Abstract
Calcium ions are predicted to be key signaling entities during biotic interactions, with calcium signaling forming an established part of the plant defense response to microbial elicitors and to wounding caused by chewing insects, eliciting systemic calcium signals in plants. However, the role of calcium in vivo during biotic stress is still unclear. This protocol describes the use of a genetically-encoded calcium sensor to detect calcium signals in plants during feeding by a hemipteran pest. Hemipterans such as aphids pierce a small number of cells with specialized, elongated sucking mouthparts, making them the ideal tool to study calcium dynamics when a plant is faced with a biotic stress, which is distinct from a wounding response. In addition, fluorescent biosensors are revolutionizing the measurement of signaling molecules in vivo in both animals and plants. Expressing a GFP-based calcium biosensor, GCaMP3, in the model plant Arabidopsis thaliana allows for the real-time imaging of plant calcium dynamics during insect feeding, with a high spatial and temporal resolution. A repeatable and robust assay has been developed using the fluorescence microscopy of detached GCaMP3 leaves, allowing for the continuous measurement of cytosolic calcium dynamics before, during, and after insect feeding. This reveals a highly-localized rapid calcium elevation around the aphid feeding site that occurs within a few minutes. The protocol can be adapted to other biotic stresses, such as additional insect species, while the use of Arabidopsis thaliana allows for the rapid generation of mutants to facilitate the molecular analysis of the phenomenon.
Highlights
Cboinyfartlahecesicupofmoimrns(ptsClereaext2so+pn)woeisnotwsuoeonnsrdekitnooogffmtdchioacewuromsnbesoidatsrltebeuaylbmicciqihtcoueoriwtsmoi,nupfgsoornismneigsinnentgscatlaasin,ngcsdouemicslehmimnaovesnonlltpvesaepirdindt ioopnpflattthehneretasr.pneAlsas6pn,t7rot.andHnsesoefiewetnnoestvbereiosrr,teehtshiapnebocpinoyosttioteces3n,ao4tn,li5iacd.lRCbroiiasole2et+iscocfisonptnr[leacCsenasnt2etC+rs]aca1yt,22ito+.hnAsaiv(gr[eiCnsaaeall2ssi+no]inc[ybCtr)eeaiess2n+pd]ocoeynbtcsisoseedortevondeed live biotic threats that cause damage to only a few cells has not been explored
The green peach aphid Myzus persicae is a hemipteran insect that represents a significant threat to world agriculture[8,9], and Ca2+ efflux from the extracellular space has been observed in leaves infested with M. persicae[10].This protocol outlines a robust and repeatable method for measuring plant Ca2+ signals while M. persicae feed from leaves using a fluorescent Ca2+ biosensor, with both aphids and GCaMP3 offering novel tools with which to dissect the role of Ca2+ during biotic interactions
This study demonstrated that the R-GECO biosensor outperformed the fluorescence resonance energy transfer (FRET) Cameleon YC3.6 in terms of maximal signal change and signal-to-noise ratio[5]
Summary
Cboinyfartlahecesicupofmoimrns(ptsClereaext2so+pn)woeisnotwsuoeonnsrdekitnooogffmtdchioacewuromsnbesoidatsrltebeuaylbmicciqihtcoueoriwtsmoi,nupfgsoornismneigsinnentgscatlaasin,ngcsdouemicslehmimnaovesnonlltpvesaepirdindt ioopnpflattthehneretasr.pneAlsas6pn,t7rot.andHnsesoefiewetnnoestvbereiosrr,teehtshiapnebocpinoyosttioteces3n,ao4tn,li5iacd.lRCbroiiasole2et+iscocfisonptnr[leacCsenasnt2etC+rs]aca1yt,22ito+.hnAsaiv(gr[eiCnsaaeall2ssi+no]inc[ybCtr)eeaiess2n+pd]ocoeynbtcsisoseedortevondeed live biotic threats that cause damage to only a few cells has not been explored. The GCaMP3 biosensor can be visualized under a fluorescence microscope rather than a costlier confocal system This protocol allows for whole-tissue imaging, essential when conducting experiments with live biotic stresses. The experiment is designed such that detached leaves from 35S::GCaMP3 plants are floated in water, to prevent insect escape and to restrict feeding to a specific tissue. The method outlined in this paper allows for the analysis of leaf Ca2+ dynamics during feeding by M. persicae, resulting in the characterization of a novel plant signaling response This method can be adapted to work with other biotic stresses, such as additional insect species and microbial pathogens, and with other plant tissues, such as roots. 1. On day 19 of the experiment, remove the 35S::GCaMP3 seedlings from the CER and detach the largest leaf from each plant using a pair of sharp scissors. Repeat the experiment with further pairs of leaves; imaging can be extended to image 2 pairs of leaves at once, allowing for the simultaneous imaging of 2 genotypes
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