Visualization and Quantification of Apoplastic ROS by Plant Immune NADPH Oxidase Utilizing Chemiluminescence

Area of Science:

• Plant biology
• Molecular plant pathology
• Biochemistry

Background:

• Reactive oxygen species (ROS) play a key role in plant innate immunity.
• NADPH oxidases (RBOH) produce ROS, like superoxide (O₂^-) and hydrogen peroxide (H₂O₂), in response to pathogen recognition.
• Specific RBOH proteins in *Nicotiana benthamiana* and *Solanum tuberosum* are linked to ROS production during pattern-triggered immunity (PTI) and effector-triggered immunity (ETI).

Purpose of the Study:

• To develop and present a novel protocol for detecting apoplastic ROS.
• To investigate the co-expression of potato StRBOHC and StCDPK5 in *N. benthamiana* for ROS detection.

Main Methods:

• Utilized L-012-derived chemiluminescence for ROS detection.
• Employed Agrobacterium-mediated co-expression of *Solanum tuberosum* StRBOHC and StCDPK5 in *Nicotiana benthamiana* leaves.

Main Results:

• Successfully established a chemiluminescence-based protocol for detecting apoplastic ROS.
• Demonstrated the feasibility of using *N. benthamiana* as a model system to study potato RBOH and CDPK interactions.

Conclusions:

• The developed protocol provides a sensitive method for studying ROS dynamics in plant immunity.
• This research facilitates further investigation into the roles of RBOH and calcium-dependent protein kinases in plant defense responses.