13 ERA-CAPS: Biosynthesis, transport and exudation of 1,4-benzoxazin-3-ones as determinants of plant biotic interactions

Lead Research Organisation: University of Sheffield
Department Name: Animal and Plant Sciences


The establishment of a suitable biotic niche is essential for plant survival and agricultural productivity. One important mechanism by which plants shape their environment is the release of phytochemicals. Low molecular weight compounds in particular can initiate the interaction with beneficial microbes in the soil and ward off herbivores. However, the same signalling molecules may also be exploited by specialized pests and pathogens. A detailed understanding of their biosynthesis, transport and release will be essential to disentangle these seemingly contradicting effects and to harness the full potential of secondary metabolite exudation in sustainable cropping systems. Yet to date, no secondary metabolite exporters have been identified in crop model systems.
Here we propose to unite the expertise of different research groups across Europe to study the molecular basis of 1,4-benzoxazin-3-one (BX) exudation in maize. Previous work by the consortium members has identified BXs as important resistance factors in maize and other grasses and has elucidated their biosynthesis in detail. We have also shown that BXs are the dominant secondary metabolites in maize root exudates and the leaf-apoplast and that they are important recruitment signals for beneficial microbes as well as for one of the most damaging pests of maize, the western corn rootworm Diabrotica virgifera. Given their obvious importance for crop productivity and their strong involvement in extracellular signalling, BXs are an ideal and relevant model to study the molecular ecology of secondary metabolite exudation.

The overall aim of BENZEX is to create the most advanced molecular toolkit in extracellular plant-environment interactions to date. First, a comprehensive population of maize genotypes that are altered in BX biosynthesis will be established by interrupting or enhancing the expression of three important biosynthetic enzymes. Second targeted and untargeted reference methods to identify and quantify BX production, storage, exudation and transformation will be established using HPLC-MS and 13C-labelled BX precursors. Third, BX transporters will be identified by a combination of proteomics, quantitative PCR and phytochemically guided QTL-mapping. Fourth, the generated resources will be used to analyse the role of extracellular BXs in the interaction with the soil microbiome, plant growth promoting bacteria, arbuscular mycorrhizal fungi, root herbivores and leaf-feeding aphids. Taken together, our project will substantially increase knowledge about the biological relevance of secondary metabolite exudation. It will furthermore enhance the competitiveness of European molecular plant sciences by providing an array of new tools and resources, including transgenic plants and mutants, analytical methods as well as molecular and microbial markers for maize as an important agricultural model system.

Technical Summary

This technical summary concerns work package 4 of the consortium project, which addresses the role of benzoxazinoids in biotic interactions at the root-soil interface:

I. Targeted approach: we will investigate how BXs influence the plant's interaction with 1) the plant growth promoting rhizobacterial (PGPR) strain Pseudomonas putida KT2440, which is a competitive colonizer of the maize rhizosphere, 2) the arbuscular mycorrhizal fungi (AMF) of the family Glomeraceae, and 3) the specialist root herbivore and important invasive pest Diabrotica virgifera. Both D. virgifera and P. putida KT2440 are attracted to BX-producing roots, while G. intraradices has been shown to boost BX production. Different sets of experiments will be conducted to study the role of BXs in interactions with the above rhizoshere organisms. involving chemical profiling of benzoxazinoids and corresponding breakdown products in the (mycor)rhizosphere, transcirptional profiling of BX biosynhtesis genes, and tripartite root colonization assays using transgenic maize lines affected in different steps of the BX biosynhtesis pathway.

II. Global approach: we will determine the global impact of BXs on the microbial composition of the (mycor)rhizosphere. Using a deep-sequencing approach of operational taxonomic units (OTUs) from 16S rRNA sequences, we will compare dominant microbial OTUs present in the rhizosphere of mycorrhizal and non-mycorrhizal roots of BX-producing maize and BX-deficient maize lines.

Planned Impact

The project will achieve impact via the following pathways:

1. Peer reviewed publication and presentation: Given the nature of the proposed research, the most efficient way of communicating the findings of BENZEX will be through i) the presentation at scientific conferences (including for instance the Gordon Research Conference on Plant-Herbivore Interactions 2016; the 57th Maize Genetics Conference 2015; and the XVI International Congress of Molecular Plant-Microbe Interactions 2014) and ii) the publication in major peer-reviewed journals. Preference will be given to journals that support an open access option (e.g. Plant Physiology, Plant Cell, PLoS Biology). All group members who have made significant research contributions will be included as authors, with the order of authorship according to the relative contributions. Publications will include detailed methods sections that allow other researchers to reproduce the experiments.

2. Deposition of materials in repositories, databases and germplasm banks: The resources generated by BENZEX will be made freely available to the scientific community, except in the case of patented material (see below), which will require a Material Transfer Agreement. The following existing data repositories will be employed for this purpose:
- Gene expression data: SRA (Short Read Archive; http://www.ncbi.nlm.nih.gov/sra) and GEO (Gene Expression Omnibus; http://www.ncbi.nlm.nih.gov/geo/)
- 16sRNA sequences: European Nucleotide Archive (ENA) (http://www.ebi.ac.uk/ena/about/submit_and_update)
- Metabolite and metabolomics data: METLIN (http://metlin.scripps.edu/) and Fiehn Lib (http://fiehnlab.ucdavis.edu/projects/FiehnLib/index_html )
- Seed stocks: Maize genetics cooperation stock centre (http://maizecoop.cropsci.uiuc.edu/). T1-seeds of transgenic plants will be made available directly from MPI-CE upon request.
- Cloned DNA in bacterial vectors: Plasmid DNA or stocks of Escherichia coli and Agrobacterium tumefaciens carrying the cloned DNA will be made available to other researchers by the consortium partners.

3. Communication through public media: To reach a wider audience, the findings of BENZEX will be communicated using the following media:
- Concerted press releases on outstanding papers and publications of BENZEX by the MPI-CE's press information officer (PIO) Angela Overmeyer M.A., and distributed to more than 18.000 journalists and subscribers e.g. via "Informationsdienst Wissenschaft (idw)" for Germany, Switzerland and Austria and EurekAlert, the idw's equivalent in the USA.
- Launching of all relevant information and the outcome of BENZEX projects via the Max Planck Societies "Sci. & Corp. Com." and "Research Marketing, Events& Exhibition" Depts.
- Articles in the bi-annual open access PULS/CE ("Public Understanding of Life Science/Chemical Ecology") newsletter of the MPI-CE (Edited by Angela Overmeyer and Jan Kellman)
- Public lectures, including the "German Talks" and the "Ernst-Stahl Seminars" at the MPI-CE
Description The project has made significant scientific impact. The key findings of the project have been:

- The project led to the development of a comprehensive mass spectrometry-based method for metabolic profiling of rhizosphere soil. This method was of direct relevance to the project, which allowed us to study direct and indirect impacts of mutations in the maize benzoxazinoid pathway on rhizosphere chemistry and associated microbiota. A manuscript describing this method has been published in The Plant Journal (Petriacq et al. 2017; 92(1):147-162).

- The main objective of the project, i.e. to determine the global impacts of maize benzoxazinoid (BX) metabolism on rhizosphere chemistry and microbiota, has revealed that mutations in the early steps in the BX pathway (bx1 and bx2) have detectable impacts on bacterial communities in the rhizosphere (as determined by next-gen 16S rRNA gene sequencing). By contrast, a downstream mutation in the BX6 gene, which leads to accumulation of DIBOA in maize roots, did not have a significant effect on rhizosphere communities. This suggests that DIBOA and DIMBOA (or breakdown products thereof) have similar activities in the rhizosphere. We also analyzed results of fungal ITS sequences, which revealed that the bx mutations have a relatively subtle impact on fungal communities compared to that on bacterial communities.

- In order to confirm that BX are responsible for the observed impacts on microbial communities, we profiled the chemical composition of roots of wild-type, biz1, bx2 and bx6 mutants. A surprising result from this un-targeted analysis was that the bx1 and bx2 mutations do not only prevent production of rhizosphere-active BX compounds in the rhizosphere, but also affect accumulation of other plant-derived chemicals in the rhizosphere. This suggests that the bx1 and bx2 mutations might have wider regulatory impact on root exudation chemistry. This is in agreement with earlier findings from my lab, which revealed that BXs can have a within-plant signalling role. Subsequent correlation analysis between BX-controlled root metabolites and bacterial taxa revealed a dominant role for BX-dependent controlled metabolites, particularly flavonoids, in constraining a range of soil microbial taxa, while specifically stimulating methylophilic bacteria. Together, our study supports a multilateral model by which BXs control root-microbe interactions via a global regulatory function in root secondary metabolism. This study was recently published in The ISME Journal (Cotton et al. 2019; 13: 1647-1658).

- As a general outcome of this study, two PIs of this grant (Jurriaan Ton and Stephen Rolfe) wrote a review about the importance of root exudation chemistry in the establishment of a plant health-promoting plant microbiome (Rolfe et al 2019; Current Opinion in Microbiology, 49: 73-82), which provides further context to the results of the project and also outlines future directions of research.

- Based on the scientific impact and outcome of this project, we have started a collaboration with a biocontrol company (Koppert Biocontrol Systems), which sells agricultural formations of beneficial microbes that promote crop growth and disease protection. So far, the company has agreed to give us in kind support for a follow-on project, for which we have submitted a grant proposal to BBSRC/NSF (BB/T015306/1, Cereal Soil Legacies: Genetic, metabolic and microbial mechanisms generating disease suppressive soil microbiomes). This grant is currently under review.
Exploitation Route Progress of our project was shared with other members of the ERA-CAPS consortium that this grant is part of.

Furthermore, we have used the scientific impact and outcome of this project to apply for a follow-up grant from BBSRC/NSF to support a joined collaborative project with the laboratory of Georg Jander at BTI, Cornell: BB/T015306/1, Cereal Soil Legacies: Genetic, metabolic and microbial mechanisms generating disease suppressive soil microbiomes. This grant application also involves support by an industrial partner (Koppert Biocontrol Systems) but was unfortunately not successful. We are considering alternative sources of funding for follow-up research.
Sectors Agriculture, Food and Drink,Chemicals,Environment

URL https://www.nature.com/articles/s41396-019-0375-2
Description The first outcome of this study has been the delivery of a novel method to comprehensively profile chemistry of non-sterile rhizosphere soil (described in the Petriacq et al. Plant Journal paper). This outcome has been identified by potentially future commercial partners to assess impacts of cover crops on soil health. The second outcome of based on our most recent publication from this grant (Cotton et al. 2019; ISME J - 13: 1647-1658), which revealed that the effects of benxozazinoid (BX) synthesis genes if maize have regulate the composition of the root metabolome, including the production of rhizosphere-active semiochemcials, such as flavonoids. We have shown that these BX-dependent metabolites correlate closely with certain members of the maize root-associated microbiome, including potentially beneficial microbes that may in the future be exploited as biocontrol organisms. This outcome also provides further incentive for crop breeding companies to select for cereal varieties with high benzoxazinoid root content.
Sector Agriculture, Food and Drink,Chemicals,Education,Environment
Impact Types Societal,Economic,Policy & public services

Description ERA-CAPS: Biosynthesis, transport and exudation of 1,4-benzoxazin-3-ones as determinants of plant biotic interactions 
Organisation University of Bern
Country Switzerland 
Sector Academic/University 
PI Contribution The BB/L027925/1 is a research consortium involving different partners from Europe
Collaborator Contribution Exchange of knowledge and resources
Impact the collaboration under this grant has just started, so no outcomes yet
Start Year 2014