Elicitation of hypersensitive cell death by extracellularly targeted HrpZPsph produced in planta

The ability of the Pseudomonas syringae pv. phaseolicola harpin (HrpZPsph) to elicit hypersensitive response was investigated in three Nicotiana genotypes. The hrpZPsph gene was placed under chemical regulation (tetracycline induction) in TetR+ Nicotiana tabacum cv. Wisconsin 38 (W38) or was transiently expressed in N. benthamiana following infection with a PVX-derived vector and in three Nicotiana genotypes by agroinfiltration. The constructs were designed to express either the canonical form of harpin (HrpZPsph) or an N-terminally extended version of the protein carrying the signal peptide portion of the tobacco pathogenesis-related protein PR1a (SP-HrpZPsph). Stable transformants of N. tabacum cv. W38 did not develop necrosis upon induction with tetracycline, probably as a result of insufficient harpin accumulation. In contrast, N. benthamiana plants infected with the PVX constructs produced high concentrations of harpin in biologically active form, but only those expressing the secretable form of harpin developed necrotic symptoms. These symptoms were less severe than those caused by PVX::avrPto; however, they were accompanied by induction of hsr203J, a hypersensitive response-specific gene transcript. These results suggest that the plant cellular receptor(s) for harpin is extracellular.