BiFC Assay to Detect Calmodulin Binding to Plant Receptor Kinases

Abstract

Plant receptor-like kinases (RLKs) are regulated at various levels including posttranscriptional modification and interaction with regulatory proteins. Calmodulin (CaM) is a calcium-sensing protein that was shown to bind to some RLKs such as the PHYTOSULFOKINE RECEPTOR1 (PSKR1). The CaM-binding site is embedded in subdomain VIa of the kinase domain. It is possible that many more of RLKs interact with CaM than previously described. To unequivocally confirm CaM binding, several methods exist. Bimolecular fluorescence complementation (BiFC) and pull-down assays have been successfully used to study CaM binding to PSKR1 and are described in this chapter (BiFC) and in Chapter 15 (pull down). The two methods are complementary. BiFC is useful to show localization and interaction of soluble as well as of membrane-bound proteins in planta.

A Plant Phytosulfokine Peptide Initiates Auxin-Dependent Immunity through Cytosolic Ca2+ Signaling in Tomato

“…This suggests that the observed increases in cytosolic [Ca 2+ ] may be generated through the PSKR1 GC activity-induced opening of the cyclic nucleotidegated channel (Ladwig et al, 2015). It should be noted that tomato CaMs not only act as Ca 2+ sensors that bind YUCs, but they can also bind directly to the PSKR1 receptor at the kinase subdomain (Hartmann et al, 2014;Fischer et al, 2017). Binding of At-PSKR1 to CaMs within the kinase subdomain is required for At-PSKR1 functioning in the regulation of growth responses of Arabidopsis roots (Hartmann et al, 2014).…”

LORE receptor homomerization is required for 3‐hydroxydecanoic acid‐induced immune signaling and determines the natural variation of immunosensitivity within the Arabidopsis genus

“…Yellow fluorescent protein (YFP) fluorescence complementation was visually assessed via confocal laser scanning microscopy. For quantification, YFP fluorescence of leaf disks from transformed areas was measured with a plate reader as described previously (Fischer et al ., 2017). Protein expression was validated by immunoblot.…”

“…Antibodies are listed in Table S6. After washing of membranes (39 10 min, TBS-T), chemiluminescence was detected with a CCD camera (Fusion SL System; Vilber Lourmat Deutschland GmbH, Eberhardzell, Germany) upon incubation with peroxidase substrates (Super-Signal (Fischer et al, 2017). Protein expression was validated by immunoblot.…”

LORE receptor homomerization is required for 3-hydroxydecanoic acid-induced immune signaling and determines the natural variation of immunosensitivity within the Arabidopsis genus

“…YFP fluorescence complementation was visually assessed via confocal laser scanning microscopy. For quantification, YFP fluorescence of leaf discs from transformed areas was measured with a plate reader as described (Fischer et al ., 2017). Protein expression was validated by immunoblot.…”