Combining a Simple Method for DNA/RNA/Protein Co-Purification and Arabidopsis Protoplast Assay to Facilitate Viroid Research

Abstract

Plant–viroid interactions represent a valuable model for delineating structure–function relationships of noncoding RNAs. For various functional studies, it is desirable to minimize sample variations by using DNA, RNA, and proteins co-purified from the same samples. Currently, most of the co-purification protocols rely on TRI Reagent (Trizol as a common representative) and require protein precipitation and dissolving steps, which render difficulties in experimental handling and high-throughput analyses. Here, we established a simple and robust method to minimize the precipitation steps and yield ready-to-use RNA and protein in solutions. This method can be applied to samples in small quantities, such as protoplasts. Given the ease and the robustness of this new method, it will have broad applications in virology and other disciplines in molecular biology.

A nuclear import pathway exploited by pathogenic noncoding RNAs

“…Arabidopsis thaliana contains the necessary machinery to support PSTVd nuclear import and replication but repels PSTVd systemic infection ( Daros and Flores, 2004 ; Jiang et al, 2019 ). To test whether any IMPa protein(s) is responsible for viroid nuclear import, we employed the RNA immunoprecipitation assay to test the possibility for PSTVd associating with any of the nine Arabidopsis IMPa proteins in a complex.…”

“…After SDS–PAGE electrophoresis, we followed a previously described protocol for immunoblotting ( Jiang et al, 2019 ). IMPas were detected by a monoclonal mouse anti-Myc antibody (catalog #M5546; MilliporeSigma; 1:3,000 dilution).…”

Evidence Supporting That RNA Polymerase II Catalyzes De Novo Transcription Using Potato Spindle Tuber Viroid Circular RNA Templates

“…Arabidopsis thaliana wildtype and tfiis mutant (SALK_027258C) plants were grown in a growth chamber with an 8/16 h light/dark cycle at 22 • C. Both lines were obtained from Arabidopsis Biological Resource Center (Ohio State University, Columbus, OH, USA). Using 4-week old plants, protoplasts were isolated following an established protocol [49,50]. Briefly, we included 3% cellulose (Onozuka Yakult Pharmaceutical IND., Tokyo, Japan) and 0.8% macerase (MilliporeSigma, Burlington, MA, USA) in the digestion buffer (0.4 M mannitol, 20 mM KCl, 20 mM MES, 10 mM CaCl 2 , 0.1% BSA, 5 mM β-mercaptoethanol, pH 5.7) for digesting leaf blades with the lower epidermis layer removed by tapes.…”

“…The products were purified using the MEGAclear kit (Thermo Fisher Scientific, Waltham, MA, USA). The products contain four RNA species, including the unit-length (+)-PSTVd as shown in Jiang et al [50]. To prepare the size markers, the PSTVd RZ-Int plasmids [51,52] were digested with HindIII (New England Biolabs, Ipswich, MA, USA) and then were subjected to in vitro transcription using T7 MEGAscript (Thermo Fisher Scientific, Waltham, MA, USA).…”

“…As shown in Figure 5, genomic PCR confirmed that our protoplasts were generated from homozygous tfiis loss-of-function plants. Using a previously established protoplast transfection system [50], we transfected wildtype and mutant protoplasts with linear PSTVd RNA inoculum and harvested the total RNA two days post inoculation. As shown in Figure 5, we could observe the accumulation of circular PSTVd in both wildtype and tfiis protoplasts, which reflects the successful replication of PSTVd based on previous studies [45,50,56,67,68].…”