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Spatial regulation of rice D14L for pre-symbiotic perception of beneficial fungi

Reference	BB/P003419/1
Principal Investigator / Supervisor	Professor Uta Paszkowski
Co-Investigators / Co-Supervisors	Dr Jeremy Neil Skepper
Institution	University of Cambridge
Department	Plant Sciences
Funding type	Research
Value (£)	587,124
Status	Completed
Type	Research Grant
Start date	15/11/2016
End date	12/05/2020
Duration	42 months

Abstract

We follow a multidisciplinary approach to maximize the resolution of our analysis by exploiting the resources available in rice. Rice offers a high attractive plant system for an integrated combination of genetic, transcriptomic, proteomic and cell biological approaches, having a well annotated genome, efficient transformation protocols and its roots being amenable to confocal microscopy. With the proposal aiming at the elucidation of the spatial regulation of D14L signaling activities across root tissues and subcellular compartments the technical workplan largely builds on Agrobacterium-mediated plant transformation, confocal microscopy, and also RNA seq and LC-MS/MS as central methodologies. Efficient protocols for the transformation of cereal crops have been established in the team of the joint-applicant Emma Wallington (EW) who routinely deliver a high number of transformants with, in rice, at least 40% of lines carrying single copy T-DNA integrations. We base all our constructs for plant transformation on the GAL4-VP16/UAS transactivation system to increase transgene transcription (and thereby sensitivity) without losing cell- or treatment specificity. Multiphoton and lightsheet confocal microscopy will enable the deep root tissue analysis of fluorescent protein accumulation and where desired in real time. These microscopes are used on a daily basis to monitor fluorescent proteins in rice roots by another PDRA in my group (Ronelle Roth). The proposed work is thus feasible in rice. RNAseq and LC-MS/MS analyses will be performed on tissue from the same individuals allowing for the direct comparison where desired. These efforts follow established protocols at the respective platforms at the University of Cambridge and enable the quantitative and qualitative estimate of transcripts and proteins.

Summary

A key event in the evolution of higher life on earth is the transition of plant life from water to land. To explore the terrestrial environment plants had to develop strategies for the acquisition of soil nutrients with initially primitive root precursors. Ever since, plants have lived in symbioses with nutrient-delivering beneficial fungi. Today, the arbuscular mycorrhizal (AM) symbiosis is the most commonly occurring beneficial plant-fungal interaction on earth, contributing to plant fitness, plant biodiversity and global nutrient cycles. Yet, we are only beginning to unveil the molecular processes leading to mutual recognition in the rhizosphere. Colonization of plant roots by AM fungi requires the reciprocal exchange of diffusible molecules before fungal attachment to the root surface occurs. Chitin-related compounds secreted by AM fungi trigger plant molecular and developmental responses. Receptor kinase proteins detect these fungal molecules in the extracellular space and initiate a cellular reprogramming leading to a change in root system development. Mutational analyses showed that these receptor kinases are indeed required for development of AM symbioses but appear not to be essential. How plants perceive these prevalent beneficial fungi has therefore still been an unanswered question. My group has found that an intracellular alpha/beta hydrolase type protein, called DWARF 14 LIKE (D14L) is crucial for fungal perception by rice. In our recent Science publication we report our finding that the deletion of this gene from the rice genome rendered the plant unable to sense the fungus. Interestingly, the protein has additional roles in detecting the smoke constituent karrikin and in mediating developmental responses to light. It is evolutionarily conserved and may have therefore served similar functions in early terrestrial plants. The recognition of the fungus by the root and the subsequent establishment of primary contact involves distinct root tissue andcell types. The precise coordination of signaling events in space and time is thus essential for the successful development of AM symbioses. With this proposal we wish to determine where in the root tissue (relative to the approaching fungus) the protein needs to be present, and by magnifying onto the subcellular level, where within the cell the protein functions in initiating signal transduction and what are the respective 'molecular translations' of signalling. Given the central role of D14L in a variety of responses to environmental stimuli elucidating the tissue/cell type contributing to the signaling is vital for understanding coordination and specificity of this general and ancient plant symbiosis.

Impact Summary

The project will achieve academic, economic and social impacts. Academic impact will predominantly be on research in the area of plant-microbe interaction. Its impact on symbiosis research will be profound as our findings cause a shift in the understanding of plant recognition of AM fungi from the rather receptor-like kinase focused recognition paradigm to an alpha-beta hydrolase driven intracellularly mediated signaling activation model. Its impact on plant developmental biology is linked with the dual function of both D14L in symbiotic and developmental processes so that investigation addressing specificity for one signaling branch (in our case mainly the symbiotic) produce information immediately relevant to define specificity in the others (karrikin and strigolactone signaling). The whole plant science community will be impacted because of the elucidation of a novel yet plant kingdom-wide conserved signaling pathway. Finally, the identification of an endogenous signaling node mediating perception of rhizosphere organisms constitutes a novelty within the plant science community with the strongest impact on inter-organismic plant communications studies. Economic impact will be accomplished through the immediate relevance of the knowledge developed in this project to cereal science. Additional impact will derive from the parallel exploration of our approach in rice and wheat with potential to extrapolate further to other cereal species such as maize or barley. Dissemination of our research to a broader community of farmers and breeders will create impact via exchange of information and facts, and ultimately knowledge transfer. Societal impact of the proposed work will be achieved by employing our findings to stimulate the public's attention to plant research and to issues related to plant and crop science. Our project can serve as an example case to illustrate in a public friendly fashion the efforts of addressing fundamental biological questions to create theunderstanding required for advancing modern agricultural to grant sustainable food security. The project will provide benefits to the UK Competitiveness in (a) global science as the discovery of D14L as a critical plant determinant for the recognition of AM fungi uniquely positions my group to lead the field; (b) in sustainable food production as plant symbioses will potentially be of huge importance for future agriculture e.g. by reducing the demands on chemical fertilizers. The proposed work is at the leading edge of research aimed at unravelling the key mechanisms that lead to plant symbioses. The UK is a world leader in this area (e.g. https://www.ensa.ac.uk/home/) and this programme will allow important advances to be made that will ensure it remains at the forefront of plant symbiosis research.

Committee	Research Committee B (Plants, microbes, food & sustainability)
Research Topics	Crop Science, Microbiology, Plant Science
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

Associated awards:

BB/P003176/1 Spatial regulation of rice D14L for pre-symbiotic perception of beneficial fungi

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