Community Research and Development Information Service - CORDIS

Periodic Report Summary 1 - PLANTMYCCELLWALL (Plant genetic determinants controlling arbuscular mycorrhizal fungal growth through the plant cell wall.)

Summary description of the project objectives.
Plant cells are surrounded by a complex matrix of polysaccharides, the plant cell wall. This cell wall provides the cell with a first barrier against pathogens and its environment, and is essential for plant growth and development. Beneficial fungi, such as arbuscular mycorrhiza fungi (AMF), also run into the plant cell wall on their way to colonize the root cortical cells, in which they form a highly branched hyphae called arbuscule. It is in these arbuscules, where the main exchange of nutrients takes place. This beneficial association is characterized by the exchange of mineral nutrients, mainly phosphate, from the fungi to the plant and in return the plant provides the fungi with carbohydrate and lipids. This association has great potential use in agriculture to improve nutrient uptake from soil to the plant. In this project, by using reverse genetic, cellular and microscopy analyses, we aim to decipher how the AMF are able to cross and grow through and between the plant cell wall to reach the cortical cell and establish the symbiosis. The plant genetic and cellular program controlling the establishment of the symbiosis has been well characterized but relatively little is known about how the plant addresses the fungal growth through the cell wall. We hypothesize that this process is controlled and guided by the plant by modifications to its cell wall. We take advantage of RNAseq data from the lab showing that several cell wall-related genes are differentially expressed during symbiosis and could be involved in controlling events at the cell wall. To accomplish the proposed objective, we generated CRISPR/Cas9-mediated mutants of these AM-related cell wall genes in the grass species Brachypodium distachyon to unravel their molecular function during AM symbiosis. As Brachypodium distachyon shows mostly intracellular fungal growth through the plant cell wall we propose to study the symbiotic association of Brachypodium distachyon and the fungus Glomus versiforme. Increased knowledge of how the plant controls these events will enhance the use of AMF in sustainable agriculture improving mineral nutrients uptake and avoiding the use of excessive fertilizer.

Description of the work performed since the beginning of the project.
CRISPR/Cas9 genome editing for Brachypodium distachyon was set-up taking advantage of a vector system developed for rice. To first test the performance of the vectors, we generated CRISPR/Cas9 lines of the Brachypodium phytoene desaturase gene (BdPDS). Most of these lines showed an expected albino phenotype indicating that the vectors system was suitable. As a second baseline control, we generated CRISPR/Cas9 mutant lines for different known AM-related genes in Brachypodium distachyon. Preliminary results showed that the mutant lines obtained showed similar AM-phenotype to mutants previously described in other plants species. Both of these evaluation experiments gave us evidence that the CRISPR/Cas9 system is suitable for generating loss-of-function mutant lines and that we can effectively assess AM-related phenotypes. Due to these successful results, and in order to gain a better knowledge about how the plant controls AM fungal growth through the cell walls, we generated CRISPR/Cas9 mutant lines for the cell wall candidate genes. Again genotyping indicated a high efficiency of gene editing and we have obtained single mutant lines for all the candidate genes. Double and triple mutants have been obtained for the BdGLPs lines and BdGLPs quadruple lines are expected soon. Together with the mutant lines, promoter fusions to GUS and protein fusions with mCherry have been generated and will allow analysis of the expression patterns and protein localization of the candidate genes respectively during symbiosis. All these lines together will allow us to better decipher the molecular function of the candidate genes during AM symbiosis, and to identify their roles in cell wall-related fungal growth. Additionally, a Brachypodium transient root transformation system has been generated, which is suitable for analysis of subcellular reporter genes and will also assist in the analyses of cell wall-related genes. Finally, we also surveyed the symbiosis phenotypes of 17 different Brachypodium accessions to determine whether there was natural variation in cell wall colonization patterns. All of the accessions 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.

Description of the main results achieved so far.
Due to the lack of available Brachypodium mutants in our candidate cell wall-related genes, we generated our own mutants. To achieve this, we took advantage of the CRISPR/Cas9 genome editing approach. This had not been previously established for Brachypodium but using a vector system developed for rice, we were able to obtain genome editing with high efficiency. 16 out of 18 guide RNAs from 11 different target genes have resulted in positive genome edits. At least 2 or more alleles per guide RNA have been obtained, and at least three independent homozygous edited plants per construct have been obtained directly in T0 lines. Double and triple mutants were also obtained directly from the first generation. Interesting preliminary results have emerged from the AM-related mutants and results from the cell wall-related genes are in progress. These mutant lines are critical to decipher cell wall events during the fungal growth. Furthermore, by microscopy analysis we studied the intracellular fungal growth in Brachypodium lines expressing a plasma membrane-GFP marker gene, which allowed us to follow fungal growth into the plant cells. Focusing our attention on the cell wall crossing points we reveal that the fungal hyphae swell at the crossing point and show a morphology similar to that observed in during the interaction of the fungus Magnaporthe grisea with rice plants. Callose staining was performed but the results were not highly conclusive. Therefore, the nature of this specific crossing points and the way the crossing is regulated remain unknown. But with the help of the cellular marker genes identified and the Brachypodium root transformation that we developed, we expect to obtain more insight into the specific fungal crossing points in the near future. The Brachypodium root transformation developed here provides an easy and reproducible technique that allows, in less than 6 weeks, the localization of the protein of interest in Brachypodium roots cells. Similar to other transient techniques, this system allows to visualize by microscopy in which cellular compartment your protein is acting, with the benefit that you are using directly roots of the plants of interest and not a heterologous system.

The expected final results and their potential impact and use.
With this project, we expect to identify and characterize the first plant cell wall proteins controlling the AM symbiosis in Brachypodium distachyon. By developing several novel genetic, molecular and cell biology methods for Brachypodium distachyon we provide valuable tools to aid and enhance grass and cereal symbiosis research. Brachypodium distachyon is emerging as an important model species for agronomically and economically important cereals crops such as wheat and barley. Gaining further insight into the regulation of the AM symbiosis will help in its deployment in sustainable agriculture where it has the potential to improve nutrient acquisition and enhance crop performance. Results obtained here will open new avenues for understanding the AM symbiosis and could also be extrapolated to better understand plant-microbe interactions and the control of important plants diseases.

Reported by

CENTRE DE RECERCA EN AGRIGENOMICA CSIC-IRTA-UAB-UB
Spain

Subjects

Life Sciences
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