Periodic Reporting for period 5 - RECEIVE (Regulatory networks of plant cell rearrangement during symbiont accommodation)
Período documentado: 2023-02-01 hasta 2024-07-31
Land plants carry a genetic heritage from the earliest conquerors of land: the ability to form an intimate interaction with beneficial fungi that deliver mineral nutrients to the plant. The earliest land plants did not form roots. During the early colonisation stages of the land, the absence of roots was likely compensated with the help of a far-reaching hyphal network that scavenges nutrients in return for plant produced carbon sources that enable the fungus to grow. Despite the evolution of roots, AM fungi remained essential to bridge depletion zones of mineral nutrients such as phosphate that form around plant roots due to limited diffusion rates. This long distance symbiotic mineral nutrient delivery is likely important for plant growth and survival in natural environments, which explains why the AM symbiosis has been a stable feature throughout the evolution of the land plants and secondary loss of the symbiosis is the exception. Furthermore, AM has the potential to improve plant performance in sustainable agricultural practices with reduced mineral fertilizer input. However, classical crop breeding programs under high fertilizer conditions are excluding the potential of AM from the selection process of high-performance genotypes and may even result in cultivars with reduced AM compatibility, which are less suitable for sustainable agriculture. Thus, understanding the molecular underpinnings of AM development and function represents an important challenge for breeding AM-optimized crops.
For symbiosis to occur, the fungus colonizes the interior of plant roots and forms highly branched, tree-like hyphal structures, the arbuscules, inside root cortical cells. Facilitated by their strongly enlarged membrane surface area, the arbuscules are the key structures, which release mineral nutrients to plant cells, that have been scavenged by the extended extraradical hyphal network from the soil. Arbuscule formation is under the control of the host and involves cellular remodelling of already differentiated cortex cells within the tissue context. Reorganization of plant cells to host arbuscules seems to occur in distinct steps, which can be dissected by plant mutants and are accompanied by expression of distinct marker genes (reviewed by Gutjahr and Parniske, 2013), suggesting that the plant cell has to fulfil distinct tasks in a spatio-temporally coordinated manner to host the arbuscule.
RECEIVE rests on the central hypothesis that the steps of plant cell rearrangement allowing arbuscule formation are accompanied by distinct transcriptional waves, which crucially determine the developmental progress from stage to stage. The characterisation of these waves and the identification of the underlying transcriptional regulons is the main focus of RECEIVE.
For symbiosis to occur, the fungus colonizes the interior of plant roots and forms highly branched, tree-like hyphal structures, the arbuscules, inside root cortical cells. Facilitated by their strongly enlarged membrane surface area, the arbuscules are the key structures, which release mineral nutrients to plant cells, that have been scavenged by the extended extraradical hyphal network from the soil. Arbuscule formation is under the control of the host and involves cellular remodelling of already differentiated cortex cells within the tissue context. Reorganization of plant cells to host arbuscules seems to occur in distinct steps, which can be dissected by plant mutants and are accompanied by expression of distinct marker genes (reviewed by Gutjahr and Parniske, 2013), suggesting that the plant cell has to fulfil distinct tasks in a spatio-temporally coordinated manner to host the arbuscule.
RECEIVE rests on the central hypothesis that the steps of plant cell rearrangement allowing arbuscule formation are accompanied by distinct transcriptional waves, which crucially determine the developmental progress from stage to stage. The characterisation of these waves and the identification of the underlying transcriptional regulons is the main focus of RECEIVE.
With the project RECEIVE we adopted methods new to the laboratory and to the field of arbuscular mycorrhiza, required for characterizing and identifying transcriptional and translational waves underlying plant cell rearrangement for arbuscule development. Furthermore, we conducted experiments corresponding to the four work packages of the project and discovered new players involved in transcriptional regulation of arbuscule accommodation inside plant cells.
In WP1 we have established a protocol for spatio-temporally resolved translating ribosome affinity purification (TRAP) for arbuscular mycorrhizal Lotus japonicus roots. We habe characterized translational events in plant cell rearrangement during arbuscule development. Furthermore, we have conducted transcriptome profiling of Lotus japonicus mutants stalled at different stages of arbuscule development to identify the gene cohorts transcribed at each developmental step and cis-elements in promoters of co-expressed genes.
In WP2, we have performed reverse genetics and identified transcription factors important for arbusuclar mycorrhiza and arbuscule development. One family of them called PHOSPHATE STARVATION RESPONSE (PHR) regulates arbuscular mycorrhiza development in response to phosphate starvation (Das et al 2022).
In WP3 we have found transcriptional targets (using DAP-Seq, ChIP-Seq and Cut&Run) and interacting proteins (using proximity labelling) of important transcriptional regulators of arbuscule development and are currently characterizing the function of interactors and targets.
In WP4 we have established markers for stages of arbusucle development based on fluorescent reporters.
The newly identified transcription factors can be used in the future for synthetically manipulating arbuscular mycorrhiza development to gain further insights into this process and potentially for application in agriculture or fungal inoculum production.
In WP1 we have established a protocol for spatio-temporally resolved translating ribosome affinity purification (TRAP) for arbuscular mycorrhizal Lotus japonicus roots. We habe characterized translational events in plant cell rearrangement during arbuscule development. Furthermore, we have conducted transcriptome profiling of Lotus japonicus mutants stalled at different stages of arbuscule development to identify the gene cohorts transcribed at each developmental step and cis-elements in promoters of co-expressed genes.
In WP2, we have performed reverse genetics and identified transcription factors important for arbusuclar mycorrhiza and arbuscule development. One family of them called PHOSPHATE STARVATION RESPONSE (PHR) regulates arbuscular mycorrhiza development in response to phosphate starvation (Das et al 2022).
In WP3 we have found transcriptional targets (using DAP-Seq, ChIP-Seq and Cut&Run) and interacting proteins (using proximity labelling) of important transcriptional regulators of arbuscule development and are currently characterizing the function of interactors and targets.
In WP4 we have established markers for stages of arbusucle development based on fluorescent reporters.
The newly identified transcription factors can be used in the future for synthetically manipulating arbuscular mycorrhiza development to gain further insights into this process and potentially for application in agriculture or fungal inoculum production.
Although a small number of transcription factors regulating arbuscule development has been identified, prior to the project many transcriptional regulators of arbuscule development and their exact function remained unknown. Furthermore, it was not understood how they interact in a network. By deciphering the transcription factors and in particular their target genes, driving arbuscule development from step to step we increased the knowledge base on transcritptional regulators, their complexes and their targets that drive arbuscule development and identified cellular functions (represented by the target genes), required for steps in plant cell remodelling.
By establishing spatio-temporally resolved TRAP-Seq to characterize translatomes associated with different steps of arbuscule development, we moved TRAP-seq beyond the state of the art, as to our knowledge this technique has been applied in plants at spatial tissue-specific but not at “spatial and temporal” resolution.
Arbuscular mycorrhiza (AM) symbiosis of most land plants with fungi of the glomeromycotina improves plant nutrition and can have a strong impact on growth and yield. Thereby it bears the potential to become an important addition to sustainable agricultural practices with reduced chemical fertilizer input. Understand the molecular basis of the development of this symbiosis can provide important knowledge for the development of AM-optimized crops and for engineering robustness of the symbiosis to perturbing environmental conditions, which normally lead to inhibition of symbiosis development. Thereby, our research can contribute to sustainable agriculture and food security.
By establishing spatio-temporally resolved TRAP-Seq to characterize translatomes associated with different steps of arbuscule development, we moved TRAP-seq beyond the state of the art, as to our knowledge this technique has been applied in plants at spatial tissue-specific but not at “spatial and temporal” resolution.
Arbuscular mycorrhiza (AM) symbiosis of most land plants with fungi of the glomeromycotina improves plant nutrition and can have a strong impact on growth and yield. Thereby it bears the potential to become an important addition to sustainable agricultural practices with reduced chemical fertilizer input. Understand the molecular basis of the development of this symbiosis can provide important knowledge for the development of AM-optimized crops and for engineering robustness of the symbiosis to perturbing environmental conditions, which normally lead to inhibition of symbiosis development. Thereby, our research can contribute to sustainable agriculture and food security.