Genetic Determinants of Microbiome Assembly on Plant Roots

Lead Research Organisation: University of Oxford
Department Name: Biology

Abstract

Plant roots are critical for the uptake of mineral nutrients by plants. In addition, they interact with the soil environment and a complex assemblage of bacteria, fungi, single celled animal cells, nematodes and other organisms. The area directly around roots that is occupied by these organisms is known as the rhizosphere and the collective name for the organisms is the rhizosphere microbiota. Microorganisms also reside inside plant roots, usually between plant cells and are knows as endophytes. Together the rhizosphere and endosphere microbiotas makes up the root microbiota of a plant. It has been shown over the last few years that the root microbiota is critical for the health and growth of plants, with many microorganisms shown to be plant growth promoting. Bacteria are simple single celled microorganisms that lack the membrane bound structures found in higher cells of plants and animals. However, while bacteria may have a less complex cellular organisation they carry out a huge range of chemical reactions not found in plants and animals. Bacteria are responsible for the cycling of many nutrients such as N2 (N2 is also known as nitrogen gas and consists of two nitrogen atoms bound by a strong triple bond), which is a very inert atmospheric gas. N2 makes up 78% of the atmosphere but is very unreactive and cannot be used directly as a source of nitrogen, which is needed for amino acid, protein and DNA synthesis. However, a small number of bacteria can reduce (add hydrogen) to N2 and convert it into ammonia (NH3), which is readily incorporated into amino acids and then all the other building blocks of life, by a wide range of organisms including bacteria and plants. Other bacteria are crucial to make phosphate available to plants which with nitrogen are the two main nutrients limiting plant growth. However, we now realise that bacteria do not work alone but rather they work as complex communities, also known as a microbiome, with hundreds or even thousands of members interacting with each other and host plants. Just as for the human gut and body the health and growth of plants is profoundly altered by the bacterial microbiome. A recent breakthrough is the demonstration that we can study how microbiomes develop using just 6 or 7 key members which simplifies the analysis. In this proposal we have developed ways to track how 7 bacteria interact and to study the underlying genetic causes. This enables us to move beyond simple characterisation of the components of the microbiota to examine the genetic mechanisms of control and how a microbiome stably colonises plant roots. This research will lead to a step change in characterisation of plant microbiota of agriculturally critical crops, including cereals such as barley and wheat.

Technical Summary

Bacterial communities are crucial to plant growth as shown by numerous phylogenetic analyses of plant roots revealing bacterial communities are strongly shaped by the plant and in turn bacteria act at the community level to promote growth, and for example, can establish suppressive communities that inhibit pathogens. What is missing are molecular studies on how bacterial microbiomes assemble on roots and interact with each other and the plant, leading to mechanistic understanding of microbiome assembly. The problem is the scale of complexity and diversity of bacteria that colonise roots. However, this has been transformed by recent advances showing that the underlying principles of microbiome assembly can be determined using bacterial Synthetic Communities (SynComs) of 6-7 members. However, for molecular analysis of even 7 membered SynComs there needed to be a new raft of imaging and analysis techniques to facilitate their study. We therefore developed differential fluorescence measurement (DFM), of up to 7 different bacteria, adapted the global bacterial mutagenesis strategy, Barseq, and established methods for meta-transcriptional analysis of bacterial communities enabling elucidation of the genetic determinants of microbiome assembly during root colonisation This transforms our ability to study communities of bacteria with profound implications for understanding how bacteria promote or inhibit plant growth and colonise roots. It can lead to understanding of how to rationally develop inocula for plants that will be stable in the environment. Given the recent enormous international investment in bacterial inoculants for plant growth makes understanding the basic biology of microbiome assembly particularly timely. However, the techniques will be applicable to all areas of animal, plant and human microbiome analysis so that its importance to both fundamental and applied biology is difficult to overstate

Planned Impact

Within this proposal, we will extend established work within our groups to develop techniques to identify the genetic determinants of root colonisation and stability of the microbiome. This is particularly important to plant growth, nitrogen and phosphate utilisation, but also has relevance to disease resistance and herbicide and pesticide use. In the bigger context the importance of the microbiome in plant growth and for agrochemical exploitation has recently been recognised. Large scale international funding has gone into this area with companies such as Bayer and Ginko forming Joyn Bio and start-ups including AgBiome and PIVOT focussed on microbial inocula and consortia for plants in agriculture. This is particularly important to nitrogen and phosphate utilisation and also to disease resistance and herbicide and pesticide use in plants. Nitrogen is at double its preindustrial level and now beyond the safe operating boundary of the earth, with widespread pollution of groundwater and ocean coastal zones by nitrates and phosphates leading to eutrophication and costal dead zones. These nutrients are leading players in the perfect storm, demanding increased agricultural production but requiring changes in agricultural practice to avoid environmental carnage. Understanding how synthetic community of bacteria (SynComs) assemble and are stabilised is a powerful resource for the use of microbes to help tackle these issues and therefore has over-arching relevance to society and government policy. Furthermore, in a regulatory environment where less fertilizer and pesticide use are becoming mandatory, this work will offer tangible results to help meet these targets and assist the competitiveness of UK industry. Currently we have strong links with UK Legume technology, including two iCase Ph.D students, working on legume and other inoculants. We will maximize the potential impact of our research by directly engaging with a range of stakeholders, including crop breeders, policymakers and farmers via our existing knowledge transfer networks, including the UK Wheat Genetic Improvement Network (WGIN), landowner/farmers groups (e.g. NFU, SNFU, NFUW, HCC, Growers association, Soil Association), academic societies (BES, BSSS), conservation bodies and local/national government departments (Defra; EA), agencies (SEPA; Natural England; Natural Resources Wales, SNH), who will directly benefit from our findings. Reducing inputs into agriculture while maintaining yields has direct benefits to British farming but also to maintenance of the countryside and its use and recreation by the British public. It will help the UK meet local and European environmental targets and help the long-term sustainability and stability of our environment. We will also be training the next generation of scientists to develop practical solutions to environmental problems and develop stable microbial inoculants.

Publications

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Bozhilova LV (2021) COGENT: evaluating the consistency of gene co-expression networks. in Bioinformatics (Oxford, England)

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Haskett TL (2022) Engineered plant control of associative nitrogen fixation. in Proceedings of the National Academy of Sciences of the United States of America

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Knights HE (2021) Deciphering bacterial mechanisms of root colonization. in Environmental microbiology reports

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Mendoza-Suárez MA (2020) Optimizing Rhizobium-legume symbioses by simultaneous measurement of rhizobial competitiveness and N2 fixation in nodules. in Proceedings of the National Academy of Sciences of the United States of America

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Pardo-Diaz J (2021) Robust gene coexpression networks using signed distance correlation. in Bioinformatics (Oxford, England)

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Poole PS (2022) Maintaining osmotic balance in legume nodules. in Journal of experimental botany

 
Description A grand challenge in microbiology is to go beyond the study of single organisms to understand how bacterial microbiomes assemble. In the case of plant roots, its microbiome is formed by a vast diversity of microorganisms, where plants shape the root microbiome and microorganisms alter plant growth. So far, plant studies have analysed the microbiome composition under different conditions by DNA sequencing. However, the cutting-edge challenge is to move from counting and classifying to understanding the underlying genetic determinants of microbiome assembly. Nevertheless, due to the huge number of different microorganisms this has proved technically and logistically extremely difficult. A key strategy is to define a simpler synthetic community (SynCom) to overcome these issues.

We defined a model SynCom (OxCom6) formed by six different members of the alpha- beta- and gamma-proteobacteria which are well-known root colonisers, whose genomes are sequenced and can be genetically modified. We applied the differential fluorescent marker tool (DFM) to track OxCom6 on plant roots.

Our main discovery is that OxCom6 differentially assembles on pea and barley roots, where Pseudomonas and Enterobacter are the main colonisers (more 70%), respectively. OxCom6 is a SynCom which can be drastically disturbed by the presence of a different plant, and therefore can be used as a model SynCom to study the disturbance of biotic and abiotic stress in microbiomes (assembly and stability).
The composition analysis of OxCom6 has allowed us to examine bacteria-bacteria interaction on pea roots. One member of OxCom6 is Rhizobium leguminosarum 3841, a strain that establish symbiosis with peas and giving nitrogen to the plant in exchange for a niche (nodule) and carbon. As aforementioned, Pseudomonas is the main coloniser on pea roots, however, in the absence of Rhizobium it is overtaken by Enterobacter. The pairwise colonisation of Pseudomonas-Enterobacter confirmed this result. Moreover, the pairwise colonisation of Pseudomonas-Rhizobium showed an increase in Pseudomonas compared to when it is colonising by itself. This can be explained by the fact that the presence of Rhizobium triggers induction of symbiotic signals in planta, likely to change the carbon profile of the rhizosphere, and therefore increase the competitiveness of Pseudomonas.
Exploitation Route In order to apply the DFM tool to OxCom6 we develop a new family of modular plasmids that allow us to change antibiotic resistance marker, promoter and fluorescent proteins easily to adapt to different bacteria. This modular plasmid family is used regularly in our laboratory and already appears in multiple publications.

In addition, OxCom6 is been usied in our laboratory as a model SynCom to study its assembly on different plants, in free-living growth and for metabolic modelling.
Sectors Agriculture, Food and Drink,Environment,Manufacturing, including Industrial Biotechology

URL https://www.nature.com/webcasts/event/probing-root-associated-microbiome-assembly-with-fluorescently-labelled-synthetic-communities/
 
Description Continuation of ENSA (RL)
Amount $226,932 (USD)
Organisation Foreign Commonwealth and Development Office (FCDO) 
Sector Public
Country United Kingdom
Start 01/2021 
End 03/2024
 
Description Factors controlling N2-fixing ability and competitiveness of rhizobia to nodulate legumes
Amount £784,606 (GBP)
Funding ID BB/W006219/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 02/2022 
End 01/2025
 
Description Marie Curie (IA)
Amount € 224,933 (EUR)
Organisation Marie Curie 
Sector Charity/Non Profit
Country United Kingdom
Start 05/2021 
End 06/2023
 
Description 5th Annual Missouri University Plant Research Symposium 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Phil gave a talk entitled 'Improving legume symbiosis or engineering cereals' in this virtual symposium: The Next Green Revolution: Challenges and Strategies towards a Sustainable Agriculture.
Year(s) Of Engagement Activity 2021
URL https://www.corteva.com/our-impact/innovation/symposiaseries.html
 
Description Microbiome Capability Workshop (March 2020) - AT and PSP 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Phil gave a talk to other interested parties at this BBSRC workshop
Year(s) Of Engagement Activity 2020
URL https://rhizosphere.org/lab-news/
 
Description Nature webcast on fluorescently labelled bacteria (BJ) 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Bea gave a presentation via a webcast entitled 'Probing root associated microbiome assembly with fluorescently labelled synthetic communities'.
Year(s) Of Engagement Activity 2020
URL https://www.nature.com/webcasts/event/probing-root-associated-microbiome-assembly-with-fluorescently...
 
Description Organise and run OxBacNet meeting - Alison East 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact I plan and design the programme, inviting all the speakers and organising the whole meeting (from tea and coffee, projectors, display of posters and pizza delivery for the networking sessions). This is an extremely successful initiative reaching the bacteriologists working in the many different locations around Oxford.
Year(s) Of Engagement Activity 2020,2021
URL https://rhizosphere.org/oxbacnet/
 
Description OxBacNet presentation May 2021 (CCMS) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact Presentation (via Zoom) to members of OxBavNet ( a networking group for Bacteriologists based in Oxford).
Year(s) Of Engagement Activity 2021
URL https://rhizosphere.org/lab-news/
 
Description Plant Genomes, Systems Biology and Engineering, Cold Spring Harbor, 1st-3rd Dec 2021 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact On-line conference. Plant Genomes, Systems Biology and Engineering.
Year(s) Of Engagement Activity 2021
URL https://meetings.cshl.edu/meetings.aspx?meet=PLANTS&year=21
 
Description Plant-Microbe Interactions Symposium, Society for Applied Microbiology, Oct 2020 (PSP) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact Phil gave a talk entitled 'Rhizobium from rhizosphere to root nodule' at this online conference. It reached a wide audience who asked a series of questions after the talk.
Year(s) Of Engagement Activity 2020
URL https://sfam.org.uk/career/ems-event-calendar/plant-microbe-interactions-series-part-1.html