The 2a protein of Cucumber mosaic virus induces a hypersensitive response in cowpea independently of its replicase activity

Resistance in cowpea to infection with strains of Cucumber mosaic virus (CMV) involves a local hypersensitive response (HR), and previous studies indicated that the 2a replicase of CMV is involved in HR induction. In this study, we confirmed and extended this observation by demonstrating that the nonviral expression of the 2a protein encoded by CMV is able to induce a cell death response in cowpea plants, whereas no other CMV-encoded proteins elicits such response. The 2a single-amino acid mutant, F631Y, no longer induces the necrosis response, yet the A641S mutant still induces cell death. The 2a double mutant, F631Y and A641S, does not induce HR. However, the three 2a mutants have comparable replicase activities in a fluorescence reporter assay. The 2aD610A mutant that alters the highly conserved GDD motif abolishes the replicase activity, however it does not affect HR induction in cowpea. The 2a301-778aa fragment introduced with the same D610A mutation in the GDD motif is also capable of inducing HR in cowpea. Collectively, these findings suggest that the 2a protein of CMV is sufficient to induce HR in cowpea independently of its replicase activity.