Increased cytosine methylation at promoter of the NB-LRR class R gene RCY1 correlated with compromised resistance to cucumber mosaic virus in EMS-generated src mutants of Arabidopsis thaliana

•RCY1-conferred resistance to CMV is compromised in EMS-induced src mutant.•Paramutation appears to occur in src mutants.•Cytosines in the RCY1 promoter are hypermethylated.•Treatment with 5-azacytidine restores RCY1 expression and CMV resistance.

In Arabidopsis thaliana, RESISTANCE TO CMV(Y) (RCY1), which encodes a CC-NB-LRR class protein, confers the hypersensitive response (HR) to a yellow strain of cucumber mosaic virus [CMV(Y)]. A. thaliana ecotype Col-0 overexpressing RCY1 transgenes (Col::pRCY1-HA#13) shows extreme resistance (ER) to CMV(Y). To identify novel regulatory factors involved in high RCY1 expression and ER, suppressor of RCY1-mediated resistance to CMV(Y) (src) mutants were screened from Col::pRCY1-HA#13 mutagenized with ethyl methanesulfonate. The amounts of RCY1 transcript and RCY1 protein in five src mutant lines was significantly lower than in Col::pRCY1-HA#13. In src mutants, virus systemically spread without formation of necrotic local lesions in the inoculated leaves. Genetic analysis of progenies of crosses between the src mutant and Col::pRCY1-HA#13 suggested that paramutation might have occurred in these src mutants. Indeed, cytosines in the promoters but not in other genomic regions of the RCY1 transgenes are hypermethylated in these src mutant lines. Moreover, decreased RCY1 expression was correlated with increased methylation of the RCY1 promoter region. Treatment with 5-azacytidine decreased methylation in the promoter region and partially restored RCY1 expression. Taken together, SRC may be involved in RCY1-conferred resistance to CMV(Y) through the regulation of cytosine methylation of the RCY1 promoter.