Development of an assay system for the analysis of host RISC activity in the presence of a potyvirus RNA silencing suppressor, HC-Pro

Abstract

Background: To investigate the mechanism of RNA silencing suppression, the genetic transformation of viral suppressors of RNA silencing (VSRs) in Arabidopsis integrates ectopic VSR expression at steady state, which overcomes the VSR variations caused by different virus infections or limitations of host range. Moreover, identifying the insertion of the transgenic VSR gene is necessary to establish a model transgenic plant for the functional study of VSR.

Methods: Developing an endogenous AGO1-based in vitro RNA-inducing silencing complex (RISC) assay prompts further investigation into VSR-mediated suppression. Three P1/HC-Pro plants from turnip mosaic virus (TuMV) (P1/HC-Pro^Tu), zucchini yellow mosaic virus (ZYMV) (P1/HC-Pro^Zy), and tobacco etch virus (TEV) (P1/HC-Pro^Te) were identified by T-DNA Finder and used as materials for investigations of the RISC cleavage efficiency.

Results: Our results indicated that the P1/HC-Pro^Tu plant has slightly lower RISC activity than P1/HC-Pro^Zy plants. In addition, the phenomena are consistent with those observed in TuMV-infected Arabidopsis plants, which implies that HC-Pro^Tu could directly interfere with RISC activity.

Conclusions: In this study, we demonstrated the application of various plant materials in an in vitro RISC assay of VSR-mediated RNA silencing suppression.

Keywords: AGO1 degradation; HC-pro; In vitro RISC assay; T-DNA insertion.

Fig. 1

α-AGO1 antibody generation. a Amino acid alignment of the N-termini of AGO1, AGO2, AGO4, and AGO10. The gray box indicates the N-terminal region of AGO1 for antigen. b Evaluation of the α-AGO1 antibody for detection of endogenous AGO1 in Col-0 plants. The null ago1-36 mutant serves as the negative control. The asterisk indicates tubulin, which was used as a loading control

Fig. 2

Establishment of an in vitro RISC assay. a Sequence pairing of MYB33 RNA and miR159 isoforms. The mismatched nucleotides are highlighted in red. b The endogenous AGO1-IP products from Col-0 plant and ago1-27 mutant were incubated with MYB33-230 RNA substrate to perform an in vitro RISC assay. The RNA substrate and cleaved RNA fragments are indicated with arrows. c The AGO1-IP amounts from Col-0 plant and ago1-27 mutant were evaluated by western blotting with α-AGO1 IgG for normalization of the RISC cleavage efficiency. In: input; IP: immunoprecipitation; FT: flow through. d Normalized RISC cleavage efficiency for AGO1-IP from Col-0 plant and ago1-27 mutant. e Sequencing pairing between miR159a and the MYB33^mSeed or MYB33^mCenter. The mismatched nucleotides are highlighted in red. f In vitro RISC assay for MYB33-230, MYB33^mSeed, and MYB33^mCenter RNA substrates. The RNA substrate and cleaved RNA fragments are indicated with arrows. (g) In vitro RISC assay for various miRNA-target substrates. h Normalized RISC cleavage efficiency for CSD-230, NAC1-230, and ARF16-230 RNA substrates (i). Normalized RISC cleavage efficiency for ARF10-230 and ARF10-191 RNA substrates (ii). The MYB33-230 RNA substrate served as a positive control

Fig. 3

The functional studies of HC-Pro for AGO1 regulation. a Phenotypes of P1/HC-Pro plants. The photographs were obtained from 14-day-old seedlings. Bar, 0.2 cm. b T-DNA insertions of various P1/HC-Pro plants. The diagrammatic chromosomes were labeled with the T-DNA insertion positions and critical RNA silencing component genes. Bar, 100,000 bp. c AGO1 levels in various P1/HC-Pro plants. RUBISCO (asterisk) and tubulin were used as loading controls. d Immunoprecipitation for evaluating the interactions of AGO1 with various HC-Pros. The asterisks indicate the heavy chain of IgG. Tubulin was used as a loading control

Fig. 4

Evaluation of various HC-Pros in terms of their efficiency in inhibiting RISC activity in vitro. a In vitro RISC assay (i) and evaluation of the AGO1-IP efficiency (ii) for P1/HC-Pro^Tu and P1/HC-Pro^Tu/atg8a^ge plants. In: input; IP: immunoprecipitation; FT: flow through. b Comparison of the in vitro RISC efficiency among the Col-0, P1/HC-Pro^Tu, and P1/HC-Pro^Tu/atg8a^ge plants. c Represented in vitro RISC assay with the P1/HC-Pro^Tu, P1/HC-Pro^Zy, P1/HC-Pro^Te, and P1/HC-Pro^Tu−K plants from 3 biological repeats. d Relative RISC cleavage efficiency of the P1/HC-Pro^Tu, P1/HC-Pro^Zy, and P1/HC-Pro^Te plants (i) and comparison of the RISC efficiency between Col-0 and P1/HC-Pro^Tu−K plants (ii). The bars represent the standard deviations (n = 3). Means were compared by Tukey’s honestly significance test. Different letters above the bars indicate significant differences

Fig. 5

The AGO1 levels and evaluation of miRNA-mediated target cleavage in TuMV-infected plants. a Symptoms of TuGR- and TuGK-infected Col-0 plants at 12 dpi. Bar, 1 cm. The red arrowheads indicate the curing and serrated systemic leaves after TuGR infection. b Endogenous AGO1 and HC-Pro levels in mock, TuGR-, and TuGK-infected plants. The CP levels were used to confirm TuMV infection. RUBISCO (asterisk) was used as loading controls. c miRNA target gene expression of AGO1 (miR168), ARF16 (miR160), and MYB33 (miR159) in mock, TuGR-, and TuGK-infected plants. The bars represent the standard errors (n = 3). The statistical significance was assessed based on Student’s t test. ** indicates P values < 0.01

Fig. 6

Evaluation of the in vitro RISC cleavage efficiency using TuMV-infected plants. a MYB33-230 RNA substrates were cleaved by AGO1-IP from mock, TuGR-, and TuGK-infected Col-0 plants. The numbers indicate the amounts of AGO1-IP that were used for cleavage. b The AGO1-IP amounts from mock, TuGR-, and TuGK-infected Col-0 plants were evaluated by western blotting with α AGO1 IgG to normalize the RISC cleavage efficiency. In: input; IP: immunoprecipitation; FT: flow through. c Normalized RISC cleavage efficiency of AGO1-IP from mock, TuGR-, and TuGK-infected Col-0 plants

Fig. 7

The working hypothesis for various HC-Pros in inhibiting the RISC activity. a The model of RISC inhibition by HC-Pro^Tu. b The model to explain no effect on RISC inhibition by HC-Pro^Zy and HC-Pro^Te. The dark red lines represent as mature miR159. The blue lines represent other small RNAs (sRNAs). The gray lines indicate the miRNA*. The dark red color indicates the methylated miRNAs. The pink color indicates the unmethylated miRNAs. Me, the methylated miRNA