Single amino acid alterations in Arabidopsis thaliana RCY1 compromise resistance to Cucumber mosaic virus, but differentially suppress hypersensitive response-like cell death

Abstract

Resistance to an yellow strain of Cucumber mosaic virus [CMV(Y)] in Arabidopsis thaliana ecotype C24 is conferred by the CC-NBS-LRR type R gene, RCY1. RCY1-conferred resistance is accompanied by a hypersensitive response (HR), which is characterized by the development of necrotic local lesion (NLL) at the site of infection that restricts viral spread. To further characterize the role of RCY1 in NLL formation we have identified six recessive CMV(Y)-susceptible rcy1 mutants, four of which contain single amino acid substitutions in RCY1: rcy1-2 contains a D to N substitution in the CC domain, rcy1-3 and rcy1-4 contain R to K and E to K substitutions, respectively, in the LRR domain, and rcy1-6 contains a W to C substitution in the NBS domain. The rcy1-5 and rcy1-7 contain nonsense mutations in the LRR and NBS domains, respectively. Although the virus systemically spread in all six rcy1 mutants, HR-associated cell death was differentially induced in these mutants. In comparison to the wild type C24 plant, HR was not observed in the CMV(Y)-inoculated leaves of the rcy1-3, rcy1-5, rcy1-6 and rcy1-7 mutants. In contrast, delayed NLL development was observed in the virus inoculated leaves of the rcy1-2 and rcy1-4 mutants. In addition, necrosis accompanied by elevated accumulation of PR gene transcript also appeared in the non-inoculated leaves of the rcy1-2 and rcy1-4 mutants. Trans-complementation was observed between the rcy1-2 and rcy1-4 alleles; in F1 plants derived from a cross between rcy1-2 and rcy1-4, HR associated cell death was accelerated and systemic spread of the virus was partially suppressed than in the homozygous rcy1-2 and rcy1-4 plants. Our results suggest that the CC, NBS and LRR domains of RCY1 are required for restriction of virus spread but differentially impact the induction of HR-like cell death. Furthermore, these results also predict inter-molecular interaction involving RCY1 in Arabidopsis resistance to CMV(Y).