Live-cell imaging of ligand-activated receptor kinases that are tagged with a fluorescent protein can provide valuable information about the mechanism by which such a receptor transduces the signal it perceives at the cell surface into a cellular response. This approach has been used for analysis of several plant receptor-like kinases of the leucine-rich repeat class of receptors. However, it has not, as of yet, been applied successfully to the S-locus receptor kinase or any other member of the S-domain class of RLKs.
A recent paper in Annals of Botany describes the expression and live-cell imaging of functional FP-tagged versions of the A. lyrata SRKb variant in A. thaliana stigma epidermal cells. The successful FP tagging of SRK and its visualization in live stigma epidermal cells suggest new approaches for future analysis of the dynamics of SRK and SRK–SCR protein complexes. For example, it should be possible to visualize cYFP-tagged full-length SRK in conjunction with SCR proteins tagged with a different fluorescent label, and thus determine conclusively if SRK is indeed internalized subsequent to its interaction with its SCR ligand.
Rea, A.C., and Nasrallah, J.B. (2015) In vivo imaging of the S-locus receptor kinase, the female specificity determinant of self-incompatibility, in transgenic self-incompatible Arabidopsis thaliana. Annals of Botany, 24 February 2015 doi: 10.1093/aob/mcv008
The S-locus receptor kinase (SRK), which is expressed in stigma epidermal cells, is responsible for the recognition and inhibition of ‘self’ pollen in the self-incompatibility (SI) response of the Brassicaceae. The allele-specific interaction of SRK with its cognate pollen coat-localized ligand, the S-locus cysteine-rich (SCR) protein, is thought to trigger a signalling cascade within the stigma epidermal cell that leads to the arrest of ‘self’ pollen at the stigma surface. In addition to the full-length signalling SRK receptor, stigma epidermal cells express two other SRK protein species that lack the kinase domain and whose role in the SI response is not understood: a soluble version of the SRK ectodomain designated eSRK and a membrane-tethered form designated tSRK. The goal of this study was to describe the sub-cellular distribution of the various SRK protein species in stigma epidermal cells as a prelude to visualizing receptor dynamics in response to SCR binding.
The Arabidopsis lyrata SRKb variant was tagged with the Citrine variant of yellow fluorescent protein (cYFP) and expressed in A. thaliana plants of the C24 accession, which had been shown to exhibit a robust SI response upon transformation with the SRKb–SCRb gene pair. The transgenes used in this study were designed for differential production and visualization of the three SRK protein species in stigma epidermal cells. Transgenic stigmas were analysed by pollination assays and confocal microscopy.
Pollination assays demonstrated that the cYFP-tagged SRK proteins are functional and that the eSRK is not required for SI. Confocal microscopic analysis of cYFP-tagged SRK proteins in live stigma epidermal cells revealed the differential sub-cellular localization of the three SRK protein species but showed no evidence for redistribution of these proteins subsequent to incompatible pollination.