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. 2020 Apr 3;21(7):2498.
doi: 10.3390/ijms21072498.

Mapping the Gene Expression Spectrum of Mediator Subunits in Response to Viroid Infection in Plants

Vishnu Sukumari Nath  1 , Ankita Shrestha  1 , Praveen Awasthi  1 , Ajay Kumar Mishra  1 , Tomáš Kocábek  1 , Jaroslav Matoušek  1 , Andrej Sečnik  2 , Jernej Jakše  2 , Sebastjan Radišek  3 , Vipin Hallan  4
Affiliations

Mapping the Gene Expression Spectrum of Mediator Subunits in Response to Viroid Infection in Plants

Vishnu Sukumari Nath et al. Int J Mol Sci. .

Abstract

The mediator (MED) represents a large, conserved, multi-subunit protein complex that regulates gene expression through interactions with RNA polymerase II and enhancer-bound transcription factors. Expanding research accomplishments suggest the predominant role of plant MED subunits in the regulation of various physiological and developmental processes, including the biotic stress response against bacterial and fungal pathogens. However, the involvement of MED subunits in virus/viroid pathogenesis remains elusive. In this study, we investigated for the first time the gene expression modulation of selected MED subunits in response to five viroid species (Apple fruit crinkle viroid (AFCVd), Citrus bark cracking viroid (CBCVd), Hop latent viroid (HLVd), Hop stunt viroid (HSVd), and Potato spindle tuber viroid (PSTVd)) in two model plant species (Nicotiana tabacum and N. benthamiana) and a commercially important hop (Humulus lupulus) cultivar. Our results showed a differential expression pattern of MED subunits in response to a viroid infection. The individual plant MED subunits displayed a differential and tailored expression pattern in response to different viroid species, suggesting that the MED expression is viroid- and plant species-dependent. The explicit evidence obtained from our results warrants further investigation into the association of the MED subunit with symptom development. Together, we provide a comprehensive portrait of MED subunit expression in response to viroid infection and a plausible involvement of MED subunits in fine-tuning transcriptional reprogramming in response to viroid infection, suggesting them as a potential candidate for rewiring the defense response network in plants against pathogens.

Keywords: Nicotiana benthamiana; Nicotiana tabacum; differential expression; hop; mediator complex; pathogen; quantitative reverse transcription PCR; viroid.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Schematic representation of dimeric infectious constructs, detection and quantification of viroids. (A) Schematic diagram of a plasmid containing the shown viroid (+) dimer created by cDNA cloning in SacI restriction site. The viroid (+) dimer was re-cloned from pPCR-Script to XhoI–XbaI sites of intermediary vector pLV-68. The final modified binary expression cassette harboring CaMV 35S promoter, viroid cDNA and CaMV terminator was cloned into PacI and AscI sites of the plasmid pLV-07. ori: origin of replication; kanR: kanamycin resistance gene; RB: left border of T-DNA; RB: right border of T-DNA; T CaMV: terminator from cauliflower mosaic virus; Pnos: nopalin synthase promoter; nptII: neomycin phosphotransferase II. RT-PCR-based detection and strand-specific real-time RT-qPCR quantification of viroids in single infected Nicotiana tabacum (B), N. benthamiana (C) and hop (D) plants. The gel picture shows three biological replicates of infected samples (with amplification) and a negative control (without amplification). The numbers under the bar indicate plant sample codes. All samples were normalized to the strand with a higher level (100%) and relative quantities were calculated using target-specific amplification efficiencies. Each column represents the mean ± SD of three technical replicates of single infected plants.
Figure 2
Figure 2
Reverse transcriptase quantitative real-time PCR (RT-qPCR)-based expression profiling of selected mediator subunits in response to the viroid infection in N. tabacum (A), N. benthamiana (B) and hop (C) plants. RT-qPCR analyses were normalized using DRH1 as an internal control gene and the fold change in each gene in viroid infected/transformed plants was calculated with respect to mock-inoculated control plants by the (2−ΔΔCt) method. The data were obtained from three independent experiments; bars show ± SD. Comparison between groups was assessed by a two-way ANOVA followed by Tukey’s test, an asterisk denotes statistically significant differences (* p < 0.1, ** p < 0.05, and *** p < 0.01).
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