Unravelling the barley genetic control of the rhizosphere microbiota

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


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.
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.

Despite disruptions caused by the SARS-CoV-2 pandemic, specifically the interruption of several months of laboratory activities, we were able to gather novel insights into the genetic mechanisms shaping the rhizosphere microbiome mediated by the locus 3H. First, by interrogating a collection of multiple barley genomes, which scientists call pangenome, we discovered that approximately 60 genes are present in the region of locus 3H. Similar to the information contained in a dictionary, the pangenome informed us also on the processes these genes are implicated in which include, among others, plant immune responses. As any of those 60 genes (or indeed a combination of them) can be implicated in microbial recruitment in the rhizosphere, we performed what scientists call comparative RNA-seq analysis. In this analysis, we compared the profiles of the messenger RNAs produced by the roots of the introgression lines once exposed to microbes in soil. The messenger RNAs translate DNA information and are needed for the production of e.g., proteins and enzymes. We reasoned that a gene in locus 3H implicated in shaping soil microbes will be likely translated by roots as this will enable plants to produce proteins and enzymes close to where soil microbes proliferate. A prediction of this consideration is that if some of those genes display differences at the messenger RNA level between introgression lines hosting contrasting microbiotas, those genes will be ideal candidates for shaping the rhizosphere microbiome. We finalised this analysis and identify three candidate genes to prioritise for further investigations, including the aforementioned plant immune receptor. We complemented this investigation using a two-pronged approach. First, using a dedicated piece of equipment akin to a scanner, we reconstructed the so called root architecture of the introgression lines, i.e. the three dimensional way roots explore the soil around them. In parallel, we characterised what scientists define rhizodeposition, i.e., the array of organic compounds secreted by plants in the vicinity of their roots, which is considered a driver for the microbial communities inhabiting the rhizosphere. The experience we gathered in this experimentation placed us in the ideal position to collaborate with an oversea research group to investigate the effect of a specific organic compound secreted by barley in the rhizosphere. Findings of this parallel line of investigation, aligned with objective 2 of the award I am reporting on, have now been published in a peer-reviewed journal. Taken together, this multidisciplinary experimentation allowed us to achieve objective 2 of the award I am reporting on.
In the last year of the award we also analysed the grain yield of the introgression lines, to establish a causal relationships between the locus shaping the microbiota and crop performance which represents Objective 3 of the award I am reporting on. We measured yield parameters, such gran weight, of the introgression lines and their domesticated donor (i.e., the cultivar Barke) over the course of four independent experiments. These latter were performed in our reference soil, which is naturally limited in nitrogen for plant uptake, and without the addition of fertilisers. This revealed that the yield parameters measured do not differ between the introgression lines and and that these latter are outperformed by the donor parent. At first glance, these observation predicts that the locus 3H is decoupled from yield. However, we made a striking discovery: the locus of interest is genetically linked to another piece of the barley genome designated Btr, for brittle rachis, and responsible in wild genotypes (and in our introgression lines) for the so called seed shattering, i.e., the dispersal of seeds at maturity. This confounds the interpretation of the results and suggests that, to infer casual relationships between microbiota and yield, it will be necessary to resolve this genetic association, by "separating" the two loci. This experimentation is beyond the scope of the award I am reporting on and will feature centre-stage in follow-up grant applications. Regardless of that, it is fair to consider objective 3 of the proposal achieved.

These data were collate in an original manuscript submitted to a premier, peer-reviewed, scientific journal. We received a positive feedback from the reviewers and we are using the last months of the award to address their comments. For further information please refer to the link at the pre-print version of the manuscript mentioned below.
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...).

The sequencing information of locus 3H can now be used to develop molecular makers for plant breeders to follow the distribution of putative genes implicated in shaping the rhizosphere microbiome.
Sectors Agriculture, Food and Drink

URL https://www.biorxiv.org/content/10.1101/2021.12.20.472907v1
Description UKRI-BBSRC joint BIUH and SAF Strategy Advisory Panel meeting
Geographic Reach National 
Policy Influence Type Participation in a guidance/advisory committee
Description HORIZON-CL6-2021-BIODIV-01
Amount € 7,999,997 (EUR)
Funding ID 101060124 
Organisation European Commission 
Sector Public
Country European Union (EU)
Start 08/2022 
End 08/2027
Description UKRI-BBSRC East of Scotland Doctoral Training Partnership 3
Amount £17,009,440 (GBP)
Funding ID BB/T00875X/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 08/2020 
End 09/2028
Description Dundee Women's Festival 2020 - Family Fun with Women in STEM 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact The Family Fun with Women in STEM was a free drop in event held at Charleston Community Centre - Sunday 1st March 2020 11am-2pm. It was aimed at families to allow the visitors to meet women scientists to find out about the amazing research work in STEM taking place at the University of Dundee. This was predominately hands on activities for all ages, what they get up to everyday in their job in STEM and what discoveries they have made. My research was represented in this program of work by Dr Carmen Escudero-Martinez, currently employed by the award I am reporting of, and aimed at increasing the awareness of the general public of how plant sciences impact on daily life.
There were approximately 98 visitors who were generally family groups with young children (below 10 years of age). The event had the following learning outcomes; share the research and achievements of local women in STEM and show the diversity of scientists - buck the stereotype of what a scientist looks like.
Feedback from scientists indicating enjoyment and engagement with the event:
"They were interested to learn more, general comments were very positive on what a lovely atmosphere there was during the event."
"I didn't think knowing what medicine you got made a difference to you thinking it would work or not. But I can see me seeing a marshmallow is pink makes me think it will be tastier, when it actually didn't taste different when I didn't know what colour it was."
"questions about the brain"
Year(s) Of Engagement Activity 2020
URL https://discovery.dundee.ac.uk/en/activities/family-fun-with-women-in-stem-dundee-womens-festival-20...
Description Dundee Women's Festival 2021 
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 For Dundee Women's Festival 2021, a week of content focused on Women in STEM from the University of Dundee was shared on Dundee Science Centre's Home Learning Programme. Women from many different areas of STEM from biology to engineering and forensics to medicine were featured. The content was divided into topic themes that contained information and fun activities related to the research of the women featured. The other part featured career profiles.
The content is accessible here: https://www.dundeesciencecentre.org.uk/women-in-stem
The content was created for schools and young people. The activities generally had a focus of primary age children while the careers profiles were generally aimed at secondary age children. The content could be enjoyed by any age group and activities could be completed as a family.
The research award I am reporting for was represented by Dr Escudero-Martinez (herself employed in the award) who co-developed an activity called 'Soil Safari' to increase the awareness of the general public on the significance of soil microbes and molecular plant-microbe interactions.
Members of Dundee Science Centre created the webpages to feature the content and to promote the content on social media.
Learning outcomes
People who engage with the content will learn about:
• the diversity of research areas that women in STEM work in
• different types of jobs in STEM and the routes to them
• the types of research undertaken by women in STEM at Dundee through engagement with simple activities, text narrative and videos
Those that align with Dundee Women's Festival Values (to meet criterion to be included in Festival programme):
• Women centric
• Promoting women achievers
Analytics: For the specific "Women In STEM" page, about 870 page views in total, of which 669 are unique page views (up to the current 1 April 2021).
Dundee's Wonder Women of Science Activity Pack: 350 downloads as of 16/03/2021.
Majority of people accessing the material was from the UK.
Legacy: Some content has already been reused for other events. For example, the Soil Safari activity was used as part of the Plant Power 2021 online content to accompany a self-guided trail in the University of Dundee Botanic Garden. Follow on plans are for the activities developed by SLS researchers to be transformed into simple activity sheets (for a new public facing website).
Year(s) Of Engagement Activity 2021
URL https://www.dundeesciencecentre.org.uk/women-in-stem
Description How can understanding plants and microorganisms help feed the world? 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Schools
Results and Impact This a teaching resource developed by Futurum Careers explaining the work in my laboratory, with emphasis for the activities of the grant I am reporting of. This teaching resource is an additional public engagement output of the grant I am reporting for, conceived to replace a cancelled face-to-face initiative scheduled for May 2020 and aimed at secondary school pupils. The article is suitable for 14-19-year olds (secondary and high schools, and college) and alongside the accompanying activity sheet, can be used in the classroom, STEM/Biology clubs and at home. This resource links to KS4/Grades 9 & 10 and KS5/Grades 11 & 12 Biology. It can also be used as a careers resource and links to Gatsby Benchmarks (UK):
Gatsby Benchmark 2: Learning from career and labour market information
Gatsby Benchmark 4: Linking curriculum learning to careers.

The teaching resource is freely available on dedicated repositories for teaching material. The number of people reached by the activity depicts the cumulative number of downloads from said repositories of the past three months.

On-line feedback posted on the website of Futurum include:
"Five-star rating from Kathy Louis on Teachers Pay Teachers, 6 September, 2020: "I like your presentation as these can easily be shared with students for their readings. Thank you!"
Year(s) Of Engagement Activity 2020
URL https://futurumcareers.com/how-can-understanding-plants-and-microorganisms-help-feed-the-world
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
Description Plant Power 2021 
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 showcases the fascinating plants at the University of Dundee Botanic Garden and highlights exciting plant science work being done in Dundee. This year, staff at the Garden worked with the University's School of Life Sciences and the James Hutton Institute to create an exciting new trail that celebrates the importance of plants in our everyday life. The self-guiding trail took visitors on an adventure through nature and offers an opportunity to learn more about the plants on display. Information boards along the trail will lead visitors to a dedicated website (https://ics.hutton.ac.uk/pp21/#/) with online interactive activities.
The event was advertised with free entry tickets (bookable via Eventbrite) from Saturday 15 May - Sunday 23 May (included Fascination in Plants Day on 18 May). Goodie bags were handed out to the first 200 children in attendance. The trail will remain in place for further visitors to enjoy.
The research of the award I am reporting of was represented in this programme of work by Dr Escudero-Martinez who developed an activity to increase the awareness of the general public towards the molecular basis of plant-microbe interactions in soil.
Approximately 1069 people came to the Botanic Garden from Saturday 15 May - Sunday 23 May. They are generally family groups with young children (below 10 years of age). From the registrations through Eventbrite, we had responses from 124 households (327 visitors). From those, 66 provided full postcodes that could be analysed by SMID 2020 from the Scottish Government. Approx a third of people came from the lower 60% of most deprived zones in Scotland while approx 2/3 came from the 40% least deprived zones. We had new visitors to our event (new to the garden, Plant Power and UoD events in general). Also, half of the visitors had no relationship to the University.

Legacy: Some activities were repurposed for a Plant Week kit for the Dundee Science Centre Science @ Home kits in Summer 2021. Follow on plans are for the activities developed for Plant Power to be transformed into simple activity sheets (for a new public facing website) and borrow boxes. They will be aligned with the Curriculum for Excellence and investigating formally sharing activities via publications would be a subsequent step. The current website content will also be transferred to the new website to allow analytics to be captured.
The work undertaken fits with the public engagement strategy that has four main aims:
1. Build on our creative partnerships to deliver a high quality, innovative engagement programme. This will inspire participation in and understanding of our research, making science relevant for everyone.
2. Engage a diverse range of people with our research.
3. Consult with our local communities to widen our reach and meet their needs.
4. Promote and support a culture of active participation in public engagement within our life sciences community.
Year(s) Of Engagement Activity 2021
URL https://ics.hutton.ac.uk/pp21/#/
Description Work Experience Week 2020 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact The Work Experience Week ran from July 27-31, 2019 for 36 S6-7 pupils from a variety of Scottish secondary schools. In response to the coronavirus pandemic sessions were moved to an online Blackboard Collaborate space, with time allocated for Q&A sessions with researchers, group work and independent study. Pupils engaged with careers stories from students and researchers, scientific experiments and data analysis activities, and workshops on animals in research and science in the media. I took part in the careers speed dating videos, sharing my personal journey through study and work, including general information of the award I am reporting for.
Feedback was overwhelmingly positive, including one student who said: "I personally found this week really insightful and informative. It was well put together and every day was packed with information about the different course's content. It has overall made me really look forward to hopefully one day becoming a student at university and has inspired me to work hard in my final year at school."
Year(s) Of Engagement Activity 2020
URL https://discovery.dundee.ac.uk/en/activities/life-sciences-virtual-work-experience-week