RNAi-Mediated Down-Regulation of Dicer-Like 2 and 4 Changes the Response of 'Moneymaker' Tomato to Potato Spindle Tuber Viroid Infection from Tolerance to Lethal Systemic Necrosis, Accompanied by Up-Regulation of miR398, 398a-3p and Production of Excessive Amount of Reactive Oxygen Species.

Takahiro Suzuki,Kohei Sugawara,Maki Okuta,Misato Fujibayashi,Teruo Sano,Atsushi Kasai,Sho Ikeda,Akito Taneda,Hayato Maeda

doi:10.3390/v11040344

Takahiro Suzuki, Kohei Sugawara + Show 7 more

Open Access

https://doi.org/10.3390/v11040344

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Journal: Viruses	Publication Date: Apr 13, 2019
Citations: 21	License type: CC BY 4.0

Affiliation: Hirosaki University

Abstract

To examine the role of RNA silencing in plant defenses against viroids, a Dicer-like 2 and 4 (DCL2&4)–double knockdown transgenic tomato plant line, 72E, was created. The expression of endogenous SlDCL2s and SlDCL4 in line 72E decreased to about a half that of the empty cassette line, EC. When challenged with potato spindle tuber viroid (PSTVd), line 72E showed significantly higher levels of PSTVd accumulation early in the course of the infection and lethal systemic necrosis late in the infection. The size distribution of PSTVd-derived small RNAs was significantly different with the number of RNAs of 21 and 22 nucleotides (nt) in line 72E, at approximately 66.7% and 5% of those in line EC, respectively. Conversely, the numbers of 24 nt species increased by 1100%. Furthermore, expression of the stress-responsive microRNA species miR398 and miR398a-3p increased 770% and 868% in the PSTVd-infected line 72E compared with the PSTVd-infected EC. At the same time, the expression of cytosolic and chloroplast-localized Cu/Zn-superoxide dismutase 1 and 2 (SOD1 and SOD2) and the copper chaperon for SOD (CCS1) mRNAs, potential targets of miR398 or 398a-3p, decreased significantly in the PSTVd-infected line 72E leaves, showing necrosis. In concert with miR398 and 398a-3p, SODs control the detoxification of reactive oxygen species (ROS) generated in cells. Since high levels of ROS production were observed in PSTVd-infected line 72E plants, it is likely that the lack of full dicer-likes (DCL) activity in these plants made them unable to control excessive ROS production after PSTVd infection, as disruption in the ability of miR398 and miR398a-3p to regulate SODs resulted in the development of lethal systemic necrosis.

Highlights

Viroids are the smallest known pathogens of higher plants [1]
The hairpin RNA produced by transcription of the SlartDCL2&4 IR transgene in tomato cells should activate the RNAi machinery, induce the production of short interfering RNA (siRNA) complementary to SlDCL2a, SlDCL2b, and SlDCL4 transcripts, and suppress endogenous SlDCL2s and SlDCL4 expression
Presence of the transgene was examined by PCR amplification of CaMV-35S promoter sequence from genomic DNA extracted from transgenic lines

Summary

Introduction

Viroids are the smallest known pathogens of higher plants [1]. They consist solely of a highlystructured, covalently closed, circular RNA molecule that ranges between 246 and 434 nucleotides (nt) in length. In light of the non-coding nature of the viroid genome, all the factors necessary to replicate in invaded host cells (i.e., to recruit transcription machinery such as RNA polymerase II and transcription factors, to move from cell to cell, and to spread systemically, and even those necessary to cause disease symptoms) must be embedded in the highly base-paired stem-loop structure [3]. Many of these properties are yet to be studied and described

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Conclusion