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

The inhibition of self-pollination in self-incompatible Brassicaceae is based on allele-specific trans-activation of the highly polymorphic S-locus receptor kinase (SRK), which is displayed at the surface of stigma epidermal cells, by its even more polymorphic pollen coat-localized ligand, the S-locus cysteine-rich (SCR) protein. In an attempt to achieve constitutive activation of SRK and thus facilitate analysis of self-incompatibility (SI) signaling, we coexpressed an Arabidopsis lyrata SCR variant with its cognate SRK receptor in the stigma epidermal cells of Arabidopsis (Arabidopsis thaliana) plants belonging to the C24 accession, in which expression of SRK and SCR had been shown to exhibit a robust SI response. Contrary to expectation, however, coexpression of SRK and SCR was found to inhibit SRK-mediated signaling and to disrupt the SI response. This phenomenon, called cis-inhibition, is well documented in metazoans but has not as yet been reported for plant receptor kinases. We demonstrate that cis-inhibition of SRK, like its trans-activation, is based on allele-specific interaction between receptor and ligand. We also show that stigma-expressed SCR causes entrapment of its SRK receptor in the endoplasmic reticulum, thus disrupting the proper targeting of SRK to the plasma membrane, where the receptor would be available for productive interaction with its pollen coat-derived SCR ligand. Although based on an artificial cis-inhibition system, the results suggest novel strategies of pollination control for the generation of hybrid cultivars and large-scale seed production from hybrid plants in Brassicaceae seed crops and, more generally, for inhibiting cell surface receptor function and manipulating signaling pathways in plants.Ligand receptor signaling plays important roles in cell-cell communication between neighboring cells in a variety of developmental and physiological processes. This communication typically relies on the interaction of transmembrane receptors displayed on the surface of signal-receiving cells with their cognate ligands derived from signal-sending neighboring cells, which, in turn, leads to the activation of receptor-mediated signaling cascades that modify intracellular activities of the signal-receiving cell. Such is the case with communication between pollen grains and stigma epidermal cells, a process that has an important role in directing reproductive success and determining pollination modes (i.e. selfing or outcrossing) in the Brassicaceae. In this family, outcrossing is enforced by self-incompatibility (SI), a mechanism controlled by haplotypes of the S locus, by which the stigma epidermal cells of a plant recognize and reject self pollen grains (i.e. those derived from the same flower, the same plant, or plants expressing the same S-locus haplotype), thus preventing self-pollination, while allowing the growth of tubes from nonself pollen grains (i.e. those derived from plants expressing a different S-locus haplotype; Nasrallah and Nasrallah, 2014a). Inhibition of self pollen in the SI response is initiated by allele-specific interaction between two highly polymorphic proteins encoded at the S locus: the S-locus receptor kinase (SRK), which is localized at the plasma membrane of stigma epidermal cells (Stein et al., 1991, 1996), and its ligand, the S-locus cysteine-rich protein (SCR), which accumulates in the pollen coat and diffuses onto the stigma surface upon pollen-stigma contact (Schopfer et al., 1999; Takayama et al., 2000; Shiba et al., 2001). The interaction of the SRK extracellular domain, or S domain, with its cognate SCR ligand is thought to activate downstream signaling cascades in stigma epidermal cells, which lead to inhibition of pollen germination on the stigma surface and/or pollen tube penetration through the stigma epidermal cell wall. The SRK and SCR genes are the primary determinants of the transition between the outcrossing and selfing modes of mating in the Brassicaceae, as demonstrated by the observation that transformation of SRK and SCR gene pairs derived from self-incompatible Arabidopsis lyrata or Capsella grandiflora restored SI in several accessions of the normally self-fertile Arabidopsis (Arabidopsis thaliana; Nasrallah et al., 2002, 2004; Boggs et al., 2009).Tight regulation of the SI response is critical for ensuring reproductive success in self-incompatible plants. Activation of SRK signaling must be triggered only by pollen-derived cognate SCR ligand upon interaction of stigma epidermal cells with self pollen grains, because constitutive activation of SI signaling in stigma epidermal cells would result in inhibition of nonself as well as self pollen grains and would result in female sterility. This adverse outcome is averted by tight regulation of the SCR gene, which is expressed exclusively in the anthers of self-incompatible plants and whose protein products are localized exclusively in the pollen coat (Schopfer and Nasrallah, 2000; Shiba et al., 2001). For experimental studies of SI, however, constitutive activation of SRK-mediated signaling in stigma epidermal cells would be useful, as it might provide a convenient means of identifying components of the poorly understood SRK-mediated signaling pathway.A reaction that resembles a constitutive SI response, in which stigma epidermal cells inhibit both self and nonself pollen grains, has been obtained by manual application of purified recombinant SCR proteins produced in bacteria (Kachroo et al., 2001; Chookajorn et al., 2004) or synthetic SCR (Takayama et al., 2001) to stigmas that express their cognate SRK receptors. Unlike the highly localized activation induced by pollen-derived SCR at the site of pollen-stigma contact, treatment of the stigma surface with SCR protein can clearly cause global activation of SRK in most, if not all, epidermal cells of a stigma. However, treating stigmas in the numbers required for analysis of SRK signaling is extremely laborious, can damage stigmas, and produces inconsistent results. Therefore, a method that circumvents these problems would be advantageous. In metazoans, constitutive activation of receptor kinases has been shown to result not only from receptor mutations that cause constitutive kinase activity (Webster and Donoghue, 1996; Hirota et al., 1998) and mutations in signaling components that cause ligand-independent activation of downstream cascades (Wang et al., 2012, 2014; Roberts et al., 2013; Han, 2014), but also from ectopic expression of ligands within the same cells as their receptors, as occurs in several pathological conditions (Sporn and Roberts, 1985; Castellano et al., 2006; Krasagakis et al., 2011).Accordingly, an attempt was made to generate Arabidopsis plants having a stable constitutive stigma SI response by coexpressing an SRK variant and its cognate SCR in stigma epidermal cells, which should, in principle, constitutively activate the SI response in these cells. This report shows that, while pollen-derived SCR trans-activates the SRK-mediated SI response, stigma-expressed SCR inhibits the activity of its cognate SRK by causing entrapment of the receptor in the endoplasmic reticulum (ER). This phenomenon is similar in its outcome to the ligand-mediated cis-inhibition phenomenon that had previously been observed in metazoans for some signaling systems that use transmembrane proteins as ligands (Yaron and Sprinzak, 2012) but had not been described for plant receptor-like kinases. The results suggest novel strategies for control of receptor-like kinase activity and manipulation of signaling pathways in plants and for pollination control in hybrid breeding programs and seed production from hybrid plants in the Brassicaceae.