Rad54 is not essential for any geminiviral replication mode in planta.

The circular single-stranded DNA of phytopathogenic geminiviruses is propagated by three modes: complementary strand replication (CSR), rolling circle replication (RCR) and recombination-dependent replication (RDR), which need host plant factors to be carried out. In addition to necessary host polymerases, proteins of the homologous recombination repair pathway may be considered essential, since geminiviruses are particularly prone to recombination. Among several others, Rad54 was suggested to be necessary for the RCR of Mungbean yellow mosaic India virus. This enzyme is a double-stranded DNA-dependent ATPase and chromatin remodeller and was found to bind and modulate the viral replication-initiator protein in vitro and in Saccharomyces cerevisiae. In contrast to the previous report, we scrutinized the requirement of Rad54 in planta for two distinct fully infectious geminiviruses with respect to the three replication modes. Euphorbia yellow mosaic virus and Cleome leaf crumple virus were inoculated into Rad54-deficient and wildtype Arabidopsis thaliana plant lines to compare the occurrence of viral DNA forms. Replication intermediates were displayed in the time course of infection by one and two-dimensional agarose gel electrophoresis and Southern hybridization. The experiments showed that Rad54 was neither essential for CSR, RCR nor RDR, and it had no significant influence on virus titers during systemic infection.