Imaging systemic calcium response and its molecular dissection using virus-induced gene silencing
Jessica Cisneros Fernandez1, Simon Gilroy1
1Department of Botany, University of Wisconsin-Madison, Madison, WI, United States.
Methods in Enzymology
|January 29, 2023
Summary
Plants use calcium (Ca2+) signals to coordinate systemic responses to local stimuli. This study introduces virus-induced gene silencing (VIGS) as a rapid method to investigate the molecular mechanisms of these Ca2+ signals.
Area of Science:
- Plant biology
- Molecular signaling
- Biochemistry
Background:
- Plants perceive local stimuli, such as insect attacks or pathogen invasions, which can trigger whole-plant (systemic) responses.
- Cytoplasmic Ca2+ signals rapidly propagate throughout plants, coordinating these systemic responses.
- Visualizing Ca2+ signals is crucial for understanding plant communication, often using genetically encoded Ca2+-sensitive fluorescent reporters.
Purpose of the Study:
- To present virus-induced gene silencing (VIGS) as an efficient alternative to traditional mutant analysis for dissecting systemic Ca2+ signaling pathways.
- To describe the methodology for generating VIGS constructs targeting candidate genes involved in systemic signaling.
- To demonstrate the visualization of wound-induced, systemic Ca2+ signaling in plants with suppressed candidate genes.
Main Methods:
- Utilizing plants expressing Ca2+-sensitive fluorescent protein bioreporters (e.g., GCaMP, GECO).
- Generating virus-induced gene silencing (VIGS) constructs to suppress candidate genes in wild-type bioreporter lines.
- Imaging and analyzing systemic Ca2+ signal propagation in VIGS-treated plants following localized wounding.
Main Results:
- Virus-induced gene silencing (VIGS) provides a rapid screening method for identifying genes involved in systemic Ca2+ signaling.
- This approach bypasses the time-consuming process of creating and selecting stable transgenic mutant lines.
- Successful visualization of altered systemic Ca2+ responses in plants with silenced candidate genes.
Conclusions:
- Virus-induced gene silencing (VIGS) is a powerful and efficient tool for dissecting the molecular basis of systemic plant signaling.
- This method accelerates the identification of key components in Ca2+ signal propagation pathways.
- Facilitates a deeper understanding of plant-wide communication networks in response to environmental stimuli.
