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Plant genetic determinants controlling arbuscular mycorrhizal fungal growth through the plant cell wall.

Final Report Summary - PLANTMYCCELLWALL (Plant genetic determinants controlling arbuscular mycorrhizal fungal growth through the plant cell wall.)

Feeding of the future population is one of the most important challenges nowadays. For this, the actual agriculture needs to be improved and evolve to a more sustainable agriculture. The use of bio-fertilizers as Arbuscular Mycorrhizal Fungi (AMF) is one of the possible solutions. AMF colonize most of the land plants in order to provide them with a better uptake of nutrients, mainly phosphate, from the soil. During AM symbiosis there is an extensive communication between partners. To establish this interaction plant needs to modify their cell wall to allow the AMF to colonize cortical cells, where it forms the highly branched hyphae structures called arbuscules. However, even though the knowledge about the signaling pathways has increased in recent years, several biological aspects of this association are still unknown. Cell walls are a complex matrix surrounding the plant cells that provide rigidity and control cell growth, development and shape, however, little is known about the role of this first cellular barrier encountered by the AM hyphae.

Our main goal with this project is to understand the molecular mechanisms controlling the growth cell to cell of the fungal hyphae through the plant cell wall to establish a fruitful interaction. How the plant controls this entrance into the plant cell of the fungal hyphae to reach the cortical cell layer were to create the arbuscule, main phosphate exchanger. For this, different hypotheses have been here postulated and candidate genes were selected as possible regulators of the AM symbiosis in Brachypodium/Glomus versiforme interaction.

Microscopy analyses show the swelling of the hyphae in the contact point with the plant cell wall, but due to the lack of a specific PD-marker in Brachypodium cells, the identity of this contact point remains still unknown. The analysis of 17 different Brachypodium accession lines showed similar intracellular fungal growth patterns or possibly a mix of inter- and intracellular growth patterns, these lines could potentially be used to identify genes controlling growth through the cell wall and to identify the exact entrance point.

Due to the lack of specific mutants for our candidate genes, we generated CRISPR/Cas9-mediated mutants of the AM-related cell wall genes in the grass species Brachypodium distachyon. The CRISPR/Cas9 boosted by the t-RNA endogenous machinery system results in a highly efficient mutagenesis in Brachypodium plants. Different alleles were obtained, and at least three independent lines were homozygously edited in the T0 plant generation, for most of the candidate genes. To further explore into the role of cell wall-related genes an Agrobacterium rhizogenes-mediated root transformation (ARMT) has been developed. This transformation allows the study of protein localization in root cells in a relatively short time experiment. Different constructs have been successfully tested. Even if the protocol is easily reproducible and 40% of the seedlings are transformed, the system is not suitable for AM symbiosis studies; the piece of roots transformed is not big enough to be successfully colonized. Therefore, stable lines for protein localization and expression patterns studies were generated. As control, well-known AM-related genes were also edited by CRISPR/Cas9 machinery in Brachypodium plants and resulted mutants were analyzed during AM symbiosis.

During the return phase, a Brachypodium-colonization system was set up at CRAG. All the CRISPR/Cas9-mediated mutants were therefore tested and phenotypically analyzed during AM symbiosis. Quadruple mutants were also genotyped and tested. Protein localization and expression patterns studies were performed for the candidate genes. All these experiments suggest the relevance of several of our candidate genes during the AM symbiosis, showing a possible role in cell wall rearrangement and/or arbuscule recycling. Auxin studies were also in silico performed suggesting the absence of a direct role of auxin and the expression of our candidate genes.

Collectively, in this project we have generated novel and valuable information in the study of arbuscular mycorrhizal symbiosis in Brachypodium plants, genes editing, transient root transformation and mechanisms involved in cell wall reorganization during AM symbiosis.

Our findings will provide new valuable genetics and molecular tools in Brachypodium plants, enhancing and improving cereals symbiosis research. Results here obtained reveal the importance of a proper cell wall rearrangement during symbiosis and a well-coordinated interaction. These new insights into Brachypodium plants symbiosis could be extrapolated to important crops species (e.g. cereals) in the way to a more sustainable agriculture using arbuscular mycorrhizal symbiosis as a natural way to acquire more nutrient from the soil. Results here obtained will also inspire more research efforts in the plant/AM interaction and could also give awareness of new breeding programs to improve crop performance.