Unravelling the barley genetic control of the rhizosphere microbiota

Lead Research Organisation: University of Dundee
Department Name: School of Life Sciences

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

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Escudero-Martinez C (2019) Tracing the evolutionary routes of plant-microbiota interactions. in Current opinion in microbiology

 
Title Plant Microbiome to the Rescue 
Description Animating Science uses stop motion animation to create animated films about Life Science concepts and processes. This film was produced by members of the public in collaboration with my group and animator Andrew Low as part of our annual Plant Power Day 2019 at the University of Dundee Botanic Garden. This project was funded by the award I am reporting of. Plant Microbiome to the Rescue is a short animation examining the interaction between soil microbes and plants in our very own plant microbiome. It shows the "good", the "bad" and the "ugly" of the soil microbe world and how they can contribute or interfere to the health of many plants. 
Type Of Art Film/Video/Animation 
Year Produced 2019 
Impact Feedback from the public involved in the development of the animation indicated that they clearly enjoyed this activity. Researchers fed back the questions and statements from the public asked while interacting with them. A significant example from my activity is represented by people realising that not all microbes are detrimental for plants and other organisms. 
URL https://www.youtube.com/watch?time_continue=3&v=JdbDRyfiad4&feature=emb_title
 
Description One of the objectives of this award is to identify plant genes and alleles shaping the microbial communities thriving at the barley root-soil interface, collectively referred to as the rhizosphere microbiome. We previously demonstrated that wild and modern barley varieties host contrasting microbiomes and we were able to identify a region of the barley genome responsible for the observed microbial phenotype. This region, hereafter called locus 3H, has a different genetic composition in wild and modern barley. We hypothesized that by "swapping" the genetic composition at locus 3H between a wild and a modern plant would be sufficient to shape, at least in part, the rhizosphere microbiome. By combining a classical genetic approach (i.e., crosses between different plants) with genomic approach (molecular markers providing us with a precise understanding of the genetic diversity of our plants) during the first year of the award we were able to produce what geneticists call "introgression lines". These lines have a "modern" barley genome with small pieces, in our case mostly located on locus 3H, in a "wild" form. Strikingly, when we generated a census of the microbiome inhabiting the rhizosphere of this lines with an approach called 16S rRNA gene sequencing we discovered that the wild introgression was sufficient to trigger a differential selection on the microbiome. This is a confirmation that genes and alleles located on locus 3H are indeed implicated in microbiome recruitment. The development of these introgression lines is a fundamental pre-requisite to complete objective 1 and embark on objectives 2 and 3 of the award I am reporting of.
Exploitation Route The award is still active, however I can anticipate that the introgression lines we developed will be a tool that can be used by a broader scientific community interested in investigating other aspects of plant-soil interactions (e.g., resistance to abiotic stress like drought, resistance to pathogens present in soil...).
Sectors Agriculture, Food and Drink

 
Description Plant Power 2019 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Plant Power is an annual event that takes place at the University of Dundee Botanic Garden where various different groups and organisations participate with plant related activities/shows. A science strand is delivered by researchers from the Division of Plant Sciences at the University of Dundee and the James Hutton Institute. They presented different interactive hands-on activities related to their respective groups research to the visitors. These activities are either brand new or have been developed over a number of years at various events. The aim is to allow the public to learn about the research taking place locally and why this research is important. Various modes were used to communicate the research as shown by the diversity of activities e.g. use of games (pin the plant & botany trail); craft activities (chromosome modelling & lino printing); science experiments (raspberry DNA extraction); art (animating science). Approximately 970 people came to the Botanic Garden for the event. They are generally family groups with young children (below 10 years of age). We estimate that around 200-250 people visited our activities. In addition to the contribution to all activities of the event, my research was represented in this program of work by a short animated film we produced with the help of members of the public in collaboration with the my group members and animator Andrew Low. In this short animation we illustrated the interactions between soil microbes and plants and how the plant microbiome can contribute to sustainable crop production (described in a separate section).
Year(s) Of Engagement Activity 2019
URL https://www.dundee.ac.uk/events/2019/19-05-25-plant-power-day-2019.php