A self-assembling split Nano luciferase-based assay for investigating Pseudomonas syringae effector secretion.

Area of Science:

• Microbiology and Molecular Plant Pathology

Background:

• Gram-negative pathogens utilize the type III secretion system (T3SS) to inject effector proteins into host cells.
• T3SS effectors manipulate host processes and suppress immunity, aiding pathogen colonization and virulence.
• Accurate detection of T3SS-mediated translocation is crucial for understanding pathogenesis.

Purpose of the Study:

• To develop a rapid, quantitative, and straightforward method for detecting T3SS-mediated effector translocation.
• To assess the utility of a self-assembling split Nano luciferase (Nluc) reporter system for this purpose.

Main Methods:

• Engineered Pseudomonas syringae strains with effectors fused to a C-terminal Nluc fragment.
• Utilized a self-assembling split Nluc reporter system for in vitro and in planta detection.
• Assessed luminescence upon effector translocation into host cells during natural infections.

Main Results:

• The split Nluc system demonstrated high sensitivity and signal-to-noise ratio for detecting effector secretion in vitro.
• Translocated effectors retained their virulence functions in planta.
• Spontaneous Nluc assembly and bright luminescence were observed in infected plant cells, confirming T3SS activity.

Conclusions:

• The self-assembling split Nluc reporter system provides an efficient tool for detecting T3SS-mediated effector translocation.
• This system enables straightforward and rapid assessment of effector delivery both in vitro and in planta.
• The developed method is suitable for studying bacterial pathogenesis and T3SS function.