Kazal-type serine protease inhibitors from Arabidopsis thaliana and Toxoplasma gondii exhibit antimicrobial activity against plant pathogens

Kazal-type serine protease inhibitors (KPIs) interfere with microbial proteases and have been associated with antimicrobial activity, yet their specific role in plant protection remains poorly understood. In this work, we evaluated the antimicrobial potential of recombinant Arabidopsis thaliana KPI (rAtKPI-1T), recombinant Toxoplasma gondii KPI (rTgPI-1), and two truncated versions of rTgPI-1 (rTgPI-1 _NT and rTgPI-1 _CT ). Both rAtKPI-1T and rTgPI-1 exhibited inhibitory effects against Pseudomonas syringae DC3000, P. syringae (AvRpm1), and P. viridiflava in a concentration-dependent manner, with significant inhibition at 3.5 μM. The lower MIC ₅₀ values obtained for rTgPI-1 and its truncated forms compared to rAtKPI-1T suggest a higher antibacterial potency. Binding and immunofluorescence assays further revealed that rAtKPI-1T, rTgPI-1, and rTgPI-1 _NT associated with bacterial surfaces, while rTgPI-1 _CT displayed weaker or transient interactions. In addition to their antibacterial activity, rTgPI-1 _NT and rTgPI-1 _CT also inhibited the germination of Botrytis cinerea conidia. Both truncated proteins significantly reduced germination after 6 and 9 hours of incubation, with rTgPI-1 _CT exerting a markedly stronger antifungal effect than rTgPI-1 _NT . These findings suggest that specific Kazal-type domains, particularly those in the C-terminal region, could play a critical role in suppressing early infection processes of necrotrophic fungi. Overall, this study demonstrates that rKPIs display distinct and complementary antimicrobial profiles mediated by microbial binding and protease inhibition. The contrasting activities of rTgPI-1 _NT and rTgPI-1 _CT highlight the value of domain-level analysis to discern functional contributions within multidomain KPIs. These results expand current knowledge on plant-derived protease inhibitors and underscore their potential as biotechnological tools for sustainable crop protection.