Using GCaMP3 to Study Ca2+ Signaling in Nicotiana Species
Using GCaMP3 to Study Ca2+ Signaling in Nicotiana Species
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DOI：
10.1093/pcp/pcx053
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发表时间：
2017-07-01
影响因子：
4.9
通讯作者：
Yoshioka, Keiko
中科院分区：
文献类型：
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作者：
DeFalco, Thomas A.;Toyota, Masatsugu;Yoshioka, Keiko
Ca2+信号传导是植物生物学的核心组成部分;然而，直接分析体内Ca2+水平在实验上具有挑战性。近年来，基因编码的Ca 2+指示剂的使用彻底改变了植物Ca 2+信号传导的研究，尽管这些研究在很大程度上限于模式植物拟南芥。我们已经开发了稳定的转基因烟草本塞姆和烟草株系表达单波长荧光Ca2+指示剂，GCaMP3。这些植物中的Ca2+水平可以使用荧光显微镜原位成像，并且这些植物可以用于定性和半定量地评估响应于广泛的非生物或生物刺激的Ca2+信号，例如冷休克或病原体相关分子模式（PAMP）。此外，这些工具可以与成熟的N。本塞姆氏技术，如病毒诱导的基因沉默（VIGS）或瞬时异源表达，以测定功能丧失或获得对Ca2+信号传导的影响，我们分别通过PAMP受体FLS2（鞭毛蛋白传感2）或EFR（EF-Tu受体）的沉默或瞬时表达验证了这种方法。使用这些技术，沿着化学抑制剂处理，我们展示了如何使用这些植物来阐明控制Ca 2+信号传导响应特定刺激的分子组分。
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.