Using GCaMP3 to Study Ca2+ Signaling in Nicotiana Species

Abstract

Ca2+ signaling is a central component of plant biology; however, direct analysis of in vivo Ca2+ levels is experimentally challenging. In recent years, the use of genetically encoded Ca2+ indicators has revolutionized the study of plant Ca2+ signaling, although such studies have been largely restricted to the model plant Arabidopsis. We have developed stable transgenic Nicotiana benthamiana and Nicotiana tabacum lines expressing the single-wavelength fluorescent Ca2+ indicator, GCaMP3. Ca2+ levels in these plants can be imaged in situ using fluorescence microscopy, and these plants can be used qualitatively and semi-quantitatively to evaluate Ca2+ signals in response to a broad array of abiotic or biotic stimuli, such as cold shock or pathogen-associated molecular patterns (PAMPs). Furthermore, these tools can be used in conjunction with well-established N. benthamiana techniques such as virus-induced gene silencing (VIGS) or transient heterologous expression to assay the effects of loss or gain of function on Ca2+ signaling, an approach which we validated via silencing or transient expression of the PAMP receptors FLS2 (Flagellin Sensing 2) or EFR (EF-Tu receptor), respectively. Using these techniques, along with chemical inhibitor treatments, we demonstrate how these plants can be used to elucidate the molecular components governing Ca2+ signaling in response to specific stimuli.