HC-Pro inhibits HEN1 methyltransferase activity, leading to autophagic degradation of AGO1

Abstract

Helper-component proteinase (HC-Pro), encoded by potyviruses, function as viral suppressors of RNA silencing (VSRs). Despite their conserved role, HC-Pros share approximately 40% similarity, implying potential differences in VSR efficiency, particularly in their ability to inhibit HEN1 methyltransferase activity. This study investigated the inhibitory potential of HC-Pros from different potyviruses in transgenic plants. P1/HC-Pro from turnip mosaic virus (P1/HC-Pro^Tu) exhibited the most potent inhibition of HEN1, followed by P1/HC-Pro from zucchini yellow mosaic virus (P1/HC-Pro^Zy), while P1/HC-Pro from tobacco etch virus (P1/HC-Pro^Te) showed the weakest inhibitory effect. These differential effectual effects corresponded to variations in unmethylated microRNAs (unMet-miRNAs) accumulation across the transgenic lines. Fluorescence resonance energy transfer (FRET) analysis indicated that HC-Pro^Tu recruits HEN1 and ATG8a to HC-Pro bodies (H-bodies) and indirectly associates with AGO1, potentially influencing the assembly of the RNA-induced silencing complex (RISC) and leading to the accumulation of free-form miRNA duplexes. The ability of HC-Pro^Tu to sequester HEN1 and AGO1 in H-bodies may, therefore, modulate miRNA loading. This observation aligns with the finding that P1/HC-Pro^Tu plants harbored approximately 50% unMet-miRNAs and exhibited the lowest AGO1 levels, suggesting a positive correlation between HEN1 inhibition and autophagic degradation of AGO1. Interestingly, unMet-miRNAs are absent in the AGO1 of P1/HC-Pro^Tu plants but reappeared in P1/HC-Pro^Tu/hen1-8/heso1-1 plants, accompanied by signs of AGO1 recovery. These findings highlight the functional diversity of HC-Pro VSRs and provide new insights into their differential effects on miRNA methylation, RISC assembly, and the regulation of RNA silencing pathways.

Fig. 1. The common molecular features of various P1/HC-Pro variants during RNA silencing.

a Schematic binary plasmids containing the various P1/HC-Pro constructs used in this study. b Phenotypes of transgenic P1/HC-Pro plants. Photographs of 3-week-old plants were taken. A representative plant of each category is shown. Bar, 1 cm. c Abnormal miRNA/miRNA* accumulation in various P1/HC-Pro plants. U6 small nuclear RNA was used as a loading control. The numbers on the panels represent the average signal strengths of the miRNAs relative to those in the non-transformed wild-type plants (Col-0) after normalization against the loading control. The levels in the Col-0 plants were set at 1.0. Two independent lines for each construct were analyzed. NA, not available. d The SEC distributions of AGO1, HC-Pro, and HA-HEN1 in various plants. AGO1-L, the large form of AGO1. AGO1-S, the small form of AGO1 (n = 10). e The miR159 SEC distribution in various plants (n = 10). Syn-ds-miR159, the synthetic miR159 duplex (i). The miR165 and miR165* SEC distribution in P1/HC-Pro^Tu plants (ii). f The methylation status of different SEC fractions in various plants. Fraction-mix I was mixed with fractions 4 and 6; fraction-mix II was mixed with fractions 10 and 12; fraction-mix III was mixed with fractions 14 and 16; and the free-form fraction was mixed with fractions 24 and 26. Two asterisks indicate an unknown additional band (n = 3). g Heatmap showing the expression levels of 103 miRNA target genes in Col-0 and various P1/HC-Pro plants. The color scale bar indicates the fold change in target gene expression. h Principal component analysis (PCA) for Col-0 and various P1/HC-Pro plants. i H-bodies observed in HC-Pro^Tu-CFP (i) and HC-Pro^Tu-K-CFP (ii). Evaluation of the subcellular colocalization between HC-Pro^Zy-CFP (iii) and HC-Pro^Tu-YFP (iv). A representative image of each category is shown. Bars, 10 μm. In, inclusion body.

Fig. 2. HC-Pro^Tu specifically attracts AGO1 and initiates its degradation.

a Endogenous AGO1 protein levels (AGO1-L and AGO1-S) in Col-0 and various P1/HC-Pro plants (i). Actin (ACT) was used as a loading control. The fold change values are indicated below the gel image. The levels in the Col-0 plants were set at 1.0. The average signal strengths of AGO1 relative to those in the non-transformed wild-type plants (Col-0) after normalization against the ACT loading control. Bar charts showed the relative amount of AGO1-L protein quantified from western blotting (WB) (ii) and from LC-MS/MS (iii). Data are presented as mean values ± standard errors (SE, n = 5 for WB, and n = 5 for LC-MS/MS). Significant differences based on Student’s t-test; *, **, and *** indicate P values < 0.05, <0.01, and 0.001, respectively. b AGO1 expression in Col-0, P1/HC-Pro^Tu, and P1/HC-Pro^Tu-K plants was evaluated by real-time RT-PCR. Relative expression levels were normalized to the ubiquitin level. Data are presented as mean values ± standard deviations (n = 3). Significant differences were determined by Student’s t-test; * indicates P values < 0.05. c The subcellular colocalization of HC-Pro^Tu-CFP (i) or HC-Pro^Tu-K-CFP (iii) with YFP-AGO1 (ii and iv). N, nucleus; In, inclusion body. Arrowheads indicate speckle-like structures. Bar, 50 μm. d FRET efficiency of co-infiltrated plants. Data are presented as mean values ± standard deviations (n = 10). e Image size measurement for speckle-like structures. Bar, 50 μm. (i) CFP channel for HC-Pro^Tu-CFP. (ii) YFP channel for YFP-AGO1. Bar, 50 μm. f Size comparison of different foci (n = 15). Data are presented as mean values ± standard deviations. Source data are provided as a Source Data file.

Fig. 3. HC-Pro^Tu triggers autophagic AGO1 degradation.

a Heatmaps of autophagy-related genes in various P1/HC-Pro plants were generated from transcriptome profiles. The color scale bar indicates the fold change in target gene expression. b Differential expression of ATG8 family genes in various P1/HC-Pro plants relative to their expression levels. Data are presented as mean values ± standard deviations (n = 3). Significant differences were determined by Student’s t-test; * indicates P values < 0.05. ** indicates P values < 0.01. *** indicates P values < 0.001. c Diagram of the gRNA positions in the ATG8a gene (i) and the gene-editing results (ii) for P1/HC-Pro^Tu/atg8a^ge. d ATG8a transcript levels in P1/HC-Pro^Tu and P1/HC-Pro^Tu/atg8a^ge plants compared with those of Col-0. Data are presented as mean values ± standard errors (n = 3). e Hierarchical clustering dendrogram on the transcriptomes of Col-0, P1/HC-Pro^Tu and P1/HC-Pro^Tu/atg8a^ge plants. f Relative expression of autophagy-related genes in P1/HC-Pro^Tu and P1/HC-Pro^Tu/atg8a^ge plants compared to that in Col-0 plants. Data are presented as mean values ± standard deviations (n = 3). Significant differences based on Student’s t-test; * indicates P values < 0.05. ** indicates P values < 0.01. g Evaluation of the suppression efficiency of miRNA-mediated target gene regulation between P1/HC-Pro^Tu and P1/HC-Pro^Tu/atg8a^ge plants by real-time RT-PCR. Relative expression levels were normalized to the ubiquitin level. Data are presented as mean values ± standard errors (n = 6). Significant differences were determined by Student’s t-test; ** indicates P values < 0.01. h Subcellular localization of ATG8a (i), HC-Pro^Tu (ii), and AGO1 (iii) within leaf cells as revealed by immunogold labeling and transmission electron microscopy (TEM). Immunogold signals were detected within and/or surrounding autophagic structures, many of which were encased by autophagic vacuoles. Red arrowheads highlight the presence of immunogold particles within autophagic vacuoles (Av) or autophagic-like structures (Ap), while blue arrowheads denote signals found in the cytoplasm (n = 3). Cw, cell wall; Cv, central vacuole; Ch, chloroplast. Scale bars, 200 nm. i Image size measurements for speckle-like structures. Data are presented as mean values ± standard deviations (n = 10). j FRET efficiency of co-infiltrated plants in which HC-Pro^Tu-CFP or HC-Pro^Tu-K-CFP interacts with YFP-ATG8a. Data are presented as mean values ± standard deviations (n = 9). Source data are provided as a Source Data file.

Fig. 4. HC-Pro^Tu explicitly inhibits and interacts with HEN1.

a Evaluation of miRNA methylation status in various P1/HC-Pro plants by β-elimination (i). Total RNA samples were treated with β-elimination (+) or without treatment (−). The β-elimination assay was used to determine the presence of methylation in miRNAs (ii). Bar charts represent the percentage of methylated (Met) and unmethylated (unMet) miR159 and miR165 in the samples. Error bars indicate standard deviations based on three biological replicates (n = 3). b Evaluation of miRNA methylation in recombinant P1/HC-Pro plants by β-elimination. c Evaluation of the miR159 methylation status in ZYMV-infected squash plants by β-elimination (n = 3) (i). P1/HC-Pro^Tu plants were used as a positive control. HC-Pro^Zy detection in ZYMV-infected squash plants (ii). The asterisk indicates the RUBISCO loading control. d YFP channel for the YFP-HEN1 sample. The yellow arrowheads indicate HEN1 foci. In, inclusion body. Bar, 50 μm. e Image size measurements for speckle-like structures. (i) CFP channel for HC-Pro^Tu-CFP, which was co-expressed with YFP-HEN1. (ii) YFP channel for YFP-HEN1, which is co-expressed with HC-Pro^Tu-CFP. Bar, 50 μm. f The sizes of various bodies. Error bar, standard deviation Data are presented as mean values ± standard deviations (n = 10). g Subcellular colocalization of HC-Pro^Tu-K-CFP (i) or HC-Pro^Tu-CFP (iii) with YFP-HEN1 (ii and iv). Bar, 50 μm. In, inclusion body. N, nucleus. Arrowheads indicate speckle-like structures. h FRET efficiency of co-infiltrated plants in which HC-Pro^Tu-CFP or HC-Pro^Tu-K-CFP interacts with YFP-HEN1. Data are presented as mean values ± standard deviations (n = 9). Source data are provided as a Source Data file.

Fig. 5. In vitro assays for HC-Pro inhibition of HEN1 activity.

a Recombinant protein purification (n = 10). b In vitro HEN1 methylation inhibition assay with the GST-HC-Pro^Tu or GST-HC-Pro^Tu-K protein. The methylation of synthetic miRNAs was examined via oxidation/β-elimination and small RNA northern blotting assays. The methylation status is indicated on the lower side of the panel. GST served as a negative control (n = 8). c In vitro EMSA for HEN1 binding to miRNA duplexes with/without various recombinant HC-Pros (n = 5). d In vitro EMSA for various HC-Pro in inhibiting HEN1-miRNA duplex binding activity (n = 8).

Fig. 6. HC-Pro^Tu prevents unMet-miRNA loading into AGO1.

a β-elimination of the miRNA methylation status in the total RNA and AGO1-IP samples from 1-week-old Col-0, P1/HC-Pro^Tu, and P1/HC-Pro^Tu/atg8a^ge plants (n = 3). b MiRNA methylation status in total RNA and AGO1-IP samples from 1-week-old hen1-8/heso1-1 mutants and P1/HC-Pro^Tu/hen1-8/heso1-1 plants that were compared with Col-0 plants and P1/HC-Pro^Tu plants (n = 3). c In vitro RISC assay in Col-0, hen1-8/heso1-1, and ago1-27 mutants (n = 3). d The endogenous AGO1 levels in the P1/HC-Pro^Tu/hen1-8/heso1-1 plant were compared to those in Col-0 and P1/HC-Pro^Tu plants (i). Relative AGO1 protein expression levels were normalized to the actin (ACT) loading control and quantified using the bar chart (ii). Data are presented as mean values ± standard errors (SE, n = 3). Significant differences based on Student’s t-test; ** indicates P values < 0.01. e The enzyme-linked immunosorbent assay (ELISA) analysis for evaluation of the TuMV severe strain (TuGR) and mild strain (TuGK) infectivity in Col-0, dcl2-4/dcl4-1, and hen1-8/heso1-1 plants at 14 days post-inoculation (dpi) using TuMV coat protein (CP) antibody at 1/20,000 dilution. Data are presented as mean values ± standard deviations (n = 20). Source data are provided as a Source Data file.

Fig. 7. A working hypothesis model for the suppression of RNA silencing by HC-Pro^Tu.

a The inside of the H-body for HC-Pro interacts with RNA silencing components. b The working hypothesis for HC-Pro^Tu-induced degradation of AGO1 suggests that HC-Pro^Tu exhibits a heightened inhibitory effect on HEN1 methylation activity. This inhibition results in the incorporation of unmethylated miRNAs (unMet-miRNAs) into AGO1 complexes, which subsequently activate autophagy pathways, leading to the degradation of AGO1.