Physiological Conditions and dsRNA Application Approaches for Exogenously induced RNA Interference in Arabidopsis thaliana

Abstract

Recent studies have revealed that foliar application of double-stranded RNAs (dsRNAs) or small-interfering RNAs (siRNAs) encoding specific genes of plant pathogens triggered RNA interference (RNAi)-mediated silencing of the gene targets. However, a limited number of reports documented silencing of plant endogenes or transgenes after direct foliar RNA application. This study analyzed the importance of physiological conditions (plant age, time of day, soil moisture, high salinity, heat, and cold stresses) and different dsRNA application means (brush spreading, spraying, infiltration, inoculation, needle injection, and pipetting) for suppression of neomycin phosphotransferase II (NPTII) transgene in Arabidopsis thaliana, as transgenes are more prone to silencing. We observed a higher NPTII suppression when dsRNA was applied at late day period, being most efficient at night, which revealed a diurnal variation in dsRNA treatment efficacy. Exogenous NPTII-dsRNA considerably reduced NPTII expression in 4-week-old plants and only limited it in 2- and 6-week-old plants. In addition, a more discernible NPTII downregulation was detected under low soil moisture conditions. Treatment of adaxial and abaxial leaf surfaces by brushes, spraying, and pipetting showed a higher NPTII suppression, while infiltration and inoculation were less efficient. Thus, appropriate plant age, late time of day, low soil moisture, and optimal dsRNA application modes are important for exogenously induced gene silencing.

External dsRNA Downregulates Anthocyanin Biosynthesis-Related Genes and Affects Anthocyanin Accumulation in Arabidopsis thaliana

“…For external application, the synthesized dsRNAs were diluted in water to a final concentration of 0.35 µg/µL. The dsRNAs (100 µL of each dsRNA per individual plant, i.e., 35 µg) were applied on the leaf surface (on both the adaxial and abaxial sides) of four-week-old A. thaliana rosettes by spreading with sterile individual soft brushes [31] (Supplementary Video S1). Importantly, we treated the four-week-old rosettes of A. thaliana at a late day time (21:00-21:30) under low soil moisture conditions in all experiments, since the appropriate plant age, late day time, and low soil moisture at the time of dsRNA application were important parameters for successful NPTII suppression in transgenic A. thaliana according to our recent analysis [31].…”

“…The dsRNAs (100 µL of each dsRNA per individual plant, i.e., 35 µg) were applied on the leaf surface (on both the adaxial and abaxial sides) of four-week-old A. thaliana rosettes by spreading with sterile individual soft brushes [31] (Supplementary Video S1). Importantly, we treated the four-week-old rosettes of A. thaliana at a late day time (21:00-21:30) under low soil moisture conditions in all experiments, since the appropriate plant age, late day time, and low soil moisture at the time of dsRNA application were important parameters for successful NPTII suppression in transgenic A. thaliana according to our recent analysis [31]. An analysis of the dsRNA concentration effect on transgene silencing in A. thaliana was performed previously [30], indicating that 35 µg of a transgene-encoding dsRNA resulted in the highest transgene silencing efficiency as compared to other dsRNA concentrations.…”

“…An analysis of the dsRNA concentration effect on transgene silencing in A. thaliana was performed previously [30], indicating that 35 µg of a transgene-encoding dsRNA resulted in the highest transgene silencing efficiency as compared to other dsRNA concentrations. [31]. An analysis of the dsRNA concentration effect on transgene silencing in A. thaliana was performed previously [30], indicating that 35 µg of a transgene-encoding dsRNA resulted in the highest transgene silencing efficiency as compared to other dsRNA concentrations.…”

“…According to Numata et al [32] and Dalakouras et al [33], downregulation of the GFP and YFP transgenes after foliar application of siRNAs was successful only after application of accessory technologies (using carrier peptide or high-pressure spraying). According to our recent data, appropriate plant age, late time of day, low soil moisture (at the moment of dsRNA application), and optimal dsRNA application modes were important for efficient NPTII transgene suppression in A. thaliana induced by direct foliar dsRNA treatments [31].…”

“…Several studies reported that external application of dsRNAs [22,30,31] to Arabidopsis thaliana and siRNAs [32][33][34] to A. thaliana or Nicotiana benthamiana triggered silencing of common plant transgenes, such as green fluorescent protein (GFP), β-glucuronidase (GUS), yellow fluorescent protein (YFP), or neomycin phosphotransferase II (NPTII). Plant transgenes are known to be more prone for RNAi-mediated suppression in comparison with plant endogenes [35][36][37], and, therefore, targeting transgenes might be more achievable.…”