Site-SpecificN-Glycosylation of the S-Locus Receptor Kinase and Its Role in the Self-Incompatibility Response of the Brassicaceae  

Abstract

The S-locus receptor kinase SRK is a highly polymorphic transmembrane kinase of the stigma epidermis. Through allelespecific interaction with its pollen coat-localized ligand, the S-locus cysteine-rich protein SCR, SRK is responsible for recognition and inhibition of self pollen in the self-incompatibility response of the Brassicaceae. The SRK extracellular ligand binding domain contains several potential N-glycosylation sites that exhibit varying degrees of conservation among SRK variants. However, the glycosylation status and functional importance of these sites are currently unclear. We investigated this issue in transgenic Arabidopsis thaliana stigmas that express the Arabidopsis lyrata SRKb variant and exhibit an incompatible response toward SCRb-expressing pollen. Analysis of single-and multiple-glycosylation site mutations of SRKb demonstrated that, although five of six potential N-glycosylation sites in SRKb are glycosylated in stigmas, N-glycosylation is not important for SCRb-dependent activation of SRKb. Rather, N-glycosylation functions primarily to ensure the proper and efficient subcellular trafficking of SRK to the plasma membrane. The study provides insight into the function of a receptor that regulates a critical phase of the plant life cycle and represents a valuable addition to the limited information available on the contribution of N-glycosylation to the subcellular trafficking and function of plant receptor kinases.

Flowers and mycorrhizal roots – closer than we think?

“…Conserved cysteine residues are highlighted in red, predicted N-glycosylation sites in blue. Glycosylation sites in AlSRKb were experimentally confirmed [50].…”

“…The extracellular domain of SRK contains a highly conserved cysteine-rich stretch consisting of an EGF-like and a PAN_APPLE motive that are required for receptor dimerization [49], and comprises Nglycosylation sites that are essential for localization to the plasma membrane [50]. The genes encoding SCR protein and SRK are tightly linked in the S-locus, and occur in many allelic variants in natural plant populations [51], thus ensuring efficient cross-pollination.…”

Ligand-Mediated cis-Inhibition of Receptor Signaling in the Self-Incompatibility Response of the Brassicaceae

“…In brief, the cYFP coding sequence together with a linker sequence, which serves to minimize improper folding of the cYFP-tagged SRKb protein, was inserted using a recombinant PCR strategy into a pCAMBIA1300 derivative containing the AtS1pr:SRKb gene. The cYFP coding sequence was inserted either directly upstream of the stop codon to express C-terminally tagged SRKb or directly downstream of the signal sequence to express N-terminally tagged SRKb (Yamamoto et al, 2014;Rea and Nasrallah, 2015). To generate the AtS1pr:mC-HDEL construct, the previously described 35Spr:mC-HDEL construct (Nelson et al, 2007) was modified by replacing the double Cauliflower mosaic virus 35S promoter with the AtS1 promoter by recombinant PCR (Rea and Nasrallah, 2015).…”

“…To visualize only the fulllength SRKb signaling receptor without interference from the eSRKb and tSRKb proteins, a version of SRKb carrying a C-terminal cYFP tag (designated SRKbcYFP), which confers a strong incompatible response toward SCRb-pollen (Yamamoto et al, 2014;Rea and Nasrallah, 2015) was used. As shown in Figure 3A, imaging of stigma epidermal cells lacking cis-SCRb showed the SRKb-cYFP signal to be localized primarily at the cell periphery, consistent with the previously demonstrated plasma membrane localization of the receptor (Yamamoto et al, 2014;Rea and Nasrallah, 2015).…”

“…To visualize only the fulllength SRKb signaling receptor without interference from the eSRKb and tSRKb proteins, a version of SRKb carrying a C-terminal cYFP tag (designated SRKbcYFP), which confers a strong incompatible response toward SCRb-pollen (Yamamoto et al, 2014;Rea and Nasrallah, 2015) was used. As shown in Figure 3A, imaging of stigma epidermal cells lacking cis-SCRb showed the SRKb-cYFP signal to be localized primarily at the cell periphery, consistent with the previously demonstrated plasma membrane localization of the receptor (Yamamoto et al, 2014;Rea and Nasrallah, 2015). Moreover, as is typical of plasma membranelocalized proteins in plants and previously shown for SRKb-cYFP (Rea and Nasrallah, 2015), upon plasmolysis of stigma epidermal cells, the SRKb-cYFP signal was observed in the stretched portions of the plasma membrane called Hechtian strands, which form upon retraction of the protoplast from the cell wall following plasmolysis (Fig.…”