The regulation of gene expression via post-transcriptional modification by RNA-binding proteins is crucial for plant disease and innate immunity. Here, we report the identification of the pepper (Capsicum annuum) RNA-binding protein1 gene (CaRBP1) as essential for hypersensitive cell death and defense signaling in the cytoplasm. CaRBP1 contains an RNA recognition motif and is rapidly and strongly induced in pepper by avirulent Xanthomonas campestris pv. vesicatoria (Xcv) infection. CaRBP1 displays in vitro RNA- and DNA-binding activity and in planta nucleocytoplasmic localization. Transient expression of CaRBP1 in pepper leaves triggers cell-death and defense responses. Notably, cytoplasmic localization of CaRBP1, mediated by the N-terminal region of CaRBP1, is essential for the hypersensitive cell-death response. Silencing of CaRBP1 in pepper plants significantly enhances susceptibility to avirulent Xcv infection. This is accompanied by compromised hypersensitive cell death, production of reactive oxygen species in oxidative bursts, expression of defense marker genes and accumulation of endogenous salicylic acid and jasmonic acid. Over-expression of CaRBP1 in Arabidopsis confers reduced susceptibility to infection by the biotrophic oomycete Hyaloperonospora arabidopsidis. Together, these results suggest that cytoplasmic localization of CaRBP1 is required for plant signaling of hypersensitive cell-death and defense responses.
Programmed cell death (PCD) is a fundamental mechanism that permits elimination of damaged, redundant, transformed or pathogen-infected cells (Greenberg, 1996; Bursch et al., 2000). Multicellular organisms maintain both proper development and homeostasis by activating an intrinsic suicide program (Raff, 1992). In plants, PCD is required for intact developmental processes, as well as for active defense against various biotic and abiotic stresses (Greenberg, 1996; Williams and Dickman, 2008). The hypersensitive response (HR) is the most characterized plant PCD process, and involves a complex, early defense response (Dangl and Jones, 2001). The HR, a localized form of cell death that is induced by pathogen infection, results in confinement of the infection and restriction of pathogen growth in planta. In particular, plants have developed innate immune systems comprising specific defenses triggered by receptor–ligand recognition against individual pathogen invasion (effector-triggered immunity), as well as basal defense induced by sensing of common components of pathogens known as pathogen-associated molecular patterns (PAMPs) (PAMP-triggered immunity) (Dangl and Jones, 2001; Zipfel and Felix, 2005; Jones and Dangl, 2006).
Various defense-related genes are induced in plants during pathogen infection (Jung and Hwang, 2000; Eulgem, 2005; Huibers et al., 2009). Regulation of defense gene expression via post-transcriptional modification is crucial for PCD and innate immunity (Choi and Hwang, 2011). Individual mRNAs are strictly controlled by numerous RNA-binding proteins and ribonucleoprotein complexes, allowing transport, stability, intracellular localization and efficiency of translation (Varani and Nagai, 1998). RNA-binding proteins that recognize pre-mRNAs or mature mRNAs function as regulators of gene expression (Maris et al., 2005). A number of RNA-binding proteins that regulate post-transcriptional modification contain an RNA recognition motif (RRM), referred to as an RNA-binding domain (RBD), and additional domains that are necessary for mediating protein–protein interactions (Varani and Nagai, 1998). The RRMs exist as single or multiple copies in individual proteins, and function mainly in targeting specific RNAs. RNA-binding proteins containing the RRM have been shown to regulate gene expression through post-transcriptional modifications. These include auto-regulation of RNA, alternative splicing of RNA, stabilization of mRNAs and control of translation efficiency (Brennan and Steitz, 2001; Johnstone and Lasko, 2001; Mitchell and Tollervey, 2001; Zhong et al., 2001; Staiger et al., 2003; Fu et al., 2007; Schöning et al., 2007). In Arabidopsis, the glycine-rich RNA-binding protein AtGRP7 that influences circadian oscillations binds to its pre-mRNA and regulates its own gene expression by alternative splicing (Staiger et al., 2003). To date, however, the function of RNA-binding proteins in the HR defense response of plants has attracted less attention than their function in physiological processes. The Pseudomonas syringae type III effector HopU1 suppresses plant innate immunity through its mono-ADP-ribosyltransferase active site (Fu et al., 2007). The substrates of HopU1 in Arabidopsis include the glycine-rich RNA-binding protein AtGRP7 and RNA-binding proteins containing RRMs. Arabidopsis knockout lines of AtGRP7 exhibit enhanced susceptibility to Pseudomonas syringae infection (Fu et al., 2007).
The precise subcellular localization of proteins is essential for their activation and proper function. The cytoplasm, plasma membrane and nucleus all represent crucial compartments involved in disease resistance. Previous studies have established that specifically targeted localization of various defense-related proteins is required for defense responses. For example, the plant intracellular innate immune receptor RPM1 (resistance to Pseudomonas syringae pv. maculicola 1) is activated at, and functions on, the plasma membrane (Gao et al., 2011). NPR1, as a key regulator of systemic acquired resistance, is localized in the nucleus to activate PR gene expression in Arabidopsis (Kinkema et al., 2000). Pepper ABR1 (abscisic acid-responsive 1) is also targeted to the nucleus for the cell death response (Choi and Hwang, 2011). Recently, Qi et al. (2010) reported that AtRBP1-DR1 (Arabidopsis thaliana RNA-binding protein-defense related 1) positively regulates salicylic acid (SA)-mediated immunity and is localized to the cytoplasm. However, few proteins have been shown to re-localize or trans-localize from one location to another for proper function. In some cases, a small fraction of the total nucleotide-binding leucine-rich repeat (NB-LRR) members appears to re-localize to the nucleus (Caplan et al., 2008). Among the RNA-binding proteins, hnRNP A1 trans-localizes continuously between the nucleus and the cytoplasm in a temperature-sensitive manner (Michael et al., 1995; Nakielny et al., 1997). Partitioning of the potato (Solanum tuberosum) NB-LRR immune receptor Rx into the nucleus and cytoplasm is mediated by co-expression with RanGAP2 (Ran GTPase-activating protein 2) (Tameling et al., 2010). However, the mechanism underlying the re-localization of defense-related proteins remains to be clarified.
In this study, we have identified an RNA-binding protein containing an RRM from pepper (Capsicum annuum) leaves infected with Xanthomonas campestris pv. vesicatoria (Xcv). This protein, CaRBP1, exhibits in vitro RNA-binding activity and resides in both the cytoplasm and nucleus. Agrobacterium-mediated transient expression of CaRBP1 in pepper leaves results in induction of the cell-death response, as well as defense phenotypes. Notably, cytoplasmic localization of CaRBP1, which is dependent on the N-terminal region of CaRBP1, is essential for the cell-death response in pepper leaves. Silencing of CaRBP1 in pepper plants leads to enhanced susceptibility to avirulent Xcv infection. CaRBP1 over-expression in Arabidopsis confers reduced susceptibility to the biotrophic oomycete Hyaloperonospora arabidopsidis but not to Pseudomonas syringae pv. tomato. Our data provide convincing evidence that CaRBP1 induction in the cytoplasm leads to activation of cell-death and defense responses. We also suggest that CaRBP1 regulates post-transcriptional modification of certain targeted RNAs and DNAs through its RNA-binding activity in plants.
CaRBP1 encodes a pepper RNA-binding protein with an RNA recognition motif
The Capsicum annuum RNA-binding protein 1 gene, CaRBP1, was isolated from a cDNA library constructed from pepper leaves infected with avirulent strain Bv5-4a of Xanthomonas campestris pv. vesicatoria (Xcv) (Jung and Hwang, 2000). The CaRBP1 cDNA is 858 bp long, with a predicted open reading frame (ORF) of 627 bp (Figure S1). The SMART database (http://smart.embl-heidelberg.de/) predicts the presence of a eukaryotic RNA recognition motif (RRM) (residues 84–164) in CaRBP1 (Figure S1). RRM-containing proteins are also known as RNA-binding proteins, containing an RNA-binding domain (RBD) or ribonucleoprotein (RNP) domain (Maris et al., 2005). RRMs are found in RNA-binding proteins, including various heterogeneous nuclear ribonucleoproteins (hnRNP) that have been implicated in regulation of alternative splicing, and in protein components of small nuclear ribonucleoproteins (snRNP) of various eukaryotes (Adam et al., 1986; Swanson et al., 1987; Dreyfuss et al., 1988). RRMs are also present in a few single-stranded DNA-binding proteins (Ding et al., 1999).
A blast search revealed that the CaRBP1 protein shares sequence identity (55–87%) with other DNA- or RNA-binding proteins. The translated CaRBP1 amino acid sequence was 87% identical to RNA-binding protein 47 of curled-leaved tobacco (Nicotiana plumbaginifolia; accession number CAC01238; Lorkovic et al., 2000) and 55% identical to RNA-binding protein 47B of Arabidopsis (Arabidopsis thaliana; accession number NP_188544) (Figure S2a). CaRBP1 contains only one RRM, whereas other homologous proteins contain three or more RRMs. As shown in Figure S2b, phylogenetic analysis indicated that CaRBP1 is highly similar to other RNA- or DNA-binding proteins. However, the greatest similarity was found between CaRBP1 and its Solanaceae homologs, indicating that CaRBP1-like homologs are evolutionarily conserved in Solanaceae species.
CaRBP1 is strongly induced in pepper leaves by avirulent Xanthomonas campestris pv. vesicatoria (Xcv) infection
RT-PCR analysis shows that CaRBP1 is rapidly and strongly induced in leaves inoculated with the Xcv avirulent (incompatible) strain Bv5-4a. The maximum CaRBP1 level was reached 12 h after inoculation, compared to mock-treated or virulent (compatible) strain Ds1-inoculated leaves (Figure 1). CaPR1 (PR1) and CaDEF1 (defensin1), included as defense-marker genes of pepper, were distinctly up-regulated during the incompatible interactions with Xcv (Figure 1). These results indicate that CaRBP1 is strongly induced by incompatible Xcv infection, similar to CaBPR1 and CaDEF1.
CaRBP1 has in vitro RNA- and DNA-binding activity
In previous studies, RRM-containing proteins were demonstrated to have DNA- or RNA-binding properties (Karlson et al., 2002; De Gaudenzi et al., 2003; Vega-Sánchez et al., 2008). Bioinformatics analysis revealed that CaRBP1 is a member of the RRM family. To investigate the biochemical function of CaRBP1, recombinant MBP (maltose-binding protein)-fused CaRBP1 (MBP–CaRBP1) or N- and C-terminal deletion mutants (MBP–CaRBP1ΔN and MBP–CaRBP1ΔC) were expressed in Escherichia coli and purified using affinity chromatography (Figure 2a,b). Immunoblot analysis shows that MBP–CaRBP1 and MBP–CaRBP1ΔN (containing the RRM) specifically bound to RNA polymers [poly(rU) and poly(rC)] and single-stranded DNA, but not double-stranded DNA (Figure 2c). However, MBP–CaRBP1ΔC lacking the RRM did not bind to any of the homoribopolymers or DNA. Polyadenylic ribohomopolymers [poly(rA)] were not used to analyze the RNA-binding activity in vitro due to lack of availability of poly(rA) for this study. As a negative control, the fusion protein MBP alone did not show RNA- and DNA-binding activity. These results indicate that CaRBP1 has in vitro RNA- and single-stranded DNA-binding activity. Furthermore, the RRM of CaRBP1 is responsible for the RNA- and DNA-binding.
CaRBP1 is localized to the cytoplasm and the nucleus
To analyze the subcellular localization of CaRBP1, green fluorescent protein (GFP)-tagged 35S:CaRBP1 constructs were transiently expressed in Nicotiana benthamiana leaves using an Agrobacterium-mediated transient expression system (Figure 3). A control GFP construct was ubiquitously distributed throughout the cells (Figure 3a/a). The nuclear region was stained with DAPI (4′6-diamidino-2-phenylindole). The 35S:CaRBP1 protein localized to both the cytoplasm and the nucleus (Figure 3a/b-1,b-2). Despite the absence of a nuclear export signal sequence (NES) in the CaRBP1 sequence, some GFP-tagged CaRBP1 protein was visible in the cytoplasm (Figure 3a/b-1). To establish whether CaRBP1 is localized to the cytoplasm from the nucleus, we generated a NES-fused CaRBP1 construct (35S:CaRBP1:NES). CaRBP1:NES localized to the cytoplasm (Figure 3a/c), similar to CaRBP1. However, nuclear localization signal (NLS)-fused CaRBP1 (35S:CaRBP1:NLS) was observed exclusively in the nucleus (Figure 3a/d). N- and C-terminal deletion mutants were constructed to determine whether the subcellular localization of CaRBP1 requires a specific region of CaRBP1. The CaRBP1ΔN mutant was abundantly localized to the nucleus, although a small amount was detected in the cytoplasm (Figure 3a/e). In contrast, the CaRBP1ΔC mutant was mainly localized to the cytoplasm (Figure 3a/f). When analyzed by immunoblotting, CaRBP1 and its derivatives were detected in cytosolic and nuclear fractions in N. benthamiana leaves (Figure 3b), consistent with their subcellular locations shown in Figure 3(a). Collectively, these results indicate that the N- and C-terminal regions of CaRBP1 are responsible for the CaRBP1 cytoplasmic and nuclear localizations, respectively.
Transient expression of CaRBP1 triggers cell death in pepper leaves
Transient expression of 35S:CaRBP1 in pepper leaves via agro-infiltration led to accelerated cell death, as well as accumulation of UV-fluorescent phenolic compounds compared with leaves infiltrated with a vector control (35S:00) (Figure 4a). 35S:CaRBP1-8cMyc was also transiently expressed in pepper leaves. When assayed by immunoprecipitation with anti-cMyc, CaRBP1–8cMyc (approximately 48 kDa) was detected in the soluble fraction (Figure 4b). Reactive oxygen species (ROS) are proposed to play multiple roles during plant HR defense responses (Lamb and Dixon, 1997). Transient expression of CaRBP resulted in a significant increase in H2O2 production in leaf tissues compared to the vector control (Figure 4c). Next, cell-death phenotypes were quantified using an electrolyte leakage assay. CaRBP1-induced cell death in pepper leaves caused an increase in electrolyte leakage from leaf cells compared to the empty vector control (Figure 4d). Together, these results imply that transient expression of CaRBP1 induces cell death and is accompanied by a ROS burst in pepper leaves.
N-terminal region-mediated cytoplasmic localization of CaRBP1 is required to initiate the cell-death response
Agrobacterium-mediated transient expression was used to investigate the subcellular localization of CaRBP1 required for cell death induction (Figure 5). Successful transient expression of CaRBP1 and the CaRBP1 mutants was confirmed by a cell-death phenotype assay (Figure 5a) and immunoblotting analysis (Figure 5b). Interestingly, transient expression of the C-terminal deletion mutant 35S:CaRBP1ΔC induced a cell death response similar to wild-type 35S:CaRBP1 expression (Figure 5a). In contrast, transient expression of the N-terminal deletion mutant 35S:CaRBP1ΔN did not significantly initiate full cell-death phenotypes. These data indicate that the CaRBP1 N-terminal region is necessary for the cell-death response. As the CaRBP1ΔC mutant was localized to the cytoplasm (Figure 3a-f ), we postulated that cytoplasmic localization of CaRBP1 may be crucial for cell-death induction. To confirm that cytoplasmic localization of CaRBP1 is required for cell-death induction, the 35S:CaRBP1:NES and 35S:CaRBP1:NLS mutants were used for Agrobacterium-mediated transient expression. Transient expression of 35S:CaRBP1:NES resulted in induced cell-death phenotypes at relatively high levels, similar to the C-terminal deletion mutant 35S:CaRBP1ΔC (Figure 5c). In contrast, 35S:CaRBP1:NLS expression induced a significantly low level of cell death, similar to the 35S:CaRBP1ΔN level. Transient expression analysis of the CaRBP1:NES or NLS fusion proteins firmly supports our hypothesis that cytoplasmic localization of CaRBP1 plays a crucial role in triggering cell death. The observed cell-death phenotypes were also verified by measuring the amount of electrolyte leakage from pepper leaf tissues (Figure 5d). Transient expression of 35S:CaRBP1, 35S:CaRBP1ΔC or 35S:CaRBP1:NES induced significant electrolyte leakage from leaf tissues 2 days after agro-infiltration. Biochemical fractionation and immunoblotting assays also confirmed the proper localization of CaRBP1 and its various derivative constructs in the cell fractions (Figure 5e). As positive controls, histone H3 and Hsc70 proteins were immunodetected in the nuclear and cytosolic fractions, respectively. Taken together, these results suggest that the N-terminal region-mediated cytoplasmic localization of CaRBP1 is essential for the cell-death response in pepper leaves.
CaRBP1 silencing confers enhanced susceptibility to avirulent Xcv infection
RT-PCR analysis showed that CaRBP1 was expressed at very low levels in silenced (TRV:CaRBP1) plants following infection with Xcv (Figure 6a). This indicates that the virus-induced silencing of CaRBP1 was effective. The susceptibility of the CaRBP1-silenced plants to virulent Xcv infection was similar to empty vector (TRV:00) control plants. However, the silenced plants were more susceptible to avirulent Xcv infection (Figure 6). The HR induced by avirulent Xcv infection was significantly compromised in the silenced plants compared with the empty vector plants (Figure 6b). In contrast to CaRBP1 transient expression in pepper leaves, UV-fluorescent phenolic compounds, which are induced during the defense response and are used as HR markers (Gachon et al., 2004), were greatly reduced in the silenced leaves. These findings are well supported by the enhanced bacterial growth observed in CaRBP1-silenced leaves compared with empty vector control leaves (Figure 6c). However, during the virulent Xcv infection, the CaRBP1-silenced pepper plants exhibited susceptible symptoms and increased bacterial growth, similar to that observed in the empty vector control plants.
CaRBP1 silencing compromises the ROS burst and the cell-death response
As an early defense response, a ROS burst occurs in pepper leaves. Silencing of CaRBP1 compromised H2O2 accumulation during the avirulent Xcv infection as measured by 3,3-diaminobenzidine (DAB) staining and the xylenol orange assay (Figure 7a). Electrolyte leakage from leaf tissues was used to quantify the cell-death response. Similar to H2O2 production, empty vector control plants exhibited a strong increase in electrolyte leakage following infection with avirulent Xcv. The electrolyte leakage from CaRBP1-silenced plants was much lower (Figure 7b). These cell-death responses were also confirmed using trypan blue staining. However, the early defense responses, such as ROS production in oxidative bursts and HR cell death, were not significantly different in non-silenced and silenced plants following infection with virulent Xcv.
CaRBP1 silencing compromises defense-related gene expression and SA and JA accumulation
As shown in Figure 8(a), expression of CaRBP1 was very low or not detectable in CaRBP1-silenced pepper leaves. CaPR1 (an SA-dependent marker) and CaDEF1 [a jasmonic acid (JA)-dependent marker] were analyzed as marker pepper defense response genes. Induction of CaPR1 and CaDEF1 were distinctly reduced in CaRBP1-silenced leaves during infection with virulent or avirulent Xcv strains. These findings indicate that CaRBP1 silencing compromises the expression of CaPR1 and CaDEF1, possibly linked to SA and JA accumulation.
We next investigated whether CaRBP1 expression is required for accumulation of SA and JA in Xcv-infected plants. Compared with non-silenced plants, SA accumulation in CaRBP1-silenced plants was greatly reduced 24 and 48 h after inoculation with the avirulent Xcv Bv5-4a strain. However, SA accumulation was not reduced after infection with the virulent Xcv Ds1 strain (Figure 8b). The accumulation of JA in the CaRBP1-silenced plants was significantly lower than that observed in the non-silenced plants at 24 h after inoculation with the virulent and avirulent Xcv strains (Figure 8c). These findings indicate that the enhanced disease susceptibility of the CaRBP1-silenced plants results from lower SA and JA synthesis.
Over-expression of CaRBP1 in Arabidopsis confers reduced susceptibility to Hp. arabidopsidis
To investigate the effect of CaRBP1 gain-of-function, we transformed Arabidopsis plants with CaRBP1. Three CaRBP1 over-expression (CaRBP1-OX) transgenic lines (numbers 3, 6 and 7) were selected for further analysis (Figure S3a). Multiplication of Pseudomonas syringae pv. tomato DC3000 and DC3000 (avrRpm1) in the leaves of the transgenic lines was not significantly different from multiplication in wild-type control leaves (Figure S3b). This indicates that CaRBP1 over-expression does not enhance resistance to Pst infection.
To investigate whether CaRBP1 over-expression enhances resistance to a biotrophic oomycete disease, 7-day-old Arabidopsis seedlings were inoculated with a conidiospore suspension of Hyaloperonospora arabidopsidis (Hpa) isolate Noco2. The CaRBP1-OX transgenic lines exhibited symptoms of reduced susceptibility as well as retarded hyphal growth on the cotyledons (Figures 9a and S4). However, any trailing necrotic cell death was not detected along the path of the oomycete hyphae on Hpa-inoculated cotyledons stained with trypan blue. Notably, sexual oospores of Hpa were almost completely absent from cotyledons of CaRBP1-OX transgenic lines compared to the abundant formation in wild-type plants 7 days after inoculation (Figure S4). Furthermore, CaRBP1 over-expression significantly inhibited sporangiophore and conidiospore formation in the transgenic plants (Figure 9b,c). These results indicate that CaRBP1 over-expression confers reduced susceptibility to the biotrophic oomycete pathogen Hpa in Arabidopsis plants.
In this work, we report that cytoplasmic localization of the pepper RNA-binding protein CaRBP1 is required for plant signaling of hypersensitive cell death and defense responses. CaRBP1 protein contains an RNA recognition motif (RRM), referred to as an RNA-binding domain (RBD), which confers the ability to bind target RNA or DNA (Karlson et al., 2002; De Gaudenzi et al., 2003; Vega-Sánchez et al., 2008). We examined whether CaRBP1 with an RRM has RNA-binding activity in vitro (Vega-Sánchez et al., 2008). The recombinant MBP–CaRBP1 fusion protein exhibited RNA- and DNA-binding activity, suggesting that CaRBP1 may bind to certain target RNAs or DNAs in planta. This also suggests that CaRBP1 may possess RNA- or DNA-binding activity toward specific target RNAs or DNAs during post-transcriptional modification (Varani and Nagai, 1998). Modifications of specific target RNAs by the RNA-binding proteins are essential for the regulation of gene expression involved in normal growth, development, hormone signaling, abiotic stress tolerance and disease resistance in plants (Jacobsen et al.,1999; Lu and Fedoroff, 2000; Rochaix, 2001; Staiger, 2001; Fu et al., 2007; Schöning and Staiger, 2009). The Arabidopsis genome encodes over 200 putative RNA-binding proteins (Lorkovic and Barta, 2002). Among them, few defense-related proteins containing the RNA recognition motif (RRM) have been identified as being involved in disease resistance. As CaRBP1 is induced by the bacterial pathogen Xcv, we reasoned that CaRBP1 is crucial for the HR and defense responses, two processes that are controlled by RNA-binding activity.
Subcellular localization assays revealed that GFP-fused CaRBP1 was localized to both the cytoplasm and the nucleus. This finding supports the notion that CaRBP1 may function as a post-transcriptional regulator via nucleocytoplasmic trafficking (Nakielny et al., 1997). Most eukaryotic RNAs are transported to their functional cellular sites after transcriptional and post-transcriptional modification. Although localized predominantly in the nucleus, the RNA-binding protein hnRNP A1 was demonstrated to shuttle continuously between the nucleus and the cytoplasm in a temperature-sensitive manner (Michael et al.,1995; Nakielny et al., 1997). Similarly, the localization of CaRBP1 in both the cytoplasm and the nucleus suggests that CaRBP1 may also shuttle between the nucleus and the cytoplasm as it targets RNAs or DNAs, although there is no experimental evidence regarding cytoplasm–nucleus shuttling of CaRBP1. The CaRBP1 N-terminal deletion mutant (CaRBP1ΔN) carrying the RRM and the CaRBP1 C-terminal deletion mutant (CaRBP1ΔC) were mainly localized to the nucleus and the cytoplasm, respectively. These results support the possibility that CaRBP1 nuclear localization relies on its C-terminal region, the RRM, which is responsible for binding to DNA and RNA.
Transient expression of CaRBP1 significantly induced cell-death responses in pepper leaves. UV-fluorescent phenolic compounds, ROS (H2O2) bursts and electrolyte leakage (all markers for hypersensitive cell death) were all strongly induced in pepper leaves by Agrobacterium-mediated transient expression of CaRBP1. These observations support the notion that CaRBP1 expression is required to trigger HR-like cell death. Increased ROS levels may be a primary cause of hypersensitive cell death (Coll et al., 2011). We further examined whether the subcellular locations of CaRBP1 affect cell-death induction in pepper leaves by Agrobacterium-mediated transient expression. Expression of CaRBP1, CaRBP1ΔC and CaRBP1:NES distinctly triggered hypersensitive cell death in pepper leaves. This suggests that cytoplasmic localization of CaRBP1 is required for cell-death induction. Interestingly, CaRBP1:NLS expression did not significantly trigger the cell-death response. This result indicates that the nucleus-tethered CaRBP1 could not effectively induce cell death in pepper leaves. Taken together, we conclude that N-terminal region-mediated cytoplasmic localization of CaRBP1 is essential for HR-like cell death signaling in plants.
In contrast to the HR-like cell-death phenotype induced by transient CaRBP1 expression, CaRBP1-silenced pepper plants exhibited enhanced susceptibility to avirulent Xcv infection. Notably, silencing of CaRBP1 in pepper plants significantly compromised the ability to reduce bacterial growth. The silenced plants were also unable to effectively induce phenolic compounds, produce ROS bursts, or increase defense response gene expression and HR. These loss-of-function results suggest that CaRBP1 induction contributes to the R gene-mediated resistance response, thereby preventing avirulent Xcv infection in pepper. Inducible immunity in plants employs the salicylic acid (SA) and jasmonic acid (JA) pathways to defend against biotrophic and necrotophic pathogens (Tsuda et al., 2009). Expression of CaPR1, a SA-responsive marker gene, was greatly compromised in CaRBP1-silenced pepper plants following avirulent Xcv infection. Furthermore, induction of CaDEF1, a JA-responsive marker gene, was also strongly suppressed in CaRBP1-silenced pepper plants after avirulent Xcv infection. In contrast to the enhanced susceptibility of the silenced plants to avirulent Xcv infection, silencing of CaRBP1 did not significantly alter the susceptibility of pepper plants to virulent Xcv. In parallel with the compromised CaPR1 and CaDEF1 induction, SA and JA accumulation may be attenuated in CaRBP1-silenced pepper plants during avirulent Xcv infection. As expected, CaRBP1 silencing significantly compromised SA and JA accumulation in the avirulent Xcv-infected leaves. Collectively, these results suggest that CaRBP1 may positively regulate the R gene-mediated resistance response associated with the SA- and JA-dependent signaling pathways. Recently, Qi et al. (2010) demonstrated that AtRBP-DR1, a putative RNA-binding protein, activates SA-mediated immunity to Pst Dc3000 in Arabidopsis. Multiplication of Pseudomonas syringae pv. tomato (Pst) DC3000 in the CaRBP1 over-expressing (OX) transgenic Arabidopsis plants was similar to that observed in wild-type plants. In contrast, CaRBP1 over-expression lowered the sporangiophore and conidiospore production of Hyaloperonospora arabidopsidis, but did not induce a cell-death response in transgenic seedlings. These findings suggest that CaRBP1 over-expression confers quantitatively reduced susceptibility to Hp. arabidopsidis infection, but is not so effective in triggering the cell-death response in the heterologous Arabidopsis system.
Here, we have identified a pepper RNA-binding protein gene, CaRBP1, as essential for hypersensitive cell death and defense signaling in the cytoplasm. Our results provide convincing evidence regarding the biological and biochemical functions of CaRBP1 in plant cell death and defense signaling. The gain- or loss-of-function analyses reveal that CaRBP1 contributes to R gene-mediated disease resistance through induction of the hypersensitive cell-death and defense responses. This includes induction of pepper defense marker genes (CaPR1 and CaDEF1) and accumulation of endogenous SA and JA, ultimately leading to a restriction of pathogen growth in pepper plants. Notably, the cytoplasmic localization of CaRBP1, mediated by the N-terminal region of CaRBP1, plays a pivotal role in triggering HR-like cell death in pepper. We also hypothesize that target RNA binding and post-transcriptional modification of unidentified target RNAs by CaRBP1 may positively regulate defense responses in pepper. Further studies are necessary to identify the target RNAs of CaRBP1 and to define how CaRBP1 regulates post-transcriptional modification during plant immunity.
Plant materials and growth conditions
Pepper (Capsicum annuum L., cv. Nockwang) and Nicotiana benthamiana were grown in soil mix (Soil conditioners perlite and vermiculite and loam soil, 1:1:3 v/v/v) at 25°C with 16 h light per day (65 μmol photons m−2 sec−1). Arabidopsis wild-type (Arabidopsis thaliana ecotype Columbia, Col-0) and transgenic plants were grown in soil mix (vermiculite, perlite and loam soil, 1:1:2 v/v/v) in a climate-controlled chamber at 24°C with 12 h of light per day (130 μmol photons m−2 sec−1) and 60% humidity.
The strains Ds1 (virulent) and Bv5-4a (avirulent) of Xanthomonas campestris pv. vesicatoria (Xcv) were cultured in yeast nutrient broth (5 g yeast extract, 8 g nutrient broth per liter) at 28°C, and infiltrated into fully expanded pepper leaves (Choi et al., 2007). Pseudomonas syringae pv. tomato (Pst) DC3000 and DC3000 (avrRpm1) were grown in King’s B medium containing 50 mg ml−1 rifampicin and 50 mg ml−1 kanamycin (Hwang et al., 2011). Arabidopsis leaves were infiltrated with Pst suspensions. One-week-old Arabidopsis seedlings were spray-inoculated with Hyaloperonospora arabidopsidis isolate Noco2 (5 × 104 conidiospores per ml) (Hwang et al., 2011). The infected plants were incubated at 17°C in an environmentally controlled chamber.
Expression and purification of recombinant proteins
pMAL-c4x vectors (NEB, http://www.neb.com) containing recombinant protein constructs were transformed into E. coli BL21(DE2) cells to produce MBP-fused proteins. Bacteria were cultured in Luria–Bertani (LB) medium containing 100 μg ml−1 ampicillin at 37°C. Expression was induced by addition of 300 μm isopropyl-β-d-thiogalactopyranoside (IPTG) for 3 h at 37°C. Expressed fusion proteins were purified by amylase affinity chromatography chromatography using amylose resin (NEB) according to the manufacturer’s instructions.
Assay of RNA- and DNA-binding activity
Resins (Sigma-Aldrich, http://www.sigmaaldrich.com) containing polycytidylic, polyuridylic and polyguanylic ribohomopolymers, as well as calf thymus single- and double-stranded DNA, were used for RNA- and DNA-binding assays (Vega-Sánchez et al., 2008). Approximately 100 ng of recombinant proteins was incubated with the resins in 400 μl KHN buffer [150 mm KCl, 20 mm HEPES, pH 7.9, 0.01% Nonidet P-40 (Sigma-Aldrich), and 1× complete protease inhibitor cocktail (Roche, http://www.roche.com)] for 60 min at 4°C. The proteins bound to homopolymer resins were eluted using 2× SDS–PAGE loading buffer at 80°C for 10 min. Aliquots (15 μl) were eluted from each resin and analyzed by electrophoresis on 10% SDS–PAGE gels. RNA- and DNA-binding activity was identified by protein gel-blot analysis using anti-MBP antibodies (NEB) (1:10 000).
GFP-fused binary vector pBIN35S carrying CaRBP1, CaRBP1ΔN, CaRBP1ΔC, CaRBP1:NES (nuclear export signal) or CaRBP1:NLS (nuclear localization signal) were transformed into Agrobacterium tumefaciens strain GV3101 (Choi and Hwang, 2011). Agrobacterium containing the constructs was grown in liquid LB medium and resuspended in induction medium (10 mm MES, pH 5.6, 10 mm MgCl2 and 200 μm acetosyringone). Agrobacterium carrying these constructs was mixed with Agrobacterium harboring p19 expression plasmid to a final OD600 of 0.5 and 0.3, respectively, and infiltrated into fully expanded Nicotiana benthamiana leaves. Subcellular localization was observed using an LSM 5 Exciter microscope (Zeiss, http://www.zeiss.com). DAPI (4′6-diamidino-2-phenylindole; 10 μg ml−1) was infiltrated into the leaves, and the DAPI fluorescence was detected using a 375 nm filter.
Virus-induced gene silencing
For virus-induced gene silencing (VIGS) of CaRBP1 (Choi et al., 2008; Lee et al., 2008), tobacco rattle virus (TRV) vectors pTRV1 and pTRV2 were used (Liu et al., 2002; Hwang and Hwang, 2011). Full-length CaRBP1 cDNA (641 bp) was inserted into the pTRV2 vector, resulting in the pTRV2:CaRBP1 construct. pTRV1, pTRV2:00 and pTRV2:CaRBP1 constructs were transformed into Agrobacterium strain GV3101. An equal volume of pTRV1 Agrobacterium culture was mixed with one of the pTRV2 cultures prior to infiltration (OD600 = 0.2). The mixed cultures were infiltrated into the cotyledons of pepper seedlings (Hong et al., 2008). Four to 5 weeks after VIGS, the leaves of CaRBP1-silenced plants were used for quantitative RT-PCR and various disease assays.
Full-length CaRBP1 cDNA was PCR-amplified using primers 5′-TCTAGAATGAACGGTGGAGATTTGAAT-3′ (forward) and 5′- GGATCCCTACTTTGCACCTTTA-3′ (reverse), and cloned into the TOP blunt vector (Enzynomix, http://www.enzynomics.com). The cDNA fragment and pBIN35S vector were digested with XbaI and BamHI and ligated together. The resulting plasmid was transformed into A. tumefaciens strain GV3101, and used to transform Arabidopsis plants according to the floral-dip method (Clough and Bent, 1998; Choi et al., 2007).
RT-PCR and real-time RT-PCR analyses
Total RNA was prepared from pepper and Arabidopsis leaves using Trizol (Invitrogen, http://www.invitrogen.com) according to the manufacturer’s instructions. First-strand cDNA was synthesized from total RNA (2 μg) using Moloney murine eukemia virus (M-MLV) reverse transcriptase (Enzynomics). The expression levels of CaRBP1 and the pepper PR genes (CaPR1 and CaDEF1) in leaves were assayed by RT-PCR and real-time RT-PCR, as described previously (Hwang and Hwang, 2011). The gene-specific primer pairs were: 5′- CTCATGTTGGCGAGCTGGTTTC-3′ (forward) and 5′-CTACTTTGCACCTTTAAA AGATGG-3′ (reverse) for CaRBP1, 5′-CAGGATGCAACACTCTGGTGG-3′ (forward) and 5′-ATCAAAGGCCGGTTGGTC-3′ (reverse) for CaPR1, 5′-CAAGGGAGTATGTGCTAGTGAGAC-3′ (forward) and 5′-TGCACAGCACTATCATTGCATAC-3′ (reverse) for CaDEF1, and 5′-AAACGGCTAC CACATCCAAG-3′ (forward) and 5′-ACCCATCCCAAGGTTCAACT-3′ (reverse) for 18S rRNA in pepper, and 5′-CATCAGGAAGGACTTGTACGG-3′ (forward) and 5′-GATGGACCTGACTCGTCATAC-3′ (reverse) for AtACT1 in Arabidopsis.
Agrobacterium-mediated transient expression
Transient expression assays were performed in pepper leaves by infiltration with Agrobacterium strain GV3101 containing the empty vector control and vectors expressing CaRBP1 and the CaRBP1 mutants, as described previously (Kim and Hwang, 2011). Agrobacterium was grown in liquid LB medium and resuspended in induction medium (10 mm MES, pH 5.6, 10 mm MgCl2 and 200 mm acetosyringone). The cultures were injected between the lateral leaf veins (OD600 = 0.1–1.0).
To visualize callose deposition, infected pepper leaves were cleared with 95% ethanol and stained with a solution containing 150 mm K2H2PO4, pH 9.5 and 0.01% aniline blue. To detect H2O2 accumulation, inoculated leaves were detached and stained with a solution containing 1 mg ml−1 3,3-diaminobenzidine (DAB). To determine cell death, infected leaves were sampled and stained with lactophenol–trypan blue solution (10 ml lactic acid, 10 ml glycerol, 10 g phenol and 10 mg trypan blue dissolved in 10 ml distilled water). The stained leaves were cleared with chloral hydrate solution (2.5 g ml−1 chloral hydrate).
Electrolyte leakage measurement
For the cell-death assay, electrolyte leakage was measured using six leaf discs (1.2 cm diameter) from pepper leaves infiltrated with Agrobacterium or Xcv (Choi et al., 2007). The leaf discs were immersed in 20 ml double-distilled water and incubated at room temperature. Conductivity was measured using a Crison conductivity meter (HACH, http://www.hach.com).
Quantification of H2O2 by xylenol orange assay
Accumulation of H2O2 in pepper leaves was quantified using the xylenol orange assay (Choi et al., 2007; Kim and Hwang, 2011). Eight excised leaf discs were floated on 1 ml distilled water. After centrifugation at 3000 g for 30 sec, one hundred microliters of the supernatant was immediately added to 1 ml xylenol orange buffer (0.25 mm FeSO4, 0.25 mm (NH4)2SO4, 25 mm H2SO4, 10 mm sorbitol and 12.5 mm xylenol orange). Absorbance was measured at 560 nm, and H2O2 levels were calculated based on a standard curve derived from a standardized solution of H2O2.
Protein extraction and immunoblot analysis
Total proteins were extracted from pepper leaves transiently expressing 35S:CaRBP1-8cMyc constructs and used for immunoblotting (Choi and Hwang, 2011). Total protein extracts from pepper leaves were incubated with 20 μl anti-cMyc agarose beads (Sigma-Aldrich). For protein gel blots, samples were separated by 10% SDS–PAGE and transferred to membranes. The membranes were blocked for 1 h in 5% milk and 1× phosphate-buffered saline with Tween-20 (PBST). This was followed by a 1 h incubation in 5% milk and 1× TBST containing the monoclonal cMyc antibody (1:3000) (Sigma-Aldrich). The proteins on the blots were detected by chemiluminescence using WEST-ZOL (Intron, http://www.intronbio.com) according to the manufacturer’s instructions.
The biochemical fractionation and immunoblotting assays were performed as previously described (Shen et al., 2007). The leaf tissue samples were homogenized in Honda buffer [2.5% Ficoll 400 (Bio basic, http://www.biobasic.com), 5% dextran T40, 0.4 m sucrose, 25 mm Tris/HCl, pH 7.5, 10 mm MgCl2, 1 mm dithiothreitol, 1 mm phenylmethanesulfonyl fluoride and 1× complete protease inhibitor cocktail (Roche). After centrifugation at 1500 g for 5 min of the homogenates, the supernatants were pooled (cytosol fraction) and the pellets were subsequently reprocessed to further purify the nuclear fraction. Anti-histone H3 and anti-Hsc70 antibodies (Abcam, http://www.abcam.com) were used as nuclear and cytosolic markers, respectively.
Measurement of SA and JA
Salicylic acid and SA glycoside were extracted from pepper leaves and analyzed by HPLC (Waters, http://www.waters.com) as described previously (Aboul-Soud et al., 2004; Choi et al., 2011). 3-hydroxybenzoic acid dissolved in absolute methanol was included as an internal standard. SA was quantified using a fluorescence detector (excitation at 305 nm and emission at 405 nm) by reverse-phase HPLC in a Waters 515 system using a C18 column.
Jasmonic acid was extracted from pepper leaves and quantified as described previously (Hwang and Hwang, 2010). JA in the methanol extracts was purified and eluted using a Sep-Pak C18 cartridge (Waters). Eluates were fractionated using chloroform, and then methylated with hexane:tert-butyl methyl ether (1:1 v/v). Endogenous JA was determined by gas chromatography/mass spectrometry (Agilent, http://www.agilent.com) using a DB-5MS column (length 30 m, insdie diameter 0.25 mm, film thickness 0.25 μm; Agilent). Dihydrojasmonic acid was used as an internal standard.
We thank Dr S.P. Dinesh-Kumar (Department of Plant Biology, University of California at Davis, CA, USA) for providing vectors pTRV1 and pTRV2, and Dr U. Bonas (Department of Genetics, Martin Luther Universität, Germany) for Agrobacterium tumefaciens strain GV3101. This work was supported by the Next Generation BioGreen21 Program (Plant Molecular Breeding Center, grant number PJ008027), Rural Development Administration, Republic of Korea.