The overall objectives of the SSPinROOTS project are divided into four work packages (WP), and the work carried out within each of them is described below.
WP1 and WP2: Identification of N- and P-responsive SSPs in roots and nodules of Medicago truncatula
In Medicago truncatula only a small fraction of known SSP families had been annotated upon commencing the project. Therefore, a large effort has been put into identifying Medicago SSPs on a genome-wide scale. This work resulted in the identification of nearly 2,000 SSPs belonging to 46 established families, as well as the identification more than 2,000 potential SSP genes.
With this data in hand, RNA-sequencing data sets from plant tissue deficient in N and P, as well as tissue re-supplied with N and P after deficiency was produced and analyzed for SSPs responding to these conditions. In addition, RNA-sequencing data from roots inoculated with rhizobia and different nodule developmental stages were collected and analyzed. Hundreds of SSP genes were found to significantly change expression in response to nodulation and N- and P-availability. This number of SSPs by far exceeds what was previously known, and is as such a main outcome of the SSPinROOTS project. Based on RNA-sequencing expression profiles, a panel of SSPs for further studies was selected. At this stage, more than 20 different synthetic peptides have been tested for their effect on root growth and nodulation. As a main result of the peptide screening, a set of peptides from the PIP family promoting lateral root development has been identified. Treatment of seedlings with PIP peptides resulted in increased total root length and as such this family of peptides have the potential to improve nutrient uptake in plants.
WP3: Identification of SSP-receptor modules and down-stream target genes
Based on the results from the RNA-sequencing data and the effects of peptide addition on root and nodule development, three Medicago receptors have been selected for further analysis. The first receptor analyzed is the one expected to bind PIP peptides described in WP1. Analyzing the roots of the PIP receptor mutant revealed that lateral root growth is delayed, a phenotype which is opposite to the one observed when adding synthetic PIP peptides seedlings. Hence, these findings indicate that the discovered PIP-receptor signaling module is important for controlling root growth in response to nutrient availability, and thus represents a novel finding of agronomic importance.
Deviations from the original research plan
After a thorough homology analysis in Medicago, it was not possible to identify homologs to the P-responsive Arabidopsis peptide initially intended to be the starting point for receptor studies within this project.
WP4: Postdoctoral training in transferable skills
One of the main goals of WP4 was to improve my skills within project management and project leadership, of which of have gained a lot of experience up until this point. During the first four months, I worked independently with most aspects of the project, while at the same time receiving advice and supervision from my peers at the Noble Foundation. In this period, I made detailed project plans, produced sample material and developed laboratory protocols. In the following period of 7 months, I had the daily responsibility of laboratory technicians who conducted experiments under my supervision. This experience I am now utilizing at University of Copenhagen in terms of student supervision, e.g. I supervise a master student who is studying a nodule-specific SSP, as well as experimental planning with new collaborators from Institute of Plant Sciences Paris-Saclay (IPS2) at Université Paris Sur. Lastly, the part of the project related to bioinformatics is being written together by myself as the primary author, and it is expected to be published in one of the very best journals within plant science. In this process, I am receiving valuable inputs to improve the
