Unravelling the barley genetic control of the rhizosphere microbiota

In recent years it has become increasingly evident that plants and animals are not autonomous organisms but rather they are colonised by a myriad of different microorganisms, collectively referred to as the microbiota. For example, a single gram of soil tightly adhering to plant roots, and called rhizosphere, host millions of different bacteria. We want to understand how plants communicate with bacteria in the rhizosphere. This is a key area of research because bacteria in the rhizosphere can promote plant mineral uptake from soil and protect plants from diseases. However, other rhizosphere bacteria can be pathogenic and cause yield losses. Understanding the molecular basis of this communication means that we would be in the position to rewire it for the benefit of plants. Ultimately, this can help farmers to achieve profitable yields while reducing the input, and the negative impact, of agrochemicals in the environment.
In this project we will use the crop plant barley, the fourth most cultivated cereal worldwide used mainly for animal feeding and in the processes of brewing and distilling. We previously demonstrated that cultivated 'elite' varieties, selected by modern breeding to respond to chemical inputs, and wild barley plants, which have evolved in marginal lands, host distinct microbiotas. We also demonstrated that the capacity to shape the microbiota is encoded by genes in the barley genome. We recently found out that some of these genes reside in a specific portion of the genome, which scientists call a locus. Here we want to investigate this biological phenomenon further by pursuing the following objectives.
1. Find out the genes shaping the rhizosphere microbiota
We will use the power of genetics to study thousands of plants derived from a cross between an elite barley variety and a wild ancestor. We will use fantastically powerful tools for following the independent inheritance of natural versions of genes (called alleles) from all over the barley genome in each of these progeny plants. By investigating the strength of correlation between alleles from all over the barley genome and microbiota composition supported by each of the plants we will be able to identify the actual gene(s) that shape the rhizosphere microbiota.
2. Find out how these genes work at the molecular level.
Plants release a lot of molecules into the soil to interact with bacteria. We will investigate whether these molecules differ between elite and wild barleys. Likewise, we will study properties of the roots, such as their weight and length, since these influence the way roots explore the soil and interact with bacteria. Finally, we will determine how many other barley genes expressed in the roots are differentially regulated between identical pairs of lines that differ only at the locus on the genome that supports different populations of rhizosphere microbiota. Together, this will provide a picture of the biological processes modulated by the locus we are investigating which may influence microbial proliferation in the rhizosphere.
3. Find out if and when these genes promote crop yield.
We will test whether elite material carrying the wild barley locus will produce more grain. We will test two types of soil. In one type, we will mimic current agronomic practices and plants will be provided with chemical fertilisers. In another type, we will omit nitrogen, a major plant nutrient. Owing to the fact that bacteria play a crucial role in recycling nitrogen in soil, our hypothesis is that bacteria recruited by wild barley genes will provide an advantage to plants grown under limiting supplies. Whether or not this will be proved, our results will provide key information on how plants communicate with bacteria in the rhizosphere.

The rhizosphere microbiota represents the microbial communities inhabiting the rhizosphere, the thin layer of soil tightly adhering to plant roots. Bacteria are important members of the rhizosphere microbiota: for example, so-called plant growth promoting rhizobacteria can increase plant mineral uptake and protect plant from pathogens. Therefore understanding how plants and rhizobacteria interact at the genetic level is a strategic priority to underpin global food security. However, the genetic basis of plant-microbiota interactions in crops remains poorly understood. This proposal aims at filling this knowledge gap by building on preliminary results we gathered using barley (Hordeum vulgare) as an experimental system. In particular, we recently identified a major regulator of microbiota recruitment located on a single locus on barley chromosome 3H. Here we want to extend this investigation to identify and characterise barley genes shaping the microbiota. First, we will use 16S rRNA gene profiles as 'quantitative traits' to perform a fine mapping of the locus on chromosome 3H. Next, we will determine whether allelic variation at the locus of interest correlates with patterns in the exudation profiles and root morphology, two traits previously implicated in the assembly of the rhizosphere microbiota. In parallel, we will identify root genes differentially regulated between lines harbouring contrasting alleles at the locus 3H using a RNA-seq approach. Finally, we will establish whether specific bacterial configurations of the barley microbiota driven by locus 3H are causally related to crop yield when plants are exposed to sufficient and limiting nitrogen supplies. By pursuing these objectives we expect to gain novel insights into plant-bacteria interactions in the rhizosphere and their significance for crop production.

The economical and societal benefits of this proposal will impact on four major categories of beneficiaries.
1. The personnel employed in this research project. This project will recruit two post-doctoral research assistants (PDRAs), providing novel interdisciplinary opportunities for training and subsequent employment in research and development. One of the PDRA will have a leading role in designing and executing the experimental lines while the other PDRA will be tasked to analyse the sequencing information generated in this work. The project will extend existing strong collaborations between the Divisions of Plant Sciences and Computational Biology at the University of Dundee. The PDRAs will be in a unique position at this research interface to advance both the field of crop sciences and computational techniques in plant genetics and metagenomics. The PDRAs will be encouraged to attend and present findings of the proposal at national and international scientific conferences. Finally, the PDRAs will receive training and participate in public engagement activities (see point 2 below). As microbiome investigations are gaining centre stage in basic and applied research, this skill set will be an asset for future employment opportunities both in academia and industry.

2. The general public (including future academics). The PI and the staff employed in this project are committed to actively contribute to devise outreach activities of the University of Dundee. Specific activities will include demonstrations and 'hands-on' experiments for the general public at the annual open day events organised by the School of Life Sciences (e.g., 'Plant Power Day', 'Magnificent Microbes'). In addition, we will engage with pupils of primary schools in the Dundee area to develop a series of animated science projects. We will perform this initiative at the end of each year of the proposal to illustrate key findings of our work in a form accessible to the general public. The animated projects will be then posted on social media such as youtube and twitter. Finally, the PI and Co-Is will welcome in their labs summer students, honours students and interns who want to be trained in molecular and computational biology techniques, to engage potential future academics in plant- microbiota interactions.

3. Barley growers and other stakeholders. Researchers involved in this project will contribute to the International Barley Hub initiative, aimed at creating the world's leading centre translating excellence in barley research and innovation into economic, social and environmental benefits. Specifically, we will take advantage of dedicate events (e.g., 'Cereals in Practice') to present findings of our investigations and increase the awareness of barley growers and other stakeholders on how rational manipulation of plant-microbiota interactions can sustainably increase crop production.

4. Plant breeders and agro-biotech companies. The project will benefit plant breeding and agro-biotech companies in developing novel and more effective strategies to sustainable enhance crop production. Towards this objective, the proposal will reveal the genes modulating the composition of the rhizosphere microbiota and how this trait is related to crop yield. This information can be used by plant breeders to develop varieties better suited for the soil environment and, ultimately, for low-input agriculture scenarios. Of note, the choice of an established genetic material and a model cereal such us Barley makes me confident that outputs of this proposal can be exploited also for other crops, including the global staple wheat.