A new presymbiotic recognition mechanism from cereals enabling root invasion by arbuscular mycorrhizal fungi.

Symbioses are fundamental to life on the Earth. One such example, known as arbuscular mycorrhizal (AM) symbiosis, evolved between fungi and plants to facilitate mineral uptake from the soil around 450 mya. This symbiotic arrangement is so widespread that it impacts on ecosystem productivity and global nutrient cycles, and is considered of fundamental importance for crop productivity and sustainability. The development of AM symbioses relies on the well-coordinated exchange of signals to achieve perception and reprogramming of the two interacting organisms. However, due to the complex genetic architecture of the AM fungi, little is known about the components present in the fungus that allow interaction with the plant. Instead, identification and analysis of plant mutants have formed the bedrock of our understanding of molecular processes that underpin the inter-species crosstalk allowing this symbiosis. This proposal centers on presymbiotic signalling to close the gap in our understanding on how perception and reprogramming is achieved to initiate physical plant-fungal engagement in AM symbiosis.

Previously, my group had identified the maize mutant independent of arbuscular mycorrhizal symbiosis (ina) that showed a complete early block against fungal root invasion. A unique feature of this mutant is that the defect could be overcome when the fungus was either supported through hyphal connections with wild type plants, or through the addition of wild type root exudates. This suggested that the mutant exudates lacked a critical component. Supported by the BBSRC and in collaboration with the industrial partner Corteva, applying positional cloning and CRISPR/Cas9-based reverse genetics, led to the identification of an essential ABC transporter encoding gene.

AM fungi are fatty acid auxotrophs and rely on plants for the provision of lipids. Fully established AM symbioses are marked by the formation of intracellular fungal arbuscules in the inner cortex tissue. It is here where the plant delivers fatty acids to the fungus in exchange for soil minerals. Lipid production is induced in arbuscule-hosting plant cortex cell and exported towards the fungus by a heterodimer of the half-size ABCG transporters Stunted Arbuscule1 and 2 (STR1 and STR2). Consistent with its indispensable role in supporting AM fungi with essential organic carbon, the components of the lipid biosynthetic and delivery pathway are specifically conserved across the mycorrhizal plants in the plant kingdom. Unexpectedly, the maize gene that conditions initial plant-fungal engagement is STR2. STR1 on the other hand, appears not to be involved with the presymbiotic stage since str1 mutants in a variety of plant species show fungal colonisation. On the basis of the strict occurrence of STR2 in genomes of AM-competent plants and the loss of susceptibility of str2 mutants to AM fungi, we hypothesise that STR2 is a core requirement for defining AM host specificity, which would be a ground breaking discovery for this globally prevalent symbiosis.

Maize and rice offer highly attractive plant system for an integrated combination of biochemical, molecular, cell biological, genetic and metabolomic techniques. The equivalent str2 mutant phenotype obtained for both plant species permits the coordinated design of suitable experimental approaches in both of them. The methodological expertise required for all the proposed experimental work is available across the Paszkowski, Eastmond and Wallington labs.
Transformation of rice and Medicago truncatula is efficiently run by joint-applicant Emma Wallington, which is particularly important for this proposal as each of the three objectives relies on the swift and reliable generation of the transgenic plant material. We produce all our constructs by Golden Gate cloning and base the selection on hygromycin or kanamycin.
The Paszkowski lab has extensive experience with live laser scanning confocal microscopy of rice roots, including BiFC. At the Crop Science Centre, the newly installed microscopy suite enables cutting edge laser scanning confocal microscopy on site.
For the unbiased identification of STR2 interactors from rice, an estradiol-inducible system is preferred to achieve control of induction and high quantities of the GFP-tagged STR2 protein. In our hands, the presence of the estradiol does not interfere with AM symbiosis development. Immunoprecipitation follows routine protocols and the pulled-down proteins will be analysed at the Cambridge Centre for Proteomics via spectrometry.
For metabolomic analysis of str2 root exudate and surface lipids (objective 3), both polar and nonpolar metabolites will be analysed from exudate by high res. LC-MS/MS, and surface lipids by GC-MS, using pipelines that are well established at Rothamsted Research. Most metabolites of interest can be identified using accurate mass data and MS/MS fragmentation patterns, but expertise in purification and 1H-NMR will also be available should further structural analysis be required.