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

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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.

The proposed work will have extensive academic, economic, and societal impacts. The major beneficiaries of this work will be: (i) Scientists, who will derive advantage from increased basic knowledge of plant root interactions with the rhizosphere environment, namely nutrient-delivering fungi. These beneficiaries will experience multidisciplinary working in molecular biology, genetics, cell biology, and cutting edge imaging to bridge the knowledge-gap concerning the earliest stage in plant perception of mutualistic soil fungi. (ii) Farmers, who will benefit through advice on application of beneficial fungi targeted to the needs of specific crop varieties, and through a broader general appreciation of the complexity of crop-symbiont interactions in the long term. (iii) Crop breeders, who will benefit from new knowledge to enable the selection of varieties adapted to maximize responsiveness to the mineral-providing root symbiosis. (iv) Industry, who will take advantage from shared knowledge to develop strategies on the basis of the new findings. (v) Policy makers, particularly in rice-growing areas, who will benefit from shared knowledge which can be used to develop legislation limiting/regulating sustainable fertilization and (vi) Wider society, who will ultimately benefit through reduced detrimental impacts on environment by reduction in agro-chemical inputs in food production and increased food security.

This proposal will provide a new insight into the mechanisms of pre-symbiotic root signalling which will be invaluable in designing strategies for accelerated establishment of the symbiosis, informing breeding efforts towards crop varieties with optimized symbiotic responsiveness. Rice is a staple food for more than half of the human population and is therefore central for food security plus a target for the design of sustainable strategies to increase crop yield. Rice also serves as the primary model for other cereals such as wheat, barley and maize, and lessons learned from rice have the potential to guide research and breeding programmes.

We will communicate our findings to the scientific community through publication in peer-reviewed journals. Farmers and industry stakeholders will be informed through events organised by the Agri-Tech East cluster, Cambridge Partnership for Plant Science and NIAB Open Days and articles in the NIAB journal, Landmark. We will also initiate public demonstrations of the project to publicise our work, engaging in presentations at events such as the annual Cambridge Science festival, the Festival of Plants at the Botanic Garden and the National Biology Week.