Evolution of D14L signalling specificity for symbiosis and development

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Rice offers a highly attractive plant system for an integrated combination of molecular, genetic and transcriptomic approaches, having a well annotated genome, efficient transformation protocols and its roots being readily colonised by AM fungi. With the proposal aiming at the elucidation of D14L protein characteristics driving signalling specificities, the technical workplan largely builds on metagenomics, plant transformation and phenotyping, including microscopic, biochemical and molecular phenotyping.

The use of the fast and sensitive protein aligner DIAMOND2 enables another dimension of phylogenomics studies, due to the significantly accelerated analysis of the ever increasing, sequence data. In combination a newly developed algorithms to define protein similarity networks, the dynamics of evolvability of proteins, protein domains, or protein residues is revealed, and will then be overlaid with the taxonomic map of plant symbiotic competence. Computational followed by genetic validation reveals D14L sequence features that are significantly associated with the symbiosis signalling phenotype.

Transformation of cereal crops is efficiently running in the team of the joint-applicant Emma Wallington (EW) who routinely delivers 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 genetic complementation on synthesising and cloning the coding region of the D14L variants under the transcriptional control of the same native rice D14L promoter and terminator.

Central phenotyping methodologies are rice germination to measure mesocotyl length, conventional light microscopy for the quantification of root colonisation, standard protoplast transformation and Western blotting to monitor protein abundance, RNAseq (Novogene) to produce the quantitative and qualitative estimate of transcripts.