Molecular development of Arbuscular Mycorrhizal symbiosis - Molecular Biology

To live in environments where nutrients are limited, plants engage in an endosymbiosis with arbuscular mycorrhizal (AM) fungi.

These fungi colonize plant roots and are hosted inside root cortex cells, where highly branched hyphal structures called arbuscules are formed (Figure 1). There, the fungi deliver scarce minerals, especially phosphate and nitrogen sources, that they take up from the soil to the plant for which they get sugars and lipids in return. The AM symbiosis is very ancient and occurs in the vast majority of all land plants, which makes it one of the agriculturally and ecologically most important endosymbioses in plants. Understanding how AM fungi are accommodated inside plant roots and how nutrient exchange is controlled is of major importance as it determines the symbiotic efficiency of the interaction. Therefore, we use molecular, genetic- and cell-biological approaches to study the molecular mechanisms by which AM fungi are accommodated to optimize symbiotic nutrient transfer.

Thesis subjects

1. AM effectors controlling symbiosis

To get a better insight into the arbuscular mycorrhizal symbiosis, we recently sequenced the first AM fungus Rhizophagus irregularis. Using this genome sequence we are now addressing the question how these fungi manage to colonize such an exceptional wide range of plant species, which must involve an extraordinary level of compatibility. We hypothesize that secreted effector proteins, that are predicted from the genome sequence, play a role in arbuscule formation and/or function (Figure 2). Therefore, we aim to identify fungal effectors and study whether they are translocated to the host cell, with which host proteins they interact, what role they play in symbiosis and whether they help determine symbiotic efficiency.

This research combines a range of techniques; laser microdissection for cell-specific transcriptome analyses, production of AM effectors using expression systems, qPCR and RNAseq analyses, defence and symbiosis assays, plant transformation, confocal fluorescence microscopy, biochemical analyses (co-IP, mass-spec, Y2H) and various reverse genetic approaches based on Golden Gate cloning technology.

Figure 2. Working model: AM effector proteins controlling arbuscule formation and function.
Figure 2. Working model: AM effector proteins controlling arbuscule formation and function.

2. Arbuscule development

The ability to host AM fungi inside cells requires a reprogramming of root cortex cells, which is controlled by a signaling cascade initiated between fungus and plant. Several key transcription factors controlling arbuscule formation, branching and maintenance have been identified. From our cell-specific transcriptome analyses additional potential key transcriptional regulators have been identified. A major challenge will be to unravel how these factors work together in a transcriptional network to control arbuscule development.

In this research reverse genetic analyses (CRISPR/RNAi/transposon-tagged lines), protein-protein interactions and transcriptome analyses are used to get insight into the transcriptional network that controls arbuscule development.

3. Genetic variation within AM fungi

One of the key questions in AM biology, both from a fundamental as well as applied point of view, i.e. to create superior inoculum, is to understand at a molecular level what determines plant response to AM fungi. It has been shown that some fungi improve plant growth much more than others. Such variation is not only observed between AMF species or isolates of the same species but even within fungal individuals. For example, single spore lines derived from a single AM fungus were shown to enhance rice growth up to five times. However, the molecular basis for such functional diversity is still largely unknown. We try to study how genetic variation between nuclei that populate the common hyphal network (Figure 3) affects differences in plant performance. Therefore, we examine the genetic variation within a field isolate of Rhizophagus irregularis, how this variation is distributed over different nuclei, what effect plant species identity has on this genetic variation and aim to identify genetic variants (alleles) that correlate with differences in plant performance. In this project we combine next-generation sequencing with single spore and single nucleus analyses (through fluorescence associated cell sorting (FACS). In addition to wet lab experiments, the project also offers a possibility to focus more on bioinformatics analyses of next-gen sequencing data.

Figure 3. The hyphal network of an AM fungus forms a continuous cytoplasmic compartment where nuclei (stained green) move and migrate. Spores typically contain several hundreds of nuclei that migrate from the hypha into the spore and there is never a stage during the AM life cycle where only one nucleus initiates the next generation.
Figure 3. The hyphal network of an AM fungus forms a continuous cytoplasmic compartment where nuclei (stained green) move and migrate. Spores typically contain several hundreds of nuclei that migrate from the hypha into the spore and there is never a stage during the AM life cycle where only one nucleus initiates the next generation.