Investigating the PrpS-PrsS (pollen & pistil SI determinant) interaction

Self-incompatibility (SI) is the most important mechanism used by flowering plants to prevent self-fertilization, which would otherwise result in undesirable inbreeding, with consequent loss of fitness. For this reason it is widely considered to have made a significant contribution to the evolutionary success of the flowering plants. SI utilizes cell-cell recognition to prevent self-fertilization. This involves a highly specific interaction between a pistil expressed protein and a cognate pollen component that results in recognition and inhibition of self- (incompatible), but not cross- pollen. In poppy, the female part of the flower (pistil) secretes a protein (PrsS) which acts as a signalling 'ligand'. This interacts specifically with a 'self' pollen receptor (PrpS), allowing pollen to distinguish between 'self' and 'non-self' partners, through interactions analogous to a lock and key mechanism. This interaction is the critical step in determining cell-cell recognition and rejection, so the nature of the pollen S-component is of considerable interest. We know a lot about the signals and events triggered by the interaction, but only recently identified the 'missing' crucial component: the pollen S-receptor, named PrpS. SI provides an excellent model system to investigate the molecular basis of cell-cell recognition and intracellular signalling in plant cells for many reasons. The nature of S allele specificity at a highly polymorphic S-locus, containing ~60 alleles, is of considerable general interest to population and evolutionary biologists. SI systems are comparable to the human major histocompatibility complex (MHC) with respect to the level of their polymorphism. How allele specificity is encoded, and how pollen and pistil components co-evolved, are long-standing questions. Moreover, receptor-ligand interactions in plants are poorly understood. Despite genomic studies revealing the existence of several hundred putative transmembrane receptors, their biological function and, in particular, the nature of the ligands with which they interact, remains largely unknown with the exception of a few examples. Our identification of PrpS provides a system where we know a lot about the signalling network and events triggered by the interaction; now we have both ligand and receptor this opens up the field for studies in this area. And as PrpS is a novel protein, its nature is of interest in its own right. Finally, on a practical and applied note, an understanding of SI could help plant breeders develop F1 hybrids more efficiently and economically than currently. Thus we could potentially not only understand basic processes involved in plant cells, but also perhaps eventually exploit these systems. Thus, one of the attractions of studying SI is the possibility of transferring S locus genes to non-SI species to create a functional SI system. This could be of immense benefit to plant breeders in the production of F1 hybrids. Preliminary data arising from BBSRC-funded studies involving transferring the SI system to the model plant, Arabidopsis. This has provided the first experimental evidence indicating this is possible, indicating that the transferred Papaver SI system is functional in other species. This discovery has been filed for a patent. We anticipate that plant breeders will be extremely excited and interested in this technology. Currently plant breeders have had to use hand-emasculation to produce F1 hybrids, which is very tedious, time-consuming and expensive, as it involves a lot of labour costs. If a crop species is self-incompatible it can be crossed without any emasculation, as no self-pollen can fertilize these plants. Thus, all crosses will result in cross seed. Excitingly, a company is interested in marketing our poppy PrpS-PrsS SI system as a useful tool for plant breeders to make improved crops.

This project will examine the nature of PrpS and its proposed 'receptor-ligand' type interaction with PrsS, using a range of live-cell approaches. We will use a range of live-cell approaches to study the SI determinant-ligand interactions in transgenic Arabidopsis pollen protoplasts, transfected animal cells and poppy pollen tubes transiently expressing PrpS-GFP using a biolistic approach. We will also use Alexa-tagged PrsS for some experiments. This will provide a detailed determination of the nature, localization, distribution and dynamics of the receptor-ligand interaction. (A) aims to establish the nature of PrpS as an ion channel We will express PrpS in a heterologous cell system (Xenopus oocytes &/or HEK cells) to build on preliminary data to firmly establish that PrsS functions as an ion channel. Patch-clamping will allow us to identify PrsS-induced activation and kinetics, channel conductance and selectivity and pharmacological properties. (B) & (C) focus on studying PrpS dynamics and interactions with PrsS and identifying amino acids/domains involved in recognition/function. We will use live-cell imaging using fluorescence microscopy, including: Bimolecular fluorescence complementation (BiFC), Total internal reflection fluorescence microscopy (TIRF-M), FRAP and FRET to study PrpS-PrsS interactions in detail. Site-directed mutagenesis on the predicted extracellular 35 aa loop and also making PrpS chimeras by domain-swopping this region between allelic variants (e.g. replace S1 with S3) will establish which changes result in loss or altered function or S-specificity. These approaches will allow us to establish if PrpS forms a multimeric complex, establish the subcellular localization/activity of PrpS/PrsS, examine probe real-time PrpS-PrsS interaction dynamics in live cells, and establish the nature of the allele-specificity of the PrpS-PrsS interaction.

Economic & Social Impact: This project fulfils a BBSRC policy priority of having economic and social impact. Longer term, knowledge about the PrpS-PrsS interaction may provide solutions to currently expensive breeding systems. F1 hybrids are generally bigger better and more productive than their parents and F1 hybrid seed makes up around 40% of global seed sales (worth billions each year). However, currently plant breeders have to hand-emasculate to produce F1 hybrids. This is very tedious, time-consuming and expensive, due to high labour costs. Alternative methods exist, but their use is limited. SI potentially provides a good way produce F1 hybrids in crops. If a crop has an SI system it can be crossed without any emasculation, as no self-pollen can fertilize these plants, and all crosses will result in cross seed. Thus, if S locus genes can be transferred to non-SI species to create a functional SI system, this could be of immense benefit to plant breeders in the production of F1 hybrids. We recently obtained evidence indicating that the transferred Papaver SI system is functional in Arabidopsis. This discovery has been filed for a patent. A company is already interested in marketing our poppy PrpS-PrsS SI system as a tool for plant breeders to make improved crops. Building further knowledge about the PrpS-PrsS interaction could benefit the patent and commercialization. We will actively pursue the exploitation of new data as a basis for consolidating the patent. Fulfilling BBSRC strategic aims: As explained above, use of SI could impact on the BBSRC research priority of Global Security. Providing enough food for the world in the future will be a major challenge, and more economic ways to make F1 hybrid crops, which have better yields, could provide an important contribution to this problem. Thus this project contributes to one of BBSRC's principal strategic aims; 'underpinning practical solutions to major challenges such as food security' and by 'securing national research capability in a strategically important area', namely, food security. Although not strictly 'Systems Biology', this project also fulfils a strategic aim: 'developing and embedding a Systems approach to Biosciences in order to advance fundamental understanding of complex biological processes'. Research on SI has consistently been focused on integrating information about this complex signalling network. Thus, this project in its own way fulfils a BBSRC research priority of 'Systems approach to biological research'. Another BBSRC strategic aim is 'maintaining world-class UK bioscience by supporting the best people and best ideas'. This project emerges from a lab which has produced three Nature papers in the last five years; the current proposal is based on the latest Nature paper (June 2009) and promises to potentially provide data for another one. 'Providing skilled researchers needed for academic research' is part of BBSRC Strategic aims: NF-T has an excellent track record for training and mentoring of post-doctoral fellows. 7 former PDFs remain in academic research; four have achieved group leader status. This grant proposal promises to help BBSRC achieve this aim, especially as it will provide the postdoc with training in a wide range of cutting edge live-cell imaging technologies. Public/Education: NF-T is actively involved in interacting with the media, public and schools to make science more accessible. NF-T has been involved in the BBSRC Schools Liaison Scheme, running projects with numerous schools and as a coordinator. She has lectured at a 6th form college; organized science activities at 'ThinkTank'. NF-T has contributed to writing press releases, featured in BBSRC Business; participated in phone interviews. Her work was discussed in layman's terms on Science's news website; she has contributed an article 'How Plants Avoid Incest' for ScienceNOW.