Genome-Wide Analysis of DCL, AGO, and RDR Gene Families in Pepper (Capsicum Annuum L.)

Abstract

RNA silencing is an evolutionarily conserved mechanism that regulates variety of cellular processes in plants. Argonaute protein (AGO), Dicer-like protein (DCL) and RNA-dependent RNA polymerase (RDR) are critical components of RNA silencing. These efficient and indispensable components of the RNAi pathway have not been identified and characterized in pepper. In this study, we identified 12 CaAGO, 4 CaDCL and 6 CaRDR genes in pepper and compared them with those of Arabidopsis, tobacco, potato and tomato. Detailed phylogenetic analyses revealed that each CaAGO, CaDCL and CaRDR protein family were classified into four clades. The tissue specific expression and respond to abiotic or biotic stress were studied. The real-time quantitative polymerase chain reaction (PCR) results demonstrated that CaAGO2, CaAGO10b, CaDCL2 and CaDCL4 were upregulated with cucumber mosaic virus (CMV), potato virus Y (PVY) and tobacco mosaic virus (TMV) infections, whereas they showed difference expression patterns in response to abiotic stress. In addition, we found that many of the candidate genes were induced by phytohormones and H₂O₂ treatment. Our results provide useful information for further elucidation of gene silencing pathways and RNAi-mediated host immunity in pepper.

Keywords: Argonaute (AGO); Dicer-like (DCL); RNA-dependent RNA polymerase (RDR); pepper.

Figure 1

Structural analysis of CaAGOs (A), CaRDRs (B) and CaDCLs (C) in pepper. Domains are indicated as boxes in different colors.

Figure 2

Phylogenetic analysis of putative Argonaute protein (AGO), RNA-dependent RNA polymerase (RDR) and Dicer-like protein (DCL) proteins of pepper. Unrooted neighbor-joining trees constructed from multiple alignments of total (A) AGO, (B) DCL and (C) RDR protein sequences of pepper, tomato, Arabidopsis, tobacco and potato. Bootstrap support values from 1000 replications are indicated above the branches. Each gene family is divided into different clades as shown in the figure. Sequences of tomato, Arabidopsis, tobacco and potato were downloaded from the NCBI database. The red star indicated the proteins in pepper.

Figure 3

Heatmap showing the expression pattern of CaAGO, CaDCL and CaRDR genes in various organs. Relative expression levels of CaAGO, CaDCL and CaRDR genes in pepper were determined by real-time quantitative polymerase chain reaction (qRT-PCR) at corresponding organs, including roots, leaves, flowers, stems and fruit. The CaUbi3 was used as the reference gene. The color scale for each value is shown on the down pane.

Figure 4

qRT-PCR analyses of CaAGOs (A), CaDCLs (B) and CaRDRs (C) expression in response to viral infections. The pepper CaUbi3 was used as the reference gene, and three biological replicates were performed for these experiments. Error bars indicate the standard errors. Asterisks indicate the significant differences (p < 0.05) between control and treatment.

Figure 5

qRT-PCR analyses of CaAGOs (A), CaDCLs (B) and CaRDRs (C) expression under abiotic stress. The pepper CaUbi3 was used as the reference gene, and three biological replicates were performed for these experiments. Error bars indicate the standard errors. Asterisks indicate the significant differences (p < 0.05) between control and treatment.

Figure 6

qRT-PCR analyses of CaAGOs (A), CaDCLs (B) and CaRDRs (C) expression under phytohormone and H₂O₂ treatment. The pepper CaUbi3 was used as the reference gene, and three biological replicates were performed for these experiments. Error bars indicate the standard errors. Asterisks indicate the significant differences (p < 0.05) between control and treatment.