Transcriptional regulation of RAM1, a central regulator ofarbuscule branching in arbuscular mycorrhiza symbiosis

Arbuscular mycorrhiza (AM) is an ancient symbiosis, established between 80% of land plants and obligate biotrophic fungi belonging to the class glomeromycotina. AM is an essential component in natural ecosystems, as it plays a major role in the global carbon cycle, enhances plant growth in nutrient deficient soil and is thus believed to sustain whole environ such as tropical rain forests. It also has a great fertilizing potential for sustainable practices in agriculture. Crucial for this symbiosis is the formation of highly branched tree-like structures called arbuscules by the fungus, inside root cortical cells of the host plant. These fungal structures deliver mineral nutrients after taking them up from the soil via extraradical hyphae, mainly phosphate and nitrogen, which are difficult to access for the plant. In turn, the fungus receives up to 20% of photosynthetically fixed carbon. Arbuscule formation is accompanied by massive transcriptional changes in the colonized cell. In addition, the cell undergoes subcellular rearrangements to accommodate the arbuscule. This is associated with the formation of a plant-derived membrane, called peri-arbuscular membrane, which surrounds the arbuscule and separates the fungal hyphae from the plant cytoplasm. The well-ordered and complex AM developmental steps, are regulated by the plant and depend on its nutritional status. Although arbuscule development is crucial for this symbiosis, the molecular basis of its development is poorly understood. A Lotus japonicus plant mutant reduced and degenerate arbuscules (red) found in a former study by forward genetics screen is perturbed in arbuscule development. To identify plant genes essential for arbuscule development, we investigated genes perturbed in red. Rough mapping indicated presence of two mutations in red, causative for the arbuscule phenotype. Complementation analysis confirmed causative mutations in a gene encoding a GRAS-type transcription factor named REDUCED ARBUSCULAR MYCORRHIZA 1 (RAM1) and in a gene encoding a lipid biosynthesis enzyme GLYCEROL 3-PHOSPHATE ACYL TRANSFERASE 6 (GPAT6/RAM2). In this doctoral thesis, I found that the AM symbiosis-specifically induced gene RAM1, is a principal regulator of arbuscule development. It is directly regulated by a complex of CYCLOPS and DELLA. CYCLOPS, is a DNA-binding transcription factor and a central regulator of symbiotic signaling and DELLA is a negative regulator of hormonal gibberellic acid (GA) signaling. The CYCLOPS-DELLA complex activates RAM1 expression via binding of CYCLOPS to a novel cis-element in the RAM1 promoter. Thus, we presented for the first time a target gene of CYCLOPS in AM symbiosis and a regulatory node integrating symbiosis (CYCLOPS) and hormonal GA signaling (DELLA). This direct connection may be important for the plant to connect symbiosis with its nutritional and therefore physiological status. Further, I revealed that RAM1 acts as a transcriptional activator of genes required for AM development, downstream of CCaMK and CYCLOPS. Ectopic expression of RAM1 induced AM-specific genes such as RAM2 in absence of AM-fungi. In frame of another thesis, they showed that RAM2 participate in an AM-specific lipid biosynthesis pathway and is essential for arbuscule development. RAM2 acts downstream of another lipid biosynthetic gene DIS (encoding s-keto-acyl ACP synthase I), which is also indispensable for arbuscule development. RAM2 uses C16:0 fatty acids synthesized by DIS as substrates for synthesis of s-monoacylglycerol. C16:0 is the predominant form of fatty acid found in AM fungi. Textbook knowledge exhibited carbohydrate as the only form of carbon supplied to the AM fungus, which is subsequently used to synthesis lipids. However, whole genome sequence analysis indicated that AM fungi lack genes encoding protein responsible for the de novo synthesis of C16:0 fatty acid. They further showed that the lipid containing C16:0 fatty acid synthesized by RAM2 is supplied to AM-fungi as a plant-derived carbon source. Arbuscule development can be conceptually divided into distinct steps by plant mutants, indicating that the respective gene product regulates the step-wise development of arbuscule. Accumulating evidences indicate transcriptional changes during arbuscule development occurs in successive but overlapping waves. For example, genes upregulated in the arbuscule containing cells might be also activated in neighboring cells preparing to accommodate arbuscule. These cells undergoing subcellular rearrangement forming a pre-penetration apparatus (PPA) do not have visible fungal structures. Transcriptomic analysis from cells containing only visible fungal structure, limit to relate the gene activation to individual stages of arbuscule development and PPA formation. To correlate the promoter activity of genes with the precise stages of arbuscule development, I designed a construct which allows visualization of the fungus in living roots due to accumulation of fluorescent protein mCherry in the apoplastic space surrounding the fungal hyphae. AM specific SbtM1 promoter used to drive mCherry is active across all stages of arbuscule development including cells undergoing rearrangement to form PPA. Using this construct, I showed that DIS and RAM2 promoters are activated during all the stages of arbuscule maturation, but become inactive during arbuscule degeneration.