Fig. 4.

NbPR3 requires its active site to reduce bacterial growth but lacks chitinase and lysozyme activity. (a) Transient NbPR3 expression decreases susceptibility to Pseudomonas syringae pathovars tomato DC3000(ΔhQ) and tabaci 6605, but not syringae B728a. NbPR3 and the empty vector (EV) control were transiently expressed in Nicotiana benthamiana by agroinfiltration. Two days later, the same leaves were infiltrated with 10⁶ CFU ml⁻¹ bacteria and bacterial population densities in log₁₀CFU cm⁻² were determined after 3 d. Bars show the mean value of 18 biological replicates performed over three separate experiments, and error intervals represent the SE. P‐values were calculated by two‐way ANOVA followed by post hoc comparison using the Dunnett test to examine the effect of NbPR3 overexpression on bacterial growth. (b) The NbPR3(E92) mutant accumulates in the apoplast of agroinfiltrated leaves. NbPR3 and its E92A mutant and the empty vector (EV) control were agroinfiltrated and apoplastic fluid (AF) was isolated at Day 4 from four different replicates, separated on protein gel and analysed by Coomassie staining. The red arrowhead indicates NbPR3 protein. *, Endogenous PR2 protein, induced by agroinfiltration. (c) Transient expression of NbPR3 but not its catalytic mutant suppresses bacterial growth of PtoDC3000(ΔhQ). NbPR3, its catalytic site E92A mutant, and the empty vector (EV) control were transiently expressed by agroinfiltration. Two days later, the same leaf was infiltrated with 10⁶ CFU ml⁻¹ PtoDC3000(ΔhQ) and bacterial population densities in log₁₀CFU cm⁻² were determined after 3 d. Bars show the mean value of 18 biological replicates performed over three separate experiments, and error intervals represent the SE. P‐values were calculated by two‐way ANOVA followed by post hoc comparison using the Dunnett test to examine the effect of NbPR3 overexpression on bacterial growth. **, P < 0.01. (d) NbPR3 lacks endochitinase activity. AF from plants transiently expressing NbPR3 or its E92A mutant were incubated with 4‐MU‐GlcNac3, and the rate of hydrolysis was measured at 355ex/450em and calculated per microgram NbPR3 protein estimated by Coomassie staining. The endochitinase of Trichoderma viride (TvEC) was included as a positive control. (e) NbPR3 lacks lysozyme activity. AF from plants transiently expressing NbPR3 or its E92A mutant were incubated with Micrococcus lysodeikticus cells. The change in A₄₅₀ was measured and converted to units per μg NbPR3 protein estimated by Coomassie staining. The lysozyme of chicken egg white (LCEW) was included as a positive control.