Exogenous dsRNA Induces RNA Interference of a Chalcone Synthase Gene in Arabidopsis thaliana

Abstract

Recent investigations have shown the possibility of artificial induction of RNA interference (RNAi) via plant foliar treatments with naked double-stranded RNA (dsRNA) to silence essential genes in plant fungal pathogens or to target viral RNAs. Furthermore, several studies have documented the downregulation of plant endogenous genes via external application of naked gene-specific dsRNAs and siRNAs to the plant surfaces. However, there are limited studies on the dsRNA processing and gene silencing mechanisms after external dsRNA application. Such studies would assist in the development of innovative tools for crop improvement and plant functional studies. In this study, we used exogenous gene-specific dsRNA to downregulate the gene of chalcone synthase (CHS), the key enzyme in the flavonoid/anthocyanin biosynthesis pathway, in Arabidopsis. The nonspecific NPTII-dsRNA encoding the nonrelated neomycin phosphotransferase II bacterial gene was used to treat plants in order to verify that any observed effects and processing of AtCHS mRNA were sequence specific. Using high-throughput small RNA (sRNA) sequencing, we obtained six sRNA-seq libraries for plants treated with water, AtCHS-dsRNA, or NPTII-dsRNA. After plant foliar treatments, we detected the emergence of a large number of AtCHS- and NPTII-encoding sRNAs, while there were no such sRNAs after control water treatment. Thus, the exogenous AtCHS-dsRNAs were processed into siRNAs and induced RNAi-mediated AtCHS gene silencing. The analysis showed that gene-specific sRNAs mapped to the AtCHS and NPTII genes unevenly with peak read counts at particular positions, involving primarily the sense strand, and documented a gradual decrease in read counts from 17-nt to 30-nt sRNAs. Results of the present study highlight a significant potential of exogenous dsRNAs as a promising strategy to induce RNAi-based downregulation of plant gene targets for plant management and gene functional studies.

Keywords: RNA interference; exogenous dsRNA; gene silencing; plant foliar treatment; plant gene regulation; small RNAs.

Figure 1

Schematic representation of the experiment conducted in the study to verify the effects of external dsRNA treatments in Arabidopsis thaliana on small RNAs profiles and AtCHS gene expression. (a) Representation of AtCHS- and NPTII-coding regions with positions of the AtCHS- and NPTII-specific dsRNA; (b) PCRs with the T7 promoter appended to both PCR primers for AtCHS and NPTII partial amplification, followed by in vitro dsRNA production; (c) plant foliar treatments with AtCHS- and NPTII-specific dsRNA; (d) low- and high-molecular-weight RNA isolation and processing. 2xP35S—the double 35S promoter of the cauliflower mosaic virus (CaMV); AtCHS—the chalcone synthase gene from A. thaliana; NPTII—the neomycin phosphotransferase II (NPTII) gene; Tnos—nopaline synthase terminator. T7–T7 promoter.

Figure 2

The effect of external AtCHS- and NPTII-encoding dsRNAs on AtCHS mRNA level in Arabidopsis thaliana analyzed by quantitative real-time PCR. WC—A. thaliana treated with sterile water; dsCHS—A. thaliana treated with AtCHS-dsRNA; dsNPTII—A. thaliana treated with NPTII-dsRNA; dpt—days post-treatment. A. thaliana plants were grown under anthocyanin-inducing (+7 °C, 23 h light) conditions for two days after treatment with sterile water or synthetic dsRNA. qRT-PCR data are presented as the mean ± SE. **—significantly different from WC at p ≤ 0.01 according to Student’s t-test.

Figure 3

Length size distribution of total small RNAs in Arabidopsis thaliana after treatment with water or dsRNA. WC—A. thaliana plants treated with sterile water; dsCHS—A. thaliana treated with AtCHS-dsRNA; dsNPTII—A. thaliana treated with NPTII-dsRNA. The data are presented as the mean ± SE. Means followed by the same letter were not different using Student’s t-test. p < 0.05 was considered statistically significant.

Figure 4

The effect of AtCHS-dsRNA and NPTII-dsRNA on the proportion and length size distribution of AtCHS-encoding 17–30-nt sRNAs among all detected small RNAs. (a) The proportion of AtCHS- and NPTII-encoding 17–30-nt sRNAs among all detected small RNAs. WC—A. thaliana plants treated with sterile water; dsCHS—A. thaliana treated with AtCHS-dsRNA; dsNPTII—A. thaliana treated with NPTII-dsRNA; (b) length size distribution of AtCHS-encoding 17–30-nt sRNAs; (c) length size distribution of NPTII-encoding 17–30-nt sRNAs. 1a_WC, 1b_WC—two A. thaliana plants treated with sterile water; 2a_dsCHS, 2b_dsCHS—two A. thaliana plants treated with AtCHS-dsRNA; 3a_dsNPTII, 3b_dsNPTII—two A. thaliana plants treated with NPTII-dsRNA.

Figure 5

Strand-specific distribution of the 17–30-nt AtCHS- and NPTII-encoding sRNAs detected in Arabidopsis thaliana after dsRNA treatments along the AtCHS (a) and NPTII (b) gene coding regions. Read depth was counted by the number of reads on each position. Sense strand is plotted with a solid line above the x-axis, and antisense strand is plotted with a dotted line below the x-axis. Green and orange colors indicate two biological replicates.