Project 2: Innovation by nature

Plant traits involved in maximizing growth and protective functions of root microbiota

Project leaders: Dr. Peter A.H.M. Bakker & Prof.dr.ir. Corné M.J. Pieterse, Utrecht University

I. SCIENTIFIC DESCRIPTION

Introduction

In natural and agricultural ecosystems, plant health and productivity is greatly influenced by microbiota in the soil (Berendsen et al., 2012; Philippot et al., 2013). To establish, maintain, and shape their microbiome, plants secrete up to 40% of their photosynthetically fixed carbon into the rhizosphere (Bais et al., 2006). Plant growth-promoting rhizobacteria (PGPR) are known to antagonize soil-borne pathogens, improve plant growth and nutrition, and prime the plant immune system (De Vleesschauwer and Höfte, 2009; Lugtenberg and Kamilova, 2009). However, these microbiome traits have not been major targets of classical breeding. Detailed knowledge on the molecular and genetic basis of plant-beneficial microbe communication will be essential for the development of biological control agents and sustainable future crops that better maximize profitable and protective functions from their root microbiome.

The rhizosphere microbiome promotes plant health and nutrition

The UU group pioneered the research field of PGPR-mediated plant responses. Using various crop species (e.g. tomato, radish, cucumber, wheat, flax, rice, and bean) and Arabidopsis as a model, important microbial determinants and major components of the PGPR-induced systemic resistance (ISR) signaling pathway have been uncovered (Bakker et al., 2007; Pieterse et al., 2014). We demonstrated that ISR is controlled by the hormones jasmonic acid and ethylene and depends on the transcriptional (co)activators NPR1, MYB72, and MYC2 (Pieterse et al., 1998; Pozo et al., 2008; Van der Ent et al., 2008). In addition, we showed that ISR-inducing rhizobacteria do not directly activate the plant immune system, but primes plants for enhanced defense resulting in a broad-spectrum resistance (Pieterse et al., 2014). Soil-borne microbes also have critical roles in improving plant mineral nutrition by enhancing bioavailability of minerals or improving the root’s exploratory capacity (Zamioudis et al., 2013). By studying cell type-specific markers, we uncovered a crucial role of auxin in PGPR-elicited changes in the root system architecture (Zamioudis et al., 2013). Moreover, we discovered a novel iron uptake mechanism in plants that is stimulated by PGPR (Zamioudis et al., 2014). Hence, members of root microbiota evolved sophisticated strategies that simultaneously stimulate systemic immunity, change root architecture, and improve iron nutrition in their hosts (Fig. 3).

Major aims of the project

The proposed research follows a sophisticated, multidisciplinary approach with model and crop species to reach to following major goals:

Characterize early root cell-type specific molecular changes that are initiated by beneficial microbes and are crucial for stimulating plant immunity and/or plant growth.
Identify microbials that are selectively enriched in the root microbiome when plants are under pathogen pressure, and study their role in enhancing plant growth and protection.
Generate knowledge that: a) provides insight into novel plant traits/genes that maximize profits from the plant’s microbiome and can be utilized in plant breeding; and b) can be used to develop high-throughput tests for the functionality of microbial products that stimulate plant growth and health via its microbiome.

Research plan

Workpackage 2.1. Systems biology of root-beneficial microbe interactions:

Beneficial plant responses to root-colonizing microbes start with recognition at the root-soil interface. We aim to understand how signals from beneficials are perceived, sensed and transduced in the different root cell types to activate responses that drive the whole-plant body towards enhanced growth and immunity.
To map root cell-type-specific molecular responses, we will analyze the dynamics of gene regulatory networks of distinct cell types of Arabidopsis roots in response to PGPR using Fluorescens Activated Cell Sorting (FACS) and next-generation genomics and bioinformatics technology (Van Verk et al., 2013). A set of Arabidopsis reporters lines expressing GFP in defined root cell types (Birnbaum et al., 2005)) is available. Discrete cell populations will be isolated by bulk root protoplasting followed by FACS (Birnbaum et al., 2005). Sorted protoplasts will be subjected to RNA-seq for transcriptomics.
Pseudomonas fluorescens WCS417 will be used as a model strain as it exerts multiple positive effects on plant growth and health (Fig. 3). In addition, highly effective microbial strains and consortia identified in the other projects will be tested in collaboration with the industrial partners.
The biological function of identified genes and gene clusters will be studied using the molecular genetic toolbox of Arabidopsis, which will allow e.g. verification of cell type-specific expression, gene knockout analysis, and ectopic overexpression of identified genes. Our extensive expertise on the molecular biology of ISR and plant growth promotion will allow a detailed functional characterization of the identified genes.
A selection of identified PGPR-induced genes/processes will be further characterized/ validated in PGPR-crop combinations (to be determined in conjunction with the industrial partners).
Knowledge gained in this project will be used to develop marker-gene-based high-throughput tests to evaluate beneficial microbes for their effectiveness in stimulating plant health and growth. These tests will also be used for beneficial microbes or microbial consortia identified in projects 1 and 3.
Identified genes represent plant traits that may be used in plant breeding programs to select for plant traits that enable plants to better profit from their natural or introduced (e.g. via bio-osmopriming) microbiota.

Workpackage 2.2. Directed evolution of the microbiome under pathogen pressure:

PGPR harness a great capacity for agriculture as they are able to control disease and increase crop yields in an environmentally-friendly manner. Development of effective biocontrol agents can be accelerated by a thorough understanding of how plants shape their microbiome. Upon pathogen attack, plants actively recruit bacteria that can assist in fending off its attacker (Berendsen et al., 2012). In addition, plants can recruit bacteria that promote plant growth and mitigate abiotic stresses (Marasco et al., 2012). We aim to explore the dynamic changes of the root microbiome in response to foliar pathogen infection, and functionally analyze PGPRs that are recruited to the rhizosphere.
We will investigate the evolution of functions and composition of the root microbiome of plants that are under pathogen attack. Arabidopsis (grown in its natural soil), and 2 crop plants (grown in soil or other growth substrates), selected in collaboration with the industrial partners, will be infected with necrotrophic and biotrophic pathogens. This will be done in multiple consecutive growth cycles. After each plant growth cycle the conditioned soils will be monitored functionally for their abilities to promote plant immunity by quantifying expression levels of the pMYB72:GFP-GUS reporter in Arabidopsis (Zamioudis et al., 2014) (Fig. 3). The evolution of root microbiome composition will be analyzed by pyrosequencing of 16S rRNA gene amplicons (Bulgarelli et al., 2012) (with project 1.1). Based on the soil functional and the pyrosequencing data, specific members of the root microbiome that are associated with promoting plant immunity will be identified and tested. In a high-throughput system selected strains isolated from the three plant species will be tested for triggering ISR and iron uptake mechanisms, using the pMYB72:GFP-GUS reporter, and for stimulating enhanced root branching (Fig. 3) using the DR5:vYFP reporter (Zamioudis et al., 2013). Additional reporter lines may be developed and used based on the results of project 2.1.
Effects of bacterial strains that are positive in the reporter line screening will be validated in seedling growth and ISR assays in crop plants (with the industrial partners). New bacterial strains and consortia that stimulate plant growth and protect against disease and that are specifically selected by plants under stress will be identified. The identified strains will also be tested in the abiotic stress assays of project 3.
The reporter system will be used to cross-test microbial strains and consortia identified in projects 1 and 3. Moreover, they can also be used for high-throughput screening of existing microbial libraries for their capacity to induce plant immunity, enhance root architecture, or stimulate iron uptake.
In parallel changes in the chemical composition of root exudates of Arabidopsis, and the crop plants upon pathogen infection will be analyzed (project 1) to identify specific cues for attraction/activation of PGPR. Components of the root exudate that correlate with the directed evolution of a plant beneficial rhizosphere microbiome will be investigated for effects on chemotaxis and growth of the selected bacteria in vitro. Furthermore we will feed such key components to the seeds or roots of plants to investigate if the evolution of a plant beneficial root microbiome can be accelerated.

II. UTILISATION PLAN

Problem

In natural ecosystems, plants have co-evolved with complex microbial communities that fulfill important plant functions related to plant growth, vigor and defense. However, these traits provided by microbiome have not been major targets of classical breeding programs. By putting beneficial microbes to work in microbial agriculture, crop production can be improved in a sustainable manner.

Solution and economic value

By investigating the nature of the evolutionary partners of plants (the beneficial microbes), the mechanisms by which they are recruited to the root system, and how they promote plant growth and health will be highly instrumental in biocontrol and plant breeding approaches for beneficial plant-microbe interactions. Combining optimized microbial communities with crop genotypes that are better able to profit from beneficial functions in their microbiome will allow crop plants to produce and grow better, also under suboptimal climatic conditions. Hence, roots and their plant health-supporting microbiome hold the key to the next green revolution.

Translation and implementation

Depending on the crop, markers for maximizing profits from beneficial microbes may be integrated in breeding programmes within 5 years. Introduction time of mutant alleles that confer enhanced stress tolerance by the root microbiome into cultivars or breeding material, e.g. via TILLING, is dependent upon the crop and cultivar, but is also envisaged in 5 years. In the first two years of WP 2.2., beneficial microbes and microbial consortia will be identified and characterized. The robustness of the protective and plant growth promoting effects of these microbes will be validated in close collaboration with the industrial partners. Thus implementation of new bacterial products is expected during the final stages of the programme. Identification of plant traits and metabolites that contribute to the assembly of plant beneficial microbiomes will be established towards the end of the programme and thus implementation of the results in breeding programmes or the direct application of plant cues to accelerate the establishment of beneficial microbiomes is expected at the end of the programme. The supporting industrial partners are Corbion, BDS/MLS, Bejo seeds, RijkZwaan, ENZA, IncoTec, and Koppert. Their in kind and cash contributions will facilitate the use of experimental systems that are of relevance to the market situation. Moreover, promising results under controlled conditions can be quickly validated under commercial conditions. The applicability of the findings in the target crops will largely depend on the nature of the findings. Therefore, progress will be intensively monitored by the users committee. Target crops for utilization are selected in close collaboration with the industrial partners. The crop-specific expertise (e.g. marker development, disease resistance assays, breeding germplasm, implementation in breeding programmes) will be provided by the breeding companies.

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