Fig. 5

The kinase catalytic active site of NbERK1 is essential for cell death-inducing/plant resistance-enhancing activity and protein stability. a The kinase catalytic active site of NbERK1 affects its cell death-inducing activity. Transient expression of NbERK1 and NbERK1-D644N in Nicotiana benthamiana by agroinfiltration. INF1 and green fluorescent protein (GFP) were used as controls. Photos were captured under UV light. b Cell death was quantified by measuring electrolyte leakage. Error bars indicate ± SD (Student’s t-test, ***P < 0.001). c Immunoblotting analysis of NbERK1 and NbERK1-D644N expressed in N. benthamiana. d, e The kinase catalytic active site of NbERK1 affects its plant resistance-enhancing activity. Representative leaves showing lesions of N. benthamiana caused by Sclerotinia sclerotiorum. Leaves were infiltrated by Agrobacterium tumefaciens strains harboring GFP, NbERK1, or NbERK1-D644N 24 h before S. sclerotiorum inoculation. Photos were captured 24 hpi. Lesion areas were counted by ImageJ. Error bars indicate ± SD (Student’s t-test, *P < 0.05). f The kinase catalytic active site of NbERK1 affects protein stability. NbERK1-HA and NbERK1-D644N-HA were transiently expressed in N. benthamiana for 2 d. Leaves were treated with 50 μM CHX for 0, 2, and 4 h before harvesting. Protein abundances were detected by immunoblotting using anti-HA antibodies