grapevine callus 3 dpi, as compared to the controls. Similar results were also obtained in grapevine protoplasts (Supplemental Figure S8B). Subsequently, grapevine callus was agroinfiltrated with pRAV1–hp to silence VvRAV1. The transcript level of VvRAV1 in pRAV1–hp-infiltrated callus 2 dpi was ∼30% of that in controls. As expected, VvPR1 level in VvRAV1-silenced calli was 60% of that in controls (Figure 7C). These results indicated that VvRAV1 positively regulates VvPR1 in grapevine.

The VvPR1 promoter (VvPR1p) region was PCR-cloned to generate the reporter construct VvPR1p::GUS (Supplemental Figure S9). Effector construct pMyc–RAV1 and VvPR1p::GUS were co-transfected into grapevine callus and incubated for 3 d. When GFP was expressed, GUS activity was not affected; however, the relative GUS activity increased by ∼70% (Figure 7D) upon expression of Myc–VvRAV1 (Supplemental Figure S10), indicating that VvRAV1 can activate the transcription of GUS reporter gene driven by the native VvPR1p.

Analysis of the VvPR1p region revealed the B3 domain-recognition sequences “YRYCTS” (Y, C, or T; R, A, or G; S, G, or C; Yamasaki et al., 2004) (TACCTG), with 5 of the 6 nt matching the motif “CACCTG” targeted by the B3 domain of RAV (Kagaya et al., 1999). Thus, the DNA fragment of VvPR1p encompassing “TACCTG” sequences (Supplemental Figure S9A) was biotin-labeled and used as a probe. VvRAV1C–His specifically bound to “TACCTG” probe (Figure 7E). However, interestingly, no band shift was detected when GST–VvRAV1 was incubated with this probe, possibly because VvRAV1’s B3 domain was masked by the relatively big size of the GST tag.

These results demonstrated that VvRAV1 positively regulates VvPR1 by acting directly on its promoter.

Our results also revealed that SA promotes VvRAV1-mediated transcriptional activation of VvPR1p. Grapevine callus agroinfiltrated with VvPR1p::GUS and pMyc–RAV1 was treated with SA 3 dpi, and the Myc–VvRAV1-induced GUS activity was further increased by 80% 2 h after SA treatment (Figure 8A).

Figure 8.

Figure 8

Open in a new tab

Transcriptional activation of VvPR1 by VvRAV1 is enhanced by SA in a VvNPR1-independent manner. A, SA promotes transcriptional activation of VvPR1p by VvRAV1. Grapevine callus was agrotransfected with the indicated construct(s). SA (0.1 mM) treatment was performed at 3 dpi, and treatment with ddH2O served as the control. GUS activity was measured 2 h after SA treatment. B, In-vitro pull-down assay showing that GST–VvRAV1 does not interact with VvNPR1–His. Combination of p24–His/GST–VvRAV1 and p24G1–His/GST–VvRAV1 served as positive and negative controls, respectively. Arrows, asterisks, and arrowheads indicate the detected proteins of p24–His, p24G1–His, and VvNPR1–His, respectively. C, Effect of VvNPR1 on VvRAV1-mediated transcriptional activation of VvPR1p. Grapevine callus was agroinfiltrated with the indicated construct(s). GUS activity was measured 3 dpi. D, VvNPR1 does not affect the binding of VvRAV1C to “TACCTG” motif from VvPR1p. The indicated fusion protein or protein mixture was incubated with “TACCTG” probe, and GFP–His served as a control. GUS activity induced by VvPR1p::GUS reporter alone was considered full promoter activity (set at 100%), and results are shown as mean ± sd of three independent experiments (A and C). sd is denoted by error bars. Statistically significant differences between means were determined by employing Tukey’s multiple range test. **P < 0.01. Different letters indicate significant differences among different treatments (P < 0.05). Each assay was repeated three times.

SA-induced expression of PR1 in plants is dependent on, or independent of nonexpressor of PR1 (NPR1) (Zhang et al., 1999; van Verk et al., 2008; Hussain et al., 2018). The sequence of VvNPR1, an Arabidopsis NPR1 homolog in grapevine (Henanff et al., 2011), was cloned. Pull-down assay showed that GST–VvRAV1 was pulled down by p24–His, whereas no signal was observed when p24–His was replaced by VvNPR1–His or p24G1–His in the assay (Figure 8B), indicating that VvRAV1 does not interact with VvNPR1.

In addition, VvNPR1 neither affected VvPR1p activity nor influenced the GUS activity induced by VvRAV1 (Figure 8C). EMSA also showed that VvNPR1 could not bind the “TACCTG” probe and it had no effect on the binding affinity of VvRAV1C–His to the “TACCTG” probe (Figure 8D).

Together, our results demonstrated that SA-inducible VvRAV1 directly and positively regulates SA-responsive marker gene VvPR1 in a VvNPR1-independent manner, and SA enhances VvRAV1-mediated transcriptional activation of VvPR1p, indicating the role of VvRAV1 in the regulation of host defense responses.

GLRaV-2 p24 suppresses VvRAV1-mediated transcriptional activation of VvPR1 by reducing the binding of VvRAV1 to VvPR1p

Since some p24 proteins localized to the nucleus (Figure 4), might p24 influence the VvRAV1-mediated transcriptional activation of VvPR1? To explore this possibility, we first investigated whether SA is involved in grapevine defense against GLRaV-2. Grapevine callus was agroinfiltrated with GLRaV-2 infectious clone, and SA was administered 3 dpi. GLRaV-2 RNA accumulation in SA-treated grapevine callus was 50% lower than that in mock-treated controls 2 h posttreatment (Figure 9A), indicating that SA treatment enhances plant resistance to GLRaV-2.

Figure 9.

Figure 9

Open in a new tab

GLRaV-2 p24 attenuates VvRAV1-mediated defense responses. A, Effect of SA treatment on GLRaV-2 replication. Grapevine callus was agroinfiltrated with GLRaV-2 infectious clone and SA treatment was performed 3 dpi. Treatment with ddH2O served as a control. B and C, GLRaV-2 infection and p24 expression suppress VvPR1 expression. Grapevine callus was agroinfiltrated with GLRaV-2 infectious clone (B) or pGD–p24 (C); agroinfiltration of pGD–GUS served as a control. D, Transient transactivation assay showing that p24, but not GINV CP, reduces VvRAV1’s activation of VvPR1p transcription. E, p24 reduces VvRAV1 binding to “TACCTG” motif from VvPR1p. The indicated fusion protein(s) with His-tag or GST-tag (for VvRAV1) was incubated with “TACCTG” probe; GFP–His served as a control. F, p24 attenuates the promotive effect of SA on VvRAV1-mediated activation of VvPR1p. For transient transactivation assay (D and F), grapevine callus was transfected with the indicated construct(s) and incubated for 3 d. Relative GUS activity was normalized to the activity induced by VvPR1p::GUS, which was considered the full promoter activity (set at 100%). Calli from each treatment in each experiment were sampled 3 dpi (B–D) or 2 h after SA treatment (A and F) and pooled for RNA and protein extraction. RT–qPCR results are shown as mean ± sd of three independent experiments. sd is denoted by error bars. Statistically significant differences between means were determined by employing Tukey’s multiple range test. Different letters indicate significant differences among different treatments (P < 0.05). **P < 0.01. Each assay was repeated three times.

Grapevine callus was agroinfiltrated with the infectious cDNA clone vector of GLRaV-2 or pGD–p24. Upon GLRaV-2 infection or p24 expression (Supplemental Figure S11), VvRAV1 transcript at 3 dpi increased by ∼210% and 144%, respectively, while VvPR1 expression was only slightly increased, compared to the controls (Figure 9, B and C). These results indicated that GLRaV-2 infection or p24 expression might suppress VvRAV1-mediated transcriptional activation of VvPR1.

Transactivation assays showed that p24 by itself did not affect VvPR1p activity. However, GUS activity induced by Myc–VvRAV1 decreased by 43% in the presence of p24 (Figure 9D). The expression of VvRAV1, p24, and GFP was verified (Supplemental Figure S12). However, the presence of GINV CP did not affect the GUS activity triggered by Myc–VvRAV1 (Figure 9D, right panel).

In addition, incubation of p24–His with “TACCTG” probe did not result in a retarded band in the EMSA. However, when protein mixture VvRAV1C–His/p24–His was incubated with the probe, the retarded band became shallow, while this phenomenon was not observed when p24–His was replaced by GFP–His (Figure 9E). These results indicated that p24 reduces the DNA-binding affinity of VvRAV1 to VvPR1p.

Grapevine callus was then co-transfected with reporter and effector constructs, and treated with SA after incubation for 3 d. As compared to the ddH2O treatment, GUS activity induced by Myc–VvRAV1 or the combination of Myc–VvRAV1/GFP was further increased by ∼83% and 76%, respectively, after SA treatment; however, this increase was relatively low (50%) in the presence of p24 (Figure 9F). These results indicated that p24 attenuates the promotive effect of SA on VvRAV1-mediated activation of VvPR1p.

Together, our findings demonstrated that during GLRaV-2 infection, p24 attenuates VvRAV1-mediated activation of VvPR1 through a reduction in the DNA-binding affinity of VvRAV1 to VvPR1p by an unknown mechanism.

Discussion

Viruses employ mechanisms to subvert or manipulate host antiviral responses, and this usually relies on the interactions between viral RSSs and host proteins. Here, we report that GLRaV-2 p24 interacts with grapevine TF VvRAV1; it recruits VvRAV1 for its suppression of RNA silencing and attenuates VvRAV1-mediated defense responses, thereby promoting GLRaV-2 accumulation.

GLRaV-2 p24 hijacks VvRAV1 in the cytoplasm for its suppression of RNA silencing

Subcellular localization of host factors can be changed by interactions with viral proteins (Chen et al., 2017; Mei et al., 2018). VvRAV1 is also partially relocalized to the cytoplasm by p24 (Figure 4, C–E). p24 accumulates in the cytoplasm (Figure 4B), where antiviral silencing activity of RNA-induced silencing complex presumably takes place. Moreover, p24 also interacts with AtRAV2 and NtRAV (Figure 2C), which are required for RSS activities of HC-Pro and p38 (Endres et al., 2010). Therefore, one might wonder whether the relocalization of VvRAV1 to the cytoplasm affects p24’s suppression function. Our results showed that VvRAV1C–His, but not GST–VvRAV1, can bind 21-nt ds siRNA, whereas 21-nt ds siRNA binding by the protein mixtures p24–His/VvRAV1C–His and p24–His/GST–VvRAV1 was more efficient than the control (Figure 5A). On the other hand, neither VvRAV1C–His nor GST–VvRAV1 was able to bind the 24-nt ds siRNA and accordingly, the binding affinities of protein mixtures p24–His/VvRAV1C–His and p24–His/GST–VvRAV1 to 24-nt ds siRNA were similar to that of p24–His/GFP–His (Figure 5B). Moreover, VvRAV1 by itself could not suppress RNA silencing, whereas p24’s silencing suppression activity was notably enhanced in the presence of VvRAV1 (Figure 5, C and D). Based on these findings, we speculate that the C-terminus of VvRAV1 contains a domain that can bind 21-nt ds siRNA, but its surface may be hidden in the folded VvRAV1 protein under normal conditions, and therefore VvRAV1 alone cannot bind siRNA and suppress RNA silencing. However, during GLRaV-2 infection, p24 hijacks VvRAV1 to the cytoplasm and exposes its RNA-binding surface by an unknown mechanism, thereby engaging VvRAV1 to sequester 21-nt ds siRNA for its suppression of RNA silencing. Results from experiments using two p24 mutants further supported this hypothesis. R2A and R86A cannot efficiently suppress local (Li et al., 2018) and systemic (Figure 1) RNA silencing, possibly due to a mutation that leads to substantially reduced binding affinity to 21-nt ds siRNA and inability to bind 24-nt ds siRNA (Figure 1D). However, dependent on their interactions with VvRAV1 (Figure 2C), the binding affinity of protein mixtures R2A–His/GST–VvRAV1, R2A–His/VvRAV1C–His, R86A–His/GST–VvRAV1, and R86A–His/VvRAV1C–His to 21-nt ds siRNA was substantially increased (Figure 5A). Since the 21-nt class of siRNAs is the most abundant species and directs potent antiviral defense (Ding and Voinnet, 2007), the RSS activity of R2A or R86A was largely recovered in the presence of VvRAV1, comparable to that of WT p24 (Figure 5D).

Some plant factors have been identified to interact with viral RSSs and attenuate their RSS activities (Canto et al., 2006; Nakahara et al., 2012; Chen et al., 2017). In contrast, AtRAV2 and NtRAV have been shown to be required for RSS activities of potyvirus HC-Pro and carmovirus p38, though the mechanism is largely unclear (Endres et al., 2010). Based on experimental results, Lakatos et al. (2006) speculated that there is a plant factor that can increase the affinity of HC-Pro to siRNA; however, that factor was not identified. Our results provide the example of a viral RSS employing a host protein to bind 21-nt ds siRNA possibly by changing its intracellular localization and exposing its RNA-binding surface for suppression of RNA silencing.

In addition, VvRAV1N was not expressed (Supplemental Figures S2B and S5). Our results also demonstrate that VvRAV1C can efficiently bind 21-nt ds siRNA in vitro but attenuates p24’s RSS activity (Figure 5), although the reason behind this phenomenon is unclear. Nevertheless, these findings underlie the importance of VvRAV1C and VvRAV1N for the stability and function of VvRAV1, respectively.

GLRaV-2 p24 Attenuates VvRAV1-mediated defense responses

Increasing evidence indicates that PR1 gene activation is influenced at multiple levels. For example, NPR1 activates the expression of PR1 through interaction with TGA TFs (Zhang et al., 1999). The A. thaliana TF WRKY50 activates PR1 expression independent of TGAs or NPR1 (Hussain et al., 2018). An Arabidopsis plasma membrane-bound NAC TF induces PR1 expression independent of NPR1 and SA (Seo et al., 2010). Here, we present several lines of evidence to support the role of VvRAV1 in SA-induced expression of VvPR1. First, rapid activation of VvRAV1 by SA preceded the induction of VvPR1 (Figure 7A), suggesting that VvRAV1 may function as an upstream component of the SA signaling pathway. Second, transient overexpression and silencing of VvRAV1 led to upregulated and downregulated VvPR1 expression, respectively (Figure 7, B and C). Third, VvRAV1 activated the transcription of GUS reporter gene driven by VvPR1p (Figure 7D), and SA promoted VvRAV1-mediated transcriptional activation of VvPR1p (Figure 8A). Finally, VvRAV1C bound to the fragment of VvPR1p containing “TACCTG” in vitro, although GST-VvRAV1 could not (Figure 7E). Considering that Myc–VvRAV1 binds to VvPR1p and activates its transcription (Figure 7D), the inability of GST–VvRAV1 to bind the DNA probe might be due to masking of its B3 DNA-binding domain by the relatively large GST tag. Our results also suggest that VvRAV1 regulation of VvPR1 is independent of VvNPR1, because VvRAV1 did not interact with VvNPR1, and VvNPR1 had no obvious effect on the VvRAV1-mediated transcription activation of VvPR1p or the binding of VvRAV1C to the “TACCTG” probe (Figure 8, B–D). Therefore, these findings provide a mechanism for PR1 gene regulation. Future studies are needed to investigate whether other TFs are involved in transcriptional activation of VvPR1 by VvRAV1. Our results also suggest that VvRAV1 participates in the regulation of host defense responses, similar to the previous report that tomato RAV2 is a pivotal modulator involved in the AP2/ERF-mediated defense pathway (Li et al., 2011).

Consistent with previous observations for other viruses (Mayers et al., 2005; Alamillo et al., 2006; Wang et al., 2014), our results also revealed that SA is involved in grapevine defense against GLRaV-2 (Figure 9A). Considering that VvRAV1 directly upregulates VvPR1 expression, we hypothesize that the p24–VvRA1 interaction interferes with VvRAV1-mediated defense responses. Several lines of evidence in this study support this hypothesis. First, similar to the relocation of TBSV p19 from the cytoplasm to the nucleus by its interacting partner (Canto et al., 2006), a fraction of p24 entered into the nucleus (Figure 4, D and E), which raises the possibility that p24 may affect the transcriptional activity of VvRAV1. Second, both VvRAV1 and VvPR1 were induced in response to SA treatment (Figure 7A), whereas upon GLRaV-2 infection or p24 expression, VvPR1 expression was not obviously changed, despite the increased VvRAV1 expression (Figure 9, B and C). Third, p24 reduced the VvRAV1-mediated transcriptional activation of VvPR1p (Figure 9D), and the promotive effect of SA on VvRAV1 transactivation of the VvPR1p was also attenuated by p24 (Figure 9F). Finally, similar to several reported instances in which an interacting partner affected DNA-binding activity of a TF (Kim et al., 2012; Townsend et al., 2018), p24 obviously reduced the binding of VvRAV1 to “TACCTG” motif in VvPR1p (Figure 9E). Based on these results, together with findings that p24 interacts with VvRAV1 in the cytoplasm (Figure 2C), we speculate that p24 enters the nucleus via heterodimerization with the B3 domain of VvRAV1, and then quickly separates from the heterodimer, resulting in free VvRAV1 with a conformational change in its B3 domain that confers reduced DNA-binding activity on VvPR1p, thereby attenuating the VvRAV1-activated VvPR1 expression.

Evidence of the biological relevance of the specific interaction between p24 and VvRAV1

Both AtRAV2 and NtRAV interacted with p24 in the cytoplasm (Figure 2C). Furthermore, neither AtRAV2 nor NtRAV could suppress RNA silencing, but their presence obviously enhanced p24’s RSS activity (Figure 6B). Moreover, GST–NtRAV could not bind 21-nt ds siRNA, whereas binding of the protein mixture p24–His/GST–NtRAV to 21-nt ds siRNA was more efficient than that of the control (Figure 6A). These results suggest that p24 can also target and employ AtRAV2 and NtRAV for the benefit of its silencing suppression. AtRAV2 and NtRAV are targeted by HC-Pro and p38 and are required for their RSS activity (Endres et al., 2010). Our findings then raise the possibility that HC-Pro and p38 use a mechanism similar to that employed by p24. The identity of viral RSSs that can interact with and recruit VvRAV1 homologs for suppression of RNA silencing remains an interesting question for future research.

On the other hand, GLRaV-1 p24G1 or GINV CP did not interact with VvRAV1 (Figure 2C). Moreover, the RSS activity of p24G1 and CP, and CP’s siRNA binding affinity were not affected by VvRAV1 (Figure 6). Furthermore, GINV CP did not affect the transcriptional activation of VvPR1p by VvRAV1 (Figure 9D). In addition, transient overexpression or silencing of VvRAV1 slightly inhibited or promoted GINV RNA accumulation (Figure 3D).

Therefore, the results presented here highlight the biological importance of the specific p24–VvRAV1 interaction.

Proposed model for the p24–VvRAV1 interaction

We, therefore, propose a working model for how p24 promotes GLRaV-2 accumulation via interaction with VvRAV1 (Figure 10). VvRAV1 is induced in response to GLRaV-2 infection or treatment with SA. However, p24 employs VvRAV1 to bind 21-nt ds siRNA via changing VvRAV1’s subcellular localization and exposing the RNA-binding surface of VvRAV1, thereby enhancing its own RSS activity. Meanwhile, some of the p24 proteins enter the nucleus by binding to VvRAV1, and attenuate VvRAV1-mediated transcriptional activation of VvPR1p via reduced binding between the two. Thus, through interaction with VvRAV1, p24 suppresses RNA silencing and VvRAV1-mediated defense responses to promote GLRaV-2 accumulation.

Figure 10.

Figure 10

Open in a new tab

Working model for the p24/VvRAV1 interaction. A, VvRAV1 in the nucleus binds to “TACCTG” element of VvPR1p in response to SA in grapevine plants that are not infected with GLRaV-2. B, In the presence of GLRaV-2 infection, p24 recruits VvRAV1 in the cytoplasm to bind 21-nt ds siRNA for suppression of RNA silencing, and p24 reduces VvRAV1’s DNA-binding affinity to VvPR1p in the nucleus for suppression of VvRAV1-mediated defense responses, thereby promoting GLRaV-2 accumulation. VvRAV1F, VvRAV1 that has been released from the p24–VvAV1 complex.

Materials and methods

Plant material and growth conditions

Grapevine (V. vinifera) cv. Summer Black in vitro-grown plantlets and cv. Cabernet Sauvignon fruit-originated callus was free of grapevine viruses. Grapevine calli were cultured in B5 medium supplemented with 30 g L−1 sucrose, 0.1 mg L−1 naphthylacetic acid, 0.1 mg L−1 kinetin and 2,500 mg L−1 casein hydrolysates. Plantlets were grown on WPM (Woody Plant medium) supplemented with 30 g L−1 sucrose and 0.1 mg L−1 indole-3-butyric acid.

Seeds of two T6 generation p24-transgenic N. benthamiana lines (p24-6/Nb and p24-8/Nb) were kept in our laboratory. N. benthamiana GFP-transgenic line 16c, and p24-expressing transgenic and WT N. benthamiana plants were grown in individual pots containing commercial soil, and 4-week-old plants were used.

Plant materials were grown at 25°C–31°C with a 16-h light regime, except grapevine callus which was cultured at 25°C in the dark.

Preparation of plasmids

The primers used in this study are listed in Supplemental Table S1. The sequence of VvRAV1 was RT–PCR amplified from grapevine callus with primer pair F1/R1, and inserted into pMD19-T to produce pT–RAV1. Sequences of VvRAV1, VvRAV1N, VvRAV1C, and VvRAV1B3 were PCR amplified from pT–RAV1 with primer pairs F2/R2, F2/R3, F3/R2, and F4/R4, respectively. The PCR products, digested with BamHI/XhoI, were subcloned into pGADT7 (Clontech). The sequence of VvRAV1 was also PCR amplified with primer pair F5/R5, and PCR products digested with BamHI/PstI were subcloned into pGBKT7 (Clontech, Mountain View, CA, USA).

Sequences of VvRAV1, VvRAV1C, VvRAV1N, and VvRAV1B3 were PCR-cloned with primer pairs F6/R6, F7/R6, F6/R7, and F8/R8, respectively. The CP sequence was PCR amplified from an infectious cDNA clone vector of GINV with primer pair F9/R9. Sequences of NtRAV and AtRAV2 were RT-PCR amplified from tobacco (N. tabacum) and Arabidopsis (A. thaliana) leaves with primer pairs F10/R10 and F11/R11, respectively. The PCR products were digested with BamHI/XhoI or SpeI/SmaI (for AtRAV2) and inserted into the BiFC vectors pSPYNE-35S and pSPYCE-35S (Walter et al., 2004).

VvNPR1 was RT–PCR amplified from grapevine callus with primer pair F12/R12, and inserted into pMD19-T to produce pT–NPR1. Sequences of GUS and VvNPR1 were PCR amplified from pBI121 and pT–NPR1 using primers F13/R13 and F14/R14, respectively. Sequences of VvRAV1, VvRAV1C, VvRAV1B3, GINV CP, NtRAV, and AtRAV2 were PCR amplified with primer pairs F15/R15, F16/R15, F17/R16, F18/R17, F19/R18, and F20/R19, respectively. PCR products were digested with BamHI/SalI or HindIII/PstI (for AtRAV2) and subcloned into pGD (Goodin et al., 2002). Sequences of VvRAV1, VvRAV1N, and VvRAV1C were PCR amplified with primer pairs F21/R20, F21/R21, and F22/R20, respectively. PCR products, digested with BamHI/SalI, were subcloned into pCAMBIA1305-Myc.

VvRAV1 was PCR amplified with primer pair F23/R22 or F24/R23. PCR products digested with BamHI/SalI or XhoI/BamHI were inserted into pCAM35S–GFP or pGDG (Goodin et al., 2002). The sequence of p24 was PCR amplified with primer pair F25/R24, and PCR products digested with XhoI/BamHI were inserted into the pGDR vector (Goodin et al., 2002) to generate RFP fusion construct.

Sequences of p24, R2A, R86A, VvRAV1, VvRAV1C, VvRAV1N, VvRAV1B3, GINV CP, VvNPR1, and GFP were PCR amplified with primer pairs F26/R25, F27/R25, F26/R25, F28/R26, F29/R26, F28/R27, F30/R28, F31/R29, F32/R30, and F33/R31, respectively. PCR products were digested with BamHI/XhoI or BamHI/SalI (for VvNPR1) and cloned into pET28a. Sequences of VvRAV1 and NtRAV were amplified with primer pairs F34/R32 and F35/R33, and PCR products digested with BamHI/XhoI were inserted into the pEGX-4T-1 to generate GST-fusion constructs.

Two copies of the partial sequences of VvRAV1 were cloned using primer pairs F36/R34 and F37/R35, respectively. PCR products were digested with XhoI/SalI and BamHI/PstI, respectively, and inserted into pCsuper1300-hp as an inverted repeat to produce RNAi hairpin construct pRAV1–hp.

The promoter region of VvPR1 was PCR amplified from grapevine callus using primer pair F38/R36. The PCR products were digested with HindIII/XbaI and inserted into pBI121 to produce VvPR1p::GUS. All other constructs used in this study were described previously (Li et al., 2018; Zhang et al., 2020).

Yeast co-transformation assay

Each pair of plasmids was co-transformed into the yeast strain AH109 by small-scale yeast transformation method according to the manufacturer’s protocol (Clontech).

Protein expression and purification

Fusion constructs were respectively transformed into E. coli (BL21). The expression of fusion proteins was induced by adding 0.2-mM isopropyl β-d-thiogalactoside to the culture medium. The GST- or His-tagged fusion proteins were purified using GST- or His-Trap (Novagen, Madison, WI, USA) according to the manufacturer’s instructions.

Total plant protein extraction and western blot assay

Protein extraction and western blot assay were performed as described previously (Li et al., 2018). Dilutions of antibodies or antiserum (noncommercial antibody) were 1:5,000 (anti-Myc, anti-HA, anti-GFP, anti-GST, and anti-His) or 1:2,000 (anti-histone H3, anti-cFBPase, and anti-p24 antiserum). The hybridization signals were detected by using an enhanced chemiluminescence system (eECL Western Blot Kit) (ComWin, Jiangsu, China).

In-vitro pull-down assay

Fusion proteins were incubated in binding buffer (50-mM Tris–HCl at pH 7.5, 100-mM NaCl, 0.25% w/v Triton X-100, 35-mM β-mercaptoethanol) for 2 h at 4°C, then 25 μL of His-Trap (Novagen) was added and the mixture was incubated for an additional 1 h. The pulled-down proteins were detected using anti-GST antibody.

Agrobacterium-mediated transient expression of protein in N. benthamiana leaves

Agroinfiltration was performed essentially as described previously (Li et al., 2018). Agrobacterium tumefaciens GV3101 was transformed with each plasmid, and separately cultured to an optical density (600 nm) of ∼0.8. Agrobacterium cells were harvested by centrifugation and resuspended in 5 mL of infiltration buffer (10-mM MES/NaOH pH 5.6, 10-mM MgCl2, 150-μM acetosyringone), with equal densities of each A. tumefaciens carrying each construct in infiltration buffer. Each treatment within each experiment consisted of 7–21 plants.

Confocal microscopy and Northern blot analysis

Northern blot and laser scanning confocal microscopy were performed as previously described (Zhang et al., 2020). The sequences of the digoxigenin (DIG)-labeled cDNA probes used for detection of GFP mRNA are given in Supplemental Table S1. Generation of DIG-labeled cDNA probes and northern blots were conducted with the DIG High Prime DNA Labeling and Detection Starter Kit II (Roche, Mannheim, Germany).

Fluorescence signals were visualized and captured under an Olympus FluoView 3000 confocal microscope equipped with Olympus FluoView FV10-ASW 4.0 Viewer Software. GFP and YFP were excited at 488 nm and the emitted signal was captured at 500–540 nm. RFP was excited at 543 nm and the emitted signal was captured at 570–670 nm. Images were captured using 5% of the maximum light intensity value and gain of 600–650.

Nuclear–cytoplasmic fractionation

Infiltrated leaf patches from the same treatment were sampled 3 dpi and pooled. Nuclear–cytoplasmic fractionation was performed using Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, China) following the manufacturers’ protocols.

Agroinfiltration of callus and in vitro plantlets

Agrobacterium tumefaciens EHA105 was transformed with each plasmid. Grapevine callus (6 g) was incubated in 30 mL of Agrobacterium suspension (resuspended with MMA buffer) at 28°C for 30 min with shaking at 100 rpm. Callus was then removed from the Agrobacterium suspension using two layers of sterile gauze and blotted dry on sterile filter paper, transferred to B5 medium and kept in the dark at 25°C until sampling time or SA treatment. About 18–20 g callus was used for each treatment in each experiment.

Agroinfiltration of in-vitro grapevine plantlets was performed essentially as described previously (Kurth et al., 2012). Whole plantlets were submerged in an Agrobacterium suspension carrying various constructs and vacuum infiltrated (−83 kPa) for ∼20 min. The infiltrated grapevine plantlets were rinsed with sterile water, dried with sterile filter paper, and cultured on WPM at 25°C until sampling time. We used 8–12 plantlets for each treatment in each experiment.

Protoplast transfection

Isolation of grapevine (in vitro-grown plantlets) leaf protoplasts and polyethylene glycol-mediated transfection were performed essentially as described previously (Zhu et al., 2014) using Arabidopsis Protoplast Preparation and Transformation Kit (Beijing Kulaibo Technology Co., China). Approximately 100 μL of protoplasts was generally mixed with the constructs. The amount for each construct used in protoplast transfection was 10 μg. Nine Eppendorf tubes with 1 × 105 protoplasts each were used for each treatment within each experiment.

Promoter transactivation assay

Constructs were introduced into grapevine callus by agroinfiltration. A total of 18–20 g callus was used for each treatment within each experiment. The quantitative GUS-activity assay was performed essentially as described by Blázquez (2007) using 4-methylumbelliferyl-β-d-glucuronide as the substrate.

SA treatment

Grapevine callus (6 g) was sprayed with 5 mL of SA solution (0.1 mM) or ddH2O and excess fluid was absorbed by gentle contact with filter paper. Each whole plantlet was sprayed with 7 mL SA solution (0.1 mM). A total of 18–20 g callus or 9–12 plantlets were used for each treatment in each experiment.

EMSA

The VvPR1p fragment was synthesized by Sangon Biotech (Beijing, China) using biotin-labeled and unlabeled oligonucleotides. Two biotin-labeled 21-nt (sRNA1 and sRNA2) or 24-nt (sRNA3 and sRNA4) single-stranded RNA oligos synthesized by Sangon Biotech were annealed to form 21- or 24-nt ds siRNA probes as previously described (Duan et al., 2012). EMSA experiments were conducted using the LightShift Chemiluminescent EMSA Kit (Thermo Fisher Scientific, Waltham, MA, USA) or EMSA/Gel-Shift Kit (Beyotime, Jiangsu, China) following the manufacturers’ protocols.

RT–PCR and RT–qPCR

RT–PCR and RT–qPCR were performed as previously described (Zhang et al., 2020). Primer pairs F39/R37, F40/R38, F41/R39, F42/R40, F43/R41, and F44/R42 were used for analysis of the relative levels of VvRAV1, GFP, VvPR1, and VvActin (served as an internal control) mRNA and viral RNA (GLRaV-2 and GINV), respectively.

Statistical and phylogenetic analyses

The statistical software package SPSS (SPSS Inc., Chicago, IL, USA, 2001) was used for statistical analyses and significant differences between treatments and controls were determined using Tukey’s multiple range test. The phylogenetic tree was constructed by the Neighbor-Joining method with 1,000 bootstrap replicates in MEGA version 7 software package.

Accession numbers

Sequence data from this article can be found in the GenBank/Genoscope data libraries under the following accession numbers: NtRAV (ACF74549.1), AtRAV2 (AF360312_1), VvRAV1 (XP_002281709.2), VvPR1 (XM_002274032.3), VvNPR1 (Genoscope Vitis 8× accession number GSVIVT00031933001), and VvActin (XM_002282480.4).

Supplemental data

The following materials are available in the online version of this article.

Supplemental Figure S1. Sequence analysis and phylogenetic relationship between VvRAV1 and other plant RAV proteins.

Supplemental Figure S2. Transactivation analysis of VvRAV1 and detection of the expression of VvRAV1 and its mutants.

Supplemental Figure S3. GINV CP inhibits local RNA silencing and binds 21-nt ds siRNA.

Supplemental Figure S4. Subcellular localization of free GFP and RFP proteins and the phenotype of p24-transgenic N. benthamiana plants.

Supplemental Figure S5. Expression of His- and GST-tagged fusion proteins in E. coli.

Supplemental Figure S6. VvRAV1 enhances p24’s RSS activity in V. vinifera callus.

Supplemental Figure S7. Verification of the expression of VvRAV1, p24, and p24 mutants in N. benthamiana leaves.

Supplemental Figure S8. Analysis of VvRAV1 expression in response to MeJA, the effect of VvRAV1 on VvPR1 expression, and the role of VvPR1 in GLRaV-2 infection.

Supplemental Figure S9. DNA probe sequence in promoter of VvPR1 and schematic map of GUS reporter construct.

Supplemental Figure S10. Expression of Myc–VvRAV1 in V. vinifera callus.

Supplemental Figure S11. The accumulation of GLRaV-2 and p24.

Supplemental Figure S12. Verification of the expressed proteins.

Supplemental Table S1. Primers used in this study.

Supplementary Material

kiac181_Supplementary_Data
Click here for additional data file. (2.4MB, zip)

Acknowledgments

We thank Prof. Valerian V. Dolja (Oregon State University, Corvallis, OR) for providing the GLRaV-2 infectious clone, Prof. Andrew O. Jackson (University of California, Berkeley) for providing pGD and pGDR vectors, Prof. Jörg Kudla (Universität Münster, Germany) for providing the vectors used in the BiFC assay, Prof. Chengui Hang (Department of Plant Pathology, China Agricultural University) for providing the vector pCAMBIA1305-Myc, and Prof. Yafeng Dong (Research Institute of Pomology, Chinese Academy of Agriculture Sciences) for providing the GINV infectious clone.

Funding

This research was supported by National Natural Science Foundation of China (No. 32072552), the Earmarked Fund for Modern Agro-Industry Technology Research System (CARS-29-bc-3), and the Construction of Beijing Science and Technology Innovation and Service Capacity in Top Subjects (CEFF-PXM2019_014207_000032).

Conflict of interest statement. The authors declare no conflict of interest.

Contributor Information

Chenwei Zhang, Department of Pomology/Lab of Stress Physiology and Molecular Biology for Tree Fruits, Key Lab of Beijing Municipality, China Agricultural University, Beijing 100193, China.

Xianyou Wang, Department of Pomology/Lab of Stress Physiology and Molecular Biology for Tree Fruits, Key Lab of Beijing Municipality, China Agricultural University, Beijing 100193, China.

Hanwei Li, Department of Pomology/Lab of Stress Physiology and Molecular Biology for Tree Fruits, Key Lab of Beijing Municipality, China Agricultural University, Beijing 100193, China.

Jinying Wang, Department of Pomology/Lab of Stress Physiology and Molecular Biology for Tree Fruits, Key Lab of Beijing Municipality, China Agricultural University, Beijing 100193, China.

Qi Zeng, Department of Pomology/Lab of Stress Physiology and Molecular Biology for Tree Fruits, Key Lab of Beijing Municipality, China Agricultural University, Beijing 100193, China.

Wenting Huang, Department of Pomology/Lab of Stress Physiology and Molecular Biology for Tree Fruits, Key Lab of Beijing Municipality, China Agricultural University, Beijing 100193, China.

Haoqiang Huang, Department of Pomology/Lab of Stress Physiology and Molecular Biology for Tree Fruits, Key Lab of Beijing Municipality, China Agricultural University, Beijing 100193, China.

Yinshuai Xie, Department of Pomology/Lab of Stress Physiology and Molecular Biology for Tree Fruits, Key Lab of Beijing Municipality, China Agricultural University, Beijing 100193, China.

Shangzhen Yu, Department of Pomology/Lab of Stress Physiology and Molecular Biology for Tree Fruits, Key Lab of Beijing Municipality, China Agricultural University, Beijing 100193, China.

Qing Kan, Department of Pomology/Lab of Stress Physiology and Molecular Biology for Tree Fruits, Key Lab of Beijing Municipality, China Agricultural University, Beijing 100193, China.

Qi Wang, Department of Plant Pathology, China Agricultural University, Beijing 100193, China.

Yuqin Cheng, Department of Pomology/Lab of Stress Physiology and Molecular Biology for Tree Fruits, Key Lab of Beijing Municipality, China Agricultural University, Beijing 100193, China; Key Laboratory of Viticulture and Enology, Ministry of Agriculture, Beijing 100083, China.

 

Y.C., C.Z., and Q.W. designed the experiments. C.Z. performed most of the experiments. X.W. provided technical assistance to C.Z. H.L. performed vacuum-agroinfiltration. J.W. provided assistance with EMSA. Z.Q., H.H., and Q.K. provided the study materials. W.H. did formal analysis. Y.X. and S.Y. managed data. Y.C. wrote the paper, and all authors reviewed the manuscript.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (https://academic.oup.com/plphys/pages/general-instructions) is Yuqin Cheng (chengyuqin@cau.edu.cn).

References

  1. Alamillo JM, Saénz P, García JA (2006) Salicylic acid-mediated and RNA-silencing defense mechanisms cooperate in the restriction of systemic spread of plum pox virus in tobacco. Plant J 48: 217–222 [DOI] [PubMed] [Google Scholar]
  2. Angelini E, Aboughanem-Sabanadzovic N, Dolja VV, Meng B (2017) Grapevine leafroll-associated virus 2. InMeng B, Martelli G, Golino D, Fuchs M, eds. Grapevine Viruses: Molecular Biology, Diagnostics and Management. Springer, Cham, Switzerland, pp 141–165 [Google Scholar]
  3. Blázquez M (2007) Quantitative GUS activity assay of plant extracts. Cold Spring Harb Protoc 2007: pdb.prot4690 [DOI] [PubMed] [Google Scholar]
  4. Canto T, Uhrig JF, Swanson M, Wright KM, Macfarlane SA (2006) Translocation of Tomato bushy stunt virus P19 protein into the nucleus by ALY proteins compromises its silencing suppressor activity. J Virol 80: 9064–9072 [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chen C, Li Y, Zhang H, Ma Q, Wei Z, Chen J, Sun Z (2021) Genome-wide analysis of the RAV transcription factor genes in rice reveals their response patterns to hormones and virus infection. Viruses 13: 752. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chen L, Yan Z, Xia Z, Cheng Y, Jiao Z, Sun B, Zhou T, Fan Z (2017) A violaxanthin deepoxidase interacts with a viral suppressor of RNA silencing to inhibit virus amplification. Plant Physiol 175: 1774–1794 [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chiba M, Reed JC, Prokhnevsky AI, Chapman EJ, Mawassi M, Koonin EV, Carrington JC, Dolja VV (2006) Diverse suppressors of RNA silencing enhance agroinfection by a viral replicon. Virology 346: 7–14 [DOI] [PubMed] [Google Scholar]
  8. Ding SW, Voinnet O (2007) Antiviral immunity directed by small RNAs. Cell 130: 413–426 [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dolja VV, Kreuze JF, Valkonen JPT (2006) Comparative and functional genomics of closteroviruses. Virus Res 117: 38–51 [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Duan CG, Fang YY, Zhou BJ, Zhao JH, Hou WN, Zhu H, Ding SW, Guo HS (2012) Suppression of Arabidopsis ARGONAUTE1-mediated slicing, transgene-induced RNA silencing, and DNA methylation by distinct domains of the Cucumber mosaic virus 2b protein. Plant Cell 24: 259–274 [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Endres MW, Gregory BD, Gao Z, Foreman AW, Mlotshwa S, Ge X, Pruss GJ, Ecker JR, Bowman LH, Vance V (2010) Two plant viral suppressors of silencing require the ethylene-inducible host transcription factor RAV2 to block RNA silencing. PLoS Patho 6: e1000729. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fan X, Zhang Z, Ren F, Hu G, Li C, Zhang B, Dong Y (2020) Development of a full-length infectious cDNA clone of the grapevine berry inner necrosis virus. Plants 9: 1340. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Goodin MM, Dietzgen RG, Schichnes D, Ruzin S, Jackson AO (2002) pGD vectors: versatile tools for the expression of green and red fluorescent protein fusions in agroinfiltrated plant leaves. Plant J 31: 375–383 [DOI] [PubMed] [Google Scholar]
  14. Henanff GL, Farine S, Kieffer-Mazet F, Miclot AS, Heitz T, Mestre P, Bertsch C, Chong J (2011) Vitis vinifera VvNPR1.1 is the functional ortholog of AtNPR1 and its overexpression in grapevine triggers constitutive activation of PR genes and enhanced resistance to powdery mildew. Planta 234: 405–417 [DOI] [PubMed] [Google Scholar]
  15. Hong JK, Lee SC, Hwang BK (2005) Activation of pepper basic PR-1 gene promoter during defense signaling to pathogen, abiotic and environmental stresses. Gene 356: 169–180 [DOI] [PubMed] [Google Scholar]
  16. Hussain RMF, Sheikh AH, Haider I, Quareshy M, Linthorst H (2018) Arabidopsis WRKY50 and TGA transcription factors synergistically activate expression of PR1. Front Plant Sci 9: 930. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Jackson PA, Galinha CI, Pereira CS, Fortunato A, Soares NC, Amâncio SB, Pinto Ricardo CP (2001) Rapid deposition of extensin during the elicitation of grapevine callus cultures is specifically catalyzed by a 40-kilodalton peroxidase. Plant Physiol 127:1065–1076 [PMC free article] [PubMed] [Google Scholar]
  18. Kagaya Y, Ohmiya K, Hattori T (1999) RAV1, a novel DNA-binding protein, binds to bipartite recognition sequence through two distinct DNA-binding domains uniquely found in higher plants. Nucleic Acids Res 27: 470–478 [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kim HS, Park HC, Kim KE, Jung MS, Han HJ, Kim SH, Kwon YS, Bahk S, An J, Bae DW, et al. (2012) A NAC transcription factor and SNI1 cooperatively suppress basal pathogen resistance in Arabidopsis thaliana. Nucleic Acids Res 40: 9182–9192 [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kurth EG, Peremyslov VV, Prokhnevsky AI, Kasschau KD, Miller M, Carrington JC, Dolja VV (2012) Virus-derived gene expression and RNA interference vector for grapevine. J Virol 86: 6002–6009 [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lakatos L, Csorba T, Pantaleo V, Chapman EJ, Carrington JC, Liu YP, Dolja VV, Calvino LF, Lopez-Moya JJ, Burgyán J (2006) Small RNA binding is a common strategy to suppress RNA silencing by several viral suppressors. EMBO J 25: 2768–2780 [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Li CW, Su RC, Cheng CP, Sanjaya You SJ, Hsieh TH, Chao TC, Chan MT (2011) Tomato RAV transcription factor is a pivotal modulator involved in the AP2/EREBP-mediated defense pathway. Plant Physiol 156: 213–227 [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Li MJ, Zhang J, Feng M, Wang XY, Luo C, Wang Q, Cheng YQ (2018) Characterization of silencing suppressor p24 of Grapevine leafroll-associated virus 2. Mol Plant Pathol 19: 355–368 [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Licausi F, Ohme-Takagi M, Perata P (2013) APETALA2/Ethylene Responsive Factor (AP2/ERF) transcription factors: mediators of stress responses and developmental programs. New Phytol 199: 639–649 [DOI] [PubMed] [Google Scholar]
  25. Liu YP, Peremyslov VV, Medina V, Dolja VV (2009) Tandem leader proteases of Grapevine leafroll-associated virus 2: host-specific functions in the infection cycle. Virology 383: 291–299 [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lozano-Duran R, Rosas-Diaz T, Gusmaroli G, Luna AP, Taconnat L, Deng XW, Bejarano ER (2011) Geminiviruses subvert ubiquitination by altering CSN-mediated derubylation of SCF E3 ligase complexes and inhibit jasmonate signaling in Arabidopsis thaliana. Plant Cell 23: 1014–1032 [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Malnoy M, Viola R, Jung MH, Koo OJ, Kim S, Kim JS, Velasco R, Nagamangala Kanchiswamy C (2016) DNA-free genetically edited grapevine and apple protoplast using CRISPR/Cas9 ribonucleoproteins. Front Plant Sci 7: 1904. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Mayers CN, Lee KC, Moore CA, Wong SM, Carr JP (2005) Salicylic acid-induced resistance to Cucumber mosaic virus in squash and Arabidopsis thaliana: contrasting mechanisms of induction and antiviral action. Mol Plant Microbe Interact 18: 428–434 [DOI] [PubMed] [Google Scholar]
  29. Mei Y, Yang X, Huang C, Zhang X, Zhou X (2018) Tomato leaf curl Yunnan virus-encoded C4 induces cell division through enhancing stability of Cyclin D 1.1 via impairing NbSKη-mediated phosphorylation in Nicotiana benthamiana. PLoS Pathog 14: e1006789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Nakahara KS, Masuta C, Yamada S, Shimura H, Kashihara Y, Wada TS, Meguro A, Goto K, Tadamura K, Sueda K, et al. (2012) Tobacco calmodulin-like protein provides secondary defense by binding to and directing degradation of virus RNA silencing suppressors. Proc Natl Acad Sci USA 109: 10113–10118 [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Nakahara KS, Masuta (2014) Interaction between viral RNA silencing suppressors and host factors in plant immunity. Curr Opin Plant Biol 20: 88–95 [DOI] [PubMed] [Google Scholar]
  32. Niehl A, Wyrsch I, Boller T, Heinlein M (2016) Double-stranded RNAs induce a pattern-triggered immune signaling pathway in plants. New Phytol 211: 1008–1019 [DOI] [PubMed] [Google Scholar]
  33. Park JM, Park CJ, Lee SB, Ham BK, Shin R, Paek KH (2001) Overexpression of the tobacco Tsi1 gene encoding an EREBP/AP2-type transcription factor enhances resistance against pathogen attack and osmotic stress in tobacco. Plant Cell 13: 1035–1046 [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Poque S, Wu HW, Huang CH, Cheng HW, Hu WC, Yang JY, Wang D, Yeh SD (2018) Potyviral gene-silencing suppressor HCPro interacts with salicylic acid (SA)-binding protein 3 to weaken SA-mediated defense responses. Mol Plant Microbe Interact 31: 86–100 [DOI] [PubMed] [Google Scholar]
  35. Pré M, Atallah M, Champion A, De Vos M, Pieterse CM, Memelink J (2008) The AP2/ERF domain transcription factor ORA59 integrates jasmonic acid and ethylene signals in plant defense. Plant Physiol 147: 1347–1357 [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Seo PJ, Kim MJ, Park JY, Kim SY, Jeon J, Lee YH, Kim J, Park CM (2010) Cold activation of a plasma membrane-tethered NAC transcription factor induces a pathogen resistance response in Arabidopsis. Plant J 61: 661–671 [DOI] [PubMed] [Google Scholar]
  37. Sohn KH, Lee SC, Jung HW, Hong JK, Hwang BK (2006) Expression and functional roles of the pepper pathogen-induced transcription factor RAV1 in bacterial disease resistance, and drought and salt stress tolerance. Plant Mol Biol 61: 897–915 [DOI] [PubMed] [Google Scholar]
  38. Song Y, Hanner RH, Meng B (2021) Probing into the effects of grapevine leafroll-associated viruses on the physiology, fruit quality and gene expression of grapes. Viruses 13: 593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Townsend PD, Dixon CH, Slootweg EJ, Sukarta OCA, Yang AWH, Hughes TR, Sharples GJ, Pålsson LO, Takken FLW, Goverse A, et al. (2018) The intracellular immune receptor Rx1 regulates the DNA-binding activity of a Golden2-like transcription factor. J Biol Chem 293: 3218–3233 [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Tsuda K, Somssich IE (2015) Transcriptional networks in plant immunity. New Phytol 206: 932–947 [DOI] [PubMed] [Google Scholar]
  41. van Verk MC, Pappaioannou D, Neeleman L, Bol JF, Linthorst H (2008) A novel WRKY transcription factor is required for induction of PR-1a gene expression by salicylic acid and bacterial elicitors. Plant Physiol 146: 1983–1995 [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Voinnet O, Baulcombe  D (1997) Systemic signalling in gene silencing. Nature 389: 553. [DOI] [PubMed] [Google Scholar]
  43. Walter M, Chaban C, Schütze K, Batistic O, Weckermann K, Näke C, Blazevic D, Grefen C, Schumacher K, Oecking C, et al. (2004) Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation. Plant J 40: 428–438 [DOI] [PubMed] [Google Scholar]
  44. Wang Q, Liu Y, He J, Zheng X, Hu J, Liu Y, Dai H, Zhang Y, Wang B, Wu W, et al. (2014) STV11 encodes a sulphotransferase and confers durable resistance to rice stripe virus. Nat Commun 5: 4768. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Wang X, Goregaoker SP, Culver JN (2009) Interaction of the Tobacco mosaic virus replicase protein with a NAC domain transcription factor is associated with the suppression of systemic host defenses. J Virol 83: 9720–9730 [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Yamasaki K, Kigawa T, Inoue M, Tateno M, Yamasaki T, Yabuki T, Aoki M, Seki E, Matsuda T, Tomo Y, et al. (2004) Solution structure of the B3 DNA binding domain of the Arabidopsis cold-responsive transcription factor RAV1. Plant Cell 16: 3448–3459 [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Zhang CW, Liu Q, Zeng Q, Huang WT, Cheng YQ (2020) p24G1 encoded by grapevine leafroll-associated virus 1 suppresses RNA silencing and elicits hypersensitive response-like necrosis in Nicotiana species. Viruses 12: 1111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Zhang Y, Fan W, Kinkema M, Li X, Dong X (1999) Interaction of NPR1 with basic leucine zipper protein transcription factors that bind sequences required for salicylic acid induction of the PR-1 gene. Proc Natl Acad Sci USA 96: 6523–6528 [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Zhu M, Chen Y, Ding XS, Webb SL, Zhou T, Nelson RS, Fan Z (2014) Maize Elongin C interacts with the viral genome-linked protein, VPg, of Sugarcane mosaic virus and facilitates virus infection. New Phytol 203:1291–1304 [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

kiac181_Supplementary_Data
Click here for additional data file. (2.4MB, zip)

Articles from Plant Physiology are provided here courtesy of Oxford University Press

RESOURCES