Skip to main content
PMC home page
Journal of Experimental Botany logoLink to Journal of Experimental Botany
. 2016 Apr 19;67(11):3353–3365. doi: 10.1093/jxb/erw155

A petunia ethylene-responsive element binding factor, PhERF2, plays an important role in antiviral RNA silencing

Daoyang Sun 1,2, Raja Sekhar Nandety 3, Yanlong Zhang 1, Michael S Reid 2, Lixin Niu 1,*, Cai-Zhong Jiang 4,*
PMCID: PMC4892726  PMID: 27099376

Highlight

PhERF2, an ethylene-responsive element binding factor, acts as a positive transcriptional regulator in antiviral RNA silencing and is essential for efficient silencing of genes in plants.

Key words: Argonaute, cucumber, mosaic, virus, dicer-like enzyme, RNA-dependent RNA polymerase, tobacco, rattle, virus, transcription factor, virus-induced gene silencing.

Abstract

Virus-induced RNA silencing is involved in plant antiviral defense and requires key enzyme components, including RNA-dependent RNA polymerases (RDRs), Dicer-like RNase III enzymes (DCLs), and Argonaute proteins (AGOs). However, the transcriptional regulation of these critical components is largely unknown. In petunia (Petunia hybrida), an ethylene-responsive element binding factor, PhERF2, is induced by Tobacco rattle virus (TRV) infection. Inclusion of a PhERF2 fragment in a TRV silencing construct containing reporter fragments of phytoene desaturase (PDS) or chalcone synthase (CHS) substantially impaired silencing efficiency of both the PDS and CHS reporters. Silencing was also impaired in PhERF2- RNAi lines, where TRV-PhPDS infection did not show the expected silencing phenotype (photobleaching). In contrast, photobleaching in response to infiltration with the TRV-PhPDS construct was enhanced in plants overexpressing PhERF2. Transcript abundance of the RNA silencing-related genes RDR2, RDR6, DCL2, and AGO2 was lower in PhERF2-silenced plants but higher in PhERF2-overexpressing plants. Moreover, PhERF2-silenced lines showed higher susceptibility to Cucumber mosaic virus (CMV) than wild-type (WT) plants, while plants overexpressing PhERF2 exhibited increased resistance. Interestingly, growth and development of PhERF2-RNAi lines were substantially slower, whereas the overexpressing lines were more vigorous than the controls. Taken together, our results indicate that PhERF2 functions as a positive regulator in antiviral RNA silencing.

Introduction

Virus-induced gene silencing (VIGS) is a rapid and effective method for functional characterization of genes in a plant. A recombinant plant virus carrying a host-derived sequence fragment initiates RNA-mediated post-transcriptional gene silencing (PTGS), leading to a transient and specific degradation of the corresponding endogenous mRNA (Kumagai et al., 1995; Dinesh-Kumar et al., 2003; Di Stilio et al., 2010; Tian et al., 2014). A modified Tobacco rattle virus (TRV) vector has proved to be an excellent tool for VIGS, due to its wide host range and its ability to infect meristematic cells (Di Stilio et al., 2010). This system is composed of binary transformation plasmids, TRV1 and TRV2, with a region harboring multiple cloning sites in TRV2 (Liu et al., 2002). The TRV vector has been used successfully for gene function analysis in a number of eudicots, including the commonly used model plants Arabidopsis (Turnage et al., 2002; Burch-Smith et al., 2006; Wang et al., 2006), Nicotiana benthamiana (Jones et al., 2006; Peterson et al., 2013), N. tabacum (Lukhovitskaya et al., 2013), tomato (Solanum lycopersicum; Liu et al., 2002; Fu et al., 2005; Jiang et al., 2008; Senthil‐Kumar and Mysore, 2011; Fragkostefanakis et al., 2014) and petunia (Petunia hybrida; Chen et al., 2004; Spitzer et al., 2007; Broderick and Jones, 2014; Chang et al., 2014). In petunia, silencing of phytoene desaturase (PDS) or chalcone synthase (CHS) provides useful phenotypical markers in VIGS studies for functional characterization of genes in leaf and floral tissues, respectively (Chen et al., 2004; Reid et al., 2009; Jiang et al., 2011).

The silencing efficiency of the VIGS system is variable, largely depending on post-inoculation growth temperature and compatibility between the host and the virus. Growth temperature seems to have profound effects on the efficiency of VIGS-based gene silencing. Gene silencing efficiency with a TRV system in tomato is enhanced by low temperature and low humidity (Fu et al., 2006). Low temperature also enhances gene silencing efficiency throughout the life of cotton plants when geminivirus-mediated VIGS is employed (Tuttle et al., 2008). But in contrast, Szittya et al. (2003) reported that low temperature inhibits silencing by preventing siRNA generation in N. benthamiana protoplasts transfected with Cymbidium ringspot virus (CymRSV). The underlying mechanisms of these apparently conflicting findings are not yet well understood. Previously, we tested the effects of silencing CHS on a range of purple-flowered petunia cultivars and found significant variations in the silencing phenotypes (Chen et al., 2004; Reid et al., 2009). In studies with silencing PDS in tomato we have also observed cultivar-dependent variations in the silencing phenotype (Jiang et al., 2011). Compatibility has limited the range of taxa where TRV-VIGS has successfully been employed. The genetic basis for the variation and limitation is largely unknown.

The antiviral RNA silencing process involves a set of crucial cellular enzymes including RNA-dependent RNA polymerases (RDRs), Dicer-like RNase III enzymes (DCLs), and Argonaute proteins (AGOs). These components contribute to the generation, cleavage of template double-stranded RNA (dsRNA), and interaction with virus-derived small interfering RNA (vsiRNA), respectively. The vsiRNA is incorporated into a RNA-induced silencing complex (RISC) for post-transcriptional suppression of the homologous RNA molecules (Burch-Smith et al., 2006; Gould and Kramer, 2007; Jaubert et al., 2011; Tian et al., 2014). In Arabidopsis thaliana, four DCLs, six RDRs, and ten AGOs serve as critical components of the silencing machinery (Donaire et al., 2008; Zhang et al., 2012). DCL1 has a unique function for the production of 21-nucleotide (nt) microRNAs (miRNAs) that is required for coordination of the complex biological functions within the plant (Dunoyer et al., 2005). DCL2, DCL3, and DCL4 catalyse exogenous RNAs with double-stranded features into 22-, 24- and 21-nt siRNAs (Dunoyer et al., 2005; Donaire et al., 2008; Axtell, 2013), respectively. Double deficiency of DCL2 and DCL4 confers more susceptibility to RNA virus infection, such as TRV (Deleris et al., 2006). Among the six RDRs, RDR1, RDR2, and RDR6 participate in the defensive response against distinct positive-strand RNA viruses through the biogenesis of viral secondary siRNAs in Arabidopsis (Donaire et al., 2008; Garcia-Ruiz et al., 2010; Wang et al., 2010, 2011). Current evidence indicates that AGO1 is mainly implicated in the miRNAs silencing pathway (Baumberger and Baulcombe, 2005). Loss-of-function mutation in AGO2 results in an impaired resistance to 2b suppressor-deficient Cucumber mosaic virus (CMV) (Wang et al., 2011) and Potato virus X (PVX) (Jaubert et al., 2011) in Arabidopsis, and Tomato bushy stunt virus (TBSV) (Scholthof et al., 2011) in N. benthamiana. Both AGO1 and AGO2 proteins bind to vsiRNAs (Zhang et al., 2006; Takeda et al., 2008; Wang et al., 2011), thereby guiding the downstream silencing process in the virus-infected cells. To date, however, few transcription factors have been reported to be involved in the transcriptional regulation of these critical components.

We have used petunia as a model system for our studies of flower senescence. Transcriptome analysis has identified a cluster of genes that are strongly up-regulated during development and senescence of the flowers, including many transcription factors (Wang et al., 2013). We have successfully employed the TRV-based VIGS vector and a visual reporter such as CHS to study the function of some of these genes (Chen et al., 2004; Reid et al., 2009; Jiang et al., 2011; Chang et al., 2014; Yin et al., 2015). In the course of these studies, we have noted lack of CHS-silencing phenotype in the case of simultaneous silencing of CHS and a PhERF2 gene encoding an ethylene response transcription factor. Given the importance of ethylene in plant responses to stress, we hypothesized that this observation might indicate an important role for this transcription factor in the antiviral RNA silencing process. The experiments reported here were designed to test this hypothesis.

Materials and methods

Plant materials and growth conditions

Petunia (Petunia × hybrida, ‘Primetime Blue’, or Petunia × hybrida ‘Mitchell Diploid’) seeds were planted in a 72-well plastic tray filled with sterile UC soil mix and germinated at room temperature with a 16/8h day/night photoperiod. Seedlings at the 4-leaf stage were transferred to small pots for inoculation with Agrobacterium bearing TRV constructs, or infected with CMV, then maintained in a growth chamber at 25/20 oC day/night with a same light/dark cycle. To determine the abundance of gene transcripts in petunia plants inoculated with various TRV constructs, RNA was extracted from the inoculated leaves, the uppermost fully-expanded leaves, or from flowers at anthesis. Young leaves from petunia plants (‘Mitchell Diploid’) grown in a greenhouse under 25/20 oC day/night temperature and natural photoperiods were collected and used for stable transformation.

Isolation and sequence analysis of PhERF2

An 802-bp EST sequence was identified among up-regulated genes during petunia flower development from a microarray analysis (Wang et al., 2013). Using this partial sequence, a further BLAST search in the NCBI Genbank (http://blast.ncbi.nlm.nih.gov/) was carried out to identify a full-length sequence encoding an ethyleneresponsive element binding factor 2, annotated as PhERF2. Protein homologues of PhERF2 were identified through the BLAST search in the non-redundant GenBank protein databases. A NCBI web server (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) was used to determine the conserved domain. Multiple alignments of the ERF2 proteins and phylogenic tree analysis were carried out using the ClustalW program (http://www.genome.jp/tools/clustalw/) and MEGA4 software.

Abiotic stress and hormone treatments

To examine the effects of abiotic stress and hormone treatments on the PhERF2 expression profile, 3-week-old seedlings were used. For the salinity and drought treatments, the plants were placed in a vial with distilled water (control), water containing 100mM NaCl, or without water at room temperature (20 oC). For the cold treatment, the seedlings were placed in a cold room at 4 oC. For the ethylene treatment, the plants were placed in a sealed glass chamber and exposed to 10 µl l–1 ethylene in air. For treatments with other hormones, plants were treated with solutions containing 50 μM abscisic acid (ABA), 50 μM gibberellic acid (GA3), 200 μM salicylic acid (SA), or 200 μM methyl jasmonate (MeJA). In each case, three individual plants were collected at 0, 3, 6, 12 and 24h post-treatment, then frozen in liquid nitrogen and stored at –80 oC.

Virus-induced gene silencing

The TRV vectors TRV1 and TRV2 have been described previously (Liu et al., 2002). Our experiments were carried out with previously generated TRV-PhPDS or TRV-PhCHS constructs, in which a 138-bp PDS or 194-bp CHS fragment was cloned into the EcoRI or XbaI-EcoRI restriction sites in the TRV2 vector, respectively (Chen et al., 2004). To create the TRV-PhPDS or TRV-PhCHS construct with a fragment of the gene of interest, SacI and XhoI were used to digest the pDAH11 vector containing a 291-bp or 339-bp fragment of PhERF2 or a 246-bp fragment of PhERF3, and the resulting product was ligated into the corresponding site in TRV-PhPDS or TRV-PhCHS (Chen et al., 2004). The recombinant plasmids were transformed into Agrobacterium tumefaciens strain GV3101 using electroporation (Reid et al., 2009; Jiang et al., 2011). The Agrobacterium strains were cultured in 15ml LB media (40mg l–1 kanamycin, 20mg l–1 gentamicin, 10mM MES and 20 μM acetosyringone) at 28 oC in a growth chamber for 48h. Agrobacterium cultures were centrifuged at 3000 g for 20min, and the pelleted cells were then resuspended in infiltration buffer (10mM MgCl2, 10mM MES and 200 μM acetosyringone) to an OD600 of 4.0. The suspensions were shaken gently at room temperature for 3–5h before inoculation. Prior to inoculation, Agrobacterium cultures bearing the TRV1 and TRV2 or TRV2-derivatives were mixed together in equal volumes to a final OD600 of 2.0. A 1-ml disposal syringe was used to infiltrate the Agrobacterium mixture into the leaves of petunia seedlings (Reid et al., 2009; Jiang et al., 2011).

Semi-quantitative and real-time quantitative RT-PCR

Total RNA was extracted from the leaves and flowers of petunia plants using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and purified with RNase-free DNase I (Promega, Madison, WI, USA), according to the manufacturer’s protocol. First-strand cDNA was synthesized from 2–5 μg total RNA with M-MLV reverse transcriptase (Invitrogen, Carlsbad, CA, USA). Real-time quantitative RT-PCR was carried out using the SYBR Green PCR Master Mix (2X) in an ABI7300 instrument (Applied Biosystem, Foster City, CA, USA) as previously described (Liang et al., 2014). 26S ribosomal RNA served as an internal control for normalization of cDNA (Chen et al., 2004; Reid et al., 2009). PCR primers to sequences beyond the region of the inserted fragment for silencing were used for determination of transcript abundance of targeted genes. To confirm accumulation of TRV2 by semi-quantitative RT-PCR, two primer pairs were produced (see Supplementary Table S1 at JXB online), of which one (TRV2-1) covered the multiple cloning sites (MCS) in TRV2, so that the size of resulting product varied depending on the inserts in the site, whereas the other (TRV2-2), targeted the region upstream of the MCS and generated bands of uniform size (Supplementary Table S1) (Reid et al., 2009).

Northern blot assay

Total RNA was isolated from the newly expanded leaves and fully open flowers of inoculated petunia plants, and low-molecular-weight RNA was precipitated using a solution of polyethylene glycol (PEG8000) and NaCl (Wang et al., 2004b). Blot hybridization was carried out as previously described (Donaire et al., 2008). The fragments of PDS (138 bps) or CHS (194 bps) corresponding to the silencing region were labeled with [γ-32P] ATP to generate DNA probes for assessing abundance of PDS- or CHS-derived siRNAs.

Measurement of ethylene production

Leaves of petunia seedlings inoculated with TRV empty vector were harvested at different time intervals and sealed in a 50-ml plastic tube at 25 oC for 4h. A 3-ml gas sample was taken from the tube using a gas-tight syringe, and injected into a gas chromatograph (GC-8A; Shimadzu, Kyoto, Japan) for measurement of ethylene concentration, as previously described (Chang et al., 2014; Yin et al., 2015).

Overexpression and RNAi silencing constructs

A 1137-bp DNA fragment containing the ORF region of the PhERF2 was PCR-amplified using the primer pair oxPhERF2F1 and oxPhERF2R1 (see Supplementary Table S1). The amplified product was cloned into the pDAH11 vector and then transferred to a pGSA1403 vector in the forward orientation to produce the 35S::PhERF2 construct (Chang et al., 2014; Yin et al., 2015). To generate the RNAi construct, primers rnaiPhERF2F1 and rnaiPhERF2R1 (Supplementary Table S1), carrying a SpeI-AscI and a BamHI-SwaI adaptor, respectively, were designed to amplify a 339-bp fragment. The amplified PCR products were cloned into the pDAH11 vector. The sense and antisense fragments were released from digestion of the pDAH11 plasmid and cloned into the pGSA1285 binary vector. The constructs were sequenced to confirm their fidelity as previously described (Liang et al., 2014).

Stable transformation

The generated constructs were introduced into Agrobacterium tumefaciens strain LBA4404. The transformed bacteria were selected by incubation on an LB plate containing 25mg l–1 chloramphenicol at 28 oC for 72h. One colony was selected and cultured in YEP medium (10g l–1 yeast extract, 10g l–1 peptone and 5g l–1 NaCl) with appropriate antibiotics for 48h, and adjusted to an OD600 of 0.3 using sterile LB media without antibiotics. Leaves of ‘Mitchell Diploid’ petunias were inoculated with Agrobacterium and regenerated as previously described (Wang et al., 2013; Liang et al., 2014). Putative transgenic petunia plants were grown to flowering. The seeds were harvested and germinated in MS plates containing 100mg l–1 kanamycin for selection (Estrada-Melo et al., 2015; Yin et al., 2015).

Inoculation assay with CMV

Cucumber mosaic virus (CMV) inoculum was obtained from leaves of Oriental hybrid lily (Lilium) cultivar ‘Siberia’. Infectious sap was prepared by extracting CMV-infected leaves in 100mM phosphate buffer, pH 7.0 (1:4 w/v). The fully expanded leaves of 4-week-old healthy petunia seedlings were mechanically inoculated with virus preparations as previously described (Hull, 2009). To ensure an efficient infection, the inoculation process was carried out once more after 24h. Subsequently, inoculated plants were maintained in the growth chamber at 22 oC for evaluation of symptoms. Transcript levels for the CMV coat protein were examined by real-time quantitative RT-PCR (Liang et al., 2014; Estrada-Melo et al., 2015).

Results

Identification of PhERF2

An 802-bp EST corresponding to a putative ethylene-responsive element binding factor 2 (ERF2) gene was identified among genes up-regulated during flower development in petunia from a microarray analysis (Wang et al., 2013). A further BLAST search in the NCBI Genbank revealed a full-length mRNA sequence (accession number HQ259596, NCBI) annotated as PhERF2 (see Supplementary Fig. S1). Analysis of the deduced polypeptide encoded by PhERF2 using the BLAST search against non-redundant GenBank databases in the NCBI revealed homologous proteins from diverse plant species. Amino acid alignment and phylogenetic analysis showed that PhERF2 is relatively close to PhERF3 in petunia and to other proteins, such as Solanum lycopersicum SlJERF1 (NP_001234513), S. tuberosum StRAP2.12 (XP_006342909), Prunus salicina PsERF2a (ACM49847), Nelumbo nucifera NnRAP2.12 (XP_010273830), Malus domestica MdRAP2.12 (NP_001280975), Nicotiana tabacum NtCEF1 (Lee et al., 2005), Capsicum annuum CaPF1 (Yi et al., 2004) and Arabidopsis thaliana AtRAP2.12 (Zhao et al., 2012; Ding et al., 2013) (Supplementary Fig. S2).

Biotic, abiotic stresses and hormone treatments induce PhERF2 expression

To investigate whether transcript levels of PhERF2 are induced by TRV infection, wild-type (WT) leaves of petunia plants at the 4-leaf stage were inoculated with Agrobacterium bearing an empty TRV vector. PhERF2 transcripts increased significantly in inoculated leaves by 36h post-inoculation (hpi) (Fig. 1A). By comparison, infection with Agrobacterium without the TRV plasmid (mock control) did not affect PhERF2 transcript levels (Fig. 1A). In systemically infected upper leaves, PhERF2 transcript abundance increased dramatically between 10 and 15 d post-inoculation (dpi), and remained high thereafter and until 25 dpi (Fig. 1B). As plant hormones and stresses play important roles in the antiviral RNA silencing processes, we examined the expression pattern of PhERF2 in WT leaves in response to different plant growth regulator and abiotic stress treatments. The expression of PhERF2 increased following exposure to low temperature, salt stress, and dehydration (Fig. 1C). Transcript levels of PhERF2 also increased following treatments with SA, ABA, ethylene, and MeJA but not GA3 (Fig. 1D).

Fig. 1.

Fig. 1.

Open in a new tab

Induction of PhERF2 expression in petunia leaves infected by TRV, or treated with abiotic stresses or plant hormones. Quantitative RT-PCR analysis of PhERF2 transcript levels in (A) inoculated and (B) systemically infected (uppermost) leaves at different time points, using 3-week-old WT seedlings infiltrated with Agrobacterium bearing no TRV construct (mock control) or TRV empty vector. Quantitative RT-PCR analysis of PhERF2 transcript abundance in response to abiotic stresses (C) or plant growth regulators (D). Three-week-old WT seedlings were placed in vials with water (control), or treated with a continuous 10 μl l–1 ethylene, or with solutions containing either 100mM NaCl, 50 μM ABA, 50 μM GA3, 200 μM SA, or 200 μM MeJA, or without water (dehydration) at room temperature, or with water at 4 oC. Transcript abundances were standardized to 26S rRNA. Error bars represent SE of the means from three biological replicates.

Simultaneous silencing of PhERF2 and PDS or CHS impairs VIGS efficiency

VIGS has proved to be a fast and efficient method to silence genes in petunia. Therefore, to study the function of PhERF2, we employed a TRV-based VIGS system to silence PhERF2 in the leaves and corollas, using PDS and CHS as visual reporters. The mock-treated and empty vector-inoculated plants showed the WT phenotype with green leaves and purple flowers (Figs 2A and 3A). In plants infected with the TRV-PhPDS construct, upper leaves showed a clear PDS-silenced photobleaching phenotype by 3 weeks post-infiltration (wpi) (Fig. 2A). The flowers on the plants infected with the TRV-PhCHS construct were largely white, corresponding to the silenced phenotype of CHS (Fig. 3A).

Fig. 2.

Fig. 2.

Open in a new tab

Failed development of photobleaching phenotype in the leaves of VIGS-silenced PhERF2 plants using PDS as a visual reporter. (A) Representative phenotypes of WT plants 3 and 5 weeks post-inoculation (wpi) with non-transformed Agrobacterium (mock control), or Agrobacterium bearing a TRV empty vector, TRV-PhPDS, TRV-PhPDS/ERF2, TRV-PhPDS/ERF3, and TRV-PhPDS/ERF2 (fragment 2, F2) constructs. (B), (C) Semi-quantitative and quantitative RT-PCR analysis of transcript abundances for PhERF2, PDS, TRV RNA1 (TRV1), and TRV RNA2 (TRV2-1, -2) in uppermost younger leaves of plants 3 wpi. Primers for TRV2-1 were designed from the region outside the multiple cloning site (MCS) in the vector and produced variable sizes of products depending on the presence of the PhPDS, PhPDS/ERF2, and PhPDS/ERF3 inserts in the TRV2 vector. Primers for TRV2-2 were used to produce the same sizes of products from the TRV2 vector. Relative accumulation levels were normalized to 26S rRNA. Error bars represent SE of the means from three biological replicates. Different letters indicate statistical significance as calculated by Duncan’s multiple range test at P<0.05. (D) Quantitative RT-PCR analysis of transcript abundances for RNA silencing-related genes, including RDR1, RDR2, RDR6, DCL1, DCL2, DCL3, DCL4, AGO1, AGO2, and AGO4, in uppermost younger leaves of plants at 3 wpi. Abundance of 26S rRNA was used as an internal control. Error bars represent SE of the means from three biological replicates. Different letters denote statistical significance using Duncan’s multiple range test at P<0.05. (E) Northern-blot analysis of PDS insert-derived siRNA levels in topmost younger leaves of plants at 3 wpi. 32P-labeled oligonucleotide probes corresponding to the PDS insert sequence were used for detection. Ethidium bromide-stained 5S rRNA served as a loading control.

Fig. 3.

Fig. 3.

Open in a new tab

Failed development of the white-corollas phenotype in the flowers of VIGS-silenced PhERF2 plants using CHS as a visual reporter. (A) Representative phenotypes of WT plants 7 weeks post-inoculation (wpi) with non-transformed Agrobacterium (mock control), or Agrobacterium bearing the TRV empty vector, TRV-PhCHS, TRV-PhCHS/ERF2, TRV-PhCHS/ERF3, and TRV-PhCHS/ERF2 (fragment 2, F2) constructs. (B), (C) Semi-quantitative and quantitative RT-PCR analysis of transcript abundances for CHS, PhERF2, TRV RNA1 (TRV1), and TRV RNA2 (TRV2-1, -2) in the corollas. Flowers were harvested at anthesis 7 wpi. Primers for TRV2-1 were designed from the region outside the multiple cloning site (MCS) in the vector and produced variable sizes of products depending on the presence of the PhCHS, PhCHS/ERF2, and PhCHS/ERF3 inserts in the TRV2 vector. Primers for TRV2-2 were used for quantitative RT-PCR to produce the same sizes of products from the TRV2 vector. Abundance of 26S rRNA was used as an internal control. SE of the means from three biological replicates are indicated by the error bars. Different letters denote statistical significance using Duncan’s multiple range test at P<0.05. (D) Quantitative RT-PCR analysis of transcript abundances for RNA silencing-related genes, including RDR1, RDR2, RDR6, DCL1, DCL2, DCL3, DCL4, AGO1, AGO2, and AGO4, in the corollas. Flowers were harvested at anthesis 7 wpi. Abundance of 26S rRNA was used as an internal control. Error bars represent the SE of the means from three biological replicates. Different letters denote statistical significance using Duncan’s multiple range test at P<0.05. (E) Northern-blot analysis of CHS insert-derived siRNA levels in the corollas, harvested at anthesis, of plants at 7 wpi. 32P-labeled oligonucleotide probes corresponding to CHS insert sequence were used for detection. Ethidium bromide-stained 5S rRNA was served as a loading control.

The plants infected with the TRV-PhPDS/ERF2 or TRV-PhCHS/ERF2 construct, bearing a 291-bp sequence from the 5′ end of the PhERF2 cDNA, failed to show the normal foliar (PDS-photobleaching leaves) and floral (CHS-white flowers) silencing phenotypes (Figs 2A and 3A). These results seemed to be specific to PhERF2, since infection with a TRV-PhPDS/ERF3 or TRV-PhCHS/ERF3 construct bearing a 246-bp fragment of PhERF3, a paralog of PhERF2 (see Supplementary Fig. S2B), produced the normal silencing phenotypes in leaves and flowers (Figs 2A and 3A). Moreover, infection with a TRV-PhPDS/ERF2 or TRV-PhCHS/ERF2 vector bearing a different 339-bp fragment (Fragment 2) from the 3′ region of the PhERF2 cDNA also failed to elicit the silencing phenotypes (Figs 2A and 3A).

VIGS silencing of PhERF2 does not suppress TRV movement or replication

To understand why upper leaves of plants infected with TRV-PhPDS/ERF2 displayed none of the photobleaching seen in those from plants infected with TRV-PhPDS and TRV-PhPDS/ERF3 (Fig. 2A), we performed semi-quantitative and real-time quantitative RT-PCR to analyse transcript abundances of genes including PhERF2, PDS, CHS, TRV RNA1, and RNA2. The results revealed a 50% reduction of PhERF2 transcript levels in leaves from TRV-PhPDS/ERF2-infected plants, and 2-fold increases in transcripts in leaves from TRV-PhPDS- and TRV-PhPDS/ERF3-infected plants, compared to the mock control (Fig. 2B, C). PDS transcript abundance negatively correlated with the photobleaching in the upper leaves (Fig. 2C). Abundance of PDS transcript in the TRV-PhPDS- and TRV-PhPDS/ERF3-infected plants was reduced by over 90% whereas PDS abundance was only 20% less than the controls in the TRV-PhPDS/ERF2-infected plants (Fig. 2C). Accumulation of TRV RNA1 (TRV1) and RNA2 (TRV2) in the leaves of plants agro-infiltrated with an empty vector or TRV-PhPDS/ERF2 constructs was significantly higher than in those of TRV-PhPDS- and TRV-PhPDS/ERF3-infected plants (Fig. 2B, C). Similar results were obtained when petunia plants were infected with the TRV-PhCHS/ERF2 construct using a flower-specific visual reporter (CHS) (Fig. 3A–C).

VIGS Silencing of PhERF2 affects expression of RNA silencing-related genes

To study the role of PhERF2 in the RNA silencing process, we determined transcript abundances of a number of putative RNA silencing-related genes. Infection with TRV empty vector or silencing constructs resulted in changes in expression of RDRs (RDR1, 2, 6), DCLs (DCL1–4) and AGOs (AGO1, 2, 4) (Fig. 2D). Almost all the selected genes were up-regulated by infection with the TRV empty vector compared to the mock control (Fig. 2D). Up-regulation was considerably higher for RDR2, DCL2, and AGO2 in leaves of plants showing the PDS-silenced photobleaching phenotype (TRV-PhPDS and TRV-PhPDS/ERF3) (Fig. 2D). However, this increase was not observed in leaves from TRV-PhPDS/ERF2-infected plants, in which RDR6 expression levels were also significantly lower than in photobleached leaves (Fig. 2D).

Using the PDS silencing insert as a labeled probe for hybridization, we conducted northern blot analysis to visualize the abundance of TRV-PhPDS-derived siRNAs. Leaves from TRV-PhPDS- and TRV-PhPDS/ERF3-infected plants contained a 22-nt RNA fragment that bound to the labeled probe (Fig. 2E). But there was no detectable signal in leaves from TRV-PhPDS/ERF2-infected plants (Fig. 2E). Similar patterns from the RNA silencing-related gene expression and northern blot analyses were obtained using corollas of petunia plants inoculated with TRV-PhCHS-based constructs (Fig. 3D, E).

PhERF2-RNAi silencing impairs VIGS efficiency

To further study the function of PhERF2, we generated PhERF2-RNAi lines in petunia. Growth and development of PhERF2-RNAi lines was substantially slower than WT plants (Fig. 4A). The PhERF2-RNAi lines (#1 and #4) showed substantial reduction in PhERF2 transcript abundance (Fig. 4B). The transcript abundance of RNA silencing-related genes, including RDR2, RDR6, DCL2, and AGO2, was also significantly lower in these RNAi-silencing lines (Fig. 4C). When these lines were infected with the TRV-PhPDS silencing construct, they showed no, or much reduced, photobleaching (Fig. 4D), and a concomitant high abundance of PDS, TRV RNA1, and RNA2 transcripts in comparison to the photobleached leaves of infected WT plants (Fig. 4E).

Fig. 4.

Fig. 4.

Open in a new tab

Impairment of photobleaching phenotype in PhERF2- RNAi lines inoculated with Agrobacterium bearing TRV-PhPDS. (A) Representative growth phenotypes of WT and PhERF2-RNAi lines (#1 and #4) 40 d post-germination. Note that PhERF2-RNAi lines showed delayed growth. (B), (C) Quantitative RT-PCR analysis of transcript abundances for PhERF2, RDR1, RDR2, RDR6, DCL1, DCL2, DCL3, DCL4, AGO1, AGO2, and AGO4 in the leaves of WT and PhERF2-RNAi lines (#1 and #4). Uppermost leaves of 4-week-old plants were used. (D) Representative phenotypes of WT and PhERF2-RNAi lines (#1 and #4) inoculated with Agrobacterium bearing no TRV vector (mock control) or a TRV-PhPDS construct at 4 and 7 weeks post-inoculation. (E) Quantitative RT-PCR analysis of transcript abundances for PDS, TRV RNA1 (TRV1), and TRV RNA2 (TRV2) in uppermost leaves of WT and PhERF2-RNAi lines (#1 and #4) inoculated with Agrobacterium bearing no TRV vector (mock control) or a TRV-PhPDS construct. Transcript abundances were standardized to 26S rRNA. Error bars represent the SE of the means from three biological replicates (B, C, E). Asterisks (B, C) or different letters (E) denote statistical significance using Duncan’s multiple range test at P<0.05.

Overexpression of PhERF2 enhances silencing

To further investigate the role of PhERF2 in the antiviral RNA silencing process, we also generated PhERF2 overexpression lines in petunia. Transgenic plants overexpressing PhERF2 under the control of the Cauliflower mosaic virus (CaMV) 35S promoter grew faster (Fig. 5A), and had up to six times the PhERF2 transcript abundance found in WT plants (Fig. 5B). Transcripts of the RNA silencing-related genes including RDR2, RDR6, DCL2, and AGO2 were significantly higher in the overexpressing lines than in the WT controls (Fig. 5C). When infected with the TRV-PhPDS silencing construct, the PhERF2-overexpressing lines showed similar silencing phenotypes and reduction in PDS transcripts to those seen in TRV-PhPDS-inoculated WT plants (Fig. 5D, E). The accumulation of TRV RNA1 and RNA2 was lower in the overexpressing lines than in the WT when inoculated with TRV-PhPDS construct (Fig. 5E).

Fig. 5.

Fig. 5.

Open in a new tab

Photobleaching phenotypes of PhERF2-overexpressing lines inoculated with Agrobacterium bearing a TRV-PhPDS construct. (A) Representative growth phenotypes of WT and PhERF2-overexpressing (OE) lines (#C, #D and #I) 30 d post-germination. Note that overexpression of PhERF2 enhanced plant growth. (B), (C) Quantitative RT-PCR analysis of transcript abundances for PhERF2, RDR2, RDR6, DCL2, and AGO2 in the leaves of WT and PhERF2-OE plants (#C, #D and #I). Samples were harvested from uppermost leaves of 4-week-old plants. (D) Representative phenotypes of WT and PhERF2-OE lines (#C, #D and #I) inoculated with Agrobacterium bearing no TRV vector (mock control) or a TRV-PhPDS construct at 4 and 6 weeks post-inoculation (wpi). (E) Quantitative RT-PCR analysis of transcript abundances for PDS and TRV RNA1 (TRV1), and TRV RNA2 (TRV2) in uppermost leaves of WT and PhERF2-OE lines (#C, #D and #I) inoculated with Agrobacterium bearing no TRV vector (mock control) or a TRV-PhPDS construct at 4 wpi. Abundance of 26S rRNA was used as an internal control. Error bars represent the SE of the means from three biological replicates (B, C, E). Asterisks (B, C) or different letters (E) denote statistical significance using Duncan’s multiple range test at P<0.05.

PhERF2 affects disease susceptibility

Given that PhERF2 is an important factor in antiviral RNA silencing against TRV, the role of PhERF2 in response to Cucumber mosaic virus (CMV) was then examined. PhERF2- RNAi lines inoculated with CMV showed more distortion and yellow mottling of systemically infected leaves than WT plants (Fig. 6A). In contrast, infected PhERF2-overexpressing lines had much milder symptoms and continued growth (Fig. 6A). These phenotypes were correlated with measured accumulations of CMV coat protein (CP) transcripts, which were significantly higher in PhERF2-RNAi lines and dramatically lower in PhERF2-overexpressing lines than in WT plants (Fig. 6B).

Fig. 6.

Fig. 6.

Open in a new tab

Involvement of PhERF2 in the defense of petunia plants against CMV. (A) Disease symptoms of WT, PhERF2-RNAi (#1 and #4), and PhERF2-overexpressing (OE) (#C, #D and #I) lines inoculated with CMV at 2 weeks post-inoculation; 4-week-old seedlings were used for inoculation. (B) Quantitative RT-PCR analysis of CMV coat protein (CP) transcript abundances in systemically infected (uppermost) leaves of WT, PhERF2-RNAi (#1 and #4), and PhERF2-OE (#C, #D and #I) lines at 10 and 14 d post-inoculation. Transcript abundances were standardized to 26S rRNA. Error bars represent the SE of the means from three biological replicates. Asterisks denote statistical significance using Duncan’s multiple range test at P<0.05.

Discussion

The data presented here support our hypothesis that the ethylene-responsive element binding factor PhERF2 plays an important role in antiviral RNA silencing (see Supplementary Fig. S3). ERFs belong to the plant-specific AP2/ERF transcription factor superfamily, whose members share a highly conserved AP2/ERF DNA binding domain with a length of 57–66 amino acids (Okamuro et al., 1997). In Arabidopsis, there are a total of 122 putative ERF genes (Nakano et al., 2006). They appear to bind to a cis-acting GCC box with a core AGCCGCC motif (Hao et al., 1998) that is commonly found in the promoter region of defense-related genes downstream from the ethylene signaling pathway (Ohme-Takagi and Shinshi, 1995).

Ethylene is the key regulator in the senescence of petunia flowers, and we have been dissecting the control of the process by silencing structural and regulatory genes, including ERF genes, in the ethylene response cascade (Wang et al., 2013; Chang et al., 2014; Yin et al., 2015). When we silenced PhERF2, using a TRV vector containing a fragment of PhERF2 and either a foliar or floral reporter gene (PDS or CHS, respectively), we noted a dramatic reduction in VIGS efficiency, as demonstrated by very little photobleaching in the leaves or white corollas on the normally purple flowers (Figs 2A and 3A). PhERF2 transcripts were abundant in control plants infected with the empty TRV vector. These findings led to the hypothesis that PhERF2 might play an important role in the transcriptional regulation of the silencing response system.

Antiviral RNA silencing involves up-regulation of a number of genes, including RDRs, DCLs, and AGOs, that are required for the generation and cleavage of double-stranded RNA (dsRNA) and its processing into virus-derived small interfering RNAs (vsiRNA). In Arabidopsis, four DCLs, six RDRs and ten AGOs serve as critical components of the silencing machinery (Donaire et al., 2008; Zhang et al., 2012). DCL2, DCL3, and DCL4 catalyse the cleavage of exogenous RNAs with double-stranded features into 22-, 24- and 21-nt siRNAs, respectively (Dunoyer et al., 2005; Donaire et al., 2008; Axtell, 2013).

We found that the silencing efficiency of both PDS and CHS reporters affected by silencing or overexpression of PhERF2 was associated with substantial changes in abundance of silencing-associated genes, including RDR2, RDR6, DCL2, and AGO2. It appears likely that PhERF2 modifies the transcription of these genes by binding cis-elements in their promoters. This possibility requires further examination in the future.

Many researchers have examined the effects of modulating expression of genes in the silencing complex on disease resistance. In Nicotiana benthamiana, for example, RDR6 was shown to be critical in resistance to CMV (Mourrain et al., 2000) and Potato virus X (PVX) (Schwach et al., 2005). In Arabidopsis, DCL mutants accumulate higher titers of TCV and CMV (Bouché et al., 2006), TRV (Donaire et al., 2008), Turnip mosaic virus (TuMV) (Garcia-Ruiz et al., 2010), and Tobacco mosaic virus (TMV) (Lewsey and Carr, 2009). Our observations of increased and reduced TRV accumulations in PhERF2-silenced (Fig. 4E) and -overexpressing (Fig. 5E) petunias, respectively, and of the negative correlation between PhERF2 expression and susceptibility to CMV (Fig. 6) are consistent with these findings, and support the hypothesis that PhERF2 plays a regulatory role in the antiviral RNA silencing process. Mutants of RAP2.2, the Arabidopsis homolog of PhERF2, showed increased susceptibility to Botrytis cinerea, while its overexpression resulted in enhanced resistance to this pathogen (Zhao et al., 2012). It seems possible that PhERF2 might be involved in a general response to disease pressure, and future studies will examine the effect of up- and down-regulation of this transcription factor on susceptibility to fungal pathogens.

We were particularly interested in the roles of PhERF2 and ethylene in plant growth and development and stress responses. Expression analysis demonstrated that PhERF2 was also up-regulated in plants treated with the hormones SA, ABA, ethylene, and MeJA. These regulators sometimes function as growth inhibitors, and the interplay between PhERF2 and these regulators may explain the substantial reduction in growth rate that was observed in silenced petunias (Fig. 4A) or the improvement of plant growth in the overexpressing lines (Fig. 5A). Although petunia corollas are ethylene sensitive (Reid and Wu, 1992), there was no significant change in flower longevity in transgenic plants where PhERF2 was silenced or overexpressed (see Supplementary Table S2). Presumably PhERF2 is not involved in ethylene-triggered floral senescence. We did, however, find that abiotic factors, such as cold, salt, and drought, enhanced the levels of PhERF2 (Fig. 1C). This is consistent with studies on PhERF2 homologs in other species. NtCEF1 from tobacco is induced by cold and salt (Lee et al., 2005). Salt treatment increases mRNA levels of JERF1 (Zhang et al., 2004) and JERF3 (Wang et al., 2004a) in tobacco, and plants overexpressing these genes were more tolerant of salt stress. CaPF1 in pepper is rapidly and strongly activated under cold and salt conditions, and upregulation of this gene confers freezing tolerance (Yi et al., 2004). Future work will include a study of the response of silenced and overexpressing plants to a range of abiotic stresses.

Our results suggest that PhERF2 plays an important role in antiviral RNA silencing. The identification of PhERF2 as a positive regulator in the TRV-based VIGS system may provide a valuable solution to enhance the VIGS response when silencing efficiency is low, as has been observed in some species in which the TRV-based VIGS system has been tested (e.g. woody species) (Shen et al., 2015). Thus it may be possible to transiently overexpress the PhERF2 or homologous proteins simultaneously or prior to the inoculation of silencing vectors. Or the inoculated plants could also be treated with ethylene or SA to increase the endogenous ERF2-like expression, which may lead to enhancement of the silencing efficiency. It is also worth mentioning that enhanced PhERF2 expression by low temperature may lead to improvement of gene silencing efficiency in VIGS systems. This notion is supported by the fact that gene silencing efficiency is enhanced by low temperature with a TRV-based VIGS system in tomato (Fu et al., 2006) and with a geminivirus-mediated VIGS system in cotton plants (Tuttle et al., 2008). It would also be interesting in the future to examine whether PhERF2 plays any positive roles in other VIGS systems.

Supplementary data

Supplementary data are available at JXB online.

Figure S1. Complete sequence of petunia PhERF2 cDNA.

Figure S2. Amino acid sequence analysis of petunia PhERF2.

Figure S3. A proposed model for the roles of PhERF2 in antiviral RNA silencing.

Table S1. Primers used for semi-quantitative and real-time quantitative RT-PCR.

Table S2. The longevity of attached flowers from WT, PhERF2-RNAi and -overexpressing plants.

Supplementary Data
supp_67_11_3353__index.html (1,001B, html)

Acknowledgements

We thank Dr Bryce Falk for technical assistance in the northern blot analysis and Xiaoqing Xie for help in the transformation experiments. Dr Weixing Shan kindly hosted some of the experiments in his laboratory. We appreciate Lee Ann Richmond’s assistance with the analytical instrumentation. This work was partially supported by United States Department of Agriculture (USDA) CRIS project 5306-21000-019-00D.

References

  1. Axtell MJ. 2013. Classification and comparison of small RNAs from plants. Annual Review of Plant Biology 64, 137–159. [DOI] [PubMed] [Google Scholar]
  2. Baumberger N, Baulcombe D. 2005. Arabidopsis argonaute1 is an RNA slicer that selectively recruits microRNAs and short interfering RNAs. Proceedings of the National Academy of Sciences, USA 102, 11928–11933. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bouché N, Lauressergues D, Gasciolli V, Vaucheret H. 2006. An antagonistic function for Arabidopsis DCL2 in development and a new function for DCL4 in generating viral siRNAs. The EMBO Journal 25, 3347–3356. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Broderick SR, Jones ML. 2014. An optimized protocol to increase virus-induced gene silencing efficiency and minimize viral symptoms in petunia. Plant Molecular Biology Reporter 32, 219–233. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Burch-Smith TM, Schiff M, Liu Y, Dinesh-Kumar SP. 2006. Efficient virus-induced gene silencing in Arabidopsis . Plant Physiology 142, 21–27. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chang X, Donnelly L, Sun D, Rao J, Reid MS, Jiang CZ. 2014. A petunia homeodomain-leucine zipper protein, PhHD-Zip, plays an important role in flower senescence. PLoS One 9, e88320. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chen JC, Jiang CZ, Gookin T, Hunter D, Clark D, Reid MS. 2004. Chalcone synthase as a reporter in virus-induced gene silencing studies of flower senescence. Plant Molecular Biology 55, 521–530. [DOI] [PubMed] [Google Scholar]
  8. Deleris A, Gallego-Bartolome J, Bao J, Kasschau KD, Carrington JC, Voinnet O. 2006. Hierarchical action and inhibition of plant dicer-like proteins in antiviral defense. Science 313, 68–71. [DOI] [PubMed] [Google Scholar]
  9. Di Stilio VS, Kumar RA, Oddone AM, Tolkin TR, Salles P, McCarty K. 2010. Virus-induced gene silencing as a tool for comparative functional studies in Thalictrum . PLoS One 5, e12064. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dinesh-Kumar S, Anandalakshmi R, Marathe R, Schiff M, Liu Y. 2003. Virus-induced gene silencing. Methods in Molecular Biology 236, 287–293. [DOI] [PubMed] [Google Scholar]
  11. Ding Y, Liu N, Virlouvet L, Riethoven JJ, Fromm M, Avramova Z. 2013. Four distinct types of dehydration stress memory genes in Arabidopsis thaliana . BMC Plant Biology 13, 229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Donaire L, Barajas D, Martínez-García B, Martínez-Priego L, Pagán I, Llave C. 2008. Structural and genetic requirements for the biogenesis of Tobacco rattle virus-derived small interfering RNAs. Journal of Virology 82, 5167–5177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Dunoyer P, Himber C, Voinnet O. 2005. Dicer-like 4 is required for RNA interference and produces the 21-nucleotide small interfering RNA component of the plant cell-to-cell silencing signal. Nature Genetics 37, 1356–1360. [DOI] [PubMed] [Google Scholar]
  14. Estrada-Melo AC, Chao, Reid MS, Jiang CZ. 2015. Overexpression of an ABA biosynthesis gene using a stress-inducible promoter enhances drought resistance in petunia. Horticulture Research 2, 15013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Fragkostefanakis S, Sedeek KEM, Raad M, Zaki MS, Kalaitzis P. 2014. Virus induced gene silencing of three putative prolyl 4-hydroxylases enhances plant growth in tomato (Solanum lycopersicum). Plant Molecular Biology 85, 459–471. [DOI] [PubMed] [Google Scholar]
  16. Fu DQ, Zhu BZ, Zhu HL, Jiang WB, Luo YB. 2005. Virus-induced gene silencing in tomato fruit. The Plant Journal 43, 299–308. [DOI] [PubMed] [Google Scholar]
  17. Fu DQ, Zhu B, Zhu H, Zhang H, Xie Y, Jiang W, Zhao X, Luo Y. 2006. Enhancement of virus-induced gene silencing in tomato by low temperature and low humidity. Molecules and Cells 21, 153–160. [PubMed] [Google Scholar]
  18. Garcia-Ruiz H, Takeda A, Chapman EJ, Sullivan CM, Fahlgren N, Brempelis KJ, Carrington JC. 2010. Arabidopsis RNA-dependent RNA polymerases and dicer-like proteins in antiviral defense and small interfering RNA biogenesis during Turnip mosaic virus infection. The Plant Cell 22, 481–496. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Gould B, Kramer EM. 2007. Virus-induced gene silencing as a tool for functional analyses in the emerging model plant Aquilegia (columbine, Ranunculaceae). Plant Methods 3, 6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hao D, Ohme-Takagi M, Sarai A. 1998. Unique mode of GCC box recognition by the DNA-binding domain of ethylene-responsive element-binding factor (ERF domain) in plant. Journal of Biological Chemistry 273, 26857–26861. [DOI] [PubMed] [Google Scholar]
  21. Hull R. 2009. Mechanical inoculation of plant viruses. Current Protocols in Microbiology , 16B.16.11–16B.16.14. [DOI] [PubMed] [Google Scholar]
  22. Jaubert M, Bhattacharjee S, Mello AF, Perry KL, Moffett P. 2011. Argonaute2 mediates RNA-silencing antiviral defenses against Potato virus X in Arabidopsis . Plant Physiology 156, 1556–1564. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Jiang CZ, Chen JC, Reid MS. 2011. Virus-induced gene silencing in ornamental plants. In Kodama H, Komamin A, eds. RNAi and plant gene function analysis . Springer, 81–96. [DOI] [PubMed] [Google Scholar]
  24. Jiang CZ, Lu F, Imsabai W, Meir S, Reid MS. 2008. Silencing polygalacturonase expression inhibits tomato petiole abscission. Journal of Experimental Botany 59, 973–979. [DOI] [PubMed] [Google Scholar]
  25. Jones L, Keining T, Eamens A, Vaistij FE. 2006. Virus-induced gene silencing of argonaute genes in Nicotiana benthamiana demonstrates that extensive systemic silencing requires argonaute1-like and argonaute4-like genes. Plant Physiology 141, 598–606. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kumagai M, Donson J, Della-Cioppa G, Harvey D, Hanley K, Grill L. 1995. Cytoplasmic inhibition of carotenoid biosynthesis with virus-derived RNA. Proceedings of the National Academy of Sciences, USA 92, 1679–1683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Lee JH, Kim DM, Lee JH, Kim J, Bang JW, Kim WT, Pai HS. 2005. Functional characterization of NtCEF1, an AP2/EREBP-type transcriptional activator highly expressed in tobacco callus. Planta 222, 211–224. [DOI] [PubMed] [Google Scholar]
  28. Lewsey MG, Carr JP. 2009. Effects of dicer-like proteins 2, 3 and 4 on Cucumber mosaic virus and Tobacco mosaic virus infections in salicylic acid-treated plants. Journal of General Virology 90, 3010–3014. [DOI] [PubMed] [Google Scholar]
  29. Liang YC, Reid MS, Jiang CZ. 2014. Controlling plant architecture by manipulation of gibberellic acid signalling in petunia. Horticulture Research 1, 14061. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Liu Y, Schiff M, Dinesh-Kumar S. 2002. Virus-induced gene silencing in tomato. The Plant Journal 31, 777–786. [DOI] [PubMed] [Google Scholar]
  31. Lukhovitskaya NI, Solovieva AD, Boddeti SK, Thaduri S, Solovyev AG, Savenkov EI. 2013. An RNA virus-encoded zinc-finger protein acts as a plant transcription factor and induces a regulator of cell size and proliferation in two tobacco species. The Plant Cell 25, 960–973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Mourrain P, Béclin C, Elmayan T, Feuerbach F, Godon C, Morel JB, Jouette D, Lacombe AM, Nikic S, Picault N. 2000. Arabidopsis SGS2 and SGS3 genes are required for post-transcriptional gene silencing and natural virus resistance. Cell 101, 533–542. [DOI] [PubMed] [Google Scholar]
  33. Nakano T, Suzuki K, Fujimura T, Shinshi H. 2006. Genome-wide analysis of the ERF gene family in Arabidopsis and rice. Plant Physiology 140, 411–432. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Ohme-Takagi M, Shinshi H. 1995. Ethylene-inducible DNA binding proteins that interact with an ethylene-responsive element. The Plant Cell 7, 173–182. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Okamuro JK, Caster B, Villarroel R, Van Montagu M, Jofuku KD. 1997. The AP2 domain of APETALA2 defines a large new family of DNA binding proteins in Arabidopsis . Proceedings of the National Academy of Sciences, USA 94, 7076–7081. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Peterson RB, Eichelmann H, Oja V, Laisk A, Talts E, Schultes NP. 2013. Functional aspects of silencing and transient expression of psbS in Nicotiana benthamiana . American Journal of Plant Sciences 4, 1521–1532. [Google Scholar]
  37. Reid MS, Chen JC, Jiang CZ. 2009. Virus-induced gene silencing for functional characterization of genes in petunia. In: Gerats T, Strommer J. eds. Petunia . Springer, 381–394. [Google Scholar]
  38. Reid MS, Wu MJ. 1992. Ethylene and flower senescence. Plant Growth Regulation 11, 37–43. [Google Scholar]
  39. Scholthof HB, Alvarado VY, Vega-Arreguin JC, Ciomperlik J, Odokonyero D, Brosseau C, Jaubert M, Zamora A, Moffett P. 2011. Identification of an argonaute for antiviral RNA silencing in Nicotiana benthamiana . Plant Physiology 156, 1548–1555. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Schwach F, Vaistij FE, Jones L, Baulcombe DC. 2005. An RNA-dependent RNA polymerase prevents meristem invasion by Potato virus X and is required for the activity but not the production of a systemic silencing signal. Plant Physiology 138, 1842–1852. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Senthil-Kumar M, Mysore KS. 2011. Virus-induced gene silencing can persist for more than 2 years and also be transmitted to progeny seedlings in Nicotiana benthamiana and tomato. Plant Biotechnology Journal 9, 797–806. [DOI] [PubMed] [Google Scholar]
  42. Shen Z, Sun J, Yao J, Wang S, Ding M, Zhang H, Qian Z, Zhao N, Sa G, Zhao R. 2015. High rates of virus-induced gene silencing by Tobacco rattle virus in Populus . Tree Physiology 35, 1016–1029. [DOI] [PubMed] [Google Scholar]
  43. Spitzer B, Zvi MMB, Ovadis M, Marhevka E, Barkai O, Edelbaum O, Marton I, Masci T, Alon M, Morin S. 2007. Reverse genetics of floral scent: application of Tobacco rattle virus-based gene silencing in petunia. Plant Physiology 145, 1241–1250. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Szittya G, Silhavy D, Molnár A, Havelda Z, Lovas Á, Lakatos L, Bánfalvi Z, Burgyán J. 2003. Low temperature inhibits RNA silencing-mediated defence by the control of siRNA generation. The EMBO Journal 22, 633–640. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Takeda A, Iwasaki S, Watanabe T, Utsumi M, Watanabe Y. 2008. The mechanism selecting the guide strand from small RNA duplexes is different among argonaute proteins. Plant and Cell Physiology 49, 493–500. [DOI] [PubMed] [Google Scholar]
  46. Tian J, Pei H, Zhang S, Chen J, Chen W, Yang R, Meng Y, You J, Gao J, Ma N. 2014. TRV-GFP: a modified Tobacco rattle virus vector for efficient and visualizable analysis of gene function. Journal of Experimental Botany 65, 311–322. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Turnage MA, Muangsan N, Peele CG, Robertson D. 2002. Geminivirus-based vectors for gene silencing in Arabidopsis . The Plant Journal 30, 107–114. [DOI] [PubMed] [Google Scholar]
  48. Tuttle J, Idris A, Brown J, Haigler CD. 2008. Geminivirus-mediated gene silencing from Cotton leaf crumple virus is enhanced by low temperature in cotton. Plant Physiology 148, 41–50. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Wang C, Cai X, Wang X, Zheng Z. 2006. Optimisation of Tobacco rattle virus-induced gene silencing in Arabidopsis . Functional Plant Biology 33, 347–355. [DOI] [PubMed] [Google Scholar]
  50. Wang H, Huang Z, Chen Q, Zhang Z, Zhang H, Wu Y, Huang D, Huang R. 2004. a Ectopic overexpression of tomato JERF3 in tobacco activates downstream gene expression and enhances salt tolerance. Plant Molecular Biology 55, 183–192. [DOI] [PubMed] [Google Scholar]
  51. Wang H, Stier G, Lin J, Liu G, Zhang Z, Chang Y, Reid MS, Jiang CZ. 2013. Transcriptome changes associated with delayed flower senescence on transgenic petunia by inducing expression of etr1-1, a mutant ethylene receptor. PLoS One 8, e65800. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Wang JF, Zhou H, Chen YQ, Luo QJ, Qu LH. 2004. b Identification of 20 microRNAs from Oryza sativa . Nucleic Acids Research 32, 1688–1695. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Wang XB, Jovel J, Udomporn P, Wang Y, Wu Q, Li WX, Gasciolli V, Vaucheret H, Ding SW. 2011. The 21-nucleotide, but not 22-nucleotide, viral secondary small interfering RNAs direct potent antiviral defense by two cooperative argonautes in Arabidopsis thaliana . The Plant Cell 23, 1625–1638. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Wang XB, Wu Q, Ito T, Cillo F, Li WX, Chen X, Yu JL, Ding SW. 2010. RNAi-mediated viral immunity requires amplification of virus-derived siRNAs in Arabidopsis thaliana . Proceedings of the National Academy of Sciences, USA 107, 484–489. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Yi SY, Kim JH, Joung YH, Lee S, Kim WT, Yu SH, Choi D. 2004. The pepper transcription factor CaPF1 confers pathogen and freezing tolerance in Arabidopsis . Plant Physiology 136, 2862–2874. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Yin J, Chang X, Kasuga T, Bui M, Reid MS, Jiang CZ. 2015. A basic helix-loop-helix transcription factor, PhFBH4, regulates flower senescence by modulating ethylene biosynthesis pathway in petunia. Horticulture Research 2, 15059. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Zhang H, Huang Z, Xie B, Chen Q, Tian X, Zhang X, Zhang H, Lu X, Huang D, Huang R. 2004. The ethylene-, jasmonate-, abscisic acid-and NaCl-responsive tomato transcription factor JERF1 modulates expression of GCC box-containing genes and salt tolerance in tobacco. Planta 220, 262–270. [DOI] [PubMed] [Google Scholar]
  58. Zhang X, Yuan YR, Pei Y, Lin SS, Tuschl T, Patel DJ, Chua NH. 2006. Cucumber mosaic virus-encoded 2b suppressor inhibits Arabidopsis Argonaute1 cleavage activity to counter plant defense. Genes & Development 20, 3255–3268. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Zhang X, Zhang X, Singh J, Li D, Qu F. 2012. Temperature-dependent survival of Turnip crinkle virus-infected Arabidopsis plants relies on an RNA silencing-based defense that requires DCL2, AGO2, and HEN1. Journal of Virology 86, 6847–6854. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Zhao Y, Wei T, Yin KQ, Chen Z, Gu H, Qu LJ, Qin G. 2012. Arabidopsis RAP2.2 plays an important role in plant resistance to Botrytis cinerea and ethylene responses. New Phytologist 195, 450–460. [DOI] [PubMed] [Google Scholar]

Associated Data

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

Supplementary Materials

Supplementary Data
supp_67_11_3353__index.html (1,001B, html)
supp_erw155_supplementary_figures_S1_S3_tables_S1_S2.pdf (357.1KB, pdf)

Articles from Journal of Experimental Botany are provided here courtesy of Oxford University Press

RESOURCES