Arabidopsism⁶A demethylase activity modulates viral infection of a plant virus and the m⁶A abundance in its genomic RNAs

Abstract

N-methyladenosine (mA) is an internal, reversible nucleotide modification that constitutes an important regulatory mechanism in RNA biology. Unlike mammals and yeast, no component of the mA cellular machinery has been described in plants at present. mA has been identified in the genomic RNAs of diverse mammalian viruses and, additionally, viral infection was found to be modulated by the abundance of mA in viral RNAs. Here we show that the protein atALKBH9B (At2g17970) is a demethylase that removes mA 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 mA demethylase activity could be linked to mRNA silencing and/or mRNA decay processes. Moreover, we identified the presence of mA in the genomes of two members of the 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 mA 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, mA modification may represent a plant regulatory strategy to control cytoplasmic-replicating RNA viruses.

Alterations in cellular RNA decapping dynamics affect tomato spotted wilt virus cap snatching and infection in Arabidopsis

“…The present results showing that UPF1 promotes TSWV infection in Arabidopsis contrast with previous reports suggesting that non‐sense‐mediated decay limits infection of RNA viruses. These conclusions are based on increased expression of a PVX transgene in a upf1 mutant, transient expression assays in N. benthamiana of PVX and TCV with a dominant‐negative version of UPF1 (Garcia et al ., ; May et al ., ), and a moderate increase in susceptibility to AMV in upf1‐5 mutant Arabidopsis (Martínez‐Pérez et al ., ). To confirm and extend the role of UPF1 in virus infections, Col‐0 and upf1‐5 mutant plant were infected with PlAMV‐GFP and TuMV‐GFP.…”

“…Transient overexpression of a dominant negative mutant of the Arabidopsis UPF1 protein with potato virus X (PVX), pea enation mosaic virus 2 (PEMV‐2) and turnip crinkle virus (TCV) resulted in increased virus accumulation in N. benthamiana leaves (Garcia et al ., ; May et al ., ). Consistent with these results, upf1 mutant plants are more susceptible to alfalfa mosaic virus (AMV) (Martínez‐Pérez et al ., ).…”

N6‐methyladenosine regulatory machinery in plants: composition, function and evolution

“…However, not all RRACH motifs are associated with m6A modification, as the m6A level is much lower than the abundance of RRACH motifs, indicating that the molecular mechanism regulating m6A modification is not fully understood (Liu and Pan, ). The Arabidopsis thaliana proteins ALKBH9B and ALKBH10B function as m6A erasers (RNA demethylases) that oxidatively reverse m6A methylation from single‐stranded RNA molecules (Duan et al ., ; Martínez‐Pérez et al ., ). YTH domain proteins act as m6A readers.…”

“…Transcriptome‐wide profiling has revealed that m6A modification is also a dynamic process in Arabidopsis (Luo et al ., ). Several studies have confirmed that m6A can revert to adenosine through the action of the m6A RNA demethylases ALKBH9B and ALKBH10B (Duan et al ., ; Martínez‐Pérez et al ., ). AtALKBH9B localizes to cytoplasmic granules, which contain siRNA bodies and can be directed to P bodies.…”

“…The m6A regulatory machinery is post‐transcriptionally assembled by a conserved set of proteins at the conserved consensus sequence, RRACH. A number of proteins involved in the addition, removal and identification of m6A have been reported, which are categorized into three groups called writers, erasers and readers, respectively, and include proteins such as MTA, MTB, ALKBH9B and ECT2 (Figure ) (Arribas‐Hernández et al ., ; Fu et al ., ; Martínez‐Pérez et al ., ; Zhong et al ., ). The m6A writer complex recognizes the consensus motif RRACH (Růžička et al ., ).…”