Dataset Information

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.

SUBMITTER: Kiselev KV

PROVIDER: S-EPMC7911504 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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Physiological Conditions and dsRNA Application Approaches for Exogenously induced RNA Interference in <i>Arabidopsis thaliana</i>.

Kiselev Konstantin V KV Suprun Andrey R AR Aleynova Olga A OA Ogneva Zlata V ZV Dubrovina Alexandra S AS

Plants (Basel, Switzerland) 20210130 2

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 s ...[more]

PMID: 33573142

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Project description:The application of double-stranded RNAs (dsRNAs) to plant surfaces has emerged as a promising tool for manipulating gene expression in plants and pathogens, offering new opportunities for crop improvement. While research has shown the capability of exogenous dsRNAs to silence genes, the full spectrum of their impact, particularly on the intricate network of microRNAs (miRNAs), remains largely unexplored. Here, we show that the exogenous application of chalcone synthase (CHS)-encoding dsRNA to the rosette leaves of Arabidopsis thaliana induced extensive alterations in the miRNA profile, while non-specific bacterial neomycin phosphotransferase II (NPTII) dsRNA had a minimal effect. Two days after treatment, we detected 60 differentially expressed miRNAs among the 428 miRNAs found in the A. thaliana genome. A total of 59 miRNAs were significantly changed after AtCHS-dsRNA treatment compared with water and NPTII-dsRNA, and 1 miRNA was significantly changed after AtCHS-dsRNA and NPTII-dsRNA compared with the water control. A comprehensive functional enrichment analysis revealed 17 major GO categories enriched among the genes potentially targeted by the up- and downregulated miRNAs. These categories included processes such as aromatic compound biosynthesis (a pathway directly related to CHS activity), heterocycle biosynthesis, RNA metabolism and biosynthesis, DNA transcription, and plant development. Several predicted targets of upregulated and downregulated miRNAs, including APETALA2, SCL27, SOD1, GRF1, AGO2, PHB, and PHV, were verified by qRT-PCR. The analysis showed a negative correlation between the expression of miRNAs and the expression of their predicted targets. Thus, exogenous plant gene-specific dsRNAs induce substantial changes in the plant miRNA composition, ultimately affecting the expression of a wide range of genes. These findings have profound implications for our understanding of the effects of exogenously induced RNA interference, which can have broader effects beyond targeted mRNA degradation, affecting the expression of other genes through miRNA regulation.

Project description:In this work we developed and exploited a spray-induced gene silencing (SIGS)-based approach to deliver double-stranded RNA (dsRNA), which was found to protect potato against potato virus Y (PVY) infection. Given that dsRNA can act as a defence-inducing signal that can trigger sequence-specific RNA interference (RNAi) and non-specific pattern-triggered immunity (PTI), we suspected that these two pathways may be invoked via exogeneous application of dsRNA, which may account for the alterations in PVY susceptibility in dsRNA-treated potato plants. Therefore, we tested the impact of exogenously applied PVY-derived dsRNA on both these layers of defence (RNAi and PTI) and explored its effect on accumulation of a homologous virus (PVY) and an unrelated virus (potato virus X, PVX). Here, we show that application of PVY dsRNA in potato plants induced accumulation of both small interfering RNAs (siRNAs), a hallmark of RNAi, and some PTI-related gene transcripts such as WRKY29 (WRKY transcription factor 29; molecular marker of PTI), RbohD (respiratory burst oxidase homolog D), EDS5 (enhanced disease susceptibility 5), SERK3 (somatic embryogenesis receptor kinase 3) encoding brassinosteroid-insensitive 1-associated receptor kinase 1 (BAK1), and PR-1b (pathogenesis-related gene 1b). With respect to virus infections, PVY dsRNA suppressed only PVY replication but did not exhibit any effect on PVX infection in spite of the induction of PTI-like effects in the presence of PVX. Given that RNAi-mediated antiviral immunity acts as the major virus resistance mechanism in plants, it can be suggested that dsRNA-based PTI alone may not be strong enough to suppress virus infection. In addition to RNAi- and PTI-inducing activities, we also showed that PVY-specific dsRNA is able to upregulate production of a key enzyme involved in poly(ADP-ribose) metabolism, namely poly(ADP-ribose) glycohydrolase (PARG), which is regarded as a positive regulator of biotic stress responses. These findings offer insights for future development of innovative approaches which could integrate dsRNA-induced RNAi, PTI and modulation of poly(ADP-ribose) metabolism in a co-ordinated manner, to ensure a high level of crop protection.

Dataset Information

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

Publications

Physiological Conditions and dsRNA Application Approaches for Exogenously induced RNA Interference in <i>Arabidopsis thaliana</i>.

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