CaRDR1, an RNA-Dependent RNA Polymerase Plays a Positive Role in Pepper Resistance against TMV

Abstract

RNA silencing functions as a major natural antiviral defense mechanism in plants. RNA-dependent RNA polymerases (RDRs) that catalyze the synthesis of double-stranded RNAs, are considered as a fundamental element in RNA silencing pathways. In Arabidopsis thaliana, RDR1, 2 and 6 play important roles in anti-viral RNA silencing. Expression of RDR1 can be elevated following plant treatment with defense hormones and virus infection. RDR1 has been studied in several crop species, but not in pepper (Capsicum annuum L.). Here, a RDR1 gene was isolated from Capsicum annuum L., designated as CaRDR1. The full-length cDNA of CaRDR1 was 3,351 bp, encoding a 1,116-amino acid protein, which contains conserved regions, such as the most remarkable motif DLDGD. The transcripts of CaRDR1 could be induced by salicylic acid (SA), abscisic acid (ABA), H₂O₂, and tobacco mosaic virus (TMV). Silencing of CaRDR1 in pepper resulted in increased susceptibility to TMV as evident by severe symptom, increased of TMV-CP transcript, higher malondialdehyde (MDA) content and lower antioxidant enzymes activities compared with that of control plants. CaRDR1-overexpressing in Nicotiana benthamiana showed mild disease symptom and reduced TMV-CP transcripts than that of empty vector (EV) following TMV inoculation. The RNA silencing related genes, including NbAGO2, NbDCL2, NbDCL3, and NbDCL4 elevated expression in overexpressed plants. Alternative oxidase (AOX), the terminal oxidase of the cyanide (CN)-resistant alternative respiratory pathway, catalyze oxygen-dependent oxidation of ubiquinol in plants. It has an important function in plant defense against TMV. In addition, CaRDR1 overexpression promoted the expression of NbAOX1a and NbAOX1b. In conclusion, these results suggest that CaRDR1 plays a positive role in TMV resistance by regulating antioxidant enzymes activities and RNA silencing-related genes expression to suppress the replication and movement of TMV.

Keywords: CaRDR1; Nicotiana benthamiana; pepper; resistance; tobacco mosaic virus (TMV).

Figure 1

Sequence alignment and phylogenetic analyses of CaRDR1 and its homologs in various species. (A) Structural analysis of CaRDR1 in pepper. Exons and introns were shown in box and line, respectively. Ca, Capsicum annuum L. (B) Sequence alignment of the amino acid of CaRDR1 with other RDR1 proteins. The identical and similar residues were shown in black and gray, respectively. The highly conserved regions for RDR1s were highlighted in yellow box, and signature DLDGD was marked with red box. The highly conserved RNA recognition motif (RRM) and RNA-dependent RNA polymeras (RdRP) domains were indicated respectively in red and green lines. At, Arabidopsis thaliana; Nt, Nicotiana tobacum; Ng, Nicotiana glutinosa; Nb, Nicotiana benthamiana; Sl, Solanum lycopersicum; St, Solanum tuberosum. (C) Phylogenetic analyses of CaRDR1 and its homologs using MEGA5 software based on the neighbor joining method. CaRDR1 from pepper was indicated in red boxes.

Figure 2

qRT-PCR analyses relative expression of CaRDR1 in different tissues of pepper. Three biological replicates were performed for this experiment and the pepper Ubi3 gene was used as the reference gene. Error bars indicate the standard errors. Letters indicate the significant differences (P < 0.05) between samples. R, roots; S, stems; L, leaves; F, flowers; GF, green fruits; GF, red fruits.

Figure 3

qRT-PCR analyses of CaRDR1 expression as influenced by exogenous phytohormones and TMV inoculation in P79. (A) Effect of 2 mM SA on the expression of CaRDR1 in pepper leaves. (B) Effect of TMV on the expression of CaRDR1 in pepper leaves. (C) Effect of 100 μM MeJA on the expression of CaRDR1 in pepper leaves. (D) Effect of 100 μM ABA on the expression of CaRDR1 in pepper leaves. (E) Effect of 10 mM H₂O₂ on the expression of CaRDR1 in pepper leaves. The pepper Ubi3 was used as the reference gene, and three biological replicates were performed for these experiments. Error bars indicate the standard errors.

Figure 4

Silencing of CaRDR1 attenuated the TMV resistance of pepper. (A) Phenotype of upper un-inoculated leaves from WT, empty vector (TRV:00), and CaRDR1-silenced (TRV:CaRDR1) plants, P79 (upper) and P54 (down) at 15 dpi with TMV. (B,C) qRT-PCR was used to determine the relative level of CaRDR1 (B) and TMV-CP (C) transcript of WT, empty vector (TRV: 00), and CaRDR1-silenced (TRV: CaRDR1) plants at 7 days post-inoculation (dpi). (D) The malondialdehyde (MDA) content. (E) catalase (CAT) activities (F) peroxidase (POD) activities (G) superoxide dismutase (SOD) activities. Error bars indicate the standard errors. Asterisks indicate the significant differences (P < 0.05) between WT, TRV:00 and TRV: CaRDR1 lines.

Figure 5

Phenotype analysis of wild type, empty vector and CaRDR1-overexpressed (OE-3, OE-4, and OE-6) plants at 0, 10, and 20 dpi with TMV.

Figure 6

Overexpression CaRDR1 enhanced the TMV resistance of N. benthamiana. (A) qRT-PCR was used to determine the relative level of TMV–CP of empty vector and CaRDR1-OE plants at 0, 3, and 7 dpi. (B) qRT-PCR was used to determine the relative level of NbTOM in upper un-inoculated leaves of empty vector and CaRDR1-OE plants at 0, 1, 3, and 7 dpi. (C) The MDA content (D–F) CAT (D), SOD (E) and POD (F) activities measurement in leaves of empty vector and CaRDR1-OE plants at 0, 1, 3, and 7 dpi. (G,H) qRT-PCR was used to determine the relative level of NbAOX1a (G) and NbAOX1b (H) transcript in empty vector and CaRDR1-OE plants at 0, 1, 3, and 7 dpi. Three biological replicates were performed for these experiments. The N. benthamiana NbEF1α gene was used as the reference gene. Error bars indicate the standard errors. Asterisks indicate the significant differences (P < 0.05) between EV and CaRDR1-OE lines.

Figure 7

RNA silencing related genes expression in EV and CaRDR1-OE plants after TMV inoculation (A–F). (A) NbAGO1 (B) NbAGO2 (C) NbDCL2 (D) NbDCL3 (E) NbDCL4 (F) NbRDR6. The leaf samples were obtained from empty vector and CaRDR1-OE plants at 0, 1, 3, and 7 dpi. Three biological replicates were performed for these experiments. The N. benthamiana NbEF1α gene was used as the reference gene. Error bars indicate the standard errors. Asterisks indicate the significant differences (P < 0.05) between EV and CaRDR1-OE lines.