Investigating the Viral Suppressor HC-Pro Inhibiting Small RNA Methylation through Functional Comparison of HEN1 in Angiosperm and Bryophyte

Abstract

In plants, HEN1-facilitated methylation at 3' end ribose is a critical step of small-RNA (sRNA) biogenesis. A mutant of well-studied Arabidopsis HEN1 (AtHEN1), hen1-1, showed a defective developmental phenotype, indicating the importance of sRNA methylation. Moreover, Marchantia polymorpha has been identified to have a HEN1 ortholog gene (MpHEN1); however, its function remained unfathomed. Our in vivo and in vitro data have shown MpHEN1 activity being comparable with AtHEN1, and their substrate specificity towards duplex microRNA (miRNA) remained consistent. Furthermore, the phylogenetic tree and multiple alignment highlighted the conserved molecular evolution of the HEN1 family in plants. The P1/HC-Pro of the turnip mosaic virus (TuMV) is a known RNA silencing suppressor and inhibits HEN1 methylation of sRNAs. Here, we report that the HC-Pro physically binds with AtHEN1 through FRNK motif, inhibiting HEN1's methylation activity. Moreover, the in vitro EMSA data indicates GST-HC-Pro of TuMV lacks sRNA duplex-binding ability. Surprisingly, the HC-Pro also inhibits MpHEN1 activity in a dosage-dependent manner, suggesting the possibility of interaction between HC-Pro and MpHEN1 as well. Further investigations on understanding interaction mechanisms of HEN1 and various HC-Pros can advance the knowledge of viral suppressors.

Keywords: HEN1; HEN1-HC-Pro interaction; methylation; sRNA; viral suppressors.

Figure 1

α-AtHEN1 and α-MpHEN1 antibodies production and sensitivity analysis. (A) The SDS-PAGE analysis for the eluted fractions of the recombinant full-length his-AtHEN1. The band corresponding to 130 kDa showed the presence of the expected protein. (B) SDS-PAGE analysis for the eluted his-MTase. The prominent band at 34 kDa depicted its expression. (C) The sensitivity evaluation of α-AtHEN1 (5000× dilution) that compared with his-monoclonal antibody (10,000× dilution). (D) The SDS-PAGE analysis for the eluted fraction of the recombinant full-length his-MpHEN1. In, input. FL, flow-through, Pe, pellet. The evaluation of antibody cross-reaction between α-AtHEN1 (E) and α-MpHEN1 (F) antibodies at different dilutions of his-AtHEN1 and his-MpHEN1. Both antibodies were diluted at 10,000×. (G) The endogenous AtHEN1 detection for α-AtHEN1 antibody. WT, Col-0. hen1-8, the HEN1 mutant. HA-AtHEN1, the HA-AtHEN1 plant. (H) The endogenous MpHEN1 detection for α-MpHEN1 antibody. His-MpHEN1, the recombinant MpHEN1. TAK1, the WT of male M. polyporpha.

Figure 2

In vitro methylation activity and substrate specificity for his-AtHEN1 and his-MpHEN1. The his-AtHEN1 (A) and his-MpHEN1 (B). In vitro methyltransferase activity assay. Meth, the 21-nt position represents the methylated miRNA. UnMeth, the 20-nt position represents the unmethylated miRNA. (C) The substrate specificity evaluation of AtHEN1 and MpHEN1.

Figure 3

The HC-Pro-mediated HEN1 activity inhibition. (A) The in vivo miR159 methylation status in wild-type TuMV (TuGR) and TuMV mutant (TuGK)-infected Col-0. (B) Evaluation of miRNA methylation status in P1/HC-Pro^R and P1/HC-Pro^K plants. The miRNA methylation status of 1-week-old plants was examined by oxidation/β-elimination, followed by small-RNA Northern blotting. (C) The in vivo miR166a methylation status in various mutant and transgenic plants. TAK1, the wild-type M. polymorpha, Col-0, wild-type Arabidopsis. hen1/heso1 mutant, the HEN1, and HESO1 double mutant. P1/HC-Pro^R, P1/HC-Pro^R plant.

Figure 4

In vitro and in vivo HC-Pro^R and HEN1 interaction. (A) The SDS-PAGE for GST-HC-Pro^R/K and the band at around 75 kDa shows the expected protein size. (B) In vitro pull-down assay for the evaluation of the GST-HC-Pro^R, GST-HC-Pro^K, and his-HEN1 interaction. (C) In vivo co-IP assay to examine the binding activity between GST-HC-Pro^R or GST-HC-Pro^K, and HA-HEN1. * Heavy chain of antibody.

Figure 5

The miRNA/miRNA* and HC-Pro^R/K-binding assay. (A) EMSA to examine the miRNA/miRNA*-binding ability of his-AtHEN1 and GST-HC-Pro^R or GST-HC-Pro^K. (B) In vitro competition assay.

Figure 6

The in vitro HEN1 activity inhibition assay. (A) The in vitro HC-Pro^R-mediated his-AtHEN1 inhibition assay. The GST was used as a negative control (i). The different ratios of his-AtHEN1 vs. GST-HC-Pro^R were performed in the HEN1 inhibition assay (ii). (B) Graph depicting quantification of GST-HC-Pro^R-mediated his-AtHEN1 inhibition. (C) The in vitro HC-Pro^R-mediated his-MpHEN1 inhibition assay. The different ratios for his-MpHEN1 vs. GST-HC-Pro^R were performed in the HEN1 inhibition assay. (D) Graph depicting quantification of GST-HC-Pro^R-mediated his-MpHEN1 inhibition. Meth, the 21-nt position of methylated miRNA. UnMeth, the 20-nt position of unmethylated miRNA. Untreated miR159a is abbreviated as Unt. miR159a.

Figure 7

AtHEN1 and MpHEN1 structural analysis. (A) The domain comparison between AtHEN1 and MpHEN1. The Y-axis represents the percentage of similarity and identity. The X-axis represents the domains of HEN1. (B) Structural comparison between (i) AtHEN1 and (ii) MpHEN1 model. The dsRBD1, LCD, dsRBD2, PLD, and MTase domains on the AtHEN1 (PDB number: 3HTX) and MpHEN1 models are highlighted by red, blue, orange, green, and magenta, respectively. (C) Amino acid sequence alignment for AtHEN1 and MpHEN1. The five domains are highlighted by a box based on AtHEN1 structure studies and compared with the MpHEN1 sequence. The arrowheads, asterisks, and dots indicate the SAM-binding residues, metal-binding residues, and double-stranded RNA-interacting residues, respectively.

Figure 8

Phylogenetic tree of HEN1 and HEN1 orthologs of different species of plant kingdom. Maximum likelihood (ML) tree of 22 HEN1 orthologs of green plants. MAGNESIUM-PROTOPORPHYRIN IX METHYLTRANSFERASE (CHLM) of Arabidopsis was used as the outgroup. Abbreviation of species names: Sm, Selaginella moellendorffii (Smoe109211); At, Arabidopsis thaliana (AT4G20910); Arabidopsis thaliana (AT4G20920); Os, Oryza sativa (BAJ16352); Atr, Amborella trichopoda (evm_27.model.AmTr_v1.0_scaffold00092.83); Pab, Picea abies (PAB00043215); Mp, Marchantia polymorpha (Mp3G16010); Pp, Physcomitrella patens (Pp3c1_60V3.4); Kf, Klebsormidium flaccidum (kfl00033_0220_v1.1); Cr, Chlamydomonas reinhardtii (Cre03.g191200); Vc, Volvox carteri (Vocar.0035s0143.1); Zm, Zea mays (GRMZM2G107457_T01); Sf, Sphagnum fallax (Sphfalx0066s0042.1, Sphfalx0001s0228.1); Sl, Solanum lycopersicum (Solyc02g070030.2.1); Pt, Populus trichocarpa (Potri.001G465500, Potri.011G163600); Gr, Gossypium raimondii (Gorai.010G144100); Tc, Theobroma cacao (Thecc1EG026937); Cpa, Carica papaya (evm.model.supercontig_166.41), Vv, Vitis vinifera (GSVIVG01021670001) and Arabidopsis thaliana, CHLM (AT4G25080). The bootstrap values are shown above the branches at the nodes. Arrowheads indicate duplicated events.

Figure 9

Multiple alignments of the MTase domain among different plant species. The arrowheads and asterisks indicate the SAM-binding residues and metal-binding residues, respectively. The black and grey boxes mark the AdoMet-binding motif and the FXPP motif, respectively.

Figure 10

The working hypothesis for TuMV HC-Pro-mediated HEN1 inhibition for RNA silencing suppression in (i) wild-type healthy plants, (ii) TuGR-infected plants, and (iii) TuGK-infected plants.