Arabidopsis m6A demethylase activity modulates viral infection of a plant virus and the m6A abundance in its genomic RNAs

Abstract

N⁶-methyladenosine (m⁶A) is an internal, reversible nucleotide modification that constitutes an important regulatory mechanism in RNA biology. Unlike mammals and yeast, no component of the m⁶A cellular machinery has been described in plants at present. m⁶A has been identified in the genomic RNAs of diverse mammalian viruses and, additionally, viral infection was found to be modulated by the abundance of m⁶A in viral RNAs. Here we show that the Arabidopsis thaliana protein atALKBH9B (At2g17970) is a demethylase that removes m⁶A from single-stranded RNA molecules in vitro. atALKBH9B accumulates in cytoplasmic granules, which colocalize with siRNA bodies and associate with P bodies, suggesting that atALKBH9B m⁶A demethylase activity could be linked to mRNA silencing and/or mRNA decay processes. Moreover, we identified the presence of m⁶A in the genomes of two members of the Bromoviridae family, alfalfa mosaic virus (AMV) and cucumber mosaic virus (CMV). The demethylation activity of atALKBH9B affected the infectivity of AMV but not of CMV, correlating with the ability of atALKBH9B to interact (or not) with their coat proteins. Suppression of atALKBH9B increased the relative abundance of m⁶A in the AMV genome, impairing the systemic invasion of the plant, while not having any effect on CMV infection. Our findings suggest that, as recently found in animal viruses, m⁶A modification may represent a plant regulatory strategy to control cytoplasmic-replicating RNA viruses.

Keywords: ALKBH9B; coat protein; demethylase; m6A; plant virus.

Fig. 1.

atALKBH9B interacts with the CP of AMV in vitro and in vivo and with the viral RNA. (A) Yeast two-hybrid analysis of the interaction between AMV CP and atALKBH9B. Interacting colonies were identified by growth after 5 d on medium lacking leucine, tryptophan, histidine, and adenine (−LWHA). (B) BiFC images of epidermal cells coinfiltrated with the indicated constructs. YFP reconstitution was found to form discrete granules in the cytoplasm (arrows). Fibrillarin fused to the mCherry protein was used to identify the cell nuclei (arrowhead). Pictures are the overlapped images of green, red, and transmitted channels. (C) Analysis of the RNA binding activity of atALKBH9B by Northwestern blot assay. Duplicate membranes with purified GST:atALKBH9B (Upper) or GST proteins (Lower) (4 and 2 µg) were incubated with viral RNA 4 (Left) to show the RNA binding activity or stained with Coomassie blue (Right) to confirm the presence of the proteins. Positions of full-length GST and GST:atALKBH9B proteins are indicated by arrows. Asterisks denote truncated GST:atALKBH9B.

Fig. S1.

(A) Identification of clones interacting with the AMV CP by yeast two-hybrid screen of an Arabidopsis cDNA library. After sequencing, these clones corresponded to atALKB9B ORF. (B) Immunoblot analysis of coimmunoprecipitated products after incubation of AMV virions and the histidine-tagged proteins indicated Above the figure. Eluted (E1–E3) proteins were detected with antibodies against the His-tag (IgHis) and the CP of AMV (IgCP). The CP was only detected when virions were incubated with His-atALKBH9B.

Fig. 2.

AMV infection is impaired in atalkbh9b plants. (A) Annotated genomic atALKBH9B gene structure showing the exons (gray boxes) and location of the T-DNA insertion (SALK_015591). (B) Agarose gel electrophoresis of RT-PCR products produced with specific primers to amplify the full-length mRNA of the atalkbh9b gene from WT and atalkbh9b plants. The position of the mRNA is indicated on the Right. (C and D) Representative Northern and Western blots from inoculated leaves at 3 and 6 dpi of three WT and atalkbh9b plants. Positions of the vRNAs and CP are indicated on the Left. Ethidium bromide and Coomassie blue staining of ribosomal RNAs and total protein extracts (rRNA and cb, respectively) were used as RNA and protein loading controls. (E) Dot-blot hybridization of upper noninoculated floral stems to determine the extent of viral systemic movement. Dots in rows a and b correspond to WT plants; dots in c and d correspond to atalkbh9b plants. Samples b6 and d6 are healthy WT and atalkbh9b plants used as negative controls.

Fig. S2.

Graphic showing the average of vRNAs accumulation in WT and atalkbh9b mutant. SD values are shown. Asterisks indicate significant differences from the WT (*P < 0.05) using the t test (n = 3).

Fig. 3.

The atALKBH9B protein colocalizes with siRNA-body/P-body components in noninfected tissues. (A and B) Confocal laser scanning microscope (CLSM) images of N. benthamiana leaf epidermal cells coinfiltrated with the DNA constructs indicated Above the images. Overlay panels are the superposition of images from the green and red channels. Arrowheads indicate granules with both proteins. (C) vRNAs accumulation in upf1.5 mutant with respect to WT. SD values are shown. Asterisks indicate significant differences from the WT (**P < 0.01) using the t test (n = 20).

Fig. S3.

Confocal laser microscope scanning images of N. benthamiana cells agroinfiltrated with the GFP fused to the N- and C-terminal ends of atALKBH9B.

Fig. S4.

Rootless phylogenetic tree derived from the Clustal Omega analysis between proteins of the AlkB family belonging to different species. Basic local alignment search tool (BLAST) web tool (National Center for Biotechnology Information) was used to compare the amino acid sequence of the protein atALKBH9B against a large number of sequences available in the databases. A total of 34 protein sequences were selected with which a multiple alignment was then performed by Clustal Omega (51), available from the European Molecular Biology Laboratory–European Bioinformatics Institute website. Finally, using this alignment a rootless phylogenetic tree was constructed in the MEGA v.6.0 program (52). The minimum evolution method was selected as the statistical method, and the bootstrap (53) analysis, with 10,000 replicates, was tested as a phylogeny test. The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were calculated using the Poisson correction method and they are found in units of number of amino acid substitutions per site. The minimal evolution tree was recorded using the close neighbor interchange (CNI) algorithm at a search level of 1. The neighbor-joining algorithm was used to generate the initial tree. All ambiguous positions were removed for each pair of sequences. A pairwise deletion method was selected for the treatment of gaps.atALKBH9B protein (at2g17970), circled in red, enclosed in a small cluster that encompasses its homologous proteins from other vegetables and this, in turn, in a larger group including the ALKBH5 protein, already described and studied in mammals. The bootstrap values are indicated on each node. At, Arabidopsis thaliana; br, Brassica rapa; fc, Felis catus; hs, Homo sapiens; md, Malus domestica; nt, Nicotiana tomentosiformis; oz, Oryza sativa; and rn, Rattus norvegicus.

Fig. S5.

The atALKBH9B protein colocalizes with siRNA-body/P-body components in infected tissues. N. benthamiana leaves were inoculated with AMV viral particles and 24 h postinfection were agroinfiltrated with the indicated coinfiltrated DNA constructs indicated Above the images. Confocal laser scanning microscope images were taken 48 h postinfiltration. Overlay panels are the superposition of images from the green and red channels. Arrowheads indicate granules with both proteins.

Fig. 4.

atALKBH9B catalyzes demethylation of m⁶A in ssRNA in vitro and modulates methylation of vRNAs. (A) Representative UPLC-PDA-Q/TOF-MS chromatogram showing the retention times of the nucleosides adenosine (A) and N⁶-methyladenosine (m⁶A) after incubation of the m⁶A-containing ssRNA substrate with GST:atALKBH9B and GST as the negative control. The peak (G) corresponds to the nucleoside guanosine. The peak denoted as (*) could not be unequivocally identified although it was determined to present a molecular weight of 343 with a maximum absorption at 261 nm. (B) Graph representing the demethylation activity in three independent experiments. (C–E) Genomic AMV RNAs are m⁶A hypermethylated in atalkbh9b plants. (C) RIP of vRNAs with a specific anti-m⁶A antibody. Total RNA extracted from WT plants infected with AMV was incubated with anti-m⁶A plus IgA or IgA alone. Dilutions of the immunoprecipitated RNAs (indicated on Top) were blotted on nylon membranes and the vRNAs were detected with DigAMV. (D) Average ratios of m⁶A in vRNAs obtained by quantification of m⁶A on three different Northwestern blots from AMV-infected WT and atalkbh9b plants. (E) Graphic showing the average m⁶A/A ratios obtained by UPLC-Q-Tof-MS after digestion of vRNAs extracted from virions purified from AMV-infected WT and atalkbh9b plants. In B, D, and E error bars represent the SEM and asterisks indicate significant differences from the WT (*P < 0.05) using the t test (n = 3).

Fig. S6.

m6A antibody recognizes AMV vRNAs by Northwestern blot. (A) Representative nylon membrane stained with ethidium bromide (EtBr) and Northwestern blot with m6A antibody (m6A) of vRNAs extracted of virions purified from AMV-infected WT and alkbh9b plants. Positions of the genomic AMV RNAs are indicated on the Right of EtBr. (B) In vitro transcripts of vRNA 4 obtained with NTPs (non-methylated) or NTPs+m6A (methylated) were used as negative and positive Northwestern blot controls, respectively.

Fig. S7.

Map of m6A sites within AMV genomic RNAs by MeRIP-seq. (A) Read coverage of the three RNAs on input RNA-seq (green), bead-only control (blue), and MeRIP-seq (pink). Reads were normalized to the total number of reads mapping to the viral genome. Below, a schematic diagram of the AMV genome is presented. Red rectangles indicate the six m6A peaks identified and numbers show their nucleotide positions in each vRNA. (B) Nucleotide sequence of the six m6A peaks identified, showing in red the [(G,A,U)/(G,A)/A/C/(A,C,U)], [(A,C)/G/A/C/(G,U)], and [UGAC] consensus motifs.

Fig. 5.

CMV genomic RNAs contain the m⁶A modification. (A) Detection of the m⁶A modification in CMV by RIP of viral RNAs with a specific anti-m6A antibody plus IgA or IgA alone. Dilutions of the immunoprecipitated RNAs (indicated on Top) were blotted on nylon membranes and vRNAs were detected with DigCMV. (B) Graphic showing the average m⁶A/A ratios obtained by UPLC-Q-Tof-MS after digestion of vRNAs extracted from virions purified from CMV-infected WT and atalkbh9b plants. Error bars represent SEM; ns, no significant differences (P > 0.05) from the WT using the t test (n = 3). (C) CMV systemic infection is not affected in atalkbh9b plants. Dot-blot hybridization of floral stems to analyze systemic viral movement. Samples b6 and d6 are the negative controls. Dots in rows a and b correspond to WT plants; dots in c and d correspond to atalkbh9b plants. (D) Yeast two-hybrid analysis of the interaction between CPs of CMV and PNRSV with atALKBH9B. Interacting colonies were identified by growth after 5 d on medium lacking leucine, tryptophan, histidine, and adenine (−LWHA). ns, not significant.