MAMP-triggered Medium Alkalinization of Plant Cell Cultures
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Gabriel L Fiorin1,2,3, Andrea Sánchez-Vallet3,4, Bart P H J Thomma3
1Graduate Program in Genetics and Molecular Biology, Universidade Estadual de Campinas, Campinas, Brazil.
Abstract:
Plants recognize a wide variety of microbial molecules to detect and respond to potential invaders. Recognition of Microbe-Associated Molecular Patterns (MAMPs) by cell surface receptors initiate a cascade of biochemical responses that include, among others, ion fluxes across the plasma membrane. A consequence of such event is a decrease in the concentration of extracellular H+ ions, which can be experimentally detected in plant cell suspensions as a shift in the pH of the medium. Thus, similarly to reactive oxygen species (ROS) accumulation, phosphorylation of MAP kinases and induction of defense-related genes, MAMP-induced medium alkalinization can be used as a proxy for the activation of plant immune responses. Here, we describe a detailed protocol for the measurement of medium alkalinization of tobacco BY-2 cell suspensions upon treatment with two different MAMPs: chitohexamers derived from fungal cell walls (NAG6; N-acetylglucosamine) and the flagellin epitope flg22, found in the bacterial flagellum. This method provides a reliable and fast platform to access MAMP-Triggered Immunity (MTI) in tobacco cell suspensions and can be easily adapted to other plant species as well as to other MAMPs.
Insights
Plants detect microbial molecules using Microbe-Associated Molecular Patterns (MAMPs), triggering immune responses. A new protocol measures medium alkalinization in tobacco cells as a reliable indicator of MAMP-Triggered Immunity (MTI).
Area of Science:
- Plant immunology
- Molecular biology
- Biochemistry
Background:
- Plants possess sophisticated mechanisms to recognize microbial molecules, initiating defense responses.
- Microbe-Associated Molecular Patterns (MAMPs) are recognized by plant cell surface receptors, leading to biochemical cascades.
- MAMP recognition triggers ion fluxes, resulting in extracellular H+ decrease and medium alkalinization, a measurable indicator of plant immune activation.
Purpose of the Study:
- To describe a detailed protocol for measuring medium alkalinization in tobacco BY-2 cell suspensions.
- To establish a reliable and fast method for assessing MAMP-Triggered Immunity (MTI) in plant cell cultures.
- To provide a adaptable platform for studying plant immune responses to various MAMPs.
Main Methods:
- Utilized tobacco BY-2 cell suspensions.
- Treated cells with chitohexamers (NAG6) and the flagellin epitope (flg22) as MAMPs.
- Measured medium alkalinization as a proxy for MAMP-Triggered Immunity (MTI) activation.
Main Results:
- Successfully measured medium alkalinization in response to NAG6 and flg22 treatments.
- Demonstrated that medium alkalinization is a reliable indicator of MAMP perception and subsequent immune signaling.
- Validated the protocol's effectiveness in tobacco cell suspensions.
Conclusions:
- The described protocol offers a robust and rapid method for assessing MAMP-Triggered Immunity (MTI) in plant cell suspensions.
- This technique can be readily adapted for use with other plant species and diverse MAMPs.
- Provides a valuable tool for advancing research in plant innate immunity.
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